asms - shimadzu · 2018. 11. 1. · felbamate gabapentin lamotrigine levetiracetam nitrazepam...

ASMSPoster collection

Clinical, Forensic and Pharmaceutical Applications

• Page 4Rapid development of analytical method for anti-epileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

• Page 11Determination of ∆9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

• Page 17Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample prepa-ration

• Page 23Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

• Page 29Simultaneous screening and quantitation of amphetamines in urine by on-line SPE-LC/MS method

• 6Single step separation of plasma from whole blood without the need for centrifugation ap-plied to the quantitative analysis of warfarin

• Page 42Development and validation of direct analysis method for screening and quantitation of amphetamines in urine by LC/MS/MS

• Page 48Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

• Page 54Application of a sensitive liquid chromatography-tandem mass spectrometric method to pharma-cokinetic study of telbivudine in humans

• Page 60Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

• Page 66Highly sensitive quantitative analysis of felodip-ine and hydrochlorothiazide from plasma using LC/MS/MS

• Page 73Highly sensitive quantitative estimation of geno-toxic impurities from API and drug formulation using LC/MS/MS

• Page 80Development of 2D-LC/MS/MS method for quan-titative analysis of 1�,25-Dihydroxylvitamin D3 in human serum

• Page 86Analysis of polysorbates in biotherapeutic prod-ucts using two-dimensional HPLC coupled with mass spectrometer

• Page 93A rapid and reproducible Immuno-MS platform from sample collection to quantitation of IgG

• Page 99Simultaneous determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS

• Page 103Low level quantitation of loratadine from plasma using LC/MS/MS

PO-CON1452E

Rapid development of analyticalmethod for antiepileptic drugs inplasma using UHPLC method scoutingsystem coupled to LC/MS/MS

ASMS 2014 ThP 672

Miho Kawashima1, Satohiro Masuda2, Ikuko Yano2,

Kazuo Matsubara2, Kiyomi Arakawa3, Qiang Li3,

Yoshihiro Hayakawa3

1 Shimadzu Corporation, Tokyo, JAPAN,

2 Kyoto University Hospital, Kyoto, JAPAN,

3 Shimadzu Corporation, Kyoto, JAPAN

2

Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

IntroductionMethod development for therapeutic drug monitoring (TDM) is indispensable for managing drug dosage based on the drug concentration in blood in order to conduct a rational and ef�cient drug therapy. Liquid chromatography coupled with tandem quadrupole mass spectrometry is increasingly used in TDM because it can perform selective and sensitive analysis by simple sample pretreatment. The UHPLC method scouting system coupled to tandem

quadrupole mass spectrometer used in this study can dramatically shorten the total time for optimization of analytical conditions because this system can make enormous combinatorial analysis methods and run batch program automatically. In this study, we developed a high-speed and sensitive method for measurement of seventeen antiepileptics in plasma by UHPLC coupled with tandem quadrupole mass spectrometer.

Figure 1 Antiepileptic drugs used in this assay

Experimental

UHPLC based method scouting system (Nexera X2 Method Scouting System, Shimadzu Corporation, Japan) is configured by Nexera X2 UHPLC modules. For the detection, tandem quadrupole mass spectrometer (LCMS-8050, Shimadzu Corporation, Japan) was used. The system can be operated at a maximum pressure of 130 MPa, and it enables to automatically select up to 96 unique combinations of eight different mobile phases and six different columns. A

dedicated software was newly developed to control the system (Method Scouting Solution, Shimadzu Corporation, Japan), which provides a graphical aid to configure the different type of columns and mobile phases. The software is integrated into the LC/MS/MS workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions are seamlessly translated into method files and registered to a batch queue, ready for analysis instantly.

Instruments

N

O NH2

Carbamazepine Carbamazepine- 10,11-epoxide

N

O NH2

O

Diazepam

N

N

O

CH3

Cl

Ethomuximide

NHCH3

CH3

O

O

Felbamate

O ONH2

O

NH2

O

Gabapentin

NH2

OH

O

N

N

NCl

Cl

NH2

NH2

Lamotrigine Levetiracetam

N O

CH3

NH2

O

Phenobarbial

NH

NH

O

O

O

CH3

Primidone

NH

NH

O

CH3 O

Phenytoin

NH NH

O

O

Tiagabine

SCH3

NS OH

O

CH3

Zonisamide

ON

S

O

O

CH3O

O

OO

O

CH3

CH3

CH3

CH3

OS

O

ONH2

Topiramate Vigabatrin

CH2

NH2

OH

O

Clonazepam

NH

N

N+

O

O O

Cl

-

NH

N

N+

O

O O

Nitrazepam

-

3


Figure 2 Nexera Method Scoutuing System and LCMS-8050 triple quadrupole mass spectrometer

Result

The MS condition optimization was performed by flow injection analysis (FIA) of ESI positive and negative ionization mode, and the compound dependent parameters such as CID and pre-bias voltage were adjusted using automatic

MRM optimization function. The transition that gave highest intensity was used for quantification. The MRM transitions used in this assay are listed in Table 1.

The main standard mixture was prepared in methanol from individual stock solutions. The calibration standards were prepared by diluting the standard mixture with methanol. QC sample was prepared by adding 4 volume of acetonitrile to 1 volume of control plasma, thereby precipitating proteins, and subsequently adding the standard mixture to the supernatant to contain plasma concentration equivalents stated in Table 4. The QC samples were further diluted 100 times (10 μL sample

added to 990μL methanol) before injection. Next step of preparation procedure was divided into three groups by the intensity of each compound. For ethomuximide, phenobarbial and phenytoin, the supernatant was used for the LC/MS/MS analysis without further dilution. For zonisamide, 10 μL supernatant was further diluted with 990 μL methanol. For others, 100 μL supernatant was further diluted with 900 μL methanol. The diluted solutions were used for the LC/MS/MS analysis.

MRM condition optimization

Calibration standards and QC samples

4


Figure. 3 Schematic representation and features of the Nexera Method Scouting System.

Table 1 Compounds, Ionization polarity and MRM transition

Retaintion (min)Compound Polarity Precursor m/z Product m/z

3.84

3.24

3.93

4.79

2.50

2.86

2.27

2.96

2.32

3.90

3.06

3.64

2.83

4.28

3.14

0.82

2.58

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

-

-

237.1

253.1

316.1

284.9

239.3

172.2

256.2

171.2

281.9

219.2

376.2

130.2

213.1

140.0

231.0

337.9

143.1

194.2

180.15

269.55

154.15

117.20

154.25

211.05

126.15

236.20

162.15

111.15

71.15

132.10

42.00

42.05

78.00

143.10

Carbamazepine

Carbamazepine-10,11-epoxide

Clonazepam

Diazepam

Ethomuximide

Felbamate

Gabapentin

Lamotrigine

Levetiracetam

Nitrazepam

Phenobarbial

Phenytoin

Primidone

Tiagabine

Topiramate

Vigabatrin

Zonisamide

36 analytical conditions, comprising combinations of 9 mobile phase and 4 columns, were automatically investigated using Method Scouting System. Schematic representation of scouting system was shown in Figure 3. From the result of scouting, the combination of 10 mM

ammonium acetate water and methanol for mobile phase and Inertsil-ODS4 for separation column were selected. Using this combination of mobile phase and column, the gradient condition was further optimized. The final analytical condition was shown in Table 2.

UHPLC condition optimization

Auto SamplerLPGE Unit

Column Oven

LCMS-8050

Pump A

Pump B

1 2 3 4

1 2 3 4

(A) 1 – 10mM Ammonium Acetate 2 – 10mM Ammonium Formate 3 – 0.1%FA - 10mM Ammonium Acetate(B) 1 – Methanol 2 – Acetonitrile 3 – Methanol/Acetonitrile=1/1

Kinetex XB-C18 (Phenomenex)

Kinetex PFP (Phenomenex)

InertsilODS-4 (GL Science)

Discovery HS F5-5 (SPELCO)

2.1 x 50 mm

2.1 x 50 mm

2.1 x 50 mm

2.1 x 50 mm

5


Table.2 UHPLC analytical conditions

Figure. 4 Chromatogram of 17 AEDs calibration standards

Column : Inertsil ODS-4 (50 mmL. x 2.1mmi.d., 2um)

Mobile phase : A) 10mM Ammonium Acetate

B) Methanol

Binary gradient : B conc. 3% (0.65 min) → 40% (1.00 min) → 85% (5.00 min)

→ 100% (5.01-8.00 min) → 3% (8.01-10.00 min)

Flow Rate : 0.4 mL/min

Injection vol. : 1 μL

Column Temp. : 40 deg. C

Figure 4 shows MRM chromatograms of the 17 AEDs. It took only 10 minutes per one UHPLC/MS/MS analysis, including column rinsing.

Precision, accuracy and linearity of AEDs

0.0 1.0 2.0 3.0 4.0 5.0 min

Vigabatrin130.20>71.15(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Gabapentin172.20>154.25(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Levetiracetam171.20>126.15(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Ethomuximide140.00>42.00(-)

0.0 1.0 2.0 3.0 4.0 5.0 min

Zonisamide213.10>132.10(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Primidone 219.20>162.15(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Felbamate239.30>117.20(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Lamotrigine256.20>211.05(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Phenobarbial231.00>42.05(-)

0.0 1.0 2.0 3.0 4.0 5.0 min

Topiramate337.85>78.00(-)

0.0 1.0 2.0 3.0 4.0 5.0 min

Carbamazepine-10,11-epoxide253.10>180.15(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Phenytoin251.00>208.20(-)

0.0 1.0 2.0 3.0 4.0 5.0 min

Carbamazepine237.10>194.20(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Nitrazepam 281.90>236.20(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Clonazepam 316.10>269.55(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Tiagabine376.20>111.15(+)

0.0 1.0 2.0 3.0 4.0 5.0 min

Diazepam284.90>154.15


6

Table.3 Linearity of 17 AEDs QC sample

Compound Linarity (ng/mL) r2

0.25

0.25

0.005

0.01

25

0.5

2

0.25

0.5

0.005

5

5

0.25

0.25

0.5

0.5

0.5

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

50

50

2.5

5

2500

100

50

50

100

1

500

500

10

50

100

50

20

0.999

0.998

0.998

0.999

0.998

0.998

0.999

0.999

0.999

0.999

0.996

0.998

0.996

0.998

0.998

0.998

0.996

Carbamazepine


Clonazepam

Diazepam

Ethomuximide

Felbamate

Gabapentin

Lamotrigine

Levetiracetam

Nitrazepam

Phenobarbial

Phenytoin

Primidone

Tiagabine

Topiramate

Vigabatrin

Zonisamide

Table 3 illustrates linearity of 17 AEDs and Table 4 illustrates accuracy and precision of the QC samples at three concentration levels. Determination coefficient (r2) of all calibration curves was larger than 0.995, and the precision

and accuracy were within +/- 15%. Excellent linearity, accuracy and precision for all 17 AEDs were obtained at only 1 μL injection volume.

For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

© Shimadzu Corporation, 2014

First Edition: June, 2014

www.shimadzu.com/an/


Table.4 Accuracy and precision of 17 AEDs QC sample

Compound

Plasma concentrationequivalents (µg/mL)

Precision (%) Accuracy (%)

HighMiddleLow

1.8

1.8

0.04

0.1

18

3.6

18

1.8

3.6

0.04

3.6

3.6

1.8

1.8

3.6

8.9

36

71

71

1.8

2.9

714

179

143

71

179

1.4

143

143

45

71

143

89

179

2.2

2.4

3.3

3.2

7.8

1.7

1.3

10.5

2.1

3.3

3.5

7.8

3.2

1.8

12.5

1.4

3.3

0.9

1.9

0.7

1.7

1.5

0.4

0.7

1.2

0.5

1.4

6.2

1.9

0.7

1.8

1.5

1.1

1.3

18

18

0.9

0.7

446

89

36

45

89

0.4

71

89

18

18

36

18

89

0.9

1.3

0.5

1.4

1.4

0.8

0.7

1.7

1.1

1.5

1.6

1.2

0.7

1.0

1.2

2.1

1.6

106.1

104.2

106.7

105.8

104.3

97.1

85.8

107.7

99.5

105.0

100.9

103.2

99.5

107.6

105.4

105.9

111.7

103.9

105.0

102.1

106.6

99.9

106.3

98.8

98.4

104.9

105.2

108.4

100.1

112.6

105.7

101.6

101.6

100.4

95.8

98.2

90.1

100.6

97.0

91.7

89.5

99.2

90.4

97.9

95.8

96.2

97.1

97.5

96.1

88.8

95.2

Carbamazepine


Clonazepam

Diazepam

Ethomuximide

Felbamate

Gabapentin

Lamotrigine

Levetiracetam

Nitrazepam

Phenobarbial

Phenytoin

Primidone

Tiagabine

Topiramate

Vigabatrin

Zonisamide

HighMiddleLowHighMiddleLow

Conclusions• We could select the most suitable combination of mobile phase and column from 36 analytical condition without

time-consuming investigation.• We have measured plasma sample as it is after 100-10,000 times dilution by methanol without making tedious sample

pretreatment. Excellent linearity, precision and accuracy for all 17 AEDs were obtained at only 1 uL injection volume.

PO-CON1446E

Determination of Δ9-tetrahydrocannabinoland two of its metabolites in whole blood,plasma and urine by UHPLC-MS/MS usingQuEChERS sample preparation

ASMS 2014 ThP600

Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,

Pierre MARQUET1,3 and Stéphane MOREAU2

1 CHU Limoges, Department of Pharmacology and Toxicology,

Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador

Allende, 77448 Marne la Vallée Cedex 23 Univ Limoges, Limoges, France

2

Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

IntroductionIn France, as in other countries, cannabis is the most widely used illicit drug. In forensic as well as in clinical contexts, ∆9-tetrahydrocannabinol (THC), the main active compound of cannabis, and two of its metabolites [11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) and 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] are regularly investigated in biological �uids for example in Driving Under the In�uence of Drug context (DUID) (�gure 1). Historically, the concentrations of these compounds were determined using a time-consuming extraction procedure

and GC-MS. The use of LC-MS/MS for this application is relatively recent, due to the low response of these compounds in LC-MS/MS while low limits of quanti�cation need to be reached. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to signi�cant carry-over after highly concentrated samples. We propose here a highly sensitive UHPLC-MS/MS method with straightforward QuEChERS sample preparation (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe).

Methods and MaterialsIsotopically labeled internal standards (one for each target compound in order to improve method precision and accuracy) at 10 ng/mL in acetonitrile, were added to 100 µL of sample (urine, whole blood or plasma) together with 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium

citrate dehydrate/Sodium citrate sesquihydrate) and 200 µL of acetonitrile. Then the mixture was shaken and centrifuged for 10 min at 12,300 g. Finally, 15 µL of the upper layer were injected in the UHPLC-MS-MS system. The whole acquisition method lasted 3.4 min.

Figure 1: Structures of THC and two of its metabolites

OH

O

H

HCH3

CH3

OHO

THC-COOH

OH

O

H

H

CH2

CH3CH3

OH

11-OH-THC

OH

O

H

H

CH3

CH3CH3

THC

3


UHPLC conditions (Nexera MP system)

Column : Kinetex C18 50x2.1 mm 2.6 µm (Phenomenex)

Mobile phase A : 5mM ammonium acetate in water

B : CH3CN

Flow rate : 0.6 mL/min

Time program : B conc. 20% (0-0.25 min) - 90% (1.75-2.40 min) - 20% (2.40-3.40 min)

Column temperature : 50 °C

MS conditions (LCMS-8040)

Ionization : ESI, negative MRM mode

Ion source temperatures : Desolvation line: 300°C

Heater Block: 500°C

Gases : Nebulization: 2.5 L/min

Drying: 10 L/min

MRM Transitions:

Compound MRM Dwell time (msec)

THC 313.10>245.25 (Quan) 60

313.10>191.20 (Qual) 60

313.10>203.20 (Qual) 60

THC-D3 316.10>248.30 (Quan) 5

316.10>194.20 (Qual) 5

11-OH-THC 329.20>311.30 (Quan) 45

329.20>268.25 (Qual) 45

329.20>173.20 (Qual) 45

11-OH-THC-D3 332.30>314.40 (Quan) 5

332.30>271.25 (Qual) 5

THC-COOH 343.20>245.30 (Quan) 50

343.20>325.15 (Qual) 50

343.20>191.15 (Qual) 50

343.20>299.20 (Qual) 50

THC-COOH-D3 346.20>302.25 (Quan) 5

346.20>248.30 (Qual) 5

Pause time : 3 msec

Loop time : 0.4 sec (minimum 20 points per peak for each MRM transition)

4


Figure 1: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 50 µg/L

Results

A typical chromatogram of the 6 compounds is presented in figure 1.

Chromatographic conditions

Figure 2: in�uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.

As described by Anastassiades et al. J. AOAC Int 86 (2003) 412-31, the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only

obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 2.

Extraction conditions

A B

5


Figure 3: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 0.5 µg/L (lower limit of quanti�cation).

One challenge for the determination of cannabinoids in blood using LC-MS/MS is the low quantification limits that need to be reached. The French Society of Analytical Toxicology proposed 0.5 µg/L for THC et 11-OH-THC and 2.0 µg/L for THC-COOH. With the current application, the

lower limit of quantification was fixed at 0.5 µg/L for the three compounds (3.75 pg on column). The corresponding extract ion chromatograms at this concentration are presented in figure 3.

Validation data

The upper limit of quantification was set at 100 µg/L. Calibration graphs of the cannabinoids-to-internal standard peak-area ratios of the quantification transition versus

expected cannabinoids concentration were constructed using a quadratic with 1/x weighting regression analysis (figure 4).

Contrary to what was already observed with on-line Solid-Phase-Extraction no carry-over effect was noted using the present method, even when blank samples were

injected after patient urine samples with concentrations exceeding 2000 µg/L for THC-COOH.

THC11-OH-THCTHC-COOH

Figure 4: Calibration curves of the three cannabinoids

THC11-OH-THCTHC-COOH






Conclusions• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase

Extraction.• Low limit of quanti�cation compatible with determination of DUID.• No carry over effect noticed.

PO-CON1445E

Determination of opiates, amphetaminesand cocaine in whole blood, plasmaand urine by UHPLC-MS/MS usinga QuEChERS sample preparation

ASMS 2014 ThP599

Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,

Pierre MARQUET1,3 and Stéphane MOREAU2

1 CHU Limoges, Department of Pharmacology and Toxicology,

Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador

Allende, 77448 Marne la Vallée Cedex 23 Univ Limoges, Limoges, France

2

Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

IntroductionThe determination of drugs of abuse (opiates, amphetamines, cocaine) in biological �uids is still an important issue in toxicology, in cases of driving under the in�uence of drugs (DUID) as well as in forensic toxicology. At the end of the 20th century, the analytical methods able to determine these three groups of narcotics were mainly based on a liquid-liquid-extraction with derivatization followed by GC-MS. Then LC-MS/MS was proposed,

coupled with off-line sample preparation. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to signi�cant carry-over after highly concentrated samples. We propose here another approach based on the QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) sample preparation principle, followed by UHPLC-MS/MS.

Methods and MaterialsThis method involves 40 compounds of interest (13 opiates, 22 amphetamines, as well as cocaine and 4 of its

metabolites) and 18 isotopically labeled internal standards (designed with *) (Table1).

Table 1: list of analyzed compounds with their associate internal standard (*)

Cocaine and metabolitesAmphetamines or related

compounds Opiates

• Anhydroecgonine methylester• Benzoylecgonine*• Cocaethylene*• Cocaine*• Ecgonine methylester*

• 2-CB• 2-CI• 4-MTA• Ritalinic acid• Amphetamine*• BDB• Ephedrine*• MBDB• m-CPP• MDA*• MDEA*• MDMA*• MDPV• Mephedrone• Metamphetamine*• Methcathinone• Methiopropamine• Methylphenidate• Norephedrine• Norfen�uramine• Norpseudoephedrine• Pseudoephedrine

• 6-monoacetylmorphine*• Dextromethorphan• Dihydrocodeine*• Ethylmorphine• Hydrocodone• Hydromorphone• Methylmorphine*• Morphine*• Naloxone*• Naltrexone*• Noroxycodone*• Oxycodone*• Pholcodine

3


UHPLC conditions (Nexera MP system, �gure 1)

Column : Restek Pinnacle DB PFPP 50x2.1 mm 1.9 µm

Mobile phase A : 5mM Formate ammonium with 0.1% formic acid in water

B : 90% CH3OH/ 10% CH3CN (v/v) with 0.1 % formic acid


Time program : B conc. 15% (0-0.16 min) - 20% (1.77 min) - 90% (2.20 min) –

100% (4.00 min) – 15% (4.10-5.30 min)


MS conditions (LCMS-8040, �gure 1)

Ionization : ESI, Positive MRM mode

Ion source temperatures : Desolvation line: 300°C

Heater Block: 500°C

Gases : Nebulization: 2.5 L/min

Drying: 10 L/min

MRM Transitions : 2 Transitions per compounds were dynamically scanned for 1 min except

pholcodine (2 min)

Pause time : 3 msec

Loop time : 0.694 sec (minimum 17 points per peak for each MRM transition)

To 100 µL of sample (urine, whole blood or plasma) were added isotopically labeled internal standards (in order to improve method precision and accuracy) at 20 µg/L in acetonitrile (20 µL), and 200 µL of acetonitrile. After a 15 s shaking, the mixture was placed at -20°C for 10 min. Then approximately 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium citrate dehydrate/Sodium citrate

sesquihydrate) were added and the mixture was shaken again for 15 s and centrifuged for 10 min at 12300 g. The upper layer was diluted (1/3; v/v) with a 5 mM ammonium formate buffer (pH 3). Finally, 5 µL were injected in the UHPLC-MS/MS system. The whole acquisition method lasted 5.5 min.

Figure 1: Shimadzu UHPLC-MS/MS Nexera-8040 system

4


Figure 2: Chromatograms obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L. Order of retention - A: norephedrine and norpseudoephedrine / B: ephedrine and pseudoephedrine

Figure 3: Chromatogram obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L

Results

The analytical conditions allowed the chromatographic separation of two couples of isomers: norephedrine and norpseudoephedrine; ephedrine and pseudoephedrine

(figure 2). A typical chromatogram of the 58 compounds is presented in figure 3.

Chromatographic conditions

A B

5


Figure 4: in�uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.

As described by Anastassiades et al. J. AOAC Int 86 (2003) 412-31, the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only

obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 4.

Extraction conditions

Among the 40 analyzed compounds, 38 filled the validation conditions in term of intra- and inter-assay precision and accuracy were less than 20% at the lower limit of quantification and less than 15% at the other concentrations.Despite the quick and simple sample preparation, no significant matrix effect was observed and the lower limit of quantification was 5 µg/L for all compounds, while the upper limit of quantification was set at 500 µg/L. The

concentrations obtained with a reference (GC-MS) method in positive patient samples were compared with those obtained with this new UHPLC-MS/MS method and showed satisfactory results.Contrary to what was already observed with on-line Solid-Phase-Extraction, no carry-over effect was noted using the present method, even when blank samples were injected after patient urine samples with analytes concentrations over 2000 µg/L.

Validation data

A B






Conclusions• Separation of two couples of isomers with a run duration less than 6 minutes and using a 5 cm column.• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase

Extraction.• Lower limit of quanti�cation compatible with determination of DUID.• No carry over effect noticed.

PO-CON1442E

Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

ASMS 2014 ThP-592

Toshikazu Minohata1, Keiko Kudo2, Kiyotaka Usui3, Noriaki Shima4, Munehiro Katagi4, Hitoshi Tsuchihashi5, Koichi Suzuki5, Noriaki Ikeda2

1Shimadzu Corporation, Kyoto, Japan 2Kyushu University, Fukuoka, Japan 3Tohoku University Graduate School of Medicine, Sendai, Japan 4Osaka Prefectural Police, Osaka, Japan 5Osaka Medical Collage, Takatsuki, Japan

2


IntroductionIn Forensic Toxicology, LC/MS/MS has become a preferred method for the routine quantitative and qualitative analysis of drugs of abuse. LC/MS/MS allows for the simultaneous analysis of multiple compounds in a single run, thus enabling a fast and high throughput analysis. In this study, we report a developed analytical system using ultra-high

speed triple quadrupole mass spectrometry with a new extraction method for pretreatment in forensic analysis. The system has a sample preparation utilizing modi�ed QuEChERS extraction combined with a short chromatography column that results in a rapid run time making it suitable for routine use.

Figure 1 Scheme of the modi�ed QuEChERS procedure

[ ref.] (1) Usui K et al, Legal Medicine 14 (2012), 286-296

Methods and Materials

Whole blood sample preparation was carried out by the modified QuEChERS extraction method (1) using Q-sep™ QuEChERS Sample Prep Packets purchased from RESTEK (Bellefonte, PA).

1) Add 0.5 mL of blood and 1 mL of distilled water into the 15 mL centrifugal tube and agitate the mixture using a vortex mixer.

2) Add two 4 mm stainless steel beads, 1.5 mL of acetonitrile and 100 µL of acetonitrile solution containing 1 ng/µL of Diazepam-d5. Then agitate using the vortex mixer.

3) Add 0.5 g of the filler of the Q-sep™ QuEChERS Extraction Salts Packet.

4) Vigorously shake the tube by hand several times, agitate well using the vortex mixer for approximately 20 seconds. Then centrifuge the tube for 10 minutes at 3000 rpm.

5) Move the supernatant to a different 15 mL centrifugal tube and add 100 µL of 0.1 % TFA acetonitrile solution. Then, dry using a nitrogen-gas-spray concentration and drying unit or a similar unit.

6) Reconstitute with 200 µL of methanol using the vortex mixer. Then move it to a microtube, and centrifuge for 5 minutes at 10,000 rpm.

7) Transfer 150 µL of the supernatant to a 1.5 mL vial for HPLC provided with a small-volume insert.

Sample Preparation

Sample0.5 mL

Water 1 mL ACN 1.5 mL Diazepam-d5 (IS) 100ng Stainless-Steel Beads (4mm x 2)

[Shake] [Centrifuge]

Transfer supernatant Add 100uL of 0.1% TFA

Dry

Reconstitution with 200 uL MeOH

LC/MS/MS analysis

Q-sep QuEChERSExtraction Salts(MgSO4,NaOAc)

3


Analytical Conditions

LC-MS/MS Analysis

HPLC (Nexera UHPLC system)

Column : YMC Triart C18 (100x2mm, 1.9μm)

Mobile Phase A : 10 mM Ammonium formate - water

Mobile Phase B : Methanol

Gradient Program : 5%B (0 min) - 95%B (10 min - 13min) - 5%B (13.1 min - 20 min)

Flow Rate : 0.3 mL / min

Column Temperature : 40 ºC

Injection Volume : 5 uL

Mass (LCMS-8050 triple quadrupole mass spectrometry)

Ionization : heated ESI

Polarity : Positive & Negative

Probe Voltage : +4.5 kV (ESI-Positive mode); -3.5 kV (ESI-Negative mode)

Nebulizing Gas Flow : 3 L / min

Drying Gas Pressure : 10 L / min

Heating gas �ow : 10 L / min

DL Temperature : 250 ºC

BH Temperature : 400 ºC

MRM parameter :

Treated samples were analyzed using a Nexera UHPLC system coupled to a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) with LC/MS/MS Rapid Tox. Screening Database. The Database contains product ion scan spectra for 106 forensic and toxicology-related compounds of Abused drugs, Psychotropic drugs and Hypnotic drugs etc (Table 1) and

provides Synchronized Survey Scan® parameters (product ion spectral data acquisition parameters based on the MRM intensity as threshold) optimized for screening analysis.Samples were separated on a YMC Triart C18 column. A �ow rate of 0.3 mL/min was used together with a gradient elution.

Analytes Ret. Time Q1 m/z Q3 m/zCollisionEnergy

-27

-34

-24

-41

-23

-30

-24

-37

-30

-19

-24

-36

-24

-39

9.338

8.646

5.378

8.408

9.350

8.786

8.253

Diazepam-d5

Alprazolam

Atropine

Estazolam

Ethyl lo�azepate

Etizolam

Haloperidol

154.05

198.20

281.10

205.10

124.15

93.20

267.15

205.25

259.10

287.15

314.10

138.15

165.15

123.10

290.15

290.15

309.10

309.10

290.15

290.15

295.05

295.05

361.15

361.15

343.05

343.05

376.15

376.15

Analytes Ret. Time Q1 m/z Q3 m/zCollisionEnergy

-28

-55

-27

-25

25

14

21

15

19

14

23

16

7.993

8.573

8.093

5.243

6.762

8.883

Risperidone

Triazolam

Amobarbital(neg)

Barbital(neg)

Phenobarbital(neg)

Thiamylal(neg)

191.05

69.05

315.00

308.20

42.00

182.00

42.10

140.10

42.20

85.10

58.10

101.00

411.20

411.20

343.05

343.05

225.15

225.15

183.10

183.10

231.10

231.10

253.00

253.00

4


Results and DiscussionEtizolam Risperidone TriazolamAlprazolam

0.1ng/mL

Conc. Area Accuracy %RSD9,0048,2889,51975,23675,98374,023829,519831,098849,597

112.1105.1119.389.689.680.699.999.6

104.2

0.01

0.1

1

6.57

6.04

2.53

Conc. Area Accuracy %RSD4,8655,1094,321

48,03849,15254,497

604,640581,207579,390

114.4119.9105.784.085.187.0103.799.2101.2

0.01

0.1

1

8.71

1.82

2.22

Conc. Area Accuracy %RSD29,83232,43630,461335,202309,273343,172

3,826,3733,718,8543,705,165

108.4116.7110.891.383.785.6102.899.4101.4

0.01

0.1

1

5.14

4.74

1.66


107.0109.2118.594.885.781.599.0101.5102.9

0.01

0.1

1

5.63

7.83

1.96

negative

positive

Figure 2 LCMS-8050 triple quadrupole mass spectrometer

0.01ng/mL

S/N 39.5

309.10>281.10(+)

309.10>281.10(+)

(x103)

(x104)

2.0

1.0

0.5

0.0

1.0

0.5

0.0

8.0

0.00 0.25 0.75 Conc. Ratio0.50

8.5 9.0 9.5

1.0

0.0

Area Ratio

r2=0.998

0.00 0.25 0.75 Conc. Ratio0.50

7.5

5.0

2.5

0.0

Area Ratio (x0.1)

r2=0.998

0.00 0.25 0.75 Conc. Ratio0.50

Area Ratio

r2=0.9985.0

2.5

0.0

4.0

2.0

3.0

1.0

0.00.00 0.25 0.75 Conc. Ratio0.50

Area Ratio (x0.1)

r2=0.998

8.0 8.5 9.0 9.5 8.0 8.5 9.0 9.57.0 7.5 8.0 8.5

0.0

(x104)0.0

0.5

(x103)

1.0

343.05>314.10(+)

343.05>314.10(+)

S/N 145.5

0.0

(x104)0.0

(x103)

2.5

2.5

411.20>191.05(+)

411.20>191.05(+)

S/N 107.6

0.0

(x103)0.0

(x102)

2.5

2.5

S/N 18.8

343.05>315.00(+)

343.05>315.00(+)

5


In this experiment, two different matrices consisting of human whole blood and urine were prepared and 18 drugs were spiked into extract solution. Calibration curves constructed in the range from 0.01 to 1 ng/mL for 12 drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam, Estazolam, Ethyl lo�azepate, Etizolam, Flunitrazepam,

Haloperidol, Nimetazepam, Risperidone and Triazolam) and from 1 to 100 ng/mL for 6 drugs (Bromovalerylurea, Amobarbital, Barbital, Loxoprofen, Phenobarbital and Thiamylal). All calibration curves displayed linearity with an R2 > 0.997 and excellent reproducibility was observed for all compounds (CV < 12%) at low concentration level.


100.299.1

105.899.6

102.492.5

101.398.3

100.9

1

10

100

4.53

5.30

1.62

Conc. Area Accuracy %RSD521464509

5,0785,0335,424

55,42055,65853,484

108.796.6103.495.695.499.4101.4100.898.7

1

10

100

7.10

2.38

1.42


7,9098,5647,93981,98783,27482,656

106100.2

9198.8107.596.799.299.7100.8

1

10

100

9.82

5.82

0.85


10795.397.5101.498.3100.6100.799.3100

1

10

100

8.99

1.68

0.71

Phenobarbital (neg) Thiamylal (neg)Amobarbital (neg) Barbital (neg)

Figure 3 Results of 8 drugs spiked in human whole blood using LCMS-8050

7.5 8.0 8.5 9.0

10ng/mL

1ng/mL

2.5

(x102)

0.0(x103)

2.5

0.0

225.15>42.00(-)

225.15>42.00(-)

Area Ratio (x0.1)

r2=0.999

0.0 25.0 Conc. Ratio50.0

2.0

1.0

0.0

Area Ratio (x0.01)

0.0 25.0 Conc. Ratio50.0

5.0

2.5

0.0

r2=0.999Area Ratio (x0.1)

r2=0.999

0.0 25.0 Conc. Ratio50.00.00

0.25

0.50

0.75

1.00

0.0 25.0 Conc. Ratio50.00.0

1.0

2.0

3.0

4.0Area Ratio (x0.1)

r2=0.999

S/N 40.2 S/N 38.2 S/N 167.95.0

(x10)

0.0(x102)

5.0

2.5

0.0

183.10>42.10(-)

183.10>42.10(-)

S/N 15.3

231.10>42.20(-)

231.10>42.20(-)

1.0

(x102)

0.0

0.5

(x103)

1.0

0.5

0.0

5.0

(x102)

0.0

2.5

(x103)

5.0

2.5

0.0

253.00>58.10(-)

253.00>58.10(-)

4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5







102.286.687.6104.4114.7106.996.810397.9

1

10

100

12.73

5.10

3.34


4,9895,6135,443

55,39269,48166,327

93.696.189

105.2109.6108.692.6104

101.3

1

10

100

2.77

2.07

5.98


5,6566,6326,38471,96588,68582,091

103.689.499.397.9106.1104.495.210599.1

1

10

100

8.16

4.24

4.95


95.1100.591.494.9110.8108.598.5104.196.1

1

10

100

4.54

8.15

4.15

Figure 4 Results of 4 drugs spiked in human urine using LCMS-8050

Conclusions• The validated sample preparation protocol can get adequate recoveries in quantitative works for all compounds ranging

from acidic to basic. • The combination of the modi�ed QuEChERS extraction method and high-speed triple quadrupole LC/MS/MS with a

simple quantitative method enable to acquire reliable data easily.

7.5 8.0 8.5 9.0

Phenobarbital (neg) Thiamylal (neg)Amobarbital (neg) Barbital (neg)

Area Ratio (x0.1)




r2=0.999

2.0

3.0

1.0

0.00.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0

0.50

0.75

0.25

0.00

1.0

0.5

0.0

5.0

2.5

0.0

10ng/mL

1ng/mL

2.5

(x102)

0.0(x103)

2.5

0.0

225.15>42.00(-)

225.15>42.00(-)

S/N 14.7 S/N 9.4 S/N 18.3 S/N 97.41.0

(x102)

(x102)

5.0

2.5

0.0

183.10>42.10(-)

183.10>42.10(-)

231.10>42.20(-)

231.10>42.20(-)

253.00>58.10(-)

253.00>58.10(-)

1.0

0.0

(x102)

(x103)

1.0

0.5

0.0

2.5

5.0

0.0

(x102)

(x103)

5.0

2.5

0.0

4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

PO-CON1460E

Simultaneous Screening and Quantitationof Amphetamines in Urine by On-line SPE-LC/MS Method

ASMS 2014 ThP587

Helmy Rabaha1, Lim Swee Chin1, Sun Zhe2,

Jie Xing2 & Zhaoqi Zhan2

1Department of Scienti�c Services, Ministry of Health,

Brunei Darussalam;2Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE

2

Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

IntroductionAmphetamines belong to stimulant drugs and are also controlled as illicit drugs worldwide. The conventional analytical procedure of amphetamines in human urine includes initial immunological screening followed by GCMS con�rmation and quantitation [1]. With new SAMHSA guidelines effective in Oct 2010 [2], screening, con�rmation and quantitation of illicit drugs including amphetamines were allowed to employ LC/MS and LC/MS/MS, which usually does not require a derivatization step as used in the GCMS method [1]. The objective of this study was to develop an on-line SPE-LC/MS method for

analysis of �ve amphetamines in urine without sample pre-treatment except dilution with water. The compounds studied include amphetamine (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guideline (group A in Table 1). Four potential interferences (group B in) and PMPA (R) as a control reference were also included to enhance the method reliability in identi�cation of the �ve targeted amphetamines from those structurally similar analogues which potentially present in forensic samples.

ExperimentalThe test stock solutions of the ten compounds (Table 1) were prepared in the toxicology laboratory in the Department of Scienti�c Services (MOH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and matrix to prepare spiked amphetamine samples were not pre-treated off-line by any means except dilution of 10 times with pure water. An on-line SPE-LC/MS was set up on the LCMS-2020, a single quadrupole system, with a switching valve and a trapping column kit (Shimadzu Co-Sense con�guration) installed in the column oven and controlled by the LabSolutions workstation. The analytical column used was Shim-pack VP-ODS 150 x 2mm (5um) and the trapping column was Synergi Polar-RP 50 x 2mm (2.5um), instead of

a normal SPE cartridge. The injected sample �rst passed through the trapping column where the amphetamines were trapped, concentrated and washed by pure water for 3 minutes followed by switching to the analytical �ow line. The trapped compounds were then eluted out with a gradient program: 0.01min, valve at position 0 & B=5%; 3 min, valve at position 1; 3.01-10 min, B=5% → 15%; 10.5-12 min, B=65%; 12.1 min, B=5%; 14 min stop, valve to position 0. The mobile phases A and B were water and MeOH both with 0.1% formic acid and mobile C was pure water. The total �ow rates of the trapping line and analytical line are 0.6 and 0.3 mL/min, respectively. The injection volume was 20uL in all experiments.

3


Figure 1: Schematic diagram of on-line SPE-LC/MS system

Table 1: Amphetamines & relevant compounds

Name Abbr. Name Formula Structure

Amphetamine

Methampheta-mine

3,4-methylene-dioxyamphetamine

3,4-methylene-dioxymetham phetamine

3,4-methylene dioxy-N-ethyl amphetamine

Nor pseudo-ephedrine

Ephedrine

Pseudo-Ephedrine

Phentermine

Propyl-amphetamine

AMPH

MAMP

MDA

MDMA

MDEA

Nor pseudo-E

Ephe

Pseudo-E

Phent

PAMP

No

A1

A2

A3

A4

A5

B1

B2

B3

B4

R

C9H13N

C10H15N

C10H13NO2

C11H15NO2

C12H17NO2

C9H13NO

C10H15NO

C10H15NO

C10H15N

C12H19N

Manual injectorPump A

SPE Trapping Column

5

13

Mixer

Switching Valve

LCMS-2020

Waste

Pump B Auto sampler

Analytical column

Pump C

4


Results and Discussion

With ESI positive SIM and scan mode, all of the 10 compounds formed protonated ions [M+H]+ which were used as quantifier ions. The scan spectra were used for confirmation to reduce false positive results. Mixed standards of the ten compounds in Table 1 spiked in urine was used for method development. An initial difficulty encountered was that the normal reusable SPE cartridges

(10-30 mmL) for on-line SPE could not trap all of the ten compounds. With using a 50mmL C18-column to replace the SPE cartridge, the ten compounds studied were trapped efficiently. Furthermore, the trapped compounds were well-separated and eluted out in 8~13 minutes as sharp peaks (Figure 2) by the fully automated on-line SPE-LC/MS method established.

Calibration curves of the on-line SPE-LC/MS method were established using mixed standard samples with concentrations from 2.5 ppb to 500 ppb. Linear calibration

curves with R2> 0.999 were obtained for every compound (Figure 3 & Table 2).

Development of on-line SPE-LC/MS method

Figure 2: SIM chromatograms of urine blank (a) and �ve amphetamines and related compounds (125 ppb each) spiked in urine (b) by on-line SPE-LC/MS.

0.0 2.5 5.0 7.5 10.0 12.5 min0.0

0.5

1.0

1.5

2.0(x1,000,000)

2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)

(a) Urine blank (b) spiked samples

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0(x1,000,000)

2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)

Nor

pseu

doEp

hedr

ine

Pseu

do

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

Phen

t

AM

PH

Figure 3: Calibration curves of �ve amphetamines and �ve related compounds with concentrations from 2.5 ppb to 500 ppb by on-line SPE-LC/MS method

0 250 Conc.0.0

2.5

5.0

7.5

Area (x1,000,000)

0 250 Conc.0.0

0.5

1.0

1.5

Area (x10,000,000)

0 250 Conc.0.0

0.5

1.0Area (x10,000,000)

0 250 Conc.0.0

1.0

2.0

Area (x10,000,000)

0 250 Conc.0.0

0.5

1.0

Area (x10,000,000)

0 250 Conc.0.0

1.0

2.0

Area (x10,000,000)

0 250 Conc.0.0

1.0

2.0

3.0Area (x10,000,000)

0 250 Conc.0.0

0.5

1.0

1.5

Area (x10,000,000)

0 250 Conc.0.0

0.5

1.0

1.5

Area (x10,000,000)

0 250 Conc.0.0

2.5

5.0

Area (x1,000,000)

AMPH MAMP

Phent PAMP

MDA MDMA MDEA

Ephedrine Pseudo-ENor pseudo-E


5

Table 2: Peak detection, retention, calibration curves and method performance evaluation

NameRec. %

(62.5ppb)RSD%(n=6)(62.5ppb)

LOD/LOQ(ppb)

Norpseudo-E

Ephe

Pseudo-E

AMPH

MAMP

MDA

MDMA

MDEA

Phent

PAMP (Ref)

97.3

84.4

78.9

85.6

76.5

71.8

72.2

74.8

74.5

69.5

M.E %(62.5ppb)

69.3

111.0

109.2

71.1

96.8

70.3

116.3

107.1

69.9

96.8

Linearity(r2)

0.9982

0.9960

0.9976

0.9983

0.9968

0.9989

0.9973

0.9908

0.9960

0.9912

1.67

0.54

0.41

0.98

0.94

1.94

1.08

2.18

1.82

5.30

S/N(2.5ppb)

11.3

33.7

28.5

17.5

30.3

18.2

36.6

41.9

12.7

37.7

0.71/2.17

0.25/0.76

0.29/0.88

0.48/1.46

0.26/0.80

0.45/1.36

0.23/0.70

0.19/0.57

0.66/2.01

0.22/0.66

SIM ion(+)

152.1

166.1

166.1

136.1

150.1

180.1

194.1

208.1

150.1

178.1

RT(min)

8.0

8.4

9.0

9.6

10.2

10.4

10.8

12.2

12.4

12.7

Conc. range(ppb)

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

2.5 - 500

The trapping efficiency of the on-line SPE is critical and must be evaluated first, because it determines the recovery of the method. In this study, the recovery of the on-line SPE was determined by injecting a same mixed standard sample from a manual injector installed before the analytical column (by-pass on-line SPE) and also from the Autosampler (See Figure 1). The peaks areas obtained by the two injections were used to calculate recovery value of the on-line SPE method. As shown in Table 2, the recovery obtained with 62.5 ppb mixed standards are at 69.5% ~ 97.3%. The recovery with 250 ppb and 500 ppb mixed samples were also determined and similar results were obtained. Matrix effect was determined with 62.5 ppb and 250 ppb levels of mixed samples in clear solution and in urine. The results (Table 2) show a variation between 69.3% and 116% with compounds. The matrix effect with different

urine specimens did not show significant differences. Repeatability was evaluated with spiked mixed samples of 62.5 ppb and 250 ppb. The results of 62.5 ppb is shown in Table 2, RSD between 0.41% and 5.3%. The sensitivity of the on-line SPE-LC/MS method was evaluated with spiked sample of 2.5 ppb level. The SIM chromatograms are shown in Figure 4. The S/N ratios obtained ranged 11.3~42, which were suitable to determine LOQ (S/N = 10) and LOD (S/N = 3). Since the urine samples were diluted for 10 times with water before injection, the LOD and LOQ of the method for source urine samples were at 1.9~7.1 and 5.7~21.7 ng/mL, respectively. The confirmation cutoff values of the five targeted amphetamines (Group A) in urine enforced by the new SMAHSA guidelines are 250 ng/mL [2]. The on-line SPE-LC/MS method established has sufficient allowance in terms of sensitivity and confirmation reliability for analysis of actual urine samples.

Performance evaluation of on-line SPE-LCMS method

Figure 4: SIM chromatograms of 10 compounds with 2.5 ppb each by on-line SPE-LC/MS method.

7.5 10.0 12.5 min

1.0

2.0

3.0

4.0

5.0

6.0(x10,000)

2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)

Nor

pseu

do

Ephe

drin

e

Pseu

do

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

Phen

t

AM

PH


6

Figure 5: Durability test of on-line SPE-LC/MS method, comparison of 1st and 200th injections.

The durability of the trapping column was tested purposely by continuous injections of spiked urine samples (125 ppb) for 200 times in a few days. Figure 5 shows the chromatograms of the first and 200th injections of a same

spiked sample. The results show that the variations of peak area and retention time of the 200th injection compared to the 1st injection were at 89.5%~117.8% and 89.5%~99.8% respectively.

Durability of on-line SPE trapping column

Confirmation reliability of LC/MS and LC/MS/MS methods must be proven to be equivalent to the GCMS method according to the SMAHSA guidelines [2]. Validation of confirmation reliability of the on-line SPE-LC/MS method has not be carried out systematically. The high sensitivity of MS detection in SIM mode is a key factor to ensure no false-negative and the scan spectra acquired

simultaneously is used for excluding false-positive. In this work, the confirmation reliability was evaluated using five different urine specimens as matrix to prepare spiked samples of 2.5 ppb (correspond 25 ng/mL in source urine) and above. The results show that false-positive and false negative results were not found.

Con�rmation Reliability

ConclusionsA novel high sensitivity on-line SPE-LC/MS method was developed for screening, conformation and quanti�cation of �ve amphetamines: AMPH, MAMP, MDMA, MDA and MDEA in urines. The recovery of the on-line SPE by employing a 50mmL Synergi Polar-RP column was at 72%~86% for the �ve amphetamines, which are considerably high if comparing with conventional on-line

SPE cartridges. The method performance was evaluated thoroughly with urine spiked samples. The results demonstrate that the on-line SPE-LC/MS method is suitable for direct analysis of the amphetamines and relevant compounds in urine samples without off-line sample pre-treatment.

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0

(x1,000,000)

2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)

Nor

pseu

do Ephe

drin

ePs

eudo

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

Phen

t

AM

PH

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0(x1,000,000)

2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)

Nor

pseu

do Ephe

drin

ePs

eudo

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

Phen

t

AM

PH

1st injection spiked mixed std 125ppb in urineinj vol: 20 µL

200th injection spiked mixed std 125ppb in urineinj vol: 20 µL






References1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. SAMHSA “Manual for urine laboratories, National laboratory certi�cation program”, Oct 2010, US Department of

Health and Human Services.

PO-CON1481E

Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

ASMS 2014 MP762

Alan J. Barnes1, Carrie-Anne Mellor2,

Adam McMahon2, Neil J. Loftus1

1Shimadzu, Manchester, UK 2WMIC, University of Manchester, UK

2


IntroductionDried plasma sample collection and storage from whole blood without the need for centrifugation separation and refrigeration opens new opportunities in blood sampling strategies for quantitative LC/MS/MS bioanalysis. Plasma samples were generated by gravity �ltration of a whole blood sample through a laminated membrane stack allowing plasma to be collected, dried, transported and analysed by LC/MS/MS. This novel plasma separation card (PSC) technology was applied to the quantitative LC/MS/MS analysis of warfarin, in blood samples. Warfarin is a coumarin anticoagulant vitamin-K antagonist used for the treatment of thrombosis and thromboembolism. As a

result of vitamin-K recycling being inhibited, hepatic synthesis is in-turn inhibited for blood clotting factors as well as anticoagulant proteins. Whilst the measurement of warfarin activity in patients is normally measured by prothrombin time by international normalized ratio (INR) in some cases the quantitation of plasma warfarin concentration is needed to con�rm patient compliance, resistance to the anticoagulant drug, or diet related issues. In this preliminary evaluation, warfarin concentration was measured by LC/MS/MS to evaluate if PSC technology could complement INR when sampling patient blood.

Materials and Methods

Warfarin standard was dissolved in water containing 50% ethanol + 0.1% formic acid, spiked (60uL) to whole human blood (1mL) and mixed gently. 50uL of spiked blood was deposited onto the PSC. After 3 minutes, the primary filtration overlay was removed followed by 15 minutes air drying at room temperature. The plasma sample disc was prepared directly for analysis after drying. LC/MS/MS sample preparation involved vortexing the sample disk in

40uL methanol, followed by centrifugation 16,000g 5 min. 20uL supernatant was added directly to the LCMS/MS sample vial already containing 80uL water (2uL analysed). Control plasma comparison was prepared by centrifuging remaining blood at 1000g for 10min. 2.5uL supernatant plasma was taken, 40uL methanol added, and prepared as PSC samples. LCMS/MS sample injection volume, 2uL.

Sample preparation

Warfarin was measured by MRM, positive negative switching mode (15msec).

LC-MS/MS analysis

LC/MS/MS System : Nexera UHPLC system + LCMS-8040 Shimadzu Corporation

Flow rate : 0.4mL/min (0-7.75min), 0.5mL/min (7.5-14min), 0.4mL/min (15min)

Mobile phase : A= Water + 0.1% formic acid

B= Methanol + 0.1% formic acid

Gradient : 20% B (0-0.5 min), 100% B (8-12 min), 20% B (12.01-15 min)

Analytical column : Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A

Column temperature : 50ºC

Ionisation : Electrospray, positive, negative switching mode

Desolvation line : 250ºC

Drying/Nebulising gas : 10L/min, 2L/min

Heating block : 400ºC

3


Design of plasma separator technology

Plasma separation work�ow

Control Spot:[Determines whether enough blood was placed on the card].

Filtration Layer[Filtration layer captures blood cells by a combination of �ltration and adsorption. The average linear vertical migration rate is approximately 1um/sec].

Collection Layer[Loads with a speci�c aliquot of plasma onto a 6.35mm disc]. Although �ow through the �ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric �ow rate into the Collection Disc of 400 pL/mm2/sec.

Isolation Screen[Precludes lateral wicking along the card surface].

Spreading Layer[Lateral spreading layer rapidly spreads blood so it will enter the �ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].

1 3 42

A NoviPlex card is removed from foil packaging.

Approximately 50uL of whole blood is added to the test area.

After 3 minutes, the top layer is completely removed (peeled back).

The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes.

The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.

Figure 1. Noviplex work�ow.

4


Figure 2. Applying a blood sample, either as a �nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and �ltration whilst plasma advances through the membrane stack

by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.

Figure 3. Comparison between the warfarin response in both positive and negative ion modes for warfarin calibration standards at 2.5ug/mL and 0.4ug/mL extracted from the plasma separation cards and a conventional plasma sample. There is a broad agreement in ion signal intensity between

the 2 sample preparation techniques.

ResultsComparison between plasma separation cards (PSC) and plasma

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.000.250.500.751.001.251.501.752.002.252.502.753.00

(x100,000)

1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.00.10.20.30.40.50.60.70.80.91.01.11.2(x100,000)

1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

(x100,000)

Plasma separation cardPositive ionWarfarin m/z 309.20 > 163.05

Q1 (V) -22Collision energy -15Q3 (V) -15

Plasma separation cardNegative ionWarfarin m/z 307.20 > 161.25

Q1 (V) 14Collision energy 19 Q3 (V) 30

PlasmaNegative ionWarfarin m/z 307.20 > 161.05

Q1 (V) 14Collision energy 19 Q3 (V) 30

Plasma Positive ionWarfarin m/z 309.20 > 163.05


2.5ug/mL Calibration standard

0.4ug/mLCalibration standard







5


The drive to work with smaller sample volumes offers significant ethical and economical advantages in pharmaceutical and clinical workflows and dried blood spot sampling techniques have enabled a step change approach for many toxicokinetic and pharmacokinetic studies. However, the impressive growth of this technique in the quantitative analysis of small molecules has also discovered several limitations in the case of sample

instability (some enzyme labile compounds, particularly prodrugs, analyte stability can be problematic), hematocrit effect and background interferences of DBS. DBS also shows noticeable effects on many lipids dependent on the sample collection process. To compare PSC to plasma lipid profiles the same blood sample extraction procedure applied for warfarin analysis was measured by a high mass accuracy system optimized for lipid profiling.

Plasma separation card comparison

Figure 4. In both ion modes, the calibration curve was linear over the therapeutic range studied for warfarin extracted from PSC’s (calibration range 0-3ug/mL, single point calibration standards at each level with the exception of replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3); r2>0.99 for

PSC analysis [r2>0.99 for a conventional plasma extraction]).

Figure 5. Matrix blank comparison. In both ion modes, the MRM chromatograms for PSC and plasma are comparable. Warfarin ion signals were not detected in the any PSC or plasma matrix blank.

Plasma separation cardNegative ionWarfarin m/z 309.20 > 163.05Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3)

Plasma separation cardPositive ionWarfarin m/z 309.20 > 163.05Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3)

Linear regresson analysisy = 246527x + 14796

R² = 0.9986

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

0 0.5 1 1.5 2 2.5 3 3.5

Linear regression analysisy = 133197x + 15795

R² = 0.9954

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0 0.5 1 1.5 2 2.5 3 3.5

Blood concentration ( ug/mL) Blood concentration ( ug/mL)

0.0 2.5 5.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75(x10,000)

2.5 5.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75(x10,000)

Matrix blank comparisonPositive ionPlasma separation card matrix blankPlasma matrix blank

Matrix blank comparisonNegative ionPlasma separation card matrix blankPlasma matrix blank






Conclusions• In this limited study, plasma separation card (PSC) sampling delivered a quantitative analysis of warfarin spiked into

human blood.• PSC generated a linear calibration curve in both positive and negative ion modes (r2>0.99; n=5); • The warfarin plasma results achieved by using the PSC technique were in broad agreement with conventional plasma

sampling data.• The plasma generated by the �ltration process appears broadly similar to plasma derived from conventional

centrifugation.• Further work is required to consider the robustness and validation in a routine analysis.

References• Jensen, B.P., Chin, P.K.L., Begg, E.J. (2011) Quanti�cation of total and free concentrations of R- and S-warfarin in

human plasma by ultra�ltration and LC-MS/MS. Anal Bioanal Chem., 401, 2187-2193• Radwan, M.A., Bawazeer, G.A., Aloudah, N.M., Aluadeib, B.T., Aboul-Enein, H.Y. (2012) Determination of free and total

warfarin concentrations in plasma using UPLC MS/MS and its application to patient samples. Biochemical Chromatography, 26, 6-11

Figure 6. Lipid pro�les from the same human blood sample extracted using a plasma separation card (left hand pro�le) compared to a conventional plasma samples (centrifugation). Both lipid pro�les are comparable in terms of distribution and the number of lipids detected (the scaling has been

normalized to the most intense lipid signal).

Conventional plasma samplePositive ionLCMS-IT-TOFLipid pro�ling

Diacylglycero-phosphocholines

Ceramidephosphocholines

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

MonoacylglycerophosphoethanolaminesMonoacylglycerophosphocholines

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

Plasma separation card samplePositive ionLCMS-IT-TOFLipid pro�ling

PO-CON1462E

Development and Validation of Direct Analysis Method for Screeningand Quantitation of Amphetamines in Urine by LC/MS/MS

ASMS 2014 MP535

Zhaoqi Zhan1, Zhe Sun1, Jie Xing1, Helmy Rabaha2

and Lim Swee Chin2 1Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE;2Department of Scienti�c Services, Ministry of Health,

Brunei Darussalam

2

Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

IntroductionAmphetamines are among the most commonly abused drugs type worldwide. The conventional analytical procedure of amphetamines in human urine in forensic laboratory involves initial immunological screening followed by GCMS con�rmation and quantitation [1]. The new guidelines of SAMHSA under U.S. Department of Health and Human Services effective in Oct 2010 [2] allowed use of LC/MS/MS for screening, con�rmation and quantitation of illicit drugs including amphetamines. One of the advantages by using LC/MS/MS is that derivatization of amphetamines before analysis is not needed, which was a standard procedure of GCMS method. Since analysis speed and throughput could be enhanced signi�cantly, development and use of LC/MS/MS methods are in

demand and many such efforts have been reported recently [3]. The objective of this study is to develop a fast LC/MS/MS method for direct analysis of amphetamines in urine without sample pre-treatment (except dilution with water) on LCMS-8040, a triple quadrupole system featured as ultra fast mass spectrometry (UFMS). The compounds studied include amphetamines (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guidelines, four potential interferences as well as PMPA as a control reference (Table 1). Very small injection volumes of 0.1uL to 1uL was adopted in this study, which enabled the method suitable for direct injection of untreated urine samples without causing signi�cant contamination to the ESI interface.

ExperimentalThe stock standard solutions of amphetamines and related compounds as listed in Table 1 were prepared in the Toxicology Laboratory in the Department of Scienti�c Services (MOH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and spiked samples were not pre-treated by any means except dilution of 10 times with Milli-Q water.An LCMS-8040 triple quadrupole coupled with a Nexera UHPLC system (Shimadzu Corporation) was used. The analytical column used was a Shim-pack XR-ODS III UHPLC column (1.6 µm) 50mm x 2mm. The mobile phases used

were water (A) and MeOH (B), both with 0.1% formic acid. A fast gradient elution program was developed for analysis of the ten compounds: 0-1.6min, B=2%->14%; 1.8-2.3min, B=70%; 2.4min, B=2%; end at 4min. The total �ow rate was 0.6 mL/min. Positive ESI ionization mode was applied with drying gas �ow of 15 L/min, nebulizing gas �ow of 3 L/min, heating block temperature of 400 ºC and DL temperature of 250 ºC. Various injection volumes from 0.1 uL to 5 uL were tested to develop a method with a lower injection volume to reduce contamination of untreated urine samples to the interface.


MRM optimization of the ten compounds (Table 1) was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions were selected for each compound, one for quantitation and second one for confirmation (Table 1). The ten compounds were separated and eluted in 0.75~2.2 minutes as sharp peaks as shown in Figure 1. In addition to analysis speed and detection sensitivity, this method development was also focused on evaluation of small to ultra-small injection volumes to develop a method suitable for direct injection of urine samples without any

pre-treatment while it should not cause significant contamination to the interface. The Nexera SIL-30A auto-sampler enables to inject as low as 0.10 uL of sample with excellent precision.Figure 1 shows a few selected results of direct injection of urine blank (a) and mixed standards spiked in urine with 1 uL (c and d) and 0.1 uL (b) injection. It can be seen that all compounds (12.5 ppb each in urine) could be detected with 0.1uL injection except MDA and Norpseudo-E. With 1uL injection, all of them were detected.

Method development of direct injection of amphetamines in urine

3


Figure 1: MRM chromatograms of urine blank (a) and spiked samples of amphetamines and related compounds in urine by LC/MS/MS method with 1uL and 0.1uL injection volumes.

Table 1: MRMs of amphetamines and related compounds

Compound Abbr. RT (min) MRM

Nor pseudo ephedrine

Ephedrine

Pseudo ephedrine

Amphetamine

Methampheta-mine

3,4-methylenedi oxyamphetamine

3,4-methylene dioxymeth amphetamine

3,4-methylene dioxy-N-ethyl amphetamine

Phentermine

Propyl amphetamine

Nor pseudo-E

Ephe

Pseudo-E

AMPH

MAMP

MDA

MDMA

MDEA

Phent

PAMP

Cat.

B1

B2

B3

A1

A2

A3

A4

A5

B4

R

0.75

0.94

1.01

1.20

1.42

1.49

1.59

1.94

1.93

2.20

152>134

152>115

166>148

166>91

166>148

166>91

136>91

136>119

150>91

150>119

180>163

180>163

194>163

194>105

208>163

208>105

150>91

150>119

178>91

178>65

CE (V)

-13

-23

-14

-31

-14

-30

-20

-14

-20

-14

-12

-38

-13

-22

-12

-24

-20

-40

-22

-47

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1.0

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3.0

(x10,000)

Phen

t

Nor

pseu

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Pseu

doEp

hedr

ine

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

AM

PH

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1.0

2.0

3.0

(x100,000)

Phen

t

Nor

pseu

do

Pseu

doEp

hedr

ine

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

AM

PH

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0.5

1.0

1.5

(x1,000,000)

Phen

t

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ine

MD

EA

MD

MA

MD

A

PAM

P

MA

MP

AM

PH

(a) Urine blank, 1 uL inj (b) 12.5ppb in urine, 0.1uL inj

(c) 12.5ppb, 1uL inj (d) 62.5ppb in urine, 1uL inj

0.0 0.5 1.0 1.5 2.0 2.5 min0.0

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4


Figure 2: Calibration Curves of amphetamines spiked in urine with 0.1uL injection

Linear calibration curves were established for the ten compounds spiked in urine with different injection volumes: 0.1, 0.2, 0.5, 1, 2 and 5 uL. Good linearity of calibration curves (R2>0.999) were obtained for all injection volumes including 0.1uL, an ultra-small injection

volume. The calibration curves with 0.1 uL injection volume are shown in Figure 2. The linearity (r2) of all compounds with 0.1 uL and 1 uL injection volumes are equivalently good as shown in Table 2.

Calibration curves with small and ultra-small injection volumes

Repeatability of peak area was evaluated with a same loading amount (6.25 pg) but with different injection volumes. The RSD shown in Table 2 were 1.6% ~ 7.9% and 1.6 ~ 7.8% for 0.1uL and 1uL injection, respectively. It is worth to note that the repeatability of every compounds with of 0.1uL injection is closed to that of 1uL injection as well as 5uL injection (data not shown).Matrix effect of the method was determined by comparison of peak areas of mixed standards in pure water and in urine matrix. The results of 62.5ppb with 1uL injection were at 102-115% except norpseudoephedrine (79%) as shown in Table 2.Accuracy and sensitivity of the method were evaluated with spiked samples of low concentrations. The results of

LOD and LOQ of the ten compounds in urine are shown in Table 3. Since the working samples (blank and spiked) were diluted for 10 times with water before injection, the concentrations and LOD/LOQ of the method described above for source urine samples have to multiply a factor of 10. Therefore, the LOQs of the method for urine specimens are at 2.1-17.1 ng/mL for AMPH, PAMP, MDMA and MDEA and 53 ng/mL for MDA. The LOQs for the potential interferences (Phentermine, Ephedrine, Pseudo-Ephedrine and Norpseudo-Ephedrine) are at 17-91 ng/mL, 2.4 ng/mL for the internal reference MAMP. The sensitivity of the direct injection LC/MS/MS method are significantly higher than the confirmation cutoff (250 ng/mL) required by the SAMHSA guidelines.

Performance validation

0 250 Conc.0.0

1.0

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3.0

Area (x100,000)

0 250 Conc.0.0

2.5

5.0

Area (x100,000)

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2.5

5.0

Area (x100,000)

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2.5

5.0

7.5

Area (x100,000)

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2.5

5.0

Area (x100,000)

0 250 Conc.0.0

2.5

5.0Area (x100,000)

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Area (x1,000,000)

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5.0

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Area (x100,000)

0 250 Conc.0.00

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0.75

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0 250 Conc.0.0

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5.0

7.5

Area (x100,000)

AMPH MAMP

Phent PAMP

MDA MDMA MDEA

Ephedrine Pseudo-ENor pseudo-E


5

Table 2: Method Performance with different inj. volumes

NameCalibration curve, R2

(0.1uL)

RSD% area (n=6)

(0.1uL)

M.E. %1

(1uL)

Norpseudo-E

Ephe

Pseudo-E

AMPH

MAMP

MDA

MDMA

MDEA

Phent

PAMP

0.9992

0.9995

0.9994

0.9997

0.9998

0.9978

0.9993

0.9996

0.9998

0.9998

(1uL)

0.9996

0.9998

0.9986

0.9998

0.9999

0.9995

0.9998

0.9998

0.9998

0.9932

(ppb)2

1-500

2.5-500

1-500

1-500

1-500

2.5-500

1-500

1-500

2.5-500

1-500

4.5

3.2

3.7

3.5

1.6

7.9

1.8

3.5

4.1

2.9

(1uL)

5.7

2.9

3.3

2.4

2.3

7.8

4.5

2.9

1.6

2.0

79

115

113

102

110

103

115

115

106

102

The method operational stability with 1uL injection was tested with spiked samples of 25 ppb in five urine specimens, corresponding to 250 ng/mL in the source urine samples. Continuous injections of accumulated 120 times was carried out in about 10 hours. The purpose of the experiment was to evaluate the operational stability against the ESI source contamination by urine samples without pre-treatment. Figure 3 shows the first injection and the

120th injection of the same spiked sample (S1) as well as other spiked samples (S2, S3, S4 and S5) in between. Decrease in peak areas of the compounds occurred, but the degree of the decrease in average was about 17% from the first injection to the last injection. This result indicates that it is possible to carry out direct analysis of urine samples (10 times dilution with water) by the high sensitivity LC/MS/MS method with a very small injection volume.

Method operational stability

1: Measured with mixed stds of 62.5 ppb in clear solution and spiked in urine2: For 0.1uL injection, the lowest conc. is 2.5 or 12.5 ppb

Table 3: Method performance: sensitivity & accuracy (1uL)

NameMeas. S/N LOQ

Norpseudo-E

Ephe

Pseudo-E

AMPH

MAMP

MDA

MDMA

MDEA

Phent

PAMP

1.2

2.2

1.0

1.1

1.0

2.4

1.1

1.1

2.6

1.0

Accuracy

(%)

118.7

88.2

99.5

114.1

103.6

96.3

106.4

111.8

105.3

101.7

Conc. (ppb)

Prep.

1.0

2.5

1.0

1.0

1.0

2.5

1.0

1.0

2.5

1.0

2.3

2.7

5.9

6.7

21.8

4.5

51.9

28.5

2.9

42.2

Sensitivity (ppb)

LOD

1.53

2.41

0.50

0.51

0.14

1.60

0.06

0.12

2.73

0.07

5.09

8.04

1.67

1.71

0.47

5.34

0.21

0.39

9.10

0.24






Figure 3: Selected chromatograms of continuous injections of spiked samples (25 ppb) with 1 µL injection. Five urine specimens S1, S2, S3, S4 and S5 were used to prepare these spiked samples.

References1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. Mandatory guidelines for Federal Workplace Drug Testing Program, 73 FR 71858-71907, Nov. 25, 2008. 3. Huei-Ru Lina, Ka-Ian Choia, Tzu-Chieh Linc, Anren Hu,, Journal of Chromatogr B, 2013, 929, 133–141.

ConclusionsIn this study, we developed a fast LC/MS/MS method for direct analysis of �ve amphetamines and related compounds in human urine for screening and quantitative con�rmation. Very small injection volumes of 0.1~1.0 uL were adopted to minimize ESI contamination and enhance

operational stability. The good performance results observed reveals that screening and con�rmation of amphetamines in human urine by direct injection to LC/MS/MS is possible and the method could be an alternative choice in forensic and toxicology analysis.

0.0 1.0 2.0 min

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S1 (110th inj)

S2 (11th inj) S3 (21st inj)

S4 (31st inj) S5 (41st inj)

PO-CON1482E

Next generation plasma collectiontechnology for the clinical analysis oftemozolomide by HILIC/MS/MS

ASMS 2014 WP641

Alan J. Barnes1, Carrie-Anne Mellor2,

Adam McMahon2, Neil Loftus1

1Shimadzu, Manchester, UK 22WMIC, University of Manchester, UK

2

Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

IntroductionPlasma extraction technology is a novel technique achieved by applying a blood sample to a laminated membrane stack which allows plasma to �ow through the asymmetric �lter whilst retaining the cellular components of the blood sample.Plasma separation card technology was applied to the quantitative analysis of temozolomide (TMZ); an oral imidazotetrazine alkylating agent used for the treatment of Grade IV astrocytoma, an aggressive form of brain tumour.

Under physiological conditions TMZ is rapidly converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) which in-turn degrades by hydrolysis to 5-aminoimidazole-4-carboxamide (AIC). Storage of plasma has previously shown that both at -70C and 4C degradation still occurs. In these experiments, whole blood containing TMZ standard was applied to NoviPlex plasma separation cards (PSC). The aim was to develop a robust LC/MS/MS quantitative method for TMZ.


TMZ spiked human blood calibration standards (50uL) were applied to the PSC as described below in figure 1.

Plasma separation

1 3 42

A NoviPlex card is removed from foil packaging.

Approximately 50uL of whole blood is added to the test area.

After 3 minutes, the top layer is completely removed (peeled back).

The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes.

The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.

Figure 1. Noviplex plasma separation card work�ow

3


Control Spot:[Determines whether enough blood was placed on the card].

Filtration Layer[Filtration layer captures blood cells by a combination of �ltration and adsorption. The average linear vertical migration rate is approximately 1um/sec].

Collection Layer[Loads with a speci�c aliquot of plasma onto a 6.35mm disc]. Although �ow through the �ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric �ow rate into the Collection Disc of 400 pL/mm2/sec.

Isolation Screen[Precludes lateral wicking along the card surface].

Spreading Layer[Lateral spreading layer rapidly spreads blood so it will enter the �ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].

Figure 1. Noviplex plasma separation card work�ow (Cont'd)

Figure 2. Applying a blood sample, either as a �nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and �ltration whilst plasma advances through the membrane stack

by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.

TMZ was extracted from the dried plasma collection discs by adding 40uL acetonitrile + 0.1% formic acid, followed by centrifugation 16,000g for 5 min. 30uL supernatant was added directly to the LC/MS/MS sample vial for analysis.

As a control, conventional plasma samples were prepared by centrifuging the human blood calibration standards at 1000g for 10min. TMZ was extracted from 2.5uL of plasma using the same extraction protocol as applied for PSC.

Sample preparation

4


Figure 3. HILIC LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99).HILIC was considered in response to previous published data and to minimize potential stability issues. However, to reduce sample cycle times a reverse

phase method was also developed.

Results

Temozolomide is known to be unstable under physiological conditions and is converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) by

a nonenzymatic, chemical degradation process. Previous studies have used HILIC to analyze the polar compound and to avoid degradation in aqueous solutions.

HILIC LC/MS/MS

LC/MS/MS analysis

Ionisation : Electrospray, positive mode

MRM 195.05 >138.05 CE -10

HPLC : HILIC

Nexera UHPLC system

Flow rate : 0.5mL/min (0-7min), 1.8mL/min (7.5min-17.5min)

Mobile phase : A= Water + 0.1% formic acid

B= Acetonitrile + 0.1% formic acid

Gradient : 95% B – 30%% B (6.5 min),

30% B (7.5 min), 95% B (18 min)

Analytical column : ZIC HILIC 150 x 4.6mm 5um 200ª


Injection volume : 10uL

Reverse Phase

Nexera UHPLC system

0.4mL/min

A= Water + 0.1% formic acid

B= methanol + 0.1% formic acid

5% B – 100%% B (3 min),

100% B (7 min), 5% B (10 min)

Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A

50ºC

2µL

Desolvation line : 300ºC

Drying/Nebulising gas : 10L/min, 2L/min

Heating block : 400ºC

Linear regression analysisy = 64578x + 18473

R² = 0.9988

0

100000

200000

300000

400000

500000

600000

700000

0 2 4 6 8 10 12

Peak Area

Blood Concentration (ug/mL)

Plasma separation cardHILIC analysisTMZ Single point calibration standardsCalibration curve 0.2-10ug/mL

0.0 2.5 5.0 min

0.0

1.0

2.0

3.0

4.0

5.0(x10,000)

Plasma separation cardHILIC analysisTMZ m/z 195.05 > 138.05

Q1 (V) -20 Collision energy -10 Q3 (V) -12

8.0ug/mL calibn std

0.5ug/mL calibn std

5


Figure 4. Reverse phase LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99; replicate samples were included in the liner regression analysis at 0.5 and 8ug/mL; n=3).

Due to the relatively long cycle time (18 min), a faster reversed phase method was developed (10 min). Sample preparation was modified with PSC sample disk placed in 40uL methanol + 0.1% formic acid, followed by centrifugation 16,000g 5 min. 20uL supernatant was

added directly to the LC/MS sample vial plus 80uL water + 0.1% formic acid. In addition to reversed phase being faster, the sample injection volume was reduced to just 2uL as a result of higher sensitivity from narrower peak width (reversed phase,13 sec; HILIC, 42 sec).

Reversed Phase LC/MS/MS

Figure 5. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the con�rmatory ion transition 195.05>67.05 both the PSC and plasma sample are in broad agreement with regard to matrix ion signal distribution.

Comparison between PSC and plasma

Linear regression analysisy = 72219x - 355.54

R² = 0.9997

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

0 2 4 6 8 10 12

Peak Area

Blood Concentration (ug/mL)

Plasma separation cardRP analysisTMZ m/z 195.05 > 138.05




Plasma separation cardRP analysisTMZ calibration curveReplicate calibration points at 0.5ug/mL and 8ug/mL (n=3)

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Plasma matrix blank

500ng/mL comparisonMRM 195.05>67.05Plasma separation card 500ng/mL calibration standard

Plasma500ng/mL calibration standard






ConclusionsThis technology has the potential for a simplified clinical sample collection by the finger prick approach, with future work aimed to evaluate long term sample stability of PSC samples, stored at room temperature. Quantitation of drug metabolites MTIC and AIC also could help provide a measure of sample stability.

References• Andrasia, M., Bustosb, R., Gaspara,A., Gomezb, F.A. & Kleknerc, A. (2010) Analysis and stability study of

temozolomide using capillary electrophoresis. Journal of Chromatography B. Vol. 878, p1801-1808• Denny, B.J., Wheelhouse, R.T., Stevens, M.F.G., Tsang, L.L.H., Slack, J.A., (1994) NMR and molecular modeliing

investigation of the mechanism of activation of the antitumour drug temozolomide and its Interaction with DNA. Biochemistry, Vol. 33, p9045-9051

Figure 6. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the quantitation ion transition 195.05>138.05 both the PSC and plasma sample are in broad agreement in signal distribution and intensity including the presence of

a matrix peak at 2.4mins.

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0.75

1.00

1.25

1.50

(x10,000) Matrix blank comparisonMRM 195.05>138.05Plasma separation card matrix blank

Plasma matrix blank

500ng/mL comparisonMRM 195.05>138.05Plasma separation card 500ng/mL calibration standard

Plasma500ng/mL calibration standard

TMZ

TMZRt

1.7mins

Matrix peak Matrix peak

PO-CON1475E

Application of a Sensitive Liquid Chromatography-Tandem Mass SpectrometricMethod to Pharmacokinetic Study of Telbivudine in Humans

ASMS 2014 WP 629

Bicui Chen1, Bin Wang1, Xiaojin Shi1, Yuling Song2,

Jinting Yao2, Taohong Huang2, Shin-ichi Kawano2,

Yuki Hashi2

1 Pharmacy Department, Huashan Hospital,

Fudan University,

2 Shimadzu Global COE, Shimadzu (China) Co., Ltd.

2

Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

IntroductionTelbivudine is a synthetic L-nucleoside analogue, which is phosphorylated to its active metabolite, 5’-triphosphate, by cellular kinases. The telbivudine 5’-triphosphate inhibits HBV DNA polymerase (a reverse transcriptase) by competing with the natural substrate, dTTP. Incorporation

of 5’-triphosphorylated-telbivudine into viral DNA obligates DNA chain termination, resulting in inhibition of HBV replication. The objectives of the current studies were to develop a selective and sensitive LC-MS/MS method to determine of telbivudine in human plasma.

Method

(1) Add 100 μL of plasma into the polypropylene tube, add 40 μL of internal standard working solution (33 µg/mL, with thymidine phosphorylase) to all other tubes.

(2) Incubate the tubes for 1 h at 37 ºC in dark.(3) Add 200 μL of acetonitrile to all tubes, seal and vortex for 1 minutes.(4) Centrifuge the tubes for 5 minutes at 13000 rpm.(5) Transfer 200 μL supernatant to a clean glass bottle and inject into the HPLC-MS/MS system.

Sample Preparation

The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, CTO-30A column oven, DGU-30A5 on-line egasser, and SIL-30AC autosampler. The separation was carried out on GL Sciences InertSustain C18 column (3.0 mmI.D. x 100

mmL.) with the column temperature at 40 ºC. A triple quadruple mass spectrometer (Shimadzu LCMS-8050, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface.

LC-MS/MS Analysis


HPLC (Nexera UHPLC system)

Column : InertSustain (3.0 mmI.D. x 100 mmL., 2 μm, GL Sciences)

Mobile Phase A : water with 0.1% formic acid

Mobile Phase B : acetonitrile

Gradient Program : as shown in Table 1



Injection Volume : 2 µL

Table 1 Time Program

Time (min) Module Command Value

0.00

4.00

4.10

6.00

Pumps

Pumps

Pumps

Controller

Pump B Conc.

Pump B Conc.

Pump B Conc.

Stop

5

80

5

3


Results and DiscussionHuman plasma samples containing telbivudine ranging from 1.0 to 10000 ng/mL were prepared and extracted by protein precipitation and the �nal extracts were analyzed by LC-MS/MS. MRM chromatograms of telbivudine (1 ng/mL) and deuterated internal standard are presented in Fig. 1 (blank) and Fig. 2 (spiked). The linear regression for telbivudine was found to be >0.9999. The calibration curve with human plasma as the matrix were shown in Fig. 3. Excellent precision and accuracy were maintained for four orders of magnitude, demonstrating a linear dynamic range suitable for real-world applications. LLOQ for telbivudine was 1.0 ng/mL, which met the criteria for bias (%) and precision within ±15% both within run and between run. The

intra-day and inter-day precision and accuracy of the assay were investigated by analyzing QC samples. Intra-day precision (%RSD) at three concentration levels (3, 30, and 8000 ng/mL) were below 2.5% and inter-day precision (%RSD) was below 2.5%. The recoveries of telbivudine were 100.6±2.5 %, 104.5±1.5% and 104.3±1.6% at three concentration levels, respectively. The stability data showed that the processed samples were stable at the room temperature for 8 h, and there was no signi�cant degradation during the three freeze/thaw cycles at -20 ºC. The reinjection reproducibility results indicated that the extracted samples could be stable for 72 h at 10 ºC.

MS (LCMS-8050 triple quadrupole mass spectrometer)

Ionization : ESI

Polarity : Positive

Ionization Voltage : +0.5 kV (ESI-Positive mode)

Nebulizing Gas Flow : 3.0 L/min

Heating Gas Flow : 8.0 L/min

Drying Gas Flow : 12.0 L/min

Interface Temperature : 250 ºC

Heat Block Temperature : 300 ºC


Mode : MRM

Table 2 MRM Parameters

CompoundPrecursor

m/z

243.10

246.10

Productm/z

127.10

130.10

Dwell Time(ms)

100

100

Q1 Pre Bias(V)

-26

-16

Q3 Pre Bias(V)

-13

-25

CE (V)

-10

-9

Telbivudine

Telbivudine-D3

4


Figure 1 Representative MRM chromatograms of blank human plasma(left: transition for telbivudine, right: transition for internal standard)

Figure 2 Representative MRM chromatograms of telbivudine (left, 1 ng/mL) and internal standard (right) in human plasma

Figure 3 Calibration curve of telbivudine in human plasma

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1:Telbivudine 243.10>127.10(+) CE: -10.0

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(x1,000)2:Telbivudine-D3 246.10>130.10(+) CE: -9.0

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Telb

ivud

ine

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Telb

ivud

ine-

D3

0 2500 5000 7500 Conc. Ratio0.0

0.5

1.0

1.5

2.0

2.5

Area Ratio


5

Figure 4 Representative MRM chromatograms of real-world sample

CompoundCalibration

Curve

Y = (2.77×10-4)X + (3.39×10-5)

Linear Range(ng/mL)

1~10000

Accuracy(%)

93.1~116.6%

r

0.9998Telbivudine

Table 3 Accuracy and precision for the analysis of amlodipine in human plasma(in pre-study validation, n=3 days, six replicates per day)

Added Conc.(ng/mL)

3

400

8000

Intra-day Precision(%RSD)

2.18

1.52

1.76

Inter-day Precision(%RSD)

2.11

1.58

1.68

Accuracy(%)

107.7~114.4

91.6~95.9

95.4~101.3

Table 5 Matrix effect for QC samples (n=6)

QC Level

LQC

MQC

HQC

Added Conc.(ng/mL)

3

400

8000

Matrix Factor

82.3%

81.7%

90.8%

IS-NormalizedMatrix Factor

99.0%

101.0%

101.5%

Table 4 Recovery for QC samples (n=6)

QC Level

LQC

MQC

HQC

Concentartion(ng/mL)

3

400

8000

Recovery(%)

100.6

104.5

104.3

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(x1,000,000)2:Telbivudine-D3 246.10>130.10(+) CE: -9.0






ConclusionResults of parameters for method validation such as dynamic range, linearity, LLOQ, intra-day precision, inter-day precision, recoveries, and matrix effect factors were excellent. The sensitive LC-MS/MS technique provides a powerful tool for the high-throughput and highly selective analysis of telbivudine in clinical trial study.

PO-CON1449E

Accelerated and robust monitoringfor immunosuppressants using triplequadrupole mass spectrometry

ASMS 2014 WP628

Natsuyo Asano1, Tairo Ogura1, Kiyomi Arakawa1

1 Shimadzu Corporation. 1, Nishinokyo Kuwabara-cho,

Nakagyo-ku, Kyoto 604–8511, Japan

2

Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

IntroductionImmunosuppressants are drugs which lower or suppress activity of the immune system. They are used to prevent the rejection after transplantation or treat autoimmune disease. To avoid immunode�ciency as adverse effect, it is recommended to monitor blood level of therapeutic drug with high throughput and high reliability. There are several analytical technique to monitor drugs, LC/MS is superior in terms of cross-reactivity at low level and throughput of

analysis. Therefore, it is important to analyze these drugs in blood by using ultra-fast mass spectrometer to accelerate monitoring with high quantitativity. We have developed analytical method for four immunosuppressants (Tacrolimus, Rapamycin, Everolimus and Cyclosporin A) with two internal standards (Ascomycin and Cyclosporin D) using ultra-fast mass spectrometer.

Figure 1 Structure of immunosuppressants and internal standards (IS)

O

HO

O

O OH

ON

OO

OHO

O

H O

HOO

O

O O OH

OOO

N

OO

O

HO

O

HO

O

O O OH

OOO

N

OO

O

HO

O

TacrolimusMW: 804.02

EverolimusMW: 958.22

RapamycinMW: 914.17

N

O

N O

NH

OHN

O

N

OHN

O

N

N

O

O

N

HO

HN

O

O

N

O

H

O

O

HO HO

N

O

OO

O

O

H

OH

O

HO

N N

OO

HN

O

N

O

N O

N

ON

OH

O

NH

OHN

O

NO

HNO

Cyclosporin AMW: 1202.61

Ascomycin (IS)MW: 792.01

Cyclosporin D (IS)MW: 1216.64

3


Methods and MaterialsStandard samples of each compound were analyzed to optimize conditions of liquid chromatograph and mass spectrometer. Whole blood extract was prepared based on liquid-liquid extraction described bellow.

2.7 mL of Whole blood and 20.8 mL of Water ↓Vortex for 15 seconds ↓Add 36 mL of MTBE/Cyclohexane (1:3) ↓Vortex for 15 seconds and Centrifuge with 3000 rpm at 20 ºC for 10 minutes ↓Extract an Organic phase ↓Evaporate and Dry under a Nitrogen gas stream ↓Redissolve in 1.8 mL of 80 % Methanol solution with 1 mmol/L Ammonium acetate ↓Vortex for 1 minute and Centrifuge with 3000 rpm at 4 ºC for 5 minutes ↓Filtrate and Transfer into 1 mL glass vial

Table 1 Analytical conditions

UHPLC

Liquid Chromatograph : Nexera (Shimadzu, Japan)

Analysis Column : YMC-Triart C18 (30 mmL. × 2 mmI.D.,1.9 μm)

Mobile Phase A : 1 mmol/L Ammonium acetate - Water

Mobile Phase B : 1 mmol/L Ammonium acetate - Methanol

Gradient Program : 60 % B. (0 min) – 75 % B. (0.10 min) – 95 % B. (0.70 – 0.90 min) –

60 % B. (0.91 – 1.80 min)



Injection Volume : 1.5 µL

MS

MS Spectrometer : LCMS-8050 (Shimadzu, Japan)

Ionization : ESI (negative)

Probe Voltage : -4.5 ~ -3 kV


Drying Gas Flow : 5.0 L/min

Heating Gas Flow : 15.0 L/min



HB Temperature : 390 ºC

4


ResultImmunosuppressants, which we have developed a method for monitoring of, has been often observed as ammonium or sodium adduct ion by using positive ionization. In general, protonated molecule (for positive) or deprotonated molecule (for negative) is more preferable for reliable quantitation than adduct ions such as ammonium, sodium, and potassium adduct. In this study,

each compound was detected as deprotonated molecule in negative mode by using heated ESI source of LCMS-8050 (Table 2).The separation of all compounds was achieved within 1.8 min, with a YMC-Triart C18 column (30 mmL. × 2 mmI.D.,1.9 μm) and at 65 ºC of column oven temperature.

Figure 2 MRM chromatograms of immnosuppresants in human whole blood (50 ng/mL)

Peak No.

1

2

3

4

5

6

Compound

Ascomysin (IS)

Tacrolimus

Rapamycin

Everolimus

Cyclosporin A

Cyclosporin D (IS)

Porality

neg

neg

neg

neg

neg

neg

Precursor ion (m/z)

790.40

802.70

912.70

956.80

1200.90

1215.10

Product ion (m/z)

548.20

560.50

321.20

365.35

1088.70

1102.60

Table 2 MRM transitions

0.75 1.00 1.25 min

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

(x100,000)

2

4

3

1

5

6


5

Figure 3 MRM chromatograms at LLOQ and ISTD (left), and calibration curves (right) for four immnosuppresants in human whole blood

a) Tacrolimus

0.5 – 1000 ng/mL

0.5 ng/mL

Ascomycin40 ng/mL

c) Everolimus

0.5 ng/mL

Ascomycin40 ng/mL 0.5 – 100 ng/mL

b) Rapamycin

0.5 ng/mL

Ascomycin40 ng/mL 0.5 – 500 ng/mL

d) Cyclosporin A

Cyclosporin D100 ng/mL

0.5 ng/mL

0.5 – 1000 ng/mL






Figure 3 illustrates both a calibration curve and chromatogram at the lowest calibration level for all immunosuppressants analyzed. Table 3 lists both the reproducibility and accuracy for each immunosuppressant that has been simultaneously measured in 1.8 minutes.

In high speed measurement condition, we have achieved high sensitivity and wide dynamic range for all analytes. Additionally, the accuracy of each analyte ranged from 88 to 110 % and area reproducibility at the lowest calibration level of each analyte was less than 20%.　

Conclusions• Monitoring with negative mode ionization permitted more sensitive, robust and reliable quantitation for four

immunosuppressants.• A total of six compounds were measured in 1.8 minutes. The combination of Nexera and LCMS-8050 provided a faster

run time without sacri�cing the quality of results.• Even with a low injection volume of 1.5 μL, the lower limit of quantitation (LLOQ) for all compounds was 0.5 ng/mL. • In this study, it is demonstrated that LCMS-8050 is useful for the rugged and rapid quantitation for immunosuppressants

in whole blood.

AcknowledgementWe appreciate suggestions from Prof. Kazuo Matsubara and Assoc. Prof. Ikuko Yano from the department of pharmacy, Kyoto University Hospital, and Prof. Satohiro Masuda from the department of pharmacy, Kyusyu University Hospital.

Table 3 Reproducibility and Accuracy

Compound

Tacrolimus

Concentration

Low (0.5 ng/mL) Low-Mid (2 ng/mL)High (1000 ng/mL)

CV % (n = 6)

18.013.02.87

Accuracy %

99.499.588.7

RapamycinLow (0.5 ng/mL)

Low-Mid (5 ng/mL)High (500 ng/mL)

6.872.883.41

95.6109.390.0

EverolimusLow (0.5 ng/mL)


10.45.112.26

95.3104.493.3

Cyclosporin ALow (0.5 ng/mL)


7.312.362.67

95.199.994.9

PO-CON1468E

Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazidefrom plasma using LC/MS/MS

ASMS 2014 TP497

Shailendra Rane, Rashi Kochhar, Deepti Bhandarkar,

Shruti Raju, Shailesh Damale, Ajit Datar,

Pratap Rasam, Jitendra Kelkar

Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh

Chambers, Makwana Road, Marol, Andheri (E),

Mumbai-400059, Maharashtra, India.

2

Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

IntroductionFelodipine is a calcium antagonist (calcium channel blocker), used as a drug to control hypertension[1]. Hydrochlorothiazide is a diuretic drug of the thiazide class that acts by inhibiting the kidney’s ability to retain water. It is, therefore, frequently used for the treatment of hypertension, congestive heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis and the prevention of kidney stones[2].Efforts have been made here to develop high sensitive

methods of quantitation for these two drugs using LCMS-8050 system from Shimadzu Corporation, Japan.Presence of heated Electro Spray Ionization (ESI) probe in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development quantitation method at low ppt level for both Felodipine and Hydrochlorthiazide.

Method of Analysis

To 100 µL of plasma, 500 µL of cold acetonitrile was added for protein precipitation then put in rotary shaker at 20 rpm for 15 minutes for uniform mixing. It was centrifuged

at 12000 rpm for 15 minutes. Supernatant was collected and evaporated to dryness at 70 ºC and finally reconstituted in 200 µL Methanol.

Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile

Figure 2. Structure of Hydrochlorothiazide

HydrochlorothiazideHydrochlorothiazide, abbreviated HCTZ (or HCT, HZT), is a diuretic drug of the thiazide class that acts by inhibiting the kidney‘s ability to retain water. Hydrochlorothiazide is 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide.Its empirical formula is C7H8ClN3O4S2 and its structure is shown in Figure 2.

Figure 1. Structure of Felodipine

FelodipineFelodipine is a calcium antagonist (calcium channel blocker). Felodipine is a dihydropyridine derivative that is chemically described as ± ethyl methyl 4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate. Its empirical formula is C18H19Cl2NO4 and its structure is shown in Figure 1.

3


LC/MS/MS analysisCompounds were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8050 triple quadrupole system (Shimadzu

Corporation, Japan), The details of analytical conditions are given in Table 1 and Table 2.

• Felodipine Calibration Std : 5 ppt, 10 ppt, 50 ppt, 100 ppt, 500 ppt, 1 ppb and 10 ppb• HCTZ Calibration Std : 2 ppt, 5 ppt, 10 ppt, 50 ppt, 100 ppt, and 500 ppt

To 500 µL plasma, 100 µL sodium carbonate (1.00 mol/L) and 5 mL of diethyl ether : hexane (1:1 v/v) was added. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing and centrifuged at 12000 rpm for 15

minutes. Supernatant was collected and evaporated to dryness at 60 ºC. It was finally reconstitute in 1000 µL Methanol.

Preparation of matrix matched plasma by liquid-liquid extraction method using diethyl ether and hexane mixture (1:1 v/v)

Response of Felodipine and Hydrochlorothiazide were checked in both above mentioned matrices. It was found that cold acetonitrile treated plasma and diethyl ether: hexane (1:1 v/v) treated plasma were suitable for

Felodipine and Hydrochlorothiazide molecules respectively. Calibration standards were thus prepared in respective matrix matched plasma.

Preparation of calibration standards in matrix matched plasma

Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe

LCMS-8050 triple quadrupole mass spectrometer by Shimadzu (shown in Figure 3), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity), Ultra fast scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability.

In order to improve ionization efficiency, the newly developed heated ESI probe (shown in Figure 4) combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide range of target compounds with considerable reduction in background.

4


Results

LC/MS/MS method for Felodipine was developed on ESI positive ionization mode and 383.90>338.25 MRM transition was optimized for it. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest concentrations (5 ppt) as seen in Figure 5 and Figure 6

respectively. Calibration curves as mentioned with R2 = 0.998 were plotted (shown in Figure 7). Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Also, LOD as 2 ppt and LOQ as 5 ppt was obtained.

LC/MS/MS analysis results of Felodipine

• Column : Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm)

• Flow rate : 0.3 mL/min

• Oven temperature : 40 ºC

• Mobile phase : A: 10 mM ammonium acetate in water

B: methanol

• Gradient program (%B) : 0.0 – 3.0 min → 90 (%); 3.0 – 3.1 min → 90 – 100 (%);

3.1 – 4.0 min → 100 (%); 4.0– 4.1 min → 100 – 90 (%)

4.1 – 6.5 min → 90 (%)

• Injection volume : 10 µL

• MS interface : ESI

• Nitrogen gas �ow : Nebulizing gas 1.5 L/min; Drying gas 10 L/min;

• Zero air �ow : Heating gas 10 L/min

• MS temperature : Desolvation line 200 ºC; Heating block 400 ºC

Interface 200 ºC

Table 1. LC/MS/MS conditions for Felodipine




• Mobile phase : A: 0.1% formic acid in water

B: acetonitrile

• Gradient program (%B) : 0.0 – 1.0 min → 80 (%); 1.0 – 3.5 min → 40 – 100 (%);

3.5 – 4.5 min → 100 (%); 4.5– 4.51min → 100 – 80 (%)

4.51 – 8.0 min → 90 (%)



• Nitrogen gas �ow : Nebulizing gas 2.0 L/min; Drying gas 10 L/min;

• Zero air �ow : Heating gas 15 L/min

• MS temperature : Desolvation line 250 ºC; Heating block 500 ºC

Interface 300 ºC

Table 2. LC/MS/MS conditions for Hydrochlorothiazide

5


Figure 5. Felodipine at 10 ppb in matrix matched plasma Figure 6. Felodipine at 5 ppt in matrix matched plasma

Figure 7. Calibration curve of Felodipine

LC/MS/MS method for Hydrochlorothiazide was developed on ESI negative ionization mode and 296.10>204.90 MRM transition was optimized for it. Checked matrix matched plasma standards for highest (500 ppt) as well as lowest (2 ppt) concentrations as seen in Figures 8 and 9 respectively.

Calibration curves as mentioned with R2=0.998 were plotted (shown in Figure 10). Also as seen in Table 4, % Accuracy was studied to confirm the reliability of method. Also, LOD as 1 ppt and LOQ as 2 ppt were obtained.

LC/MS/MS analysis results of Hydrochlorothiazide

Table 3: Results of Felodipine calibration curve

Nominal Concentration (ppb)

Measured Concentration (ppb)

% Accuracy(n=3)

% RSD for area counts (n=3)

0.005

0.01

0.05

0.1

0.5

1

10

Standard

STD-FEL-01

STD-FEL-02

STD-FEL-03

STD-FEL-04

STD-FEL-05

STD-FEL-06

STD-FEL-07

Sr. No.

1

2

3

4

5

6

7

0.005

0.010

0.053

0.103

0.469

0.977

10.023

97.43

103.80

104.47

103.13

94.88

97.33

100.90

9.87

8.76

2.24

1.23

1.33

0.95

0.60

0.0 2.5 5.0

0.0

2.5

5.0(x100,000)383.90>338.25(+)

FELO

DIP

INE

0.0 2.5 5.0

0.0

0.5

1.0

1.5

2.0

(x1,000)383.90>338.25(+)

FELO

DIP

INE

0.0 2.5 5.0 7.5 Conc.0.0

0.5

1.0

1.5

2.0Area (x1,000,000)

1 2 3 4 5

6

7

0.05 0.10 Conc.0.0

0.5

1.0

1.5

2.0

2.5

3.0Area (x10,000)

1 2

3

4


6

Figure 8. Hydrochlorothiazide at 500 ppt in matrix matched plasma Figure 9. Hydrochlorothiazide at 2 ppt in matrix matched plasma

Figure 10. Calibration curve of Hydrochlorothiazide

Conclusion• Highly sensitive LC/MS/MS method for Felodipine and Hydrochlorothiazide was developed on LCMS-8050 system.• LOD of 2 ppt and LOQ of 5 ppt was achieved for Felodipine and LOD of 1 ppt and LOQ of 2 ppt was achieved for

Hydrochlorothiazide by matrix matched methods.• Heated ESI probe of LCMS-8050 system enables drastic augment in sensitivity with considerable reduction in

background. Hence, LCMS-8050 system from Shimadzu is an all rounder solution for bioanalysis.

Table 4. Results of Hydrochlorothiazide calibration curve



% Accuracy(n=3)


0.002

0.005

0.01

0.05

0.1

0.5

Standard

STD-HCTZ-01

STD-HCTZ-02

STD-HCTZ-03

STD-HCTZ-04

STD-HCTZ-05

STD-HCTZ-06

Sr. No.

1

2

3

4

5

6

0.0020

0.0048

0.0099

0.0512

0.1019

0.4944

102.03

95.50

100.07

102.67

102.11

102.13

6.65

3.53

3.80

1.60

3.58

1.68

0.0 2.5 5.0 7.5

0.0

0.5

1.0

1.5

(x10,000)296.10>204.90(-)

HC

TZ

0.0 2.5 5.0 7.5

0.0

0.5

1.0

1.5

2.0

2.5(x100)

296.10>204.90(-)

HC

TZ

0.0 0.1 0.2 0.3 0.4 Conc.0.00

0.25

0.50

0.75

1.00Area (x100,000)

1 2 3

4

5

6

0.000 0.025 0.050 Conc.0.0

0.5

1.0

1.5

Area (x10,000)

1 2 3

4






References[1] YU Peng; CHENG Hang, Chinese Journal of Pharmaceutical Analysis, Volume 32, Number 1, (2012), 35-39(5).[2] Hiten Janardan Shah, Naresh B. Kataria, Chromatographia, Volume 69, Issue 9-10, (2009), 1055-1060.

PO-CON1467E

Highly sensitive quantitative estimationof genotoxic impurities from API and drug formulation using LC/MS/MS

ASMS 2014 TP496

Shruti Raju, Deepti Bhandarkar, Rashi Kochhar,

Shailesh Damale, Shailendra Rane, Ajit Datar,


Shimadzu Analytical (India) Pvt. Ltd.,

1 A/B Rushabh Chambers, Makwana Road, Marol,

Andheri (E), Mumbai-400059, Maharashtra, India.

2

Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

IntroductionThe toxicological assessment of Genotoxic Impurities (GTI) and the determination of acceptable limits for such impurities in Active Pharmaceutical Ingredients (API) is a dif�cult issue. As per European Medicines Agency (EMEA) guidance, a Threshold of Toxicological Concern (TTC) value of 1.5 µg/day intake of a genotoxic impurity is considered to be acceptable for most pharmaceuticals[1]. Dronedarone is a drug mainly used for indications of cardiac arrhythmias. GTI of this drug has been quantitated here. Method has been optimized for simultaneous analysis of DRN-IA {2-n-butyl-3-[4-(3-di-n-butylamino-propoxy)benzoyl]-5-nitro

benzofuran}, DRN-IB {5-amino-3-[4-(3-di-n-butylamino-propoxy)benzoyl}-2-n-butyl benzofuran} and BHBNB {2-n-butyl-3-(4-hydroxy benzoyl)-5-nitro benzofuran}. Structures of Dronedarone and its GTI are shown in Figure 1.As literature references available on GTI analysis are minimal, the feature of automatic MRM optimisation in LCMS-8040 makes method development process less tedious. In addition, the lowest dwell time and pause time and ultrafast polarity switching of LCMS-8040 ensures uncompromised and high sensitive quantitation.

Figure 1. Structures of Dronedarone and its GTI

O

OOH

NO2

C4H9

O

O O

C4H9

NH2

N

C4H9

C4H9

O

O O

C4H9

NO2

N

C4H9

C4H9

DRN-IA

O

O O

C4H9

NHSO2Me

N

C4H9

C4H9

Dronedarone

DRN-IB BHBNB

3


LC/MS/MS Analytical ConditionsAnalysis was performed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8040 triple quadrupole system (Shimadzu Corporation, Japan), shown in Figure 2. Limit of GTI for Dronedarone is 2 mg/kg. However, general dosage of Dronedarone is 400 mg, hence, limit for GTI is 0.8 µg/400 mg. This when reconstituted in 20 mL system, gives an

effective concentration of 40 ppb. For analytical method development it is desirable to have LOQ at least 30 % of limit value, which in this case corresponds to 12 ppb. The developed method gives provision for measuring GTI at much lower level. However, recovery studies have been done at 12 ppb level.

Figure 2. Nexera with LCMS-8040 triple quadrupole system by Shimadzu

Method of Analysis

• Preparation of DRN-IA and DRN-IB and BHBNB stock solutions 20 mg of each impurity standard was weighed separately and dissolved in 20 mL of methanol to prepare stock solutions

of each standard.

• Preparation of calibration levels GTI mix standards (DRN-IA, DRN-IB and BHBNB) at concentration levels of 0.5 ppb, 1 ppb, 5 ppb, 10 ppb, 40 ppb, 50

ppb and 100 ppb were prepared in methanol using stock solutions of all the three standards.

• Preparation of blank sample 400 mg of Dronedarone powder sample was weighed and mixed with 20 mL of methanol. Mixture was sonicated to

dissolve sample completely.

• Preparation of spiked (at 12 ppb level) sample 400 mg of sample was weighed and spiked with 60 µL of 1 ppm stock solution. Solution was then mixed with 20 mL of

methanol. Mixture was sonicated to dissolve sample completely.

Sample Preparation

4


Table 1. LC/MS/MS analytical conditions

Results

LC/MS/MS method was developed for simultaneous quantitation of GTI mix standards. MRM transitions used for all GTI are given in Table 2. No peak was seen in diluent (methanol) at the retention times of GTI for selected MRM transitions which confirms the absence of any interference from diluent (shown in Figure 3). MRM chromatogram of GTI mix standard at 5 ppb level is shown in Figure 4. Linearity studies were carried out using external standard

calibration method. Calibration graphs of each GTI are shown in Figure 5. LOQ was determined for each GTI based on the following criteria – (1) % RSD for area < 15 %, (2) % Accuracy between 80-120 % and (3) Signal to noise ratio (S/N) > 10. LOQ of 0.5 ppb was achieved for DRN-IB and BHBNB whereas 1 ppb was achieved for DRN-IA. Results of accuracy and repeatability for all GTI are given in Table 3.

LC/MS/MS analysis

• Column : Shim-pack XR-ODS II (75 mm L x 3 mm I.D.; 2.2 µm)

• Mobile phase : A: 0.1% formic acid in water

B: acetonitrile



• Gradient program (B%) : 0.0–2.0 min → 35 (%); 2.0–2.1 min → 35-40 (%);

2.1–7.0 min → 40-60 (%); 7.0–8.0 min → 60-100 (%);

8.0–10.0 min → 100 (%); 10.0–10.01 min → 100-35 (%);

10.01–13.0 min → 35 (%)


• MS interface : Electro Spray Ionization (ESI)

• MS analysis mode : MRM

• Polarity : Positive and negative

• MS gas �ow : Nebulizing gas 2 L/min; Drying gas 15 L/min

• MS temperature : Desolvation line 250 ºC; Heat block 400 ºC

Note: Flow Control Valve (FCV) was used for the analysis to divert HPLC �ow towards waste during elution of Dronedarone so as to prevent contamination of Mass Spectrometer.

Table 2: MRM transitions selected for all GTI

Name of GTI MRM transition Retention time (min) Mode of ionization

DRN-IB

DRN-IA

BHBNB

479.15>170.15

509.10>114.10

338.20>244.05

1.83

5.85

8.77

Positive ESI

Positive ESI

Negative ESI

Below mentioned table shows the analytical conditions used for analysis of GTI.

5


Figure 4. MRM chromatogram of GTI mix standard at 5 ppb level

Figure 5. Calibration graphs for GTI

Figure 3. MRM chromatogram of diluent (methanol)

0.0 2.5 5.0 7.5 10.0 min

0

5000

10000

15000

20000

25000

30000

35000

40000 3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0

BHBN

B 33

8.20

>24

4.05

DRN

-IA 5

09.1

0>11

4.10

DRN

-IB 4

79.1

5>17

0.15

0.0 2.5 5.0 7.5 10.0 min

0

250

500

750

1000

3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0

0.0 25.0 50.0 75.0 Conc.0

250000

500000

750000Area

DRN-IB R2-0.9989

0.0 25.0 50.0 75.0 Conc.0

250000

500000

750000

1000000

1250000

Area

DRN-IA R2-0.9943

0.0 25.0 50.0 75.0 Conc.0

50000

100000

150000

Area

BHBNB R2-0.9922


6

Figure 6. MRM chromatogram of blank sample

Table 3: Results of accuracy and repeatability for all GTI

Standard concentration (ppb)

Calculated concentration from calibration graph

(ppb) (n=6)

% Accuracy (n=6)


0.5

1

5

12

40

50

100

1

5

12

40

50

100

0.5

1

5

12

40

50

100

Name of GTI

DRN-IB

DRN-IA

BHBNB

Sr. No.

1

2

3

0.492

1.044

4.961

12.014

38.360

49.913

103.071

0.994

4.916

11.596

37.631

48.605

100.138

0.486

1.062

4.912

11.907

37.378

48.518

96.747

98.40

104.40

99.22

100.12

95.90

99.83

103.07

99.40

98.32

96.63

94.08

97.21

100.14

97.20

106.20

98.24

99.23

93.45

97.04

96.75

9.50

6.62

3.10

2.97

1.17

1.08

0.86

5.02

2.82

2.43

1.27

1.40

0.99

4.88

6.97

2.16

1.31

0.37

0.43

0.91

Recovery studiesFor recovery studies, samples were prepared as described previously. MRM chromatogram of blank and spiked samples are shown in Figures 6 and 7 respectively. Results

of recovery studies have been shown in Table 4. Recovery could not be calculated for DRN-IB as blank sample showed higher concentration than spiked concentration.

0.0 2.5 5.0 7.5 10.0 min

0

50000

100000

150000

200000

250000

300000

350000

4000003:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0

BHBN

B 33

8.20

>24

4.05

DRN

-IA 5

09.1

0>11

4.10

DRN

-IB 4

79.1

5>17

0.15






Figure 7. MRM chromatogram of spiked sample

Conclusion• A highly sensitive method was developed for analysis of GTI of Dronedarone.• Ultra high sensitivity, ultra fast polarity switching (UFswitching) enabled sensitive, selective, accurate and reproducible

analysis of GTI from Dronedarone powder sample.

References[1] Guideline on The Limits of Genotoxic Impurities, (2006), European Medicines Agency (EMEA).

Table 4. Results of the recovery studies

Concentration of GTI mix standard spiked

in blank sample (ppb)

Average concentration obtained from calibration graph for blank sample (ppb) (A) (n=3)

Average concentration obtained from calibration graph

for spiked sample (ppb) (B) (n=3)

% Recovery = (B-A)/ 12 * 100

12

12

12

Name of Impurity

DRN-IB

DRN-IA

BHBNB

94.210

3.279

1.241

NA

12.840

12.723

NA

79.678

95.689

0.0 2.5 5.0 7.5 10.0 min

0

25000

50000

75000

100000

125000 3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0

BHBN

B 33

8.20

>24

4.05

DRN

-IA 5

09.1

0>11

4.10

DRN

-IB 4

79.1

5>17

0.15

PO-CON1470E

Development of 2D-LC/MS/MS Method for Quantitative Analysis of1α,25-Dihydroxylvitamin D3 in Human Serum

ASMS 2014 WP449

Daryl Kim Hor Hee1, Lawrence Soon-U Lee1,

Zhi Wei Edwin Ting2, Jie Xing2, Sandhya Nargund2,

Miho Kawashima3 & Zhaoqi Zhan2

1 Department of Medicine Research Laboratories,

National University of Singapore, 6 Science Drive 2,

Singapore 1175462 Customer Support Centre, Shimadzu (Asia Paci�c) Pte

Ltd, 79 Science Park Drive, #02-01/08, Singapore 1182643 Global Application Development Centre, Shimadzu

Corporation, 1-3 Kanda Nishihiki-cho, Chiyoda-ku,

Tokyo 101-8448, Japan

2

Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

IntroductionDevelopments of LC/MS/MS methods for accurate quantitation of low pg/mL levels of 1α,25-dihydroxy vitamin D2/D3 in serum were reported in recent years, because their levels in serum were found to be important indications of several diseases associated with vitamin D metabolic disorder in clinical research and diagnosis [1]. However, it has been a challenge to achieve the required sensitivity directly, due to the intrinsic dif�culty of ionization of the compounds and interference from matrix [2,3]. Sample extraction and clean-up with SPE and immunoaf�nity methods were applied to remove the interferences [4] prior to LC/MS/MS analysis. However, the

amount of serum required was normally rather high from 0.5mL to 2mL, which is not favourite in the clinical applications. Direct analysis methods with using smaller amount of serum are in demand. Research efforts have been reported in literatures to enhance ionization ef�ciency by using different interfaces such as ESI, APCI or APPI and ionization reagents to form purposely NH3 adduct or lithium adduct [4,5]. Here, we present a novel 2D-LC/MS/MS method with APCI interface for direct analysis of 1α,25-diOH-VD3 in serum. The method achieved a detection limit of 3.1 pg/mL in spiked serum samples with 100 uL injection.

ExperimentalHigh purity 1α,25-dihydroxyl Vitamin D3 and deuterated 1α,25-dihydroxyl-d6 Vitamin D3 (as internal standard) were obtained from Toronto Research Chemicals. Charcoal-stripped pooled human serum obtained from Bioworld was used as blank and matrix to prepare spiked samples in this study. A 2D-LC/MS/MS system was set up on LCMS-8050 (Shimadzu Corporation) with a column switching valve installed in the column oven and controlled by LabSolutions workstation. The details of columns, mobile phases and gradient programs of 1st-D and 2nd-D LC

separations and MS conditions are compiled into Table 1. The procedure of sample preparation of spiked serum samples is shown in Figure 1. It includes protein precipitation by adding ACN-MeOH solvent into the serum in 3 to 1 ratio followed by vortex and centrifuge at high speed. The supernatant collected was �ltered before standards with IS were added (post-addition). The clear samples obtained were then injected into the 2-D LC/MS/MS system.

Table 1: 2D-LC/MS/MS analytical conditions

LC condition

1st D: FC-ODS (2.0mml.D. x 75mm L, 3μm)2nd D: VP-ODS (2.0mmI.D. x 150mm L, 4.6μm)

A: Water with 0.1% formic acidB: Acetontrile

C: Water with 0.1% formic acidD: MeOH with 0.1% formic acid

B: 40% (0 to 0.1min) → 90% (5 to 7.5min) → 15% (11 to 12min) → 40% (14 to 25min); Total �ow rate: 0.5mL/min

D: 15% (0min) → 80% (20 to 22.5min) → 15% (23 to 25min); Peak cutting: 3.15 to 3.40; Total �ow rate: 0.5 mL/min

45ºC

100 uL

Column

Mobile Phase of 1st D

Mobile Phase of 2nd D

1st D gradient pro-gram & �ow rate

2nd D gradient pro-gram & �ow rate

Oven Temp.

Injection Vol.

MS Interface condition

APCI, 400ºC

Positive, MRM

300ºC & 200ºC

Ar (270kPa)

N2, 2.5 L/min

N2, 7.0 L/min

Interface

MS mode

Heat Block & DL Temp.

CID Gas

Nebulizing Gas Flow

Drying Gas Flow

3


Figure 1: Flow chart of serum sample pre-treatment method

150µL of serum 450µL of ACN/MeOH (1:1)

Shake and Vortex 10mins

Centrifuge for 10 minutes at 13000rpm

480µL of Supernatant

0.2µm nylon �lter

400µL of �ltered protein precipitated Serum

500µL of calibrate50µL of of Std stock

50µL of IS stock


An APCI interference was employed for effective ionization of 1α,25-diOH-VitD3 (C27H44O3, MW 416.7). A MRM quantitation method for 1α,25-diOH-VitD3 with its deuterated form as internal standard (IS) was developed. MRM optimization was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions for each compound were selected

(Table 2), the first one for quantitation and the second one for confirmation. The parent ion of 1α,25-diOH-VitD3 was the dehydrated ion, as it underwent neutral lost easily in ionization with ESI and APCI [2,3]. The MRM used for quantitation (399.3>381.3) was dehydration of the second OH group in the molecule.

Development of 2D-LC/MS/MS method

Table 2: MRM transitions and CID parameters of 1α,25-diOH-VitD3 and deuterated IS

Q1 Pre Bias Q3 Pre BiasName

1α,25-dihydroxyl Vitamin D3

1α,25-dihydroxyl-d6 Vitamin D3 (IS)

RT1 (min)

22.74

22.71

Transition (m/z)

399.3 > 381.3

399.3 > 157.0

402.3 > 366.3

402.3 > 383.3

-20

-20

-20

-20

CID Voltage (V)

CE

-13

-29

-12

-15

-14

-17

-18

-27

1, Retention time by 2D-LC/MS/MS method

4


Figure 2: 1D-LC/MS/MS chromatograms of 22.7 pg/mL 1α,25-diOH-VitD3 (top) and 182 pg/mL internal

standard (bottom) in serum (injection volume: 50uL)

0.0 2.5 5.0 7.5 10.0 min0

1000

2000

3000

4000

5000 1:OH2D3 399.30>105.00(+) CE: -44.01:OH2D3 399.30>157.00(+) CE: -29.01:OH2D3 399.30>381.30(+) CE: -13.0

OH

2-V

D3

2.5 5.0 7.5 10.0 min0

100

200

300

400

500

600

700 2:OH2D3-D6 402.30>366.30(+) CE: -12.02:OH2D3-D6 402.30>383.30(+) CE: -15.0

OH

2-V

D3-

D3

Peak cutting (125 uL) in 1st D separationand transferred to 2nd D LC

The reason to develop a 2-D LC separation for a LC/MS/MS method was the high background and interferences occurred with 1D LC/MS/MS observed in this study and also reported in literatures. Figure 2 shows the MRM chromatograms of 1D-LC/MS/MS of spiked serum sample. It can be seen that the baseline of the quantitation MRM (399.3>381.3) rose to a rather high level and interference peaks also appeared at the same retention time. The 2-D LC/MS/MS method developed in this study involves “cutting the targeted peak” in the 1st-D separation precisely (3.1~3.4 min) and the portion retained in a stainless steel sample loop (200 uL) was transferred into the 2nd-D column for further separation. The operation was accomplished by switching the 6-way valve in and out by a time program. Both 1st-D and 2nd-D separations were carried out in gradient elution mode. The organic mobile phase of 2nd-D (MeOH with 0.1% formic acid) was different from that of 1st-D (pure ACN). The interference peaks co-eluted with the analyte in 1st-D were separated from the analyte peak (22.6 min) as shown in Figure 3.

Two sets of standard samples were prepared in serum and in clear solution (diluent). Each set included seven levels of 1α,25-diOH-VitD3 from 3.13 pg/mL to 200 pg/mL, each added with 200 pg/mL of IS (See Table 3). The chromatograms of the seven spiked standard samples in serum are shown in Figure 3. A linear IS calibration curve (R2 > 0.996) was established from these 2D-LC/MS/MS analysis results, which is shown in Figure 4. It is worth to

note that the calibration curve has a non-zero Y-intercept, indicating that the blank (serum) contains either residual 1α,25-diOH-VitD3 or other interference which must be deducted in the quantitation method. The peak area ratios shown in Table 3 are the results after deduction of the background peaks. The accuracy of the method after this correction is between 92% and 117%.

Calibration curve (IS), linearity and accuracy

Figure 3: Overlay of 2nd-D chromatograms of 7 levels from 3.13 pg/mL to 200 pg/mL spiked in serum.

Figure 4: Calibration curves of1α,25-diOH VD3 in serum by IS method.

0 10 20 min

0

1000

2000

3000

4000

22.0 23.0 min

1000

2000

3000

4000

1α,25-diOH-VitD3

0.00 0.25 0.50 0.75 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

Area Ratio

R2 = 0.9967

Non-zero intercept


5

Table 3: Seven levels of standard samples for calibration curve and performance evaluation

Figure 5: MRM peaks of 1α,25-diOH-VitD3 spiked in pure diluent (top) and in serum (bottom) of L1, L3, L5 and L7 (spiked conc. refer to Table 3)

Matrix effect of the 2D-LC/MS/MS method was determined by comparison of peak area ratios of standard samples in diluent and in serum at the seven levels. The results are compiled into Table 3. The matrix effect of the method are between 58% and 95%. It seems that the matrix effect is stronger at lower concentrations than at higher concentrations. Repeatability of peak area of the method was evaluated with L2 and L3 spiked serum samples for both target and IS. The Results of RSD (n=6) are displayed in Table 4. The MRM peaks of 1α,25-diOH VD3 in clear solution and in serum are displayed in pairs (top and bottom) in Figure 5. It can be seen from the first pair (diluent and serum blank) that a peak appeared at the same retention of 1α,25-diOH VD3 in the blank serum. As pointed out above, this peak is

from either the residue of 1α,25-diOH VD3 or other interference present in the serum. Due to this background peak, the actual S/N ratio could not be calculated. Therefore, it is difficult to determine the LOD and LOQ based on the S/N method. Tentatively, we propose a reference LOD and LOQ of the method for 1α,25-diOH VD3 to be 3.1 pg/mL and 10 pg/mL, respectively. The specificity of the method relies on several criteria: two MRMs (399>381 and 399>157), their ratio and RT in 2nd-D chromatogram. The MRM chromatograms shown in Figure 5 demonstrate the specificity of the method from L1 (3.1 pg/mL) to L7 (200 pg/mL). It can be seen that the results of spiked serum samples (bottom) meet the criteria if compared with the results of samples in the diluent (top).

Matrix effect, repeatability, LOD/LOQ and speci�city

Conc. Level of Std.

L1

L2

L3

L4

L5

L6

L7

1α,25-diOH VD3 (pg/mL)

3.13

6.25

12.5

25.0

50.0

100.0

200.0

Conc. Ratio1 (Target/IS)

0.0156

0.0313

0.0625

0.1250

0.2500

0.5000

1.0000

Area Ratio2

(in serum)

0.243

0.321

0.456

0.757

1.188

2.168

4.531

Area Ratio2

(in clear solu)

0.414

0.481

0.603

0.914

1.354

2.580

4.740

Accuracy3

(%)

103.8

100.0

117.3

115.9

95.5

92.15

102.0

Matrix Effect (%)

58.7

66.8

75.6

82.9

87.7

84.0

95.6

1, Target = 1α,25-diOH VD3; 2, Area ratio = area of target / area of IS; 3, Based on the data of spiked serum samples

22.5 24.7

0

250

500

7501:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.565

22.5 24.7

0

250

500

7501:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.565

22.5 24.7

0

500

1000

1:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.573

22.5 24.7

0

1000

2000

3000

4000 1:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.598

22.5 24.7

0

250

500

7501:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.595

22.5 24.7

0

250

500

7501:399.30>157.00(+)1:399.30>381.30(+)

22.5 24.7

0

250

500

750

10001:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.619

22.5 24.7

0

1000

2000

3000

4000 1:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.630

22.5 24.7

0

250

500

7501:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.622

22.5 24.7

0

250

500

7501:399.30>157.00(+)1:399.30>381.30(+)

OH

2VD

3/22

.602L1 L3 L5 L7 Diluent

L1 L3 L5 L7 Serum blank






ConclusionsA 2D-LC/MS/MS method with APCI interface has been developed for quantitative analysis of 1α,25-dihydroxylvitamin D3 in human serum without of�ine extraction and cleanup. The detection and quantitation range of the method is from 3.1 pg/mL to 200 pg/mL, which meets the diagnosis requirements in clinical applications. The performance of the method was evaluated thoroughly, including linearity, accuracy,

repeatability, matrix effect, LOD/LOQ and speci�city. The results indicate that the 2D-LC/MS/MS method is sensitive and reliable in detection and quantitation of trace 1α,25-dihydroxylvitamin D3 in serum. Further studies to enable the method for simultaneous analysis of both 1α,25-dihydroxylvitamin D3 and 1α,25-dihydroxylvitamin D2 are needed.

References1. S. Wang. Nutr. Res. Rev. 22, 188 (2009).2. T. Higashi, K. Shimada, T. Toyo’oka. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2010) 878, 1654.3. J. M. El‐Khoury, E. Z. Reineks, S. Wang. Clin. Biochem. 2010. DOI: 10.1002/jssc.20200911.4. Chao Yuan, Justin Kosewick, Xiang He, Marta Kozak and Sihe Wang, Rapid Commun. Mass Spectrom. 2011, 25,

1241–12495. Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon. Eur. J. Mass Spectrom. 2010, 16, 81.

For Research Use Only. Not for use in diagnostic procedures.

Table 4: Repeatability Test Results (n=6)

Sample

L2

L3

Compound

1α,25-diOH VD3

IS

1α,25-diOH VD3

IS

Spiked Conc. (pg/mL)

6.25

200

12.5

200

%RSD

10.10

7.66

9.33

6.28

PO-CON1450E

Analysis of polysorbates in biotherapeuticproducts using two-dimensional HPLC coupled with mass spectrometer

ASMS 2014 WP 182

William Hedgepeth, Kenichiro Tanaka Shimadzu Scienti�c Instruments, Inc., Columbia MD

2

Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

IntroductionPolysorbate 80 is commonly used for biotherapeutic products to prevent aggregation and surface adsorption, as well as to increase the solubility of biotherapeutic compounds. A reliable method to quantitate and characterize polysorbates is required to evaluate the quality and stability of biotherapeutic products. Several methods for polysorbate analysis have been reported, but most of

them require time-consuming sample pretreatment such as derivatization and alkaline hydrolysis because polysorbates do not have suf�cient chromophores. Those methods also require an additional step to remove biotherapeutic compounds. Here we report a simple and reliable method for quantitation and characterization of polysorbate 80 in biotherapeutic products using two-dimensional HPLC.

Fig.1 Typical structure of polysorbate 80

Materials

Reagents: Tween® 80 (Polysorbate 80), IgG from human serum, potassium phosphate monobasic, potassium phosphate dibasic, and ammnonium formate were purchased from Sigma-Aldrich. Water was made in house using a Millipore Milli-Q Advantage A10 Ultrapure Water Purification System. Isopropanol was purchased from Honeywell. Standard solutions: 10 mmol/L phosphate buffer (pH 6.8) was prepared by dissolving 680 mg of potassium

phosphate monobasic and 871 mg of potassium phosphate dibasic in 1 L of water. Polysorbate 80 was diluted with 10 mmol/L phosphate buffer (pH 6.8) to 200, 100, 50, 20, 10 mg/L and transferred to 1.5 mL vials for analysis.Sample solutions: A model sample was prepared by dissolving 2 mg of IgG in 0.1 mL of a 100 mg/L polysorbate 80 standard solution. The sample was centrifuged and transferred to a 1.5 mL vial for analysis.

Reagents and standards

w+x+y+z=approx. 20

OO

OH

OOH

O

OOH

O

O

CH3

yz

x

w

3


Fig.2 Flow diagram of Co-Sense for BA

The standard and sample solutions were injected into a Shimadzu Co-Sense for BA system consisting of two LC-20AD pumps and a LC-20AD pump equipped with a solvent switching valve, DGU-20A5R degassing unit, SIL-20AC autosampler, CTO-20AC column oven equipped with a 6-port 2-position valve, and a CBM-20A system controller. Polysorbate 80 was detected by a LCMS-2020 single quadrupole mass spectrometer or a LCMS-8050 triple quadrupole mass spectrometer because polysorbates do not have any chromophores and are present at low concentrations in antibody drugs. A SPD-20AV UV-VIS

detector was used to check protein removal.Fig. 2 shows the flow diagram of the Co-Sense for BA system. In step 1, a sample pretreatment column “Shim-pack MAYI-ODS” traps polysorbate 80 in the sample. Proteins (antibody) cannot enter the pore interior that is blocked by a hydrophilic polymer bound on the outer surface. Other additives and excipients such as sugars, salts, and amino acids cannot be retained by the ODS phase of the inner surface due to their polarity. In step 2, the trapped polysorbate 80 is introduced to the analytical column by valve switching.

System

Step 1 : Protein removal

Step 2 : Analyzing the trapped polysorbate

Autosampler

Valve(Position 0)

Pump 1

Pump 2

Sample pretreatment column

Analytical column

Mass spectrometer

UV-VIS detector

Mobile phase C

Mobile phase D

Mobile phase A(solution for sample injection)

Mobile phase B(solution for rinse)

Protein,Salts,

Amino acids,Sugars

Polysorbate80

Autosampler

Valve(Position 1)

Pump 1

Pump 2

Sample pretreatment column

Analytical column

Mass spectrometer

UV-VIS detector

Mobile phase C

Mobile phase D

Mobile phase B(solution for rinse)

Mobile phase A(solution for sample injection)

Polysorbate80

4


Results

A fast analysis for quantitation will be shown here. Table 1 shows the analytical conditions and Fig. 3 shows the TIC chromatogram of a 100 mg/L polysorbate 80 standard solution and the mass spectrum of the peak at 4.4 min. Polysorbates contain many by-products, so several peaks appeared on the TIC chromatogram. The peak at 4.4 min was identified as polyoxyethylene sorbitan monooleate (typical structure of polysorbate 80) based on E. Hvattum et al 2011. The ion at 783 was used as a marker for detection in selected ion mode (SIM). This ion is attributable to the 2NH4

+ adduct of polyoxyethylene sorbitan monooleate containing 25 polyoxyethylene groups. Fig. 4 shows the SIM chromatogram of the model sample (20 g/L of IgG, 100 mg/L of polysorbate 80 in 10

mmol/L phosphate buffer pH6.8). Polysorbate 80 in the model sample was successfully analyzed. The peak at 4.4 min was used for quantitation.Six replicate injections for the model sample were made to evaluate the reproducibility. The relative standard deviations of retention time and peak area were 0.034 % and 1.11 %, respectively. The recovery ratio was obtained by comparing the peak area of the model sample and a 100 mg/L polysorbate 80 standard solution and was 99 %. Five different levels of polysorbate 80 standard solutions ranging from 10 to 200 mg/L were used for the linearity evaluation. The correlation coefficient (R2) of determination was higher than 0.999.

Quantitation method

Table 1 Analytical Conditions

System : Co-Sense for BA equipped with LCMS-2020

[Sample Injection]

Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)

Mobile Phase : A: 10 mmol/L ammonium formate in water

B: Isopropanol

Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)


Valve Position : 0 (0-1 min, 7-9 min), 1 (1-7 min)


[Separation]

Column : Kinetex 5u C18 100A (50 mm L. x 2.1 mm I.D., 5 μm)


B: Isopropanol

Time Program : B. Conc 5 % (0-1 min) - 100 % (6-7 min) -5 % (7.01-9 min)



[UV Detection]

Detection : 280 nm

Flow Cell : Semi-micro cell

[MS Detection]

Ionization Mode : ESI Positive

Applied Voltage : 4.5 kV

Nebulizer Gas Flow : 1.5 mL/min


Block Heater Temp. : 400 ºC

Scan : m/z 300-2000

SIM : m/z 783

5


Fig.4 SIM chromatogram of the model sample

An analysis for characterization will be shown here. Table 2 shows the analytical conditions and Fig. 5 shows the TIC chromatogram of the model sample and mass spectra of the peaks from 10 to 30 min. A longer column and gradient were applied to obtain better resolution. Polysorbate 80 consists of not only monooleate (typical structure of polysorbate 80), but also many by-products such as polyoxyethylene, polyoxyethylene sorbitan, polyoxyethylene isosorbide, dioleate, trioleate, tetraoleate

and others. The peaks on the TIC chromatogram are assumed to correspond to those by-products. For example, the peaks from 10 to 22 min correspond to polyoxyethylene and polyoxyethylene isosorbide and the peaks from 22 to 30 min correspond to polyoxyethylene sorbitan. This method is helpful for characterization as well as checking degradation such as auto-oxidation and hydrolysis.

Characterization method

Fig.3 TIC Chromatogram of 100 mg/L polysorbate 80 standard solution and mass spectrum of the peak at 4.4 min

500 550 600 650 700 750 800 850 900 950 m/z0.0

0.5

1.0

1.5

Inten.(x100,000)

601587 616631

572645

660557

783675 805 827543849761689 739528 871 893704 915717

Doubly charged ions

Triply charged ions

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

1000000

2000000

3000000

4000000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min0

25000

50000

75000

100000


6

Fig.5 TIC chromatogram of the model sample

Table 2 Analytical Conditions

System : Co-Sense for BA equipped with LCMS-8050

[Sample Injection]

Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)


B: Isopropanol

Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)

Flow Rate : 0.6 mL/min (0-10 min, 95.01-110 min), 0.1 mL/min (10.01-95 min)

Valve Position : 0 (0-3 min, 100-110 min), 1 (3-100 min)


[Separation]

Column : Kinetex 5u C18 100A (100 mm L. x 2.1 mm I.D., 5 μm)


B: Isopropanol

Time Program : B. Conc 5 % % (0-3min) – 35% (15min) – 100% (100min) – 5% (100.01-110min)



[UV Detection]

Detection : 280 nm

Flow Cell : Semi-micro cell

[MS Detection]

Ionization Mode : ESI Positive

Applied Voltage : 4.5 kV

Nebulizer Gas Flow : 2 mL/min

Drying Gas Flow : 10 mL/min

Heating Gas Flow : 10 mL/min



Block Heater Temp. : 400 ºC

Q1 Scan : m/z 300-2000

0 10 20 30 40 50 60 70 80 90 100 min

0.0

1.0

2.0

3.0

4.0

(x100,000,000)1:TIC(+)

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

0.0

2.5

5.0

7.5

(x10,000,000)1:TIC(+)

Polyoxyethylene sorbitan

Polyoxyethylene isosorbide

Polyoxyethylene

400 500 600 700 800 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0Inten.(x100,000)

513.6528.3498.9 543.0

484.2557.6

469.5651.0673.0628.9 695.0572.3

717.1454.8 606.9 739.0587.0761.1

440.2 783.1805.1425.4 827.1

300 400 500 600 700 800 900 m/z0.0

1.0

2.0

3.0

Inten.(x100,000)

692.8648.8

736.8604.7

560.7 780.9421.7443.8399.7 465.8 564.7 608.8 652.8520.7377.6 824.9516.6

696.9445.4 740.9355.6 423.5401.6 869.0379.5

784.9913.0

O

O OOH

OOH

y

z

OHO

H

x

OO

OH

OOH

O

OOH

OH

yz

x

w






Fig.6 Chromatogram of elution from the sample pretreatment column

Fig. 6 shows the chromatogram of elution from the sample pretreatment column. Protein (IgG) was successfully removed from the sample by using the MAYI-ODS column.

Con�rmation of protein removal

E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally oxidized polysorbate 80, J Pharm Biomed Anal 62, (2012) 7-16

Reference

Conclusions1. Co-Sense for BA system automatically removed protein from the sample and enabled quantitation and characterization

of polysorbate 80 in a protein formulation.2. The quantitation method was successfully applied to the model sample with excellent reproducibility and recovery.3. The high-resolution chromatogram was obtained by the characterization method. This method is helpful for

characterization as well as checking degradation such as auto-oxidation and hydrolysis.

5uL injection of model sample

1uL injection of model sample

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

0

250000

500000

750000

1000000

1250000

uV

PO-CON1457E

A Rapid and Reproducible Immuno-MSPlatform from Sample Collection to Quantitation of IgG

ASMS 2014 WP161

Rachel Lieberman1, David Colquhoun1, Jeremy Post1,

Brian Feild1, Scott Kuzdzal1, Fred Regnier2, 1Shimadzu Scienti�c Instruments, Columbia, MD, USA 2Novilytic L.L.C, North Webster, IN, USA

2

A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

Sample Work�ow

Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-8050 allows for improved analysis of Immunoglobulin G.

Introduction

Novel Aspect

Dried blood spot analysis (DBS) has provided clinical laboratories a simple method to collect, store and transport samples for a wide variety of analyses. However, sample stability, hematocrit effects and inconsistent spotting techniques have limited the ability for wide spread adoption in clinical applications. Dried plasma spots (DPS) offer new opportunities by providing stable samples that

avoid variability caused by the hematocrit. This presentation focuses on an ultra-fast-immuno-MS platform that combines next generation plasma separator cards (Novilytic L.L.C., North Webster, IN) with fully automated immuno-af�nity enrichment and rapid digestion as an upfront sample preparation strategy for mass spectrometric analysis of immunoglobulins.

LC/MS/MSAffinitySelection

EnzymeDigestion Desalting

Automates and integrates key proteomic workflow steps: - Affinity Selection (15 min) - Trypsin digestion (1-8 min) - Online Desalting - Reversed phase LCExceptional reproducibility (CV less than 10%)

Rapid plasma extraction technology from whole blood (~ 18 minutes) - 2.5 uL of plasma collected (3 min) - Air dry for 15 minutes - Extract plamsa disc for analysis

- Ultrafast MRM methods - Up to 555 MRM transitions per run - Heated electrospray source - Scan speeds up to 30,000 u/sec - Polarity switching 5 msec

Perfinity WorkstationNoviplexTM Card LCMS-8050 Triple Quadrupole MS

BufferExchange

PlasmaGeneration

3


MethodsIgG was weighed out and then diluted in 500 μL of 0.5% BSA solution. Approximately15 uL of IgG standard was spiked into mouse whole blood and processed using the Noviplex card. The resulting plasma collection disc was extracted with 30 uL of buffer and each sample was

reduced and alkylated to yield a total sample volume of 100 uL. IgG standards and QC samples were directly injected onto the Per�nity-LCMS-8050 platform for af�nity pulldown with a Protein G column followed by trypsin digestion and LC/MS/MS analysis.

Noviplex Cards

Approximately 50 uL of the spiked whole blood was pipetted onto the Noviplex card test area (1). The spot was allowed to dry for 3 minutes (2). The top layer of the card was then peeled back (3) to reveal the plamsa collection

disc. The plasma collection disc was allowed to dry for an additional 15 minutes. Once the disc was dry (4), it was placed into an eppendorf tube for solvent extraction.

IgG concentrations for calibration levels. LCMS gradient conditions.

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

%B

Time (minutes)

MRM transitions on LCMS-8050 for two IgG peptides monitored.

Compound Name

TTPPVLDSDGSFFLYSK

VVSVLTVLHQDWLNGK

Transitions

937.70>836.25

937.70>723.95

603.70>805.7

+/-

+

+

+

Q1 Rod Bias(V)

-27

-27

-22

CE (V)

-28

-30

-16

Q3 Rod Bias(V)

-26

-22

-13

Level

1

2

3

4

5

6

7

Conc.(μg/mL)

465

315

142.5

127.5

102

60

22.5

Amount oncolumn (μg)

34.88

23.63

10.69

9.56

7.65

4.50

1.69

Time (min)

0

0.2

8

9.5

10

12.5

12.51

16

%B

2

2

50

50

90

90

2

2

Amount oncolumn (pmol)

581.25

393.75

178.13

159.37

127.50

75.00

28.12

(1)

(2) (3) (4)

4


Results - Chromatograms

Total Ion Chromatogram for IgG

Optimization of Collision Energies for peptides of interest

MRM Chromatogram for Level 4 standard of spiked IgG in whole blood.

VVSVLTVLHQDWLNGKTTPPVLDSDGSFFLYSK

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min

0

25000000

50000000

75000000

100000000

125000000

150000000

175000000

200000000

225000000

250000000

275000000

300000000

6.200 6.225 6.250 6.275 6.300 6.325 6.350 6.375 6.400 6.425 6.450 6.475 6.500 6.525 6.550 6.575 6.600 6.625 6.650 6.675 min

0

250000

500000

750000

1000000

1250000

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00Inten.

938

836915510

397

938

937

836

836

724283

891379

397

836 1046640591283

809443

352295 524

723407 851

407337 724466 756658

837

1163561397369

449

Range CE: -50 to -10 VTTPPVLDSDGFFLYSK

[M+2H]+2

[P1+2H]+2

[P2+2H]+2

Carryover Assessment

Blank InjectionControl - Mouse blood

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min

0

100

200

300

400

500

600

700

800

900

1000

1100

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min

0

10

20

30

40

50

60

70

80

90

5


Results - Calibration Curves

VVSVLTVLHQDWLNGK

Sample

QC 1

QC 2

QC 3

QC 4

Ret. Time

6.49

6.516

6.514

6.492

Area

32,492

11,726

8,507

2,727

Calc. Conc.

502.804

167.189

115.155

21.745

Std. Conc.

465

142.5

102

22.5

% Accuracy

108.1

117.3

112.9

96.6

TTPPVLDSDGSFFLYSK

Sample

QC 1

QC 2

QC 3

QC 4

Ret. Time

6.029

6.052

6.047

6.029

Area

61,525

25,355

16,900

6,502

Calc. Conc.

416.447

155.568

94.58

19.587

Std. Conc.

465

142.5

102

22.5

% Accuracy

89.6

109.2

92.7

87.1

0 100 200 300 400 Conc.0

25000

50000

Area

r2 = 0.979

TTPPVLDSDGSFFLYSK VVSVLTVLHQDWLNGK

r2 = 0.989

0 100 200 300 400 Conc.0

5000

10000

15000

20000

25000

30000

Area

Level 7

5.50 5.75 6.00 6.25 6.50

0

500

1000

1500

2000 937.70>723.95(+)937.70>836.25(+)Level 1

5.50 5.75 6.00 6.25 6.50

0

5000

10000

15000

20000

25000937.70>723.95(+)937.70>836.25(+) Level 7

6.00 6.25 6.50 6.75

0

100

200

300

400

500

600603.70>805.70(+)Level 1

6.00 6.25 6.50 6.75

0

2500

5000

7500

10000603.70>805.70(+)

Calibration Curve and MS Chromatograms

Results - Tables and Replicates

QC data and Calculations for IgG Peptides






VVSVLTVLHQDWLNGK TTPPVLDSDGSFFLYSK

Skyline Data - Retention Time Replicates

839

AM

_226

2014

...L1

...00

5

839

AM

_226

2014

...L2

...00

4

839

AM

_226

2014

...L3

...00

3

839

AM

_226

2014

...L4

...00

2

1433

PM

_225

2014

...L5

...00

8

1433

PM

_225

2014

...L6

...00

6

1433

PM

_225

2014

...L7

...00

4

Replicate

5.90

5.95

6.00

6.05

6.10

6.15

6.20

Ret

enti

on

Tim

e

y15 - 836.4169++

839

AM

_226

2014

...L1

...00

5

839

AM

_226

2014

...L2

...00

4

839

AM

_226

2014

...L3

...00

3

839

AM

_226

2014

...L4

...00

2

1433

PM

_225

2014

...L5

...00

8

1433

PM

_225

2014

...L6

...00

6

1433

PM

_225

2014

...L7

...00

46.35

6.40

6.45

6.50

6.55

6.60

6.65y14 - 805.4385++

839

AM

_226

2014

...L1

...00

5

839

AM

_226

2014

...L2

...00

4

839

AM

_226

2014

...L3

...00

3

839

AM

_226

2014

...L4

...00

2

1433

PM

_225

2014

...L5

...00

8

1433

PM

_225

2014

...L6

...00

6

1433

PM

_225

2014

...L7

...00

4

Replicate

6.35

6.40

6.45

6.50

6.55

6.60

6.65

Ret

enti

on

Tim

e

y14 - 805.4385++

Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative �gures showing the retention time reproducibility for each peptide monitored during the analysis.

ConclusionsCombining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection from whole blood, with the automated af�nity selection and trypsin digestion of the Per�nity workstation coupled to LCMS-8050, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore, this rapid immuno-MS platform can be applied to many other peptide/protein applications.

PO-CON1473E

Simultaneous Determinations of 20 kindsof common drugs and pesticides in human blood by GPC-GC-MS/MS

ASMS 2014 TP 757

Qian Sun, Jun Fan, Taohong Huang,

Shin-ichi Kawano, Yuki Hashi,

Shimadzu Global COE, Shanghai, China

2

Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS

IntroductionOn-line gel permeation chromatography-gas chromatography/mass spectrometry (GPC-GC-MS) is a unique technique to cleanup sample that reduce the time of sample preparation. GPC can ef�ciently separates fats, protein and pigments from samples, due to this advantage, on-line GPC is widely used for pesticide analysis. Meanwhile, compared to widely used GC-MS, GC-MS/MS

techniques provide much better selectivity thus signi�cantly lower detection limits. In this work, a new method was developed for rapid determination of 20 common drugs and pesticides in human blood by GPC-GC-MS/MS. The modi�ed QuEChERS method was used for sample preparation.

ExperimentalThe human blood samples were extracted with acetonitrile, then was puri�ed by PSA, C18 and MgSO4 to remove most of the fats, protein and pigments in samples, then after on-line GPC-GC-MS/MS analysis which further removed

macromolecular interference material, such as protein and cholesterol, the background interference brought about by the complex matrix in samples was effectively reduced.

Figure 1 Schematic �ow diagram of the sample preparation

Sample pretreament

PSA/C18/MgSO4

vortex

centrifuge

CH3CN

vortex

human blood 2 mL

evaporate

GPC-GC-MS/MS

supernatant

set volume using moblie phase

3


ResultsFor all of analytes, recoveries in the acceptable range of 70~120% and repeatability (relative standard deviations, RSD)≤5% (n=3) were achieved for matrices at spiking levels of 0.01 µg/mL. The limitis of detection were 0.03~4.4 µg/L.

The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements for the analysis of common drugs and pesticides in the human blood.

Figure 2 MRM chromatograms of standard mixture

Instrument

GPC

Mobile phase : acetone/cyclohexane (3/7, v/v)

Flow rate : 0.1mL/min

Column : Shodex CLNpak EV-200 (2 mmI.D. x 150 mmL.)

Oven temperature : 40 ºC

Injection volume : 10 μL

GCMS-TQ8030

Column : deactivated silica tubing [0.53 mm(ID) x 5 m(L)]

+pre-column Rtx-5ms [0.25 mm(ID) x 5 m(L)]

Rtx-5ms [0.25mm(ID) x 30 m(L), Thickness: 0.25 μm]

Injector : PTV

Injector time program : 120 ºC(4.5min)-(80 ºC/min)-280 ºC(33.7 min)

Oven temperature program : 82 ºC(5min)-(8 ºC/min)-300 ºC(7.75 min)

Linear velocity : 48.8 cm/sec

Ion Source temperature : 210 ºC

Interface temperature : 300 ºC

15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5

0.00

0.25

0.50

0.75

1.00

(x10,000,000)






Table 1 Results of method validation for drugs and pesticides(Concentration range: 5-100 μg/L, LODs: S/N≥3, LOQs: S/N≥10, RSDs: n=3)

ConclusionA very quick, easy, effective, reliable method in human blood based on modi�ed QuEChERS method was developed using GPC-GCMS-TQ8030. The performance of the method was very satisfactory with results meeting

validation criteria. The method has been successfully applied for determination of human blood samples and ostensibly has further application opportunities, e.g. biological samples.

No.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Compound Name

Dichlorvos

Methamidophos

Barbital

Sulfotep

Dimethoate

Malathion

Chlorpyrifos

Phenobarbital

Parathion

Triazophos

Zopiclone deg.

Diazepam

Midazolam

Zolpidem

Clonazepam

Estazolam

Clozapine

Alprazolam

Zolpidem

Triazolam

10.795

11.800

15.210

17.580

18.310

21.555

21.715

22.000

22.180

25.675

26.025

27.635

29.250

31.225

31.795

32.335

32.400

32.730

33.095

33.700

tR

(min)

0.9993

0.9994

0.9994

0.9995

0.9993

0.9997

0.9996

0.9995

0.9993

0.9994

0.9993

0.9992

0.9994

0.9993

0.9995

0.9994

0.9991

0.9993

0.9995

0.9992

CorrelationCoef�cient*

0.103

0.023

0.018

0.011

0.400

0.005

0.010

0.353

0.003

0.046

0.189

0.007

0.048

1.298

0.432

0.092

0.050

0.028

1.027

0.027

LOD(µg/L)

0.345

0.076

0.058

0.037

1.333

0.016

0.033

1.177

0.009

0.155

0.631

0.022

0.160

4.325

1.440

0.305

0.167

0.095

3.425

0.091

LOQ(µg/L) Recovery (%)

72.9

85.3

72.4

110.7

103.7

82.7

85.7

79.6

92.3

87.7

83.5

98.3

87.1

99.3

110.0

103.7

100.6

103.3

87.3

81.3

RSD (%)

2.99

3.58

1.72

2.27

3.10

2.52

3.57

3.25

3.17

1.32

1.28

1.55

2.01

1.01

1.57

1.37

3.12

1.48

1.75

2.56

0.01 µg/mL

PO-CON1466E

Low level quantitation of Loratadinefrom plasma using LC/MS/MS

ASMS 2014 TP498

Shailesh Damale, Deepti Bhandarkar, Shruti Raju,

Rashi Kochhar, Shailendra Rane, Ajit Datar,





2

Low level quantitation of Loratadine from plasma using LC/MS/MS

IntroductionLoratadine is a histamine antagonist drug used for the treatment of itching, runny nose, hay fever and such other allergies. Here, an LC/MS/MS method has been developed for high sensitive quantitation of this molecule from plasma using LCMS-8050, a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan. Presence

of heated Electro Spray Ionization (ESI) interface in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development of a low ppt level quantitation method for Loratadine.

Method of AnalysisThis bioanalytical method was developed for measuring Loratadine in therapeutic concentration range for the analysis of routine samples. It was important to develop a

simple and accurate method for estimation of Loratadine in human plasma.

To 100 µL of plasma 500 µL cold acetonitrile was added for protein precipitation. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing. This solution

was centrifuged at 12000 rpm for 15 minutes. Supernatant was taken and evaporated to dryness at 70 ºC . The residue was reconstituted in 200 µL Methanol.

Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile

1 ppt, 5 ppt, 50 ppt, 100ppt, 500 ppt, 1 ppb, 5 ppb and 10 ppb of Loratadine calibration standards were prepared

in cold acetonitrile treated matrix matched plasma.

Preparation of calibration standards in matrix matched plasma

Figure 1. Structure of Loratadine

LoratadineLoratadine, a piperidine derivative, is a potent long-acting, non-sedating tricyclic antihistamine with selective peripheral H1-receptor antagonist activity. It is used for relief of nasal and non-nasal symptoms of seasonal allergies and skin rashes[1,2,3]. Due to partial distribution in central nervous system, it has less sedating power compared to traditional H1 blockers. Loratadine is given orally, is well absorbed from the gastrointestinal tract, and has rapid �rst-pass hepatic metabolism; it is metabolized by isoenzymes of the cytochrome P450 system, including CYP3A4, CYP2D6, and, to a lesser extent, several others. Loratadine is almost totally (97–99 %) bound to plasma proteins and reaches peak plasma concentration (Tmax) in ~ 1–2 h[4,5].

Ethyl 4- (8-chloro-5, 6-dihydro-11H-benzo [5, 6] cyclohepta [1, 2-b] pyridin-11-ylidene) -1-piperidinecarboxylate

3


LC/MS/MS analysisLCMS-8050 triple quadrupole mass spectrometer by Shimadzu Corporation, Japan (shown in Figure 2A), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity) with Scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability.In order to improve ionization ef�ciency, the newly developed heated ESI probe combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide

range of target compounds with considerable reduction in background.Presence of heated Electro spray interface in LCMS-8050 (shown in Figure 2B) ensured good quantitative sensitivity even in presence of a complex matrix like plasma.The parent m/z of 382.90 giving the daughter m/z of 337.10 in the positive mode was the MRM transition used for quantitation of Loratadine. MS voltages and collision energy were optimized to achieve maximum transmission of mentioned precursor and product ion. Gas �ow rates, source temperature conditions and collision gas were optimized, and linearity graph was plotted for 4 orders of magnitude.

Figure 2A. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 2B. Heated ESI probe

Table 2. LCMS conditions

ESI

Positive

2.0 L / min (nitrogen)

10.0 L / min (nitrogen)

15.0 L / min (zero air)

300 ºC

250 ºC

400 ºC

382.90 > 337.10

MS Interface

Polarity

Nebulizing Gas Flow

Drying Gas Flow

Heating Gas Flow

Interface Temp.

Desolvation Line Temp.

Heater Block Temp.

MRM Transition

B conc. (%)Time (min)

60

100

100

60

0.01

1.50

4.00

4.10

13.00

A conc. (%)

40

0

0

40

Stop

Table 1. LC conditions

Shim-pack XR-ODS (100 mm L x 2.0 mm ID ; 2.2 µm)

A : 0.1% formic acid in water

B : acetonitrile

0.15 mL/min

40 ºC

20 µL

Column

Mobile Phase

Gradient Program

Flow Rate

Oven Temperature

Injection Volume


4

Figure 4A. Mass chromatogram 10 ppb Figure 4B. Mass chromatogram 0.001 ppb

Figure 5. Overlay chromatogram

Results

LC/MS/MS method for Loratadine was developed on ESI +ve ionization mode and 382.90>337.10 MRM transition was optimized for Loratadine. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest (0.001 ppb) concentrations as seen in Figures 4A and 4B respectively. Optimized MS method to ensure no plasma interference at the retention time of Loratadine (Figure 5).

Calibration curve was plotted for Loratadine concentration range. Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Linear calibration curves were obtained with regression coefficients R2 > 0.998. % RSD of area was within 15 % and accuracy was within 80-120 % for all calibration levels.

LC/MS/MS Analysis

Speci�city and interference

0.0 2.5 5.0 7.5

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5(x1,000,000)382.90>337.10(+)

LORA

TAD

INE/

3.39

1

0.0 2.5 5.0 7.5-1.0

0.0

1.0

2.0

3.0

4.0

5.0

(x10,000)382.90>337.10(+)

LORA

TAD

INE/

3.37

7

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2(x10,000)

1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_002.lcd1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_003.lcd

------ LOQ Level

------ Blank

5


Figure 6. Loratadine calibration curve

Conclusion• Highly sensitive LC/MS/MS method for Loaratadine was developed on LCMS-8050 system.• Calibration was plotted from 10 ppb to 0.001 ppb, and LOQ was computed as 0.001 ppb.

Table 3. Results of Loratadine calibration curve



% Accuracy(n=3)


0.001

0.005

0.05

0.1

0.5

1.0

5.0

10.0

Standard

STD-01

STD-02

STD-03

STD-04

STD-05

STD-06

STD-07

STD-08

Sr. No.

1

2

3

4

5

6

7

8

0.00096

0.0050

0.057

0.095

0.048

0.986

5.077

9.983

0.62

5.24

0.98

1.81

1.40

0.11

1.07

1.96

95.83

100.73

114.83

95.40

95.70

98.53

101.53

99.37

Result Table

0.0 2.5 5.0 7.5 Conc.0.0

1.0

2.0Area (x10,000,000)

1 2 3 4 5

6

7

8

0.05 0.10 Conc.0.0

1.0

2.0

Area (x100,000)

1 2

3

4






References[1] Bhavin N. Patel, Naveen Sharma, Mallika Sanyal, and Pranav S. Shrivastav, Journal of chromatographic Sciences,

Volume 48, (2010), 35-44.[2] J. Chen, YZ. Zha, KP. Gao, ZQ. Shi, XG. Jiang, WM. Jiang, XL. Gao, Pharmazie, Volume 59, (2004), 600-603.[3] M. Haria, A. Fitton, and D.H. Peters, Drugs, Volume 48, (1994), 617-637.[4] J. Hibert, E. Radwanski, R. Weglein, V. Luc, G. Perentesis, S. Symchowicz, and N. Zampaglione, J.clin. Pharmacol,

Volume 27, (1987), 694-698.[5] S.P.Clissold, E.M. Sorkin, and K.L. Goa, Drugs, Volume 37,(1989), 42-57.

• Page 111An LCMS method for the detection of cocoa butter substitutes, replacers, and equivalents in commercial chocolate-like products

• Page 116Highly sensitive and robust LC/MS/MS method for quantitative analysis of articial sweeteners in beverages

• Page 122Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

• Page 129High sensitivity analysis of acrylamide in potato chips by LC/MS/MS with modified QuEChERS sample pre-treatment procedure

• Page 135Determination of benzimidazole residues in animal tissue by ultra high performance liquid chromatography tandem mass spectrometry

• Page 141High sensitivity quantitation method of dicyandi-amide and melamine in milk powders by liquid chromatography tandem mass spectrometry

• Page 147Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

• Page 154Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

• Page 161Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

PO-CON1458E

An LCMS Method for the Detectionof Cocoa Butter Substitutes,Replacers, and Equivalents inCommercial Chocolate-like Products

ASMS 2014 ThP632

Jared Russell, Liling Fang and Willard Bankert

Shimadzu Scienti�c Instruments., Columbia, MD

2

An LCMS Method for the Detection of Cocoa Butter Substitutes,Replacers, and Equivalents in Commercial Chocolate-like Products

IntroductionThere is increasing demand for genuine cocoa butter (CB) in chocolate products in developed nations, however, this demand has created a shortage of CB and raised its costs. To overcome this, chocolate manufactures sometimes add vegetable-derived fats to some chocolate products to reduce costs while still maintaining desirable physical characteristics. It is of current interest to have a reliable method to detect, identify, and quantify the triacylglycerol (TAG) components of cocoa butter substitutes, replacers, and equivalents (CBEs) in chocolate products. Traditionally GC was used for this task, but due to the low volatility of triacylglycerides and their susceptibility to thermal decomposition, retention time is the only identifying factor

for the TAGs and typical GC analyses of this type can take 40 minutes. LCMS is able to not only provide faster throughput, but also has the additional advantage of allowing characterization of the TAG, including qualitative regiospeci�c analysis. We have developed a single, UHPLC column-based LCMS method to analyze the TAG components in commercial chocolate and chocolate-like products. This analysis has a runtime of 17minutes, making it suitable for relatively high throughput. Additionally, the method was very repeatable, with an interday variability of <7% for the absolute area counts of the three major TAGs in CB (POP,POS,SOS).

Materials and MethodA Shimadzu Nexera UHPLC coupled to a Shimadzu LCMS-8040 triple quadrupole mass spectrometer was utilized for this analysis. A pure CB standard was used as a

reference. Chocolate and chocolatey products were purchased in retail stores over a range of cocoa content.

For analysis, we slightly modified a sample preparation method originally used for algal oils. For analysis, 5mg of sample was weighed and then dissolved in a 3:1 Toluene-Isopropyl Alcohol solution. We then sonicated the

mixture for 5 minutes. The solution was filtered through a Thomson filter vial (P/N 35538-100) to remove sugars and other insoluble materials and diluted 5-fold using 3:1 Toluene-IPA and injected into the UHPLC-MS system.

Sample Preparation

Chromatography

Instrument : Shimadzu Nexera UHPLC system

Column : Shimadzu Shim-Pack XR-ODSIII (200x2.1mm,)

Mobile Phase A : LC/MS Acetonitrile

Mobile Phase B : 1:1 Dichloromethane-Isopropyl Alcohol

Gradient Program : 48% B (initially) – gradient to 51% B (0-8.0 min) – gradient to 54% B

(8.0 – 11.0 min) – gradient to 74% B (11.0-14.0 min) – hold at 74% B

(14.0-15.0 min) – reequilibrate at 48% B (15.1-17 min)


Column Temperature : 30°C

Injection Volume : 1 μL

Mass Spectrometry

Instrument : Shimadzu LCMS-8040 Triple Quadrupole Mass Spectrometer

Ionization : APCI

Polarity : Positive

Scan Mode : Q3 Scan

3


ResultsRetail Chocolates from Hershey’s, Lindt and Tcho, as well as a chocolatey candy - Charleston Chew - were compared against pure cocoa butter. The chocolates used were selected to cover a range of Cocoa content and purity. We speci�cally chose to use Hershey’s Mr. Goodbar and Charleston Chews because they listed the use of vegetable oils in their ingredients list. As you can see in the chromatograms, the products that market themselves as pure chocolate have similar chromatograms in comparison to the pure CB.We used an MS library that was provided to us by Dr. John Carney and Mona Koutchekinia to identify the types of TAGs contained in the chocolates using the spectral information captured in the Q3 scans. A minimum similarity of 70 was required for a result to be considered a

match. In order to identify usage of CBEs, we applied the equation: %POP<44.025-0.733*%SOS, which was determined by the European Commission Joint Research Centre, which can detect around 2% CBE usage in CB content, or approximately 0.4% CBE content in chocolate.The chocolate products we tested all agreed with the expected results: All of the dark chocolate products we tested passed this speci�cation, as well as Hershey’s Milk Chocolate. The two products which had a higher %POP than is allowable, Mr. Goodbar and Charleston Chew, were selected speci�cally for the inclusion of vegetable oils. It may be informative to further test the accuracy of this testing method by adulterating cocoa butter with known quantities of CBEs. The data has been summarized in Table 1.

Table 1: Percentage of the major TAGs in CB in various chocolate products

Product

Cocoa Butter

Lindt 85% Cocoa

TCHO 70% from Ghana

TCHO 65% from Ecuador

Hershey's Special Dark

Hershey's Milk Chocolate

Hershey's Mr Goodbar

Charleston Chew

%POP

23.7%

16.9%

17.8%

20.9%

20.0%

18.6%

44.8%

100.0%

%POS

46.9%

46.4%

46.1%

46.2%

47.1%

46.6%

21.1%

0.0%

%SOS

29.5%

36.6%

36.1%

32.9%

32.9%

34.8%

34.1%

0.0%

%POP needs tobe less than

43.8

43.8

43.8

43.8

43.8

43.8

43.8

44.0

4


Figure 1. Chromatograms of the various chocolate products analyzed versus pure cocoa butter

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

(x100,000,000)

1:TIC(+) Hershey's Milk Chocolate.lcd1:TIC(+) Hershey's Special Dark 45% Cacao.lcd1:TIC(+) TCHO 65% from Ecuador.lcd1:TIC(+) TCHO 70% from Ghana.lcd1:TIC(+) Lindt 85% Cocoa.lcd1:TIC(+) Cocoa Butter.lcd

PLP

OO

P

POP

OO

S

POS

SOS*

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0(x10,000,000)

1:TIC(+) Charleston Chew.lcd1:TIC(+) Hershey's Mr. Goodbar.lcd1:TIC(+) Cocoa Butter.lcd

PLP

OO

P

POP

OO

S

POS

SOS*






ConclusionsWe have developed a 17 minute method for the rapid determination of CBE usage in chocolate products by using a UHPLC column and Q3 ion scans to analyze samples and then matching spectral information with an MS library of ion ratios for identifying TAGs.Further studies could add a calibration curve to enable quanti�cation of TAGs. This method should also provide a base method which can be modi�ed to support TAG analysis in other product types.

ReferencesCo ED, Koutchekinia M, Carney J et al. Matching the Functionality of Single-Cell Algal Oils with Different Molecular Compositions. 2014. Buchgraber M and Anklam E. Validation of a Method for the Detection of Cocoa Butter Equivalents in Cocoa Butter and Plain Chocolate. 2003.

AcknowledgementsDr. John Carney and Mona Koutchekinia for the invaluable information they provided.

PO-CON1471E

Highly Sensitive and Robust LC/MS/MSMethod for Quantitative Analysis of Arti�cial Sweeteners in Beverages

ASMS 2014 MP351

Jie Xing1, Wantung Liw1, Zhi Wei Edwin Ting1,

Yin Ling Chew*2 & Zhaoqi Zhan1

1 Customer Support Centre, Shimadzu (Asia Paci�c)

Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV,

Singapore Science Park 1, Singapore 1182642 Department of Chemistry, Faculty of Science,

National University of Singapore, 21 Lower Kent

Ridge Road, Singapore 119077, *Student

2

Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti�cial Sweeteners in Beverages

IntroductionArti�cial sweeteners described as intense, low-calorie and non-nutritive are widely used as sugar substitutes in beverages and foods to satisfy consumers’ desire to sweet taste while concerning about obesity and diabetes. As synthetic additives in food, the use of arti�cial sweeteners must be approved by authority for health and safety concerns. For example, Aspartame, Acesulfame-K, Saccharin, Sucralose and Neotame are the FDA approved arti�cial sweeteners on the US market. However, there are also many other arti�cial sweeteners allowed to use in EU and many other countries (Table 2), but not in the US. In this regard, analysis of arti�cial sweeteners in beverages and foods has become essential due to the relevant regulations in protection of consumers’ bene�ts and safety concerns in many countries [1, 2]. Recently, arti�cial

sweeteners are found as emerging environmental contaminants in surface water and waste water [3]. Initially, HPLC analysis method with ELSD detection was adopted, because many arti�cial sweeteners are non-UV absorption compounds [2]. Recently, LC/MS/MS methods have been developed and used for identi�cation and quantitation of arti�cial sweeteners in food and beverages as well as water for its high sensitivity and selectivity [3, 4]. Here we report a high sensitivity LC/MS/MS method for identi�cation and quantitation of ten arti�cial sweeteners (Table 2) in beverage samples. An ultra-small injection volume was adopted in this study to develop a very robust LC/MS/MS method suitable for direct injection of beverage samples without any sample pre-treatment except dilution with solvent.

ExperimentalTen arti�cial sweeteners of high purity as listed in Table 2 were obtained from chemicals suppliers. Stock standard solutions and a set of calibrants were prepared from the chemicals with methanol/water (50/50) solvent as the diluent. Three brand soft-drinks and a mouthwash bought from local supermarket were used as testing samples in this study. The samples were not pretreated by any means

except dilution with the diluent prior to injection into LCMS-8040 (Shimadzu Corporation, Japan), a triple quadrupole LC/MS/MS system. The front-end LC system connected to the LCMS-8040 is a high pressure binary gradient Nexera UHPLC. The details of analytical conditions of LC/MS/MS method are shown in Table 1.

Table 1: LC/MC/MS analytical conditions of arti�cial sweeteners on LCMS-8040

Synergi, Polar-RP C18 (100 x 2 mm, 2.5µm )

0.25 mL/min

A: water with 0.1% Formic acid - 0.03% TA

B: MeOH with 0.1% FA - 0.03% Trimethylamine

B: 10% (0.01 to 0.5 min) → 95% (8 to 9 min) → 10% (9.01 to 11min)

ESI, MRM, positive-negative switching

Nebulizing gas: 3L/min, Drying gas: 15L/min, Heating block: 400ºC, DL: 250ºC

0.1uL, 0.5uL, 1uL, 5uL and 10uL

Column

Flow Rate

Mobile Phase

Gradient program

MS mode

ESI condition

Inj. Vol.

3


Table 2: Arti�cial Sweeteners, MRM transitions and calibration curves on LCMS-8040


First, precursor selection and MRM optimization of the ten sweeteners studied was carried out using an automated MRM optimization program of the LabSolutions. Six compounds were ionized in negative mode and four in positive mode as shown in Table2. For each compound, two optimized MRM transitions were selected and used, with the first one for quantitation and the second one for confirmation.The ten compounds were well-separated as sharp peaks between 2 min and 8.2 min as shown in Figure 1. Linear calibration curves of wide concentration ranges were established with mixed standards in diluent as summarized

in Table 2. We also investigated the performance of the LC/MS/MS method established by employing very small injection volumes (0.1, 0.5, 1 and 5 uL). This is because actual beverages usually contain very high contents of sweeteners (>>1ppm) to MS detection. Analysts normally dilute the samples before injection into LC/MS/MS. An alternative way is to inject a very small volume of samples even without dilution. Figs 2 & 3 show a chromatogram and calibration curves established with 0.1uL injection, which demonstrates the feasibility of an ultra-small injection volume combined with high sensitivity LC/MS/MS.

Method development

Compd. & Abbr. Name

Acesulfame K (Ace-K)

Cyclamate (CYC)3

Saccharin (SAC)

Sucralose2 (SUC)

Aspartame (ASP)

Neotame (NEO)

Alitame (ALI)

Dulcin (DUL)

NeohespiridinDihydrochalcone (NHDC)

Glycyrrhi-Zinate (GLY)

Pola. (+/-)

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

-

-

-

-

Q1 (V)

11

11

19

12

13

13

20

20

-19

-19

-18

-18

-23

-23

-22

-21

30

30

22

22

Trans. (m/z)

161.9 >82.1

161.9 >78.0

178.3 >80.1

178.3 >79.0

181.9 >106.1

181.9 >42.1

441.0 >395.1

441.0 >359.1

295.1 >120.1

295.1 >180.1

379.3 >172.2

379.3 >319.3

332.2 >129.1

332.2 >187.1

181.1 >108.1

181.1 >136.1

611.3 >303.1

611.3 >125.3

821.5 >351.2

821.5 >193.2

Cat1

A2

A5

A3

A4

A1

A6

B1

B3

B2

C1

CE (V)

14

32

24

27

20

36

11

15

-25

-14

-23

-18

-19

-16

-25

-18

38

47

46

52

Q3 (V)

29

28

30

10

15

13

25

23

-25

-20

-20

-24

-26

-21

-21

-26

30

20

20

19

RT (min)

1.99

2.87

3.28

4.61

5.15

7.51

5.44

5.58

6.71

8.19

Conc. R. (ug/L)

1 - 20000

5 - 20000

1 - 20000

5 - 20000

0.1 - 2000

0.05 - 1000

0.1 - 2000

5 - 10000

0.5 - 2000

5 - 1000

R2

0.9999

0.9996

0.9984

0.9983

0.9999

0.9998

0.9995

0.999

0.9988

0.9996

MRM parameter RT & Calibration Curve4

1. A1~A6: US FDA, EU and others approval; B1~B3: only EU and other countries approval. C1: natural sweetener, info not available.2. Sucralose precursor ion m/z 441.0 is formic acid adduct ion. 3. Sodium cyclamate known as “magic sugar” was initially banned in the US in 2000. FDA lifted the ban in 2013.4. Injection volume: 10 uL

4


Figure 3: Calibration curves of arti�cial sweeteners on LCMS-8040 with an ultra-small injection volume (0.1 uL) of same set of calibrants as shown in Table 2.

Figure 1: MRM Chromatogram of ten sweeteners by LC/MS/MS with 10uL injection: Asp & Ali 1ppb, Neo 0.5ppb, Dul, Gly, Ace-K, Sac, Suc and Cyc 10ppb, NHDC 1ppb.

Figure 2: MRM Chromatogram of ten sweeteners by LC/MS/MS with 0.1uL injection: Asp & Ali 0.1ppm, Neo 0.05ppm, Dul, Gly, Ace-K, Sac, Suc and Cyc 1ppm, NHDC 0.1ppm.

0.0 2.5 5.0 7.5 10.0 min

0.0

1.0

2.0

3.0

4.0

5.0

(x10,000)

Cyc

lam

ate

NH

DC

Sucr

alos

e

Sacc

harin

Ace

sulfa

me

K

Gly

cyrr

hizi

c

Dul

cin

Neo

tam

e

Alit

ame

Asp

arta

me

0 10000 Conc.0.0

1.0

2.0

Area (x100,000)

Ace-K r2=0.9977

0 10000 Conc.0.0

1.0

2.0

3.0

4.0Area (x10,000)

0 10000 Conc.0.0

2.5

5.0

Area (x10,000)

0 10000 Conc.0.0

0.5

1.0

1.5

Area (x100,000)

0 1000 Conc. 0.0

0.5

1.0

1.5 Area (x100,000)

0 500 Conc.0.0

2.5

5.0Area (x100,000)

0 1000 Conc.0.0

0.5

1.0

1.5

Area (x100,000)

0 10000 Conc.0.0

1.0

2.0

3.0

Area (x100,000)

0 1000 Conc.0.0

1.0

2.0

3.0

4.0

Area (x10,000)

0 10000 Conc.0.0

2.5

5.0

7.5Area (x10,000)

CYC r2=0.9948

SAC r2=0.9977

SUC r2=0.9991

ASP r2=0.9983

NEO r2=0.9982

ALI r2=0.9990

DUL r2=0.9987

NHDC r2=0.9991

GLY r2=0.9997

0 500 Conc.0.0

1.0

Area(x10,000)

0 500 Conc.0.0

0.5

1.0

Area(x1,000)

24 Conc.0.0

2.5

5.0Area(x1,000)

0 Conc.0.0

5.0

Area(x1,000)

0 Conc.0.0

2.5

Area(x1,000)

0.0 25.0 Conc.0.0

1.0

Area(x10,000)

0 Conc.0.0

0.5

1.0Area(x10,000)

0 500 Conc.0.0

1.0

Area(x10,000)

0.0 25.0 Conc.0.0

0.5

1.0Area(x1,000)

0 500 Conc.0.0

2.5

Area(x1,000)

0.0 2.5 5.0 7.5 10.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5(x1,000)

Cyc

lam

ate

NH

DC

Sucr

alos

e

Sacc

harin

Ace

sulfa

me

K

Gly

cyrr

hizi

c

Dul

cin

Neo

tam

e

Alit

ame

Asp

arta

me


5

Table 3 summarizes the results of repeatability and sensitivity of the method with mixed standards. The method was not evaluated with beverage spiked samples. However, because beverage samples are normally diluted many times,

matrix effect and interferences can be ignored for high sensitivity LC/MS/MS analysis. The results indicate that the method with ultra-small injection volume exhibits good linearity, repeatability and sensitivity.

Method performance

The LC/MS/MS method established was applied for screening and quantitation of the targeted sweeteners in three brand beverages: S1, S2 and S3, and a mouthwash

S4. The results are shown in Figure 4 and Table 4. It is interested to note that glycyrrizinate was found in the mouthwash.

Analysis of beverage samples

Table 3: Repeatability and Sensitivity of LC/MS/MS method of arti�cial sweeteners

RSD%

5.2

8.1

5.8

2.7

3.0

1.0

1.7

3.1

4.6

5.4

LOQ/LOD (0.1 µL inj)

200

800

250

200

80

5

40

160

100

400

Conc. (ug/L)

100

100

100

100

10

5

10

100

10

100

RSD%

5.1

11.7

8.0

7.5

7.8

5.3

8.6

7.5

9.2

8.2

Conc. (ug/L)

20

20

20

20

2

1

2

20

2

20

Name

Ace-K

CYC

SAC

SUC

ASP

NEO

ALI

DUL

NHDC

GLY

50

500

100

100

20

3

25

50

25

150

LOQ/LOD (0.5 µL inj)

40

200

50

50

20

2

10

30

40

15

10

90

20

15

4

1

5

10

6

5

LOQ/LOD 10 (µL inj)

4.0

14

4.5

2.4

0.5

0.03

0.2

1.4

0.5

5.0

1.33

4.5

1.5

0.8

0.17

N.A.

N.A.

0.5

0.18

1.8

Repeatability (peak area), 10uL Sensitivity (ug/L)

Table 4: Screening and quantitation results for ten arti�cial sweeteners in beverages and mouthwash (mg/L)

S4

ND

ND

208.7

ND

449.3

ND

S3

ND

97.2

ND

183.4

ND

ND

S2

127.9

165.9

ND

ND

ND

ND

S1

116.9

143.9

ND

55.1

ND

ND

Arti�cial Sweetener

ASP

Ace-K

Saccharin

SUC

GLY

Others

1. S2 was diluted 100 times, the rests were diluted 10 times. 1 uL injection.2. ND = not detected.






References1. http://en.wikipedia.org/wiki/Sugar_substitute and EU directive 93/35/EC, 96/83/EC, 2003/115/EC, 2006/52/EC and

2009/163/EU.2. Buchgraber and A. Wasik, Report EUR 22726 EN (2007).3. F.T. Large, M. Scheurer and H.-J Brauch, Anal Bioanal Chem, 403: 2503-2518 (2012) 4. Ho-Soo Lim, Sung-Kwan Park, In-Shim Kwak, Hyung-Ll Kim, Jun-Hyun Sung, Mi-Youn Byun and So-Hee Kim, Food Sci,

Biotechnol, 22(S):233-240 (2013)

ConclusionsA MRM-based LC/MS/MS method was developed and evaluated for screening and quantitation of ten arti�cial sweeteners in beverages. This high sensitivity LC/MS/MS method combined with small or ultra-small injection volume (0.1~1.0 uL) was proven to be feasible and reliable in actual samples analysis of the targeted sweeteners in beverages, achieving high throughput and free of sample

pre-treatment (except dilution). The method is expected to be applicable to surface water and drinking water samples. For wastewater and various foods, sample pre-treatment is usually required. However, the advantages of the method in high sensitivity and ultra-small injection volume are expected to enable it tolerates relatively simple sample pre-treatment procedures.

Figure 4: Screening and quantitation for 10 targeted arti�cial sweeteners in beverage and mouthwash samples by LC/MS/MS with 1uL injection.

0.0 2.5 5.0 7.5 10.0 min

0.0

1.0

2.0

3.0

4.0

5.0(x1,000,000)

Sucr

alos

e (x

10)

Ace

sulfa

me

K (x

10)

Asp

arta

me

S1

0.0 2.5 5.0 7.5 10.0 min

0.0

1.0

2.0

3.0

4.0

5.0

(x100,000)

Ace

sulfa

me

K (x

10)

Asp

arta

me

S2

0.0 2.5 5.0 7.5 10.0 min

0.0

1.0

2.0

3.0

(x100,000)

Sucr

alos

e

Ace

sulfa

me

K

S3

0.0 2.5 5.0 7.5 10.0 min

0.0

0.5

1.0

1.5

(x100,000)

Sacc

harin

Gly

cyrr

hizi

c

S4

PO-CON1480E

Highly sensitive and rapid simultaneous method for 45 mycotoxinsin baby food samples by HPLC-MS/MS using fast polarity switching

ASMS 2014 MP345

Stéphane MOREAU1 and Mikaël LEVI2

1 Shimadzu Europe, Albert-Hahn Strasse 6-10,

Duisburg, Germany 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador

Allende, 77448 Marne la Vallée Cedex 2, France

2

Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

IntroductionMycotoxins are toxic metabolites produced by fungal molds on food crops. For consumer food safety, quality control of food and beverages has to assay such contaminants. Depending on the potency of the mycotoxin and the use of the food, the maximum allowed level is de�ned by legislation. Baby food is particularly critical. For example, European Commission has �xed the maximum level of A�atoxin B1 and M1 to 0.1 and 0.025 µg/kg, respectively, in baby food or milk.

Therefore, a sensitive method to assay mycotoxins in complex matrices is mandatory. In order to ensure productivity of laboratory performing such assays, a unique rapid method able to measure as much mycotoxins as possible independently of the sample origin is also needed.In this study, we tested three kind of samples: baby milk powder, milk thickening cereals (�our, rice and tapioca) and a vegetable puree mixed with cereals.


Sample preparation was performed by homogenization followed by solid phase extraction using specific cartridges (Isolute® Myco, Biotage, Sweden) covering a large spectrum of mycotoxins.Sample (5g) was mixed with 20 mL of water/acetonitrile (1/1 v/v), sonicated for 5 min and agitated for 30 min at room temperature. After centrifugation at 3000 g for 10 min, the supernatant was diluted with water (1/4 v/v). Columns (60mg/3 mL) were conditioned with 2 mL of acetonitrile then 2 mL of water. 3 mL of the diluted supernatant were loaded at the lowest possible flow rate.

Then column was washed with 3 mL of water followed by 3 mL of water/acetonitrile (9/1 v/v). After drying, compounds were successively eluted with 2 mL of acetonitrile with 0.1% of formic acid and 2 mL of methanol.The eluate was evaporated under nitrogen flow at 35 ºC until complete drying (Turbovap, Biotage, Sweden).The sample was reconstituted in 150 µL of a mixture of water/methanol/acetonitrile 80/10/10 v/v with 0.1% of formic acid.

Sample preparation

Extracts were analysed on a Nexera X2 (Shimadzu, Japan) UHPLC system and coupled to a triple quadrupole mass spectrometer (LCMS-8050, Shimadzu, Japan). Analysis was

carried out using selected reaction monitoring acquiring 2 transitions for each compound.

LC-MS/MS analysis

3


Table 1 – LC conditions

Table 2 – MS/MS conditions

Analytical column : Shimadzu GLC Mastro™ C18 150x2.1 mm 3µm

Mobile phase : A = Water 2mM ammonium acetate and 0.5% acetic acid

B = Methanol/Isopropanol 1/1 + 2mM ammonium acetate

and 0.5% acetic acid

Gradient : 2%B (0.0min), 10%B (0.01min), 55%B (3.0min), 80%B (7.0 -8.0min),

2%B (8.01min), Stop (11.0min)


Injection volume : 10 µL


Ionization mode : Heated ESI (+/-)

Temperatures : HESI: 400ºC

Desolvation line: 250ºC

Heat block: 300ºC

Gas �ows : Nebulizing gas (N2): 2 L/min

Heating gas (Air): 15 L/min

Drying gas (N2): 5 L/min

CID gas pressure : 270 kPa (Ar)

Polarity switching time : 5 ms

Pause time : 1 ms

Dwell time : 6 to 62 ms depending on the number of concomitant transitions

to ensure a minimum of 30 points per peak in a maximum loop time

of 200 ms (including pause time and polarity switching)

4


Name

15-acetyldeoxynivalenol (15ADON) [M+H]+

3-acetyldeoxynivalenol (3ADON) [M+H]+

A�atoxine B1 (AFB1) [M+H]+

A�atoxine B2 (AFB2) [M+H]+

A�atoxine G1 (AFG1) [M+H]+

A�atoxine G2 (AFG2) [M+H]+

A�atoxine M1 (AFM1) [M+H]+

Alternariol [M-H]-

Alternariol monomethyl ether [M-H]-

Beauvericin (BEA) [M+H]+

Citrinin (CIT) [M+H]+

D5-OTA (ISTD)

Deepoxy-Deoxynivalenol (DOM-1) [M-H]-

Deoxynivalenol (DON) [M-CH3COO]-

Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]-

Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]-

Diacetoxyscirpenol (DAS) [M+NH4]+

Enniatin A (ENN A) [M+H]+

Enniatin A1 (ENN A1) [M+H]+

Enniatin B (ENN B) [M+H]+

Enniatin B1 (ENN B1) [M+H]+

Fumagillin (FUM) [M+H]+

Fumonisine B1 (FB1) [M+H]+

Fumonisine B2 (FB2) [M+H]+

Fumonisine B3

Fusarenone-X (FUS-X) [M+H]+

HT2 Toxin [M+Na]+

Moniliformin (MON) [M-H]-

Neosolaniol (NEO) [M+NH4]+

Nivalenol (NIV) [M+CH3COO]-

Ochratoxin A (OTA) [M+H]+

Ochratoxin B (OTB) [M+H]+

Patulin (PAT) [M-H]-

Sterigmatocystin (M+H]+

T2 Tetraol [M+CH3COO]-

T2 Toxin [M+NH4]+

Tentoxin [M-H]-

Tenuazonic acid (TEN) [M-H]-

Wortmannin (M-H)

Zearalanol (alpha) (ZANOL) [M-H]-

Zearalanol (beta) (ZANOL) [M-H]-

Zearalanone (ZOAN) [M-H]-

Zearalenol (alpha) (ZENOL) [M-H]-

Zearalenol (beta) (ZENOL) [M-H]-

Zearalenone (ZON) [M-H]-

Ret. Time (min)

3.37

3.37

3.78

3.57

3.46

3.26

3.30

4.78

5.81

8.03

4.16

5.22

3.02

2.61

2.45

2.45

1.20

8.51

8.22

7.57

7.92

6.16

4.10

4.71

4.38

2.84

4.58

1.16

2.90

2.41

5.53

4.83

2.35

5.60

1.64

4.94

4.77

4.51

3.95

5.17

4.85

5.43

5.25

4.94

5.52

MRM Quan

339 > 297.1

339 > 231.1

312.6 > 284.9

315.1 > 259

329.1 > 242.9

330.9 > 244.9

329.1 > 273

257 > 214.9

271.1 > 255.9

784 > 244.1

251.3 > 233.1

409.2 > 239.1

279.2 > 249.3

355.3 > 295.2

517.5 > 457.1

517.5 > 457.1

384 > 283.3

699.2 > 682.2

685.3 > 668.3

657 > 640.4

671.2 > 654.2

459.2 > 131.1

722.1 > 334.2

706.2 > 336.3

706.2 > 336.2

355.1 > 247

446.9 > 344.9

97.2 > 40.9

400.2 > 215

371.2 > 280.9

404.2 > 239

370.2 > 205.1

153 > 81.2

325.3 > 310

356.8 > 297.1

484.2 > 215

413.1 > 140.9

196.1 > 138.8

426.9 > 384

321.3 > 277.2

321.3 > 277.2

319 > 275.1

319.2 > 275.2

319.2 > 275.2

316.8 > 174.9

MRM Qual

339 > 261

339 > 231.1

312.6 > 240.9

315.1 > 286.9

329.1 > 199.9

330.9 > 313.1

329.1 > 229

257 > 213.1

271.1 > 228

784 > 262

251.3 > 205.1

N/A

279.2 > 178.4

355.3 > 265.1

517.5 > 427.1

517.5 > 427.1

384 > 343

699.3 > 210

685.3 > 210.1

657 > 195.9

671.2 > 196

459.2 > 338.7

722.1 > 352.2

706.2 > 318.1

706.2 > 688.1

355.1 > 175

446.9 > 285

N/A

400.2 > 185

371.2 > 311.1

404.2 > 358.1

370.2 > 187

153 > 53

325.3 > 281.1

356.8 > 59.1

484.2 > 305

413.1 > 271.1

196.1 > 112

426.9 > 282.1

321.3 > 303.2

321.3 > 303.1

319 > 301.1

319.2 > 160.1

319.2 > 160.1

316.8 > 131.1

Table 3 – MRM transitions

5


Figure 1 – Structure of the Mastro™ column

Figure 2 – Parameters selection view in the Interface Setting Support Software

Results and discussion

LC conditions were transferred from a previously described method (Tamura et al., Poster TP-739, 61st ASMS). In particularly, the column was chosen to provide very good peak shape for chelating compounds like fumonisins thanks to its inner PEEK lining.

Small adjustments in the mobile phase and in the gradient program were made to handle more mycotoxins, especially the isobaric ones. These modifications are reported in the Table 1.

Method development

Also, autosampler rinsing conditions were kept to ensure carry-over minimisation of some difficult compounds.Electrospray parameters (gas flows and temperatures) were cautiously optimized to find the optimal combination for the most critical mycotoxins (aflatoxins). Since these parameters act in a synergistic way, a factorial design experiment is needed to find it. Manually testing all combinations in the chromatographic conditions is very

time consuming. Therefore, new assistant software (Interface Setting Support) was used to generate all possible combinations and generate a rational batch analysis. Optimal combination was found in chromatographic conditions. The difference observed between optimum and default or worst parameters was of 200 and 350%, respectively.

Stationary phase

Stainless steel Body Polymer lining

Polymer frit


6

Extraction and ionisation recovery for aflatoxins was measured in the three matrices by comparing peak areas of the raw sample extract to extract spiked at 50 ppb after or before extraction and to standard solution. Results in table

4 showed that the total recovery was quite acceptable to ensure accurate quantification. Results from other matrices were not significatively different.

Results

Repeatability was evaluated at low level for aflatoxins. Figure 3 shows an overlaid chromatogram (n=4) for aflatoxins.

Table 4 – Extraction and ionisation recoveries in puree

Figure 3 – Chromatogram of a�atoxins at 0.1 ppb in milk thickening cereals

Figure 4 – Chromatogram of the 45 mycotoxins in standard at 50 ppb (2 ppb for a�atoxins and ochratoxines)

Extraction recovery

Ionisation recovery

Total recovery

AFB1

101%

49%

49%

AFB2

109%

90%

98%

AFG1

104%

96%

100%

AFG2

114%

106%

121%

AFM1

118%

91%

108%

3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

(x10,000)

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min-500000

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

6500000

7000000

7500000

8000000

8500000

9000000

9500000

10000000

10500000

11000000

11500000

12000000






Conclusion• A very sensitive method for multiple mycotoxines was set up to ensure low LOQ in baby food sample,• Thanks to high speed polarity switching, a high number of mycotoxines can be assayed using the same method in a

short time, • The extraction method demonstrate good recoveries to ensure accurate quanti�cation.

PO-CON1461E

High Sensitivity Analysis of Acrylamidein Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure

ASMS 2014 MP342

Zhi Wei Edwin Ting1; Yin Ling Chew*2;

Jing Cheng Ng*2; Jie Xing1; Zhaoqi Zhan1

1 Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE; 2 Department of Chemistry, Faculty of Science,


Ridge Road, Singapore119077, *Student

2

High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure

IntroductionAcrylamide was found to form in fried foods like potato-chips via the so-called Maillard reaction of asparagine and glucose (reducing sugar) at higher temperature (120ºC) in 2002 [1,2]. The health risk of acrylamide present in many processing foods became a concern immediately, because it is known that the compound is a neurotoxin and a potential carcinogen to humans [3]. Various analytical methods, mainly LC/MS/MS and GC/MS based methods, were established and used in analysis of acrylamide in foods in recent years [4]. We

present a novel LC/MS/MS method for quantitative determination of acrylamide in potato chips with using a modi�ed QuEChERS procedure for sample extraction and clean-up, achieving high sensitivity and high recovery. A small sample injection volume (1uL) was adopted purposely to reduce the potential contamination of samples to the interface of MS system, so as to enhance the operation stability in a laboratory handling food samples with high matrix contents.

ExperimentalAcrylamide and isotope labelled acrylamide-d3 (as internal standard) were obtained from Sigma-Aldrich. The QuEChERS kits were obtained from RESTEK. A modi�ed procedure of the QuEChERS was optimized and used in the sample extraction of acrylamide (Q-sep Q100 packet, original unbuffered) in potato chips and clean-up of matrix with d-SPE tube (Q-sep Q250, AOAC 2007.01). Acrylamide and acrylamide-d3 (IS) stock solutions and diluted calibrants were prepared using water as the solvent.

Method development and performance evaluation were carried out using spiked acrylamide samples in the extracted potato chip matrix. A LCMS-8040 triple quadrupole LC/MS/MS (Shimadzu Corporation, Japan) was used in this work. A polar-C18 column of 2.5µm particle size was used for fast UHPLC separation with a gradient elution method. Table 1 shows the details of analytical conditions on LCMS-8040 system,.

Table 1: LC/MS/MS analytical conditions of LCMS-8040 for acrylamide

LC condition

Phenomenex Synergi 2.5u Polar-Rp 100A (100 x 2.00mm)

0.2 mL/min

A: waterB: 0.1% formic acid in Methanol

Gradient elution, B%: 1% (0 to 1 min) → 80% (3 to 4.5 min) → 1% (5.5 to 10min)

40ºC

1.0 µL

Column

Flow Rate

Mobile Phase

Elution Mode

Oven Temp.

Injection Vol.

MS Interface condition

ESI

Positive, MRM, 2 transitions each compound

400ºC

200ºC

Ar (230kPa)

N2, 1.5L/min

N2, 10.0L/min

Interface

MS mode

Block Temp.

DL Temp.

CID Gas

Nebulizing Gas Flow

Drying Gas Flow

3



The details of a modified QuEChERS procedure for potato chips are shown in Figure 1. Hexane was used to defat potato chips, removing oils and non-polar components. In the extraction step with Q-sep Q100Packet extraction salt (contain 4g MgSO4 & 0.5g NaCl), additional 4g of MgSO4 was added to absorb the water completely (aqueous phase disappeared). Acrylamide is soluble in both aqueous and organic phases. With this modification, high recovery of acrylamide was obtained. It is believed that this is because complete removal of water in the mixed extract solution could promote acrylamide transferring into the organic phase. Dispersive SPE tube was used as PSA to remove organic acids which may decompose acrylamide in the process.

QuEChERS Sample Pre-treatment

As acrylamide is a more polar compound, a Polar-RP type column was selected. Isotope labeled internal standard (acrylamide-d3) was used to compensate the variation of acrylamide peak area caused by system fluctuation and inconsistency in sample preparation of different batches.The precursor ions of acrylamide and acrylamide-d3 (IS) were their protonated ions (m/z72.1 and m/z75.1). The MRM optimization was carried out using an automated program of the LabSolutions workstation, which could generate a list of all MRM transitions with optimized CID voltages accurate to (+/-) 1 volt in minutes. Two MRM transitions of acrylamide and acryl-amide-d3 were selected as quantifier and confirmation ion as shown in Table 2.The obtained extract solution of potato chips was used as “blank” and also matrix for preparation of post-spiked calibrants for establishment of calibration curve with IS (acrylamide-d3). To obtain reliable results, the blank and each post-spiked calibrant as shown in Table 3 were injected three times and the average peak area ratios were calculated and used.

Method Development

Figure 1: Flow chart of sample pre-treatment with modi�ed QuEChERS.

Table 3: Acrylamide spiked samples and peak area ratios of measured by IS method

Acrylamide post-spiked

IS post-spiked

Conc. RatioCalculated

Area Ratio measured*

L0, Blank

L1, 1ppb

L2, 5ppb

L3, 10ppb

L4, 50ppb

L5 100ppb

L6, 500ppb

50ppb

0

0.02

0.10

0.20

1.00

2.00

10.00

0.6033

0.6120

0.6786

0.8239

1.7686

2.8196

11.8330

*= Area (acrylamide) / Area (IS)

Table 2: MRM transitions and CID voltages

Name MRM (m/z)Q1 Q3

Acrylamide-d3

Acrylamide

75.1 > 58.0*

75.1 > 30.1

72.1 > 55.0*

72.1 > 27.1

-29

-29

-17

-17

CID Voltage (V)

CE

-15

-24

-16

-22

-22

-30

-24

-30

*MRM transition as quanti�er

[1] Weigh 2.0g of sample in a 50mL centrifuge tube Add 5mL hexane, 10mL water

and 10mL acetonitrile[2] Vortex and shake vigorously for 1min Add Q-sep Q100Packet salt Additional 4g MgSO4 (anhydrous)[3] Vortex and shake vigorously for 5min

[4] Discard the hexane (top layer)

[5] Transfer the solution into a 20mL volumetric �ask wash extraction salt with ACN

in the centrifuge tube[6] Combine the washing solution into the

volumetric �ask (above)

[7] Transfer 1mL of solution into the 2mL Q-sep Q250 QuEChERS dSPE tube

[8] Vortex and centrifuge for 10min at 13000rpm

[9] Transfer 500uL extract to a 1.5mL vial Evaporate to dryness by N2 blow[10] Reconstitute with 250uL of Milli Q water

[11] Analyze by Shimadzu LCMS-8040

4


It was found that the potato chips used as “blank” in this study was not free of acrylamide. Instead, it contained 27.1 ng/mL of acrylamide in the extract solution. A linear calibration curve was established with an intercept of

0.594 at zero spiked concentration (L0) as shown in Figure 2. Good linearity with correlation coefficient (R2) greater than 0.9999 across the range of 1.0 ng/mL– 500.0 ng/mL was obtained.

Figure 2: Calibration curve (left) and MRM peaks (right) of acrylamide spiked into potato chips matrix, 1-500 ppb with 50 ppb IS added.

It was hard to estimate the LOD and LOQ of the analytical method due to the presence of acrylamide (27.1 ng/mL) in the “blank” (extract of potato chips). However, as reported also by other researchers, it is difficult to obtain potato chips free of acrylamide actually. To obtain actual concentration, it is normally subtracting the background content of acrylamide of a “blank” sample used as reference from a measurement of testing sample. The same way was used to estimate actual S/N value in this work. As a result, the LOD and LOD of acrylamide of this method with 1ul injection volume were estimated to be lower than 1ng/mL and 3ng/mL, respectively. This is consistence with the results estimated with the IS.The repeatability of the method was evaluated with L2 and L4 spiked samples. The results are shown in Table 4 and

Figure 3. The peak area %RSD of acrylamide and IS were below 4%. The matrix effect (M.E.), recovery efficiency (R.E.) and process efficiency (P.E.) of the method were determined with a duplicate set of spiked samples of 50 ng/mL level except for the non-spiked sample. The chromatograms of “set 2”, i.e., non-spiked extract, pre-spiked, post-spiked and the standard in neat solution are shown in Figure 4. Noted that, the existing acrylamide in the extract of the potato chips used as reference was accounted for 27.1 ng/mL, corresponding to 135.5 ng per gram of potato chips. The average R.E, M.E and P.E of the method for extraction and analysis of acrylamide obtained are shown in Table 6.

Method Performance Evaluation

spiked Sample Compound Conc. (ng/mL) %RSD

L2

L4

Acrylamide

Acrylamide-d3

Acrylamide

Acrylamide-d3

5

50

50

50

3.5

3.8

3.9

3.6

Table 4: Repeatability Test Results (n=6)

2.5 5.0 7.5 min

0

100000

200000

3000002:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 500ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 100ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 50ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 10ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 1ppb 01a.lcd

1.5 2.0 2.5 min

0

50000

100000

150000

0.0 2.5 5.0 7.5 Conc. Ratio0.00

0.25

0.50

0.75

1.00

1.25Area Ratio (x10)

Y= 1.1239X + 0.594168 R2 = 0.9999

0.00 Conc. Ratio0.0

0.5

1.0

Area Ratio


5

Figure 3: Overlay MRM chromatograms of 5 ng/mL acrylamide spiked in potato chips extract (total: 27.1+5 = 32.1 ng/mL)

2.5 5.0 7.5 min

0

10000

20000

30000

2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R06.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R05.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R04.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R03.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R02.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R01.lcd

1.0 1.5 2.0 2.5

Figure 4: The MRM peaks of acrylamide detected in “blank” extract of potato chips (a), neat standard of 50ppb (b) post-spiked sample of 50ppb (c) and pre-spiked sample of 50ppb.

ConclusionsAcrylamide is formed unavoidably in starch-rich food in cooking and processing at high temperature like potato chips, French fries, cereals and roasted coffee etc. The analysis method established in this work can be used to monitor the levels of acrylamide in processing food accurately and reliably. The QuEChERS method is proven to be fast and effective in extraction of acrylamide from potato chips. The excellent performance of the method in terms of sensitivity, linearity, repeatability and recovery are

related to the outstanding performance of the LC/MS/MS used which features ultra fast mass spectrometry (UFMS) technology. The high sensitivity of the method allows the analysis to be performed with a very small injection volume (1µL or below), which would be a great advantage in running heavily food samples with high matrix contents and strong matrix effects. Maintenance of the interface of a mass spectrometer could also be reduced signi�cantly.

Table 6: Method evaluation of at 50.0ng/mL concentration in potato chips matrix

Parameter Set 1 Set 2 Average

R.E.

M.E.

P.E.

104.7%

96.5%

100.8%

112.0%

84.6%

94.5%

108.4%

90.5%

97.6%

(d) pre-spiked

1.5 2.0 2.50

10000

20000

30000

40000

50000

1.5 2.0 2.5

0

10000

20000

30000

40000

50000

1.5 2.0 2.50

10000

20000

30000

40000

50000

1.5 2.0 2.50

10000

20000

30000

40000

50000(a) Extract (non-spiked)

(c) post-spiked(b) standard






References[1] Swedish National Food Administration. “Information about acrylamide in food, 24 April 2002”, http://www.slv.se[2] Mottram, D.S., & Wedzicha, B.L., Nature, 419 (2002), 448-449. [3] Ahn, J.S., Castle, J., Clarke, D.B., Lloyd, A.S., Philo, M.R., & Speck, D.R., Food Additives and Contaminants, 19 (2002),

1116-1124. [4] Mastovska, K., & Lehotary, S.J., J. Food Chem., 54 (2006), 7001-7998.

PO-CON1472E

Determination of Benzimidazole Residues in Animal Tissue by Ultra High PerformanceLiquid Chromatography Tandem Mass Spectrometry

ASMS 2014 TP 281

Yin Huo, Jinting Yao, Changkun Li, Taohong Huang,

Shin-ichi Kawano, Yuki Hashi

Shimadzu Global COE, Shimadzu (China) Co., Ltd., China

2

Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

IntroductionBenzimidazoles are broad-spectrum, high ef�ciency, low toxicity anthelmintic. Because some benzimidazoles and their metabolites showed teratogenic and mutagenic effects in animal and target animal safety evaluation experiment, many countries have already put benzimidazoles and metabolites as the monitoring object.

This poster employed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method to determinate 16 benzimidazole residues in animal tissue. The method is simple, rapid and high sensitivity, which meets the requirements for the analysis of veterinary drug residue in animal tissue.

Method

(1) Animal tissue samples were extracted with ethyl acetate-50% potassium hydroxide-1% BHT(2) The samples were treated with n-hexane for defatting and further cleaned-up on MCX solid phase (SPE) cartridge. (3) The separation of benzimidazoles and their metabolites was performed on LC-MS/MS instrument.

Sample Preparation

The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, a CTO-30A column oven, a DGU-30A5 degasser, and an SIL-30AC autosampler. The separation was carried out on a Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm, Shimadzu) with the column temperature at 30 ºC. A triple quadrupole mass spectrometer (Shimadzu LCMS-8040, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface.

LC/MS/MS Analysis


UHPLC (Nexera system)

Column : Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm)

Mobile phase A : water with 0.1% formic acid

Mobile phase B : acetonitrile

Gradient program : as in Table 1


Column temperature : 30 ºC

Injection volume : 20 µL

Table 1 Time program

Time (min) Module Command Value

0.01

3.50

4.00

4.01

6.00

Pumps

Pumps

Pumps

Pumps

Controller

Pump B Conc.

Pump B Conc.

Pump B Conc.

Pump B Conc.

Stop

5

80

80

5

3


MS/MS (LCMS-8040 triple quadrupole mass spectrometer)

Ionization : ESI

Polarity : Positive

Ionization voltage : +4.5 kV

Nebulizing gas �ow : 3.0 L/min

Heating gas pressure : 15.0 L/min

DL temperature : 200 ºC

Heat block temperature : 350 ºC

Mode : MRM

Table 2 MRM parameters of 16 benzimidazoles (*: for quantitation)

CompoundPrecursor

m/z

300.10

282.00

202.00

218.00

316.20

266.30

240.30

298.30

296.30

238.30

298.30

314.30

256.30

303.20

250.30

332.20

Productm/z

268.05*

159.05

240.10*

208.05

175.10*

131.15

191.05*

147.10

159.15*

191.15

234.10*

191.10

133.20*

198.10

159.10*

224.05

264.15*

105.25

105.20*

133.20

266.10*

160.15

282.15*

123.15

123.20*

95.20

217.15*

261.10

218.15*

176.15

300.10*

159.05

Dwell Time(ms)

50

50

10

10

10

10

50

50

20

20

8

8

50

50

20

20

10

10

10

10

10

10

10

10

10

10

5

5

5

5

10

10

Q1 Pre Bias(V)

-15.0

-15.0

-14.0

-14.0

-30.0

-30.0

-30.0

-30.0

-11.0

-11.0

-30.0

-30.0

-15.0

-15.0

-13.0

-13.0

-13.0

-13.0

-15.0

-15.0

-30.0

-30.0

-14.0

-14.0

-16.0

-16.0

-30.0

-30.0

-30.0

-30.0

-15.0

-15.0

Q3 Pre Bias(V)

-18.0

-30.0

-17.0

-22.0

-18.0

-25.0

-13.0

-27.0

-30.0

-20.0

-25.0

-20.0

-24.0

-21.0

-30.0

-23.0

-27.0

-19.0

-20.0

-25.0

-18.0

-30.0

-19.0

-24.0

-22.0

-18.0

-23.0

-28.0

-23.0

-18.0

-21.0

-30.0

CE (V)

-21.0

-36.0

-12.0

-23.0

-24.0

-31.0

-23.0

-32.0

-34.0

-22.0

-19.0

-33.0

-27.0

-18.0

-37.0

-27.0

-21.0

-35.0

-26.0

-36.0

-22.0

-35.0

-22.0

-35.0

-26.0

-41.0

-28.0

-17.0

-17.0

-27.0

-22.0

-39.0

Fenbendazole

Albendazole sulfoxide

Thiabendazole

Thiabendazole-5-hydroxy

Oxfendazole

Albendazole

Albendazole -2-aminosulfone

Albendazole sulfone

Mebendazole

Mebendazole-amine

5-Hydroxymebendazole

Flubendazole

2-Amino�ubendazole

Cambendazole

Oxibendazole

Oxfendazole

4


Results and DiscussionA liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed to identify and quantify trace levels of 16 benzimidazoles residue (fenbendazole, albendazole sulfoxide, thiabendazole, thiabendazole- 5-hydroxy, oxfendazole, albendazole, albendazole-2-aminosulfone, albendazole sulfone, mebendazole, mebendazole-amine, 5-hydroxymebendazole, �ubendazole, 2-amino�ubendazole, cambendazole, oxibendazole, oxfendazole) in animal tissue. The MRM chromatograms of

16 drugs mixture are presented in Fig.1. The correlation coef�cients for 16 drugs (0.5 – 50 ng/mL) were found to 0.9993~0.9999. MRM chromatograms of pork samples and pork samples spiked with standards are shown in Fig.2. By analyzing 16 drugs at three levels including 0.5 ng/mL, 5 ng/mL, 50 ng/mL, excellent repeatability was demonstrated with the %RSD being better than 5% for all the compound within six injections as shown in Table 3. Results of recovery test were good as shown in Table 4.

Figure 1 MRM chromatograms of standard 16 drugs (1 ng/mL)(1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole;

4: Mebendazole-amine; 5: 2-Amino�ubendazole;6: 5-Hydroxymebendazole;7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole;11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole;

15: Flubendazole; 16: Fenbendazole)

0.0 1.0 2.0 3.0 4.0 min

0

10000

20000

30000

40000

50000

60000

70000

16:300.10>268.05(+)15:314.30>282.15(+)14:332.20>300.10(+)(2.00)13:296.30>264.15(+)12:266.30>234.10(+)11:298.30>159.10(+)(2.00)10:316.20>159.15(+)(2.00)9:250.30>218.15(+)8:303.20>217.15(+)7:282.00>240.10(+)6:298.30>266.10(+)5:256.30>123.20(+)(2.00)4:238.30>105.20(+)(3.00)3:202.00>175.10(+)2:240.30>133.20(+)(2.00)1:218.00>191.05(+)(10.00)

151413

12

1110

98

76

54

32

1

16


5

Figure 2 MRM chromatograms of pork sample (left) and spiked pork sample (right) (1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole;

4: Mebendazole-amine; 5: 2-Amino�ubendazole;6: 5-Hydroxymebendazole;7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole;11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole;

15: Flubendazole; 16: Fenbendazole)

Table 3 Repeatability of 16 drugs in pork sample (n=6)

CompoundArea

3.01

4.26

4.52

4.44

2.71

2.07

4.36

3.95

4.95

3.95

2.31

4.22

4.30

4.90

3.46

3.23

%RSD (0.5 ng/mL) %RSD (5.0 ng/mL) %RSD (50 ng/mL)

R.T.

0.059

0.202

0.272

0.526

0.121

0.073

0.392

0.103

0.093

0.363

0.091

0.107

0.339

0.150

0.091

0.170

R.T.

0.064

0.084

0.180

0.249

0.089

0.090

0.162

0.126

0.095

0.149

0.099

0.058

0.177

0.123

0.108

0.044

Area

1.48

2.86

2.85

3.91

2.91

1.29

2.08

0.63

1.69

2.72

0.79

1.52

2.53

3.38

1.31

3.09

Area

0.34

0.92

2.58

1.41

0.97

0.92

1.72

0.64

0.74

0.94

1.17

1.00

1.43

1.87

1.20

0.80

R.T.

0.082

0.153

0.132

0.158

0.105

0.099

0.177

0.113

0.094

0.243

0.140

0.091

0.166

0.121

0.125

0.084

Fenbendazole

Albendazole Sulfoxide

Thiabendazole


Oxfendazole

Albendazole

Albendazole -2-Aminosulfone

Albendazole sulfone

Mebendazole

Mebendazole-amine


Flubendazole


Cambendazole

Oxibendazole

Oxfendazole

0.0 1.0 2.0 3.0 4.0 min0

10000

20000

30000

40000

50000

16:300.10>268.05(+)15:314.30>282.15(+)14:332.20>300.10(+)13:296.30>264.15(+)12:266.30>234.10(+)11:298.30>159.10(+)10:316.20>159.15(+)9:250.30>218.15(+)8:303.20>217.15(+)7:282.00>240.10(+)6:298.30>266.10(+)5:256.30>123.20(+)4:238.30>105.20(+)3:202.00>175.10(+)2:240.30>133.20(+)1:218.00>191.05(+)

0.0 1.0 2.0 3.0 4.0 min0

10000

20000

30000

40000

50000

16:300.10>268.05(+)15:314.30>282.15(+)14:332.20>300.10(+)13:296.30>264.15(+)12:266.30>234.10(+)11:298.30>159.10(+)10:316.20>159.15(+)9:250.30>218.15(+)8:303.20>217.15(+)7:282.00>240.10(+)6:298.30>266.10(+)5:256.30>123.20(+)4:238.30>105.20(+)3:202.00>175.10(+)2:240.30>133.20(+)1:218.00>191.05(+)(10.00)

15

1413

12

1110

98

76

54

321

16






ConclusionThe sensitive and reliable LC/MS/MS technique was successfully applied for determination of 16 benzimidazoles residue. The calibration curves of 16 benzimidazoles ranging from 0.5 to 50 ng/mL were established and the correlation coef�cients were

0.9993~0.9999. The LODs of the 16 benzimidazoles were 1 -2.2 µg/kg. The recoveries were in the range of 80.9%~118.5% for pork samples, with relative standard deviations less than 5%.

Table 4 Recovery of 16 drugs in pork sample

CompoundSpike Conc.

(µg/kg)

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

10.0

Sample Conc.(µg/kg)

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

Measured Conc.(µg/kg)

9.5

8.1

9.8

10.0

11.4

9.6

9.6

11.8

11.3

11.8

9.8

10.4

9.3

10.8

9.6

9.1

Recovery(%)

94.5

80.9

98.2

99.8

113.8

96.3

96.1

118.5

112.8

118.3

97.8

103.6

92.6

107.8

96.1

90.7

Fenbendazole

Albendazole Sulfoxide

Thiabendazole


Oxfendazole

Albendazole

Albendazole -2-Aminosulfone

Albendazole sulfone

Mebendazole

Mebendazole-amine


Flubendazole


Cambendazole

Oxibendazole

Oxfendazole

PO-CON1459E

High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid ChromatographyTandem Mass Spectrometry

ASMS 2014 TP275

Zhi Wei Edwin Ting1, Jing Cheng Ng2*,

Jie Xing1 & Zhaoqi Zhan1

1 Customer Support Centre, Shimadzu (Asia Paci�c)

Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV,

Singapore Science Park 1, Singapore 1182642 Department of Chemistry, Faculty of Science,


Ridge Road, Singapore 119077, *Student

2

High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

IntroductionMelamine was found to be used as a protein-rich adulterant �rst in pet-food in 2007, and then in infant formula in 2008 in China [1]. The outbreak of the melamine scandal that killed many dogs and cats as well as led to death of six infants and illness of many had caused panic in publics and great concerns in food safety worldwide. Melamine was added into raw milk because of its high nitrogen content (66%) and the limitation of the Kjeldahl method for determination of protein level indirectly by measuring the nitrogen content. In fact, in addition to melamine and its analogues (cyanuric acid etc), a number of other nitrogen-rich compounds was reported

also to be potentially used as protein-rich adulterants, including amidinourea, biuret, cyromazine, dicyandiamide, triuret and urea [2]. Recently, low levels of dicyandiamide (DCD) residues were found in milk products from New Zealand [3]. Instead of addition directly as an adulterant, the trace DCD found in milk products was explained to be relating to the grass “contaminated by DCD”. Dicyandiamide has been used to promote the growth of pastures for cows grazing. We report here an LC/MS/MS method for sensitive detection and quanti�cation of both dicyandiamide (DCD) and melamine in infant milk powder samples.

ExperimentalHigh purity dicyandiamide (DCD) and melamine were obtained from Sigma Aldrich. Amicon Ultra-4 (MWCO 5K) centrifuge �ltration tube (15 mL) obtained from Millipore was used in sample pre-tretment. The milk powder sample was pre-treated according to a FDA method [1] with some

modi�cation as illustrated in Figure 1. The �nal clear sample solution was injected into LC/MS/MS for analysis. Stock solutions of DCD and melamine were prepared in pure water.

Table 1: Analytical conditions of DCD and melamine in milk powders on LCMS-8040

Fig 1: Sample pre-treatment work�ow

LC conditions

Alltima HP HILIC 3µ, 150 x 2.10mm

0.2 mL/min

A: 0.1 % formic acid in H2O/ACN (5:95 v/v)B: 20mM Ammonium Formate in H2O/ACN (50:50 v/v)

Gradient elution: 5% (0.01 to 3.0 min) → 95% (3.5 to 5.0 min) → 5% (5.5 to 9.0 min)

40ºC

5 µL

Column

Flow Rate

Mobile Phase

Elution Mode

Oven Temperature

Injection Volume

MS conditions

ESI

Positive

400ºC

300ºC

Ar (230kPa)

N2, 2.0L/min

N2, 15.0L/min

Interface

MS mode

Block Temperature

DL Temperature

CID Gas

Nebulizing Gas Flow

Drying Gas Flow

Weigh 2.0g of milk powder sample

Add 14mL of 2.5% formic acid

(1) Sonicate for 1hr

(2) Centrifuge at 6000rpm for 10min

Transfer 4mL of supernatant to Amicon Ultra-4(MWCO 5K) centrifuge �ltration tube (15mL)

Filter the �ltrate by a 0.2um PTFE syringe �lter

Collect clear �ltrate

To 50uL of �ltrate added 950uL of ACN

Further 10x dilution with ACN

LC/MS/MS analysis

Centrifuge at 7500rpm for 10min

3


An LCMS-8040 triple quadrupole LC/MS/MS (Shimadzu Corporation, Japan) was used in this work. The system is consisted of a high pressure binary gradient Nexera UHPLC coupled with a LCMS-8040 MS system. An Alltima HP HILIC column was used for separation of DCD and

melamine with a gradient program developed (Table 1). The details of the LC and MS conditions are shown in Table 1. A set of calibrants (0.5, 1.0, 2.5, 5 and 10 ppb) was prepared from the stock solutions using of ACN/water (90/10) as diluent.


MRM optimization of DCD and melamine were performed using an automated MRM optimization program of the LabSolutions. The precursors were the protonated ions of DCD and melamine. Two optimized MRM transitions of each compound were selected and used for quantitation and confirmation. The MRM transitions and parameters are shown in Table 2.

MRM optimization

A LC/MS/MS method was developed for quantitation of DCD and melamine based on the MRM transitions in Table 2. Under the HILIC separation conditions (Table 1), DCD and melamine eluted at 2.55 min and 6.29 min as sharp peaks (see Figures 4 & 5). Figures 2 and 3 show the

calibration curves of DCD and melamine standard in neat solutions and in milk matrix solutions (spiked). The linearity with correlation coefficient (R2) greater than 0.997 across the calibration range of 0.5~10.0 ng/mL was obtained for both compounds in both neat solution and matrix (spiked).

Method Development

Figure 2: Calibration curves of DCD and melamine in neat solution

Table 2: MRM transitions and optimized parameters

Name Transition (m/z)Q1 Pre Bias Q3 Pre Bias

DCD

MEL

RT (min)

2.55

6.29

85.1 > 68.1

85.1 > 43.0

127.1 > 85.1

127.1 > 68.1

-15

-15

-26

-26

Voltage (V)

CE

-21

-17

-20

-27

-26

-17

-17

-26

0.0 2.5 5.0 7.5 Conc.0.0

2.5

5.0

7.5

Area (x10,000)

0.0 2.5 5.0 7.5 Conc.0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Area(x100,000)

DCD (85.1>68.1)R2 = 0.997

Melamine (127.1>85.1)R2 = 0.999

4


Figure 3: Calibration curves of DCD and melamine spiked in milk powder matrix

Figure 4: Overlapping of six MRM peaks of 0.5 ng/mL DCD and melamine in neat solution

Figure 5: Overlapping of six MRM peaks of 0.5 ng/mL DCD and melamine in milk powder matrix

The repeatability of the method was evaluated at the levels of 0.5 ng/mL and 1.0 ng/mL. Figures 4 & 5 show the MRM chromatograms of DCD and melamine of six consecutive

injections of 0.5 ng/mL level with and without matrix. The peak area %RSD for the two analytes were lower than 9.2% (see Table 3).

Performance Evaluation

5.5 6.0 6.5 min0.0

1.0

2.0

3.0

4.0

5.0

(x1,000)

2.00 2.25 2.50 2.75 min

0.00

0.25

0.50

0.75

1.00

(x1,000)

DCD(85.1>68.1)

Melamine (127.1>85.1)

2.00 2.25 2.50 2.75 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0(x100)

5.5 6.0 6.5 min0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5(x1,000)

DCD (85.1>68.1)

Melamine (127.1>85.1)

0.0 2.5 5.0 7.5 Conc.0.0

1.0

2.0

3.0

4.0

5.0

Area(x10,000)

0.0 2.5 5.0 7.5 Conc.0.0

0.5

1.0

1.5

2.0

2.5Area(x100,000)

Melamine (127.1>85.1)

R2 = 0.997

DCD (85.1>68.1)

R2 = 0.998

Table 3: Results of repeatability and sensitivity evaluation of DCD and melamine (n=6)

Sample %RSD LOD (ng/mL) LOQ (ng/mL)

In solvent

In matrix

Compd.

DCD

MEL

DCD

MEL

Conc. (ng/mL)

0.5

1.0

0.5

1.0

0.5

1.0

0.5

1.0

5.9

5.3

5.5

2.6

5.9

8.2

9.2

2.4

0.03

0.03

0.05

0.05

0.10

0.09

0.16

0.15

5


The LOD and LOQ were estimated from the results of 0.5 ng/mL in both neat and matrix solution. The LOD and LOQ results were summarized in Table 3. The method achieved LOQs (in matrix) of 0.16 and 0.15 ng/mL (ppb) for DCD and melamine, respectively. Tables 4 & 5 show the results of matrix effect and recovery of the method. The matrix effects for DCD and melamine in the whole concentration ranges were at 64%~70%

and 62%~73%, respectively. The recovery was determined by comparing the results of pre-spiked and post-spiked mixed samples of DCD and melamine in the milk powder matrix (2.5 ng/mL each compound). The chromatograms of these samples are shown in Figure 6. The recovery of DCD and melamine were determined to be 103% and 105% respectively.

Figure 6: MRM peaks of DCD and melamine in pre- and post-spiked samples of 2.5 ng/mL (each). DCD and melamine were not detected in blank matrix of milk powder.

Table 4: Matrix effect (%) of DCD and melamine in milk powder matrix

Conc. (ng/mL) 2.5 5 10

DCD

MEL

66.9

73.1

1

65.4

62.5

0.5

70.4

62.2

64.8

68.9

66.6

68.0

Table 5: Recovery of DCD and melamine determined with spiked sample of 2.5 ng/mL

Compound Pre-spiked Area Post-spiked Area Recovery (%)

DCD

MEL

14,393

65,555

13,987

62,659

102.9

104.6

Melamine Pre-spiked

Melamine Post-spiked

DCD Post-spiked

DCD Pre-spiked

2.00 2.25 2.50 2.75 3.00

0

1000

2000

3000

4000

5000

6000

7000 1:85.10>43.00(+)1:85.10>68.05(+)

Dic

yand

iam

ide

2.00 2.25 2.50 2.75 3.00

0

1000

2000

3000

4000

5000

6000

7000 1:85.10>43.00(+)1:85.10>68.05(+)

6.00 6.25 6.50 6.75

0

2500

5000

7500

10000

12500

15000

175002:127.10>68.05(+)2:127.10>85.10(+)

6.00 6.25 6.50 6.75

0

2500

5000

7500

10000

12500

15000

175002:127.10>68.05(+)2:127.10>85.10(+)

Mel

amin

e

6.00 6.25 6.50 6.75

0

2500

5000

7500

10000

12500

15000

175002:127.10>68.05(+)2:127.10>85.10(+)

Mel

amin

e

2.00 2.25 2.50 2.75 3.00

0

1000

2000

3000

4000

5000

6000

7000 1:85.10>43.00(+)1:85.10>68.05(+)

Dic

yand

iam

ide

Blank matrix of milk powder

Blank matrix of milk powder






ConclusionsA high sensitivity LC/MS/MS method was developed on LCMS-8040 for detection and quantitation of dicyandiamide (DCD) and melamine in milk powders. The method performance was evaluated using infant milk powders as the matrix. The method achieved LOQ of 0.16

ng/mL for both compounds in the matrix, allowing its application in simultaneous analysis of melamine, a protein adulterant in relatively high concentration, and dicyandiamide residue in trace level in milk powders samples.

References1. S. Turnipseed, C. Casey, C. Nochetto, D. N. Heller, FDA Food, LIB No. 4421, Volume 24, October 2008.2. S. MachMahon, T. H. Begley, G. W. Diachenko, S. A. Stromgren, Journal of Chromatography A, 1220, 101-107 (2012).3. http://www.naturalnews.com/041834_Fonterra_milk_powder_dicyandiamide.html

PO-CON1465E

Multiresidue pesticide analysis fromdried chili powder using LC/MS/MS

ASMS 2014 WP350

Deepti Bhandarkar, Shruti Raju, Rashi Kochhar,

Shailesh Damale, Shailendra Rane, Ajit Datar,

Jitendra Kelkar, Pratap Rasam




2

Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

IntroductionPesticide residues in foodstuffs can cause serious health problems when consumed. LC/MS/MS methods have been increasingly employed in sensitive quanti�cation of pesticide residues in foods and agriculture products. However, matrix effect is a phenomenon seen in Electro Spray Ionization (ESI) LC/MS/MS analysis that impacts the data quality of the pesticide analysis, especially for complex matrix like spice/herb.Chili powder is one such complex matrix that can exhibit matrix effect (either ion suppression or enhancement). A calibration curve based on matrix matched standards can demonstrate true sensitivity of analyte in presence of

matrix. Therefore, this approach was used to obtain more reliable and accurate data as compared to quantitation against neat (solvent) standards[1].Multiresidue, trace level analysis in complex matrices is challenging and tedious. Feature of automatic MRM optimization in LCMS-8040 makes method development process less tedious. In addition, the lowest dwell time and pause time along with ultra fast polarity switching (UFswitching) enables accurate, reliable and high sensitive quantitation. UFsweeperTM II technology in the system ensures least crosstalk, which is very crucial for multiresidue pesticide analysis.

Method of Analysis

Commercially available red chili was powdered using mixer grinder. To 1 g of this chili powder, 20 mL water:methanol (1:1 v/v) was added and the mixture was sonicated for 10 mins. The mixture was centrifuged and supernatant was collected. This supernatant was used as diluent to prepare

pesticide matrix matched standards at concentration levels of 0.01 ppb, 0.02 ppb, 0.05 ppb, 0.1 ppb, 0.2 ppb, 0.5 ppb, 1 ppb, 2 ppb, 5 ppb, 10 ppb and 20 ppb. Each concentration level was then filtered through 0.2 µ nylon filter and used for the analysis.

Sample Preparation

Pesticides were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8040 triple quadrupole system (Shimadzu

Corporation, Japan), shown in Figure 1. The details of analytical conditions are given in Table 1.

LC/MS/MS Analytical Conditions

Table 1. LC/MS/MS analytical conditions


• Guard column : Phenomenex SecurityGuard ULTRA Cartridge

• Mobile phase : A: 5 mM ammonium formate in water:methanol (80:20 v/v)

B: 5 mM ammonium formate in water:methanol (10:90 v/v)



• Gradient program (B%) : 0.0–1.0 min → 45 (%); 1.0–13.0 min → 45-100 (%);

13.0–18.0 min → 100 (%); 18.0–19.0 min → 100-45 (%);

19.0–23.0 min → 45 (%)



• Polarity : Positive and negative

• Nitrogen gas �ow : Nebulizing gas 2 L/min; Drying gas 15 L/min

• MS temperature : Desolvation line 250 ºC; Heat block 400 ºC

• MS analysis mode : Staggered MRM

3


ResultsLC/MS/MS method was developed for analysis of 80 pesticides belonging to different classes like carbamate, organophosphate, urea, triazines etc. in a single run[2]. LOQ was determined for each pesticide based on the following criteria – (1) % RSD for area < 16 % (n=3), (2) % Accuracy between 80-120 % and (3) Signal to noise ratio (S/N) > 10.

LOQ achieved for 80 pesticides have been summarized in Table 2 and results for LOQ and linearity for each pesticide have been given in Table 3. Representative MRM chromatogram of pesticide mixture at 1 ppb level is shown in Figure 2. Representative MRM chromatograms at LOQ level for different classes of pesticides are shown in Figure 3.

Figure 1. Nexera with LCMS-8040 triple quadrupole system by Shimadzu

Table 2: Summary of LOQ achieved

LOQ (ppb)

Number of pesticides

0.01

1

0.02

1

0.05

3

0.1

8

0.2

17

0.5

24

1

26

Table 3. Results of LOQ and linearity for pesticide analysis

MRM Transition Polarity LOQ (ppb) Linearity (R2)

746.20>142.10

421.90>366.10

301.00>198.00

732.20>142.10

371.00>273.10

222.90>126.00

221.70>123.00

229.80>198.90

387.90>301.00

387.90>301.00

207.00>72.10

305.70>108.00

408.90>186.00

Name of compound

Spinosyn D

Fenpyroximate

Bifenazate

Spinosyn A

Spiromesifen

Acetamiprid

Carbofuran

Dimethoate

Dimethomorph I

Dimethomorph II

Isoproturon

Pirimiphos methyl

Tri�oxystrobin

Sr. No.

1

2

3

4

5

6

7

8

9

10

11

12

13

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

0.01

0.02

0.05

0.05

0.05

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.9987

0.9915

0.9947

0.9974

0.9957

0.9910

0.9971

0.9970

0.9991

0.9992

0.9984

0.9997

0.9989

4



367.70>198.85

215.90>174.00

235.90>143.00

324.85>108.10

310.60>111.00

384.70>198.80

434.70>330.00

248.80>159.90

283.90>252.00

267.90>174.90

367.80>181.90

299.90>173.90

252.90>126.00

257.90>125.10

354.90>88.00

293.90>196.90

189.90>162.90

208.10>116.05

411.10>190.10

338.00>99.10

305.70>201.00

349.90>266.00

483.75>452.90

357.90>280.80

363.70>193.90

315.90>247.00

313.90>70.10

352.90>227.90

507.70>167.00

288.70>205.00

314.90>99.00

330.90>284.90

411.90>356.20

280.00>220.10

221.70>150.00

162.90>88.00

362.15>303.00

283.90>70.10

260.80>75.00

276.80>96.90

342.90>151.00

890.30>305.10

333.70>139.00

Name of compound

Anilophos

Atrazine

Carboxin

Cyazofamid

Edifenphos

Ethion

Fipronil

Linuron

Metolachlor

Oxycarboxin

Phosalone

Phosphamidon

Thiacloprid

Thiobencarb

Thiodicarb

Triadimefon

Tricyclazole

Aldicarb

Benfuracarb

Bitertanol

Buprofezin

Clodinafop propargyl

Chlorantraniliprole

Diclofop methyl

Flufenacet

Flusilazole

Hexaconazole

Hexythiazox

Iodosulfuron methyl

Iprobenfos

Malaoxon

Malathion

Mandipropamid

Metalaxyl

Methabenzthiazuron

Methomyl

Oxadiazon

Penconazole

Phorate

Phorate sulfoxide

Thiophanate methyl

Avermectin B1a

Carpropamid

Sr. No.

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

Positive

Positive

Positive

Positive

Positive

Positive

Negative

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

1

1

0.9974

0.9985

0.9952

0.9971

0.9997

0.9957

0.9973

0.9945

0.9966

0.9995

0.9987

0.9997

0.9976

0.9977

0.9906

0.9994

0.9977

0.9962

0.9981

0.9935

0.9933

0.9978

0.9994

0.9976

0.9997

0.9983

0.9996

0.9909

0.9971

0.9981

0.9996

0.9997

0.9952

0.9996

0.9957

0.9988

0.9963

0.9992

0.9987

0.9991

0.9996

0.9990

0.9985

5


Figure 2. MRM chromatogram of pesticide mixture at 1 ppb level


241.90>127.00

415.30>186.00

198.90>128.10

385.00>329.10

310.80>158.00

228.10>60.00

886.30>158.10

311.90>236.10

330.70>268.00

306.95>57.10

229.90>202.70

680.90>254.05

247.90>129.00

331.00>116.00

293.90>70.10

328.90>125.00

281.90>212.10

372.70>302.70

368.00>231.10

209.90>110.90

414.90>182.00

321.90>96.10

201.90>103.90

246.80>89.10

Name of compound

Clomazone

Clorimuron ethyl

Cymoxanil

Diafenthiuron

Di�ubenzuron

Dodine

Emamectin benzoate

Fenamidone

Fenarimol

Fenazaquin

Flonicamid

Flubendiamide

Forchlorfenuron

Kresoxim methyl

Paclobutrazol

Pencycuron

Pendimethalin

Profenofos

Propargite

Propoxur

Pyrazosulfuron ethyl

Pyriproxyfen

Simazine

Thiomethon

Sr. No.

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Negative

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

0.9967

0.9965

0.9949

0.9961

0.9982

0.9980

0.9983

0.9997

0.9900

0.9992

0.9971

0.9993

0.9956

0.9996

0.9974

0.9943

0.9932

0.9966

0.9950

0.9987

0.9992

0.9975

0.9992

0.9989

5.0 10.0 15.0 min

0

10000

20000

30000

40000

50000


6

Figure 3. Representative MRM chromatograms at LOQ level from different classes of pesticides

Conclusion• A highly sensitive method was developed for analysis of 80 pesticides belonging to different classes, from dried chili

powder in a single run.• Ultra high sensitivity, ultra fast polarity switching (UFswitching), low pause time and dwell time along with UFsweeperTM

II technology enabled sensitive, selective, accurate and reproducible multiresidue pesticide analysis from complex matrix like dried chili powder.

10.0 11.0 12.0 13.0

0

2500

5000

7500 70:283.90>252.00(+)

Met

olac

hlor

Chloroacetanilide

11.0 12.0 13.0 14.0

0

1000

2000

3000

4000

5000

600080:283.90>70.10(+)

Penc

onaz

ole

Azole

10.0 11.0 12.0 13.0

0

1000

2000

3000

4000

5000126:680.90>254.05(-)

Flub

endi

amid

eAnthranilicDiamide

15.0 16.0 17.0 18.0

0

250

500

750

1000

1250

121:421.90>366.10(+)

Fenp

yrao

xim

atePyrazole

16.0 17.0 18.0 19.0

0

100

200

300

115:746.20>142.10(+)

Spin

osyn

D

MacrocyclicLactone

6.0 7.0 8.0 9.0

1000

2000

3000

4000 42:215.90>174.00(+)

Atr

azin

e

Triazine

7.0 8.0 9.0 10.0

0

1000

2000

3000

400044:207.00>72.10(+)

Isop

rotu

ron

Urea

2.0 3.0 4.0 5.0

0

250

500

750

1000

15:229.80>198.90(+)

Dim

etho

ate

Organophosphorus N

5.0 6.0 7.0 8.0

1000

2000

3000

4000

33:221.70>123.00(+)

Car

bofu

ran

-Methyl Carbamate






References[1] Kwon H, Lehotay SJ, Geis-Asteggiante L., Journal of Chromatography A, Volume 1270, (2012), 235–245.[2] Banerjee K, Oulkar DP et al., Journal of Chromatography A, Volume 1173, (2007), 98-109.

PO-CON1463E

Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERSas an extraction method

ASMS 2014 TP762

Durvesh Sawant(1), Dheeraj Handique(1), Ankush Bhone(1),

Prashant Hase(1), Sanket Chiplunkar(1), Ajit Datar(1),

Jitendra Kelkar(1), Pratap Rasam(1), Kaushik Banerjee(2),

Zareen Khan(2)

(1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh



(2) National Referral Laboratory, National Research

Centre for Grapes, P.O. Manjri Farm, Pune-412307,

Maharashtra, India.

2

Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

IntroductionIndia is the world’s second largest producer (after China) and consumer (after Brazil) of tobacco with nearly $ 1001.54 million revenue generated annually from its export.[1] In countries like India, with tropical-humid climate, the incidences of insect attacks and disease infestations are frequent and application of pesticides for their management is almost obligatory. Like any other crop, tobacco (Nicotiana tabacum Linn.), one of the world’s leading high-value crops, is also prone to pest attacks, and the farmers do apply various pesticides as a control measure. The residues of pesticides applied on tobacco during its cultivation may remain in the leaves at harvest that may even sustain post harvest processing treatments and could appear in the �nal product. Thus, monitoring of pesticide residues in tobacco is an important issue of critical concern from public health and safety point of view demanding implementation of stringent regulatory policies.[2] To protect the consumers by controlling pesticide residue

levels in tobacco, the Guidance Residue Levels (GRL) of 118 pesticides have been issued by the Agro-Chemical Advisory Committee (ACAC) of the Cooperation Center for Scienti�c Research Relative to Tobacco (CORESTA). Tobacco is a complex matrix and hence requires selective extraction and extensive cleanup such as QuEChERS (Quick Easy Cheap Effective Rugged Safe) to ensure trace level detection with adequate precision and accuracy. The objective of the present study was to develop an effective, sensitive and economical multi-pesticide residue analysis method for 203 pesticides in tobacco as listed in Table 1.

Figure 1. Dried tobacco

Method of Analysis

Extraction of pesticides was done using QuEChERS method, as described below.[3]

Extraction of pesticides from tobacco

Take 2 g of dry powdered tobacco leaves (Figure 1). Add 18 mL of water containing 0.5 % acetic acid. Homogenize the sample and Keep it for 30 min.

Add 10 mL ethyl acetate. Immediately, put 10 g sodium sulfate.

Homogenize it thoroughly at 15000 rpm for 2 min.

Centrifuge at 5000 rpm for 5 min for phase separation.

Draw 3 mL of ethyl acetate upper layer from the extract for further cleanup.

3


Figure 2. GCMS-TQ8030 Triple quadrupole system by Shimadzu

• ASSP™ (Advanced Scanning Speed Protocol) enables high-speed scan and data acquisition for accurate quantitation at 20,000 u/sec

• Capable of performing simultaneous Scan/MRM• UFsweeper® technology efficiently sweeps residual ions from the collision cell for fast, efficient ion transport ensuring no

cross-talk• Two overdrive lenses reduce random noise from helium, high-speed electrons and other factors to improve S/N ratio• Flexible platform with EI (Electron Ionization), CI (Chemical Ionization), and NCI (Negative Chemical Ionization)

techniques• Full complement of acquisition modes including MRM, Scan/MRM, Precursor Ion, Product Ion and Neutral Loss Scan

Key Features of GCMS-TQ8030

Add 1 mL toluene to it and vortex for 0.5 min.

Add cleanup mixture [PSA (150 mg), C18 (150 mg), GCB (75 mg) and anhydrous MgSO4 (300 mg)] and vortex for 2 min.

Centrifuge the mixture at 7000 rpm for 7 min.

Collect the supernatant and �lter through a 0.2 µm PTFE membrane �lter.

Inject 2.0 µL of the clean extract into GCMS-TQ8030 (Figure 2).

4


Table 1. List of pesticides

Pesticide

2,6-Dichlorobenzamide

2-Phenylphenol

3,4-Dichloraniline

3-Chloroaniline

4-Bromo 2-Chloro phenol

4,4-Dichlorobenzophenone

Acetochlor

Acrinathrin

Alachlor

Aldrin

Azinphos-ethyl

Azinphos-methyl

Azoxystrobin

Barban

Be�ubutamid

Ben�uralin

Benoxacor

Beta-endosulfan

Bifenox

Bifenthrin

Bitertanol

Boscalid

Bromacil

Bromophos-ethyl

Bromopropylate

Bromuconazole-1

Bromuconazole-2

Butralin

Butylate

Carbaryl

Carbofuran

Carfentrazone

Chlordane-trans

Chlordecone

Chlorfenvinphos

Chlormephos

Chlorobenzilate

Chloroneb

Chlorothalonil

Chlorpyriphos-ethyl

Chlorpyriphos-methyl

Chlorpyriphos-oxon

Chlorthal-dimethyl

Cinidon-ethyl

Cis-1,2,3,6 tetrahydrophthalimide

Clodinafop propargyl

Clomazone

Crimidine

Cyanophos

Cy�uthrin-1

Cy�uthrin-2

Pesticide

Cy�uthrin-3

Cy�uthrin-4

Cyhalofop-butyl

Cypermethrin-2

Cypermethrin-3

Cypermethrin-4

Cyprodinil

Delta-HCH

Demeton-s-methyl

Demeton-S-methyl sulphone

Dialifos

Diazinon

Dichlobenil

Dichlo�uanid

Diclofop

Dicloran

Dieldrin

Diethofencarb

Difenoconazole-1

Difenoconazole-2

Di�ubenzuron

Di�ufenican

Dimethipin

Dimethomorph-1

Dimethomorph-2

Dimoxystrobin

Diniconazole

Dinoseb

Dinoterb

Dioxathion

Edifenfos

Endosulfan sulphate

Endrin

Epoxiconazole

Ethal�uralin

Ethoprophos

Etoxazole

Etridiazole

Etrimfos

Famoxadone

Fenamidone

Fenarimol

Fenbuconazole

Fenchlorphos

Fenchlorphos oxon

Fenhexamid

Fenobucarb

Fenoxycarb

Fenthion sulphoxide

Fenvalerate

Fipronil

Pesticide

Fipronil sulphone

Flucythrinate-1

Flucythrinate-2

Flufenacet

Flumoixazine

Fluquinconazole

Flurochloridone-1

Flurochloridone-2

Flutolanil

Flutriafol

Fluxapyoxad

Folpet

Fuberidazole

Heptachlor

Hexaconazole

Iprobenfos

Isoprocarb

Isoprothiolane

Isopyrazam

Isoxaben

Lactofen

Lambda-cyhalothrin

Malaoxon

Malathion

Mepanipyrim

Mepronil

Metalaxyl

Metalaxyl M

Metazachlor

Metconazole

Methabenzthiazuron

Methacrifos

Methidathion

Methiocarb

Metholachlor-s

Methoxychlor

Metribuzin

Mevinphos

Monolinuron

Myclobutanyl

Napropamide

Nitrapyrin

Oxadiargyl

Oxadiazon

Oxycarboxin

p,p-DDE

Parathion-ethyl

Parathion-methyl

Penconazole

Pencycuron (Deg.)

Pendimethalin

Pesticide

Permethrin-1

Permethrin-2

Pethoxamid

Phosalone

Phosmet

Pirimicarb

Pretilachlor

Procymidone

Profenofos

Propanil

Propaquizafop

Propazine

Propham

Propiconazole-1

Propisoclor

Propyzamide

Proquinazid

Pyra�ufen-ethyl

Pyrazophos

Pyrimethanil

Pyriprooxyfen

Pyroquilon

Quinoxyfen

Simazine

Spirodiclofen

Sulfotep

Swep

Tebufenpyrad

Tebupirimfos

Tebuthiuron

Te�uthrin

Terbacil

Tetraconazole

Tetradifon

Thiobencarb

Tolyl�uanid

Tralkoxydim

Triadimefon

Tri-allate

Triazophos

Tricyclazole

Tri�oxystrobin

Tri�umizole

Tri�umuron

Tri�uralin

Tri�usulfuron

Triticonazole

Valifenalate

Vinclozolin

Zoxamide (Deg.)

Sr. No.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

Sr. No.

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

Sr. No.

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

Sr. No.

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

5


The analysis was carried out on Shimadzu GCMS-TQ8030 as per the conditions given below.

GCMS/MS Analytical Conditions

Chromatographic parameters

• Column : Rxi-5Sil MS (30 m L x 0.25 mm I.D.; 0.25 µm)

• Injection Mode : Splitless

• Sampling Time : 2.0 min

• Split Ratio : 5.0

• Carrier Gas : Helium

• Flow Control Mode : Linear Velocity

• Linear Velocity : 40.2 cm/sec

• Column Flow : 1.2 mL/min

• Injection Volume : 2.0 µL

• Injection Type : High Pressure Injection

• Total Program Time : 41.87 min

• Column Temp. Program : Rate (ºC /min) Temperature (ºC) Hold time (min)

70.0 2.00

25.00 150.0 0.00

3.00 200.0 0.00

8.00 280.0 10.00

Mass Spectrometry parameters

• Ion Source Temp. : 230.0 ºC

• Interface Temp. : 280.0 ºC

• Ionization Mode : EI

• Acquisition Mode : MRM

ResultsFor MRM optimisation, well resolved pesticides were grouped together. Standard solution mixture of approximately 1 ppm concentration was prepared and analyzed in Q3 scan mode to determine the precursor ion for individual pesticides. Selected precursor ions were allowed to pass through Q1 & enter Q2, also called as Collision cell. In Collision cell, each precursor ion was bombarded with collision gas (Argon) at different energies (called as Collision Energy-CE) to produce fragments (product ions). These product ions were further scanned in Q3 to obtain their mass to charge ratio. For each precursor ion, product ion with highest intensity and its

corresponding CE value was selected, thereby assigning a characteristic MRM transition to every pesticide. Based on MRM transitions, the mixture of 203 pesticides was analyzed in a single run (Figure 3). Method was partly validated for each pesticide with respect to linearity (0.5 to 25 ppb), reproducibility, LOQ and recovery. The validation summary for two pesticides namely Mevinphos and Parathion-ethyl (Sr. Nos.140 and 149 in Table 1) is shown in Figures 4 and 5. The summary data of linearity and LOQ for 203 pesticides is given in Table 2 and 3 respectively.


6

Figure 3. MRM Chromatogram for 203 pesticides mixture

Figure 4. Summary data for mevinphos

Calibration overlay Linearity curve Recovery overlay

Linearity (R2)

0.9999

LOD (ppb)

0.3

LOQ (ppb)

1

S/N at LOQ

173

% RSD at LOQ(n=6)

6.93

% Recoveryat LOQ

89.28

Figure 5. Summary data for parathion-ethyl

Calibration overlay Linearity curve Recovery overlay

Linearity (R2)

0.9993

LOD (ppb)

1.5

LOQ (ppb)

5

S/N at LOQ

93

% RSD at LOQ(n=6)

4.05

% Recoveryat LOQ

109.10

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 min-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

(x100,000)

15.0 15.5 16.0 16.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5(x10,000)

15.0 15.5 16.0 16.5

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0 (x1,000)

0.0 5.0 10.0 15.0 20.0 Conc.0.00

0.25

0.50

0.75

1.00

1.25

1.50Area (x100,000)

min min

7.25 7.50 7.75 8.00 8.25 8.50

0.00

0.25

0.50

0.75

1.00

(x10,000)

0.0 5.0 10.0 15.0 20.0 Conc.0.0

0.5

1.0

1.5

2.0

2.5Area (x100,000)

7.0 7.5 8.0 8.5 9.0

0.0

1.0

2.0

3.0

4.0

5.0

(x10,000)

min min

MRM : 192.00>127.00 MRM : 192.00>127.00

MRM : 291.10>137.00 MRM : 291.10>137.00

Post extraction spikePre extraction spike

Post extraction spikePre extraction spike






Conclusion• A highly sensitive method was developed for quantitation of 203 pesticides in complex tobacco matrix by using

Shimadzu GCMS-TQ8030. • The MRM method developed for 203 pesticides can be used for screening of pesticides in various food commodities. For

90 % of the pesticides, the LOQ of 10 ppb or below was achieved. • Ultra Fast scanning, UFsweeper® and ASSP™ features enabled sensitive, selective, fast, reproducible, linear and accurate

method of analysis.

Reference[1] Tobacco Board (Ministry of Commerce and Industry, Government of India), Exports performance during 2013-14,

(2014), 1. http://tobaccoboard.com/admin/statistics�les/Exp_Perf_Currentyear.pdf

[2] CORESTA GUIDE Nº 1, The concept and implementation of cpa guidance residue levels, (2013), 4. http://www.Coresta.org/Guides/Guide-No01-GRLs%283rd-Issue-July13%29.pdf

[3] Zareen S Khan, Kaushik Banerjee, Rushali Girame, Sagar C Utture et al., Journal of Chromatography A, Volume 1343, (2014), 3.

Table 2. Linearity Summary

Linearity (R2)

0.9950 - 1.0000

0.9880 - 0.9950

Sr. No.

1

2

Number ofpesticides

193

10

Sr. No.

1

2

3

4

LOQ (ppb)

1

5

10

25

Number ofpesticides

15

18

158

12

% RSD range(n=6)

6 – 15

3 – 15

0.95 – 15

1 – 10

S/N Ratiorange

16 – 181

19 – 502

10 – 14255

19 – 660

% Recoveryrange

70 – 130

Table 3. LOQ Summary

PO-CON1453E

Simultaneous quantitative analysis of20 amino acids in food samples withoutderivatization using LC-MS/MS

ASMS 2014 TP 510

Keiko Matsumoto1; Jun Watanabe1; Itaru Yazawa2

1 Shimadzu Corporation, Kyoto, Japan;

2 Imtakt Corporation, Kyoto, Japan

2

Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

IntroductionIn order to detect many kinds of amino acids with high selectivity in food samples, the LC/MS analysis have been used widely. Amino acids are high polar compound, so they are hard to be retained to reverse-phased column such as ODS (typical method in LC/MS analysis). It needs their derivartization or addition of ion pair reagent in mobile phase to retain them. For easier analysis of amino

acids, it is expected to develop the method without using reagents mentioned above.This time, we tried to develop a simultaneous high sensitive analysis method of 20 amino acids by LC/MS/MS with mix-mode column (ion exchange, normal-phase) and the typical volatile mobile phase suitable for LC/MS analysis.

Methods and MaterialsAmino acid standard regents and food samples were purchased from the market. Standards of 20 kinds of amino acids were optimized on each compound-dependent parameter and MRM transition. As an LC-MS/MS system, HPLC was coupled to triple

quadrupole mass spectrometer (Nexera with LCMS-8050, Shimadzu Corporation, Kyoto, Japan). Sample was eluted with a binary gradient system and LC-MS/MS with electrospray ionization was operated in multiple-reaction-monitoring (MRM) mode.

Result

First, MRM method of 20 amino acids was optimized. As a result, all compounds were able to be detected high sensitively and were detected in positive MRM transitions. As the setting temperature of ESI heating gas was found to affected on the sensitivity of amino acids, it was also

optimized. Even though amino acids were not derivartized and ion-pairing reagent wasn’t used, 20 amino acids were retained by using a mixed-mode stationary phase structure and separated excellently on the below-mentioned condition.

Method development


High Speed Mass Spectrometer

UF-MRM High-Speed MRM at 555ch/sec

UFswitching High-Speed Polarity Switching 5msec

3


Figure 2 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)

HPLC conditions (Nexera system)

Column : Intrada Amino Acid (3.0mmI.D. x 50mm, 3um, Imtakt Corporation, Kyoto, Japan)

Mobile phase

Case1

A : Acetonitrile / Formic acid = 100 / 0.1

B : 100mM Ammonium formate

Time program : B conc.14%(0-3 min) -100%(10min) - 14%(10.01-15min)

Case2 (High Resolution condition)

A : Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3

B : 100mM Ammonium formate / Acetonitrile = 80 / 20

Time program : B conc.0%(0-2 min) - 5%(3min) - 30%(6.5min) - 100%(12min)

- 0%(12.01-17min)


Injection volume : 2 uL


MS conditions (LCMS-8050)

Ionization : ESI, Positive MRM mode

MRM transition are shown in Table 1.

Case1

Asn

Phe

Trp

Ile

Met

Pro

Tyr

Val

Ara

Thr

Glu (Cys)2

ArgLys

His

Gln

GlyAspSer

Mobile PhaseA: Acetonitrile / Formic acid = 100 / 0.1

B: 100mM Ammonium formate

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min

Leu

4


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min

PheTrpLeu

Met

Pro

TyrVal

ThrGlu (Cys)2

LysHis

Gln

Arg

Asn

AraGly

Asp Ser

Thr

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min

Ile

In this study, two conditions of mobile phase were investigated. It was found that 20 amino acids were separated with higher resolution in case2. As the mobile phase condition of case1 is more simple and

the result of case1 was sufficiently well, case1 analytical condition was used for quantitative analysis. The dilution series of these compounds were analyzed. All amino acids were detected with good linearity and repeatability (Table1).

Figure 3 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)

Case2 (High Resolution condition)

Mobile PhaseA: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3

B: 100mM Ammonium formate / Acetonitrile = 80 / 20


5

Table1 Linearity and Repeatability of 20 amino acids

Trp

Phe

Tyr

Met

Lue, Lle

Val

Glu

Pro

Asp

Thr

Ala

Ser

Gln

Gly

Asn

(Cys)2

His

Lys

Arg

Range (nmol/mL)MRM Transition

205.10>188.10

166.10>120.10

182.10>136.00

150.10>56.10

132.10>86.15

118.10>72.05

148.10>84.10

116.10>70.10

134.20>74.10

120.10>74.00

90.10>44.10

106.10>60.20

147.10>84.10

76.20>29.90

133.10>74.05

241.00>151.95

156.10>110.10

147.10>84.10

175.10>70.10

Linearity

0.01-100

0.01-100

0.05-100

0.05-200

0.01-100

0.05-100

0.05-10

0.01-50

0.5-500

0.1-50

0.5-500

0.5-500

0.05-1

5-200

0.05-20

0.05-20

0.05-200

0.05-5

0.01-100

Coef�cient (r2)

0.9950

0.9971

0.9900

0.9963

0.9955

0.9991

0.9965

0.9933

0.9953

0.9923

0.9989

0.9988

0.9959

0.9974

0.9939

0.9909

0.9983

0.9908

0.9956

Repeatability*

%RSD

1.4

1.2

1.7

0.1

0.7

1.9

4.5

1.5

1.4

4.5

16.2

6.5

3.9

11.0

6.1

2.3

1.7

0.9

0.5

The analysis of the amino acids contained in sports beverage on the market was carried out. In the case of sports beverage, all amino acids written in the package were detected.

The analysis of 20amino acids in food samples

Figure 4 Mass Chromatograms of Sports Beverage (100 fold dilution with 0.1N HCl)

*@ 0.5nmol/mL : except for Gly, 5nmol/mL : for Gly

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Phe

TrpIle

Met

Pro

Tyr

Val

Ara

ThrGlu

Arg

Lys

His

Gly

AspSer

4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 min

Thr

Leu

Sports Beverage


6

Furthermore, Japanese Sake, Beer and sweet cooking rice wine (Mirin) were analyzed using this method. Japanese Sake and Beer were diluted with 0.1N HCl. Sweet cooking rice wine was diluted in the same way after a deproteinizing

preparation. These were filtered through a 0.2um filter and then analyzed. MRM chromatograms of each food samples are shown in Figure 5,6,7. Amino acids of each sample were detected with high sensitivity.

Figure 5 Mass Chromatograms of Japanese Sake (100 fold dilution with 0.1N HCl)

Figure 6 Mass Chromatograms of Beer (10 fold dilution with 0.1N HCl)

Figure7 Mass Chromatograms of Sweet Cooking Rice Wine (100 fold dilution with 0.1N HCl)

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2min

Phe

Trp IleMet

Pro

Tyr

Val

Thr

Glu(Cys)2

LysHis

Gln

Arg

Asn

Ala

Ala

Gly Ser

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

4.5 4.6 4.7 4.8 4.9 5.0 5.1 min

Phe

Trp

Ile

Met

Pro

Tyr

ValGlu

Gln

Ala

Thr

Gly

Ser

Asn

Asp

(Cys)2LysHis

Arg

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

4.5 4.6 4.7 4.8 4.9 5.0 5.1 minPhe

Trp

IleMet

Pro

Tyr

Val

AlaThr Gly

Asn

Ser

Glu Gln

Asp

(Cys)2Lys

HisArg

Leu

Leu

Leu

Sweet Cooking Rice Wine

Beer

Japanese Sake






Conclusions• 20 amino acids could be separated without derivatization using a typical volatile mobile phase suitable for LC/MS analysis

and detected with high sensitivity.• This methods was able to be applied to the analysis of amino acids in various food samples.

Environment

• Page 170

Rapid screening and confirmation

analysis of polycyclic aromatic

hydrocarbons (PAHs) with DART mass

spectrometry

• Page 176

Fast and highly sensitive analysis of

multiple drugs in ground-, surface- and

wastewater

• Page 182

Multi-residue analysis of pyrethroids in soil

and sediment using QuEChERS by LC/MS/MS

PO-CON1455E

Rapid Screening and con�rmationanalysis of polycyclic aromatichydrocarbons (PAHs) with DARTmass spectrometry

ASMS 2014 MP 551

Yu Takabayashi1, Jun Watanabe2, Motoshi Sakakura3,

Teruhisa Shiota3

1 SHIMADZU TECHNO-RESEARCH, INC., Tokyo, Japan;

2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan;

3 AMR Inc., Meguro-ku, Tokyo, Japan

2

Rapid Screening and con�rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

IntroductionRecently, the regulation of the content of the polycyclic aromatic hydrocarbons (PAHs) in goods which may put into a mouth or may contact is advanced and the technologies of measuring PAHs quickly are being developed. The ionizing principle of DART (Direct Analysis in Real Time) using the excitation helium gas is able to widely ionize the wide-range compounds and it may also be able to ionize

the compounds which are not ionized by ESI. Since PAHs is ionizable by DART, PAHs can be quickly screened by holding up a sample directly to DART. In this research, the technique detected by DART-MS was developed coupling with LC and DART analysis after carrying out LC separation was performed.

Methods and MaterialsCommercial PAHs were used for the sample. The samples were applied to DART MS with the solution formed in suitable concentration or the powder formed. Small amount of the samples were picked up and held in the DART ionization gas stream using glass capillaries. In LC-DART MS analysis, the mixed-solution of PAHs standard was prepared and applied to HPLC. After carrying out chromatogram separation using a reverse phased column,

LC-DART MS analysis was conducted by loading an eluate to a DART ionization area continuously. DART OS ion source and single/triple quadrupole type mass spectrometer were used for this experiment. PAHs measured in the detection mode which performs a full scan mode with positive and negative simultaneous ionization.

Figure 1 DART-OS ion source & LCMS-2020


Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec

MS condition (LCMS-2020; Shimadzu Corporation)

Ionization : DART (Direct Analysis in Real Time)

Heater Temperature (DART) : 300°C to 500°C

Measuring mode (MS) : Positive/Negative scanning simultaneously

3


ResultFirst, in order to verify whether PAHs ionizes in DART, PAH standard reagents were analyzed in DART-MS. Benzo[a]anthracene, acenaphthene, anthracene, etc. were used as typical PAHs. When benzo[a]anthracene was analyzed, in the positive spectrum, the signal at m/z 229 which is equivalent to [M+H]+ was detected. Moreover, in the negative spectrum, the signal at m/z 243 which is equivalent to [M+O-H]- was detected. Similarly, acenaphthene and anthracene could also be ionized by DART-MS and were able to be assigned as molecular related ion. Additionally pyrene and �uoranthene were also examined. As each of these is structural isomers mutually in structural-formula C16H10, in the negative spectrum, the signal of [M+O-H]- is detected by m/z 217 in each other, and either was not able to identify whether the detected signal is pyrene or �uoranthene in analysis by DART-MS without chromatogram separation.

Figure 2 DART mass chromatogram and mass spectrum of Benzo[a]anthraceneA: positive mass chromatogram, B: negative mass chromatogram (The area with the orange dashed line is the time when sample was held in DART.)

C: positive mass spectrum, D: negative mass spectrum

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0Inten. (x1,000,000)

229.3

245.2

261.3

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5Inten. (x100,000)

243.2

259.3 275.1 291.2277.1125.0 179.3 220.6

0

2500000

5000000

7500000

10000000

12500000 1:BPC(+)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 min0

250000

500000

7500002:BPC(-)

[M+H]+

M+

[M+O-H]-

Positive

Negative

A

B

C

D

Benzo[a]anthracene

C18H12Fw 228

Positive TIC m/z 100-300

Negative TIC m/z 100-300

4


Figure 3 DART mass spectra of acenaphthene (positive), anthracene (positive), pyrene (positive/negative) and �uoranthene (positive/negative)

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0Inten.(x100,000)

154.2

155.2

171.2

202.3220.2 253.3

102.3

130.2 187.3142.3 268.9

Positive

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

Inten.(x1,000,000)

179.2

195.2

211.2158.3 225.1

[M+H]+

M+

[M+H]+M+

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.00

0.25

0.50

0.75

1.00

Inten.(x10,000,000)

204.2

193.1 218.2

AcenaphtheneC12H10Fw 154

AnthraceneC14H10Fw 178

PyreneC16H10Fw 202

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Inten.(x100,000)

217.2

190.3

233.3179.2253.3 269.3226.3165.2

298.1

205.2

101.1

255.6 287.3115.5 137.1

[M+O-H]-

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

Inten.(x1,000,000)

208.3

194.2

122.3

220.3169.2

222.3183.2136.3108.2236.3

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Inten.(x100,000)

217.1

167.1233.3

208.3 247.0194.2 222.7181.1 256.2 270.9165.8 283.0

�uorantheneC16H10Fw 202

Positive

Positive

Negative

[M+O-H]-

Positive

Negative


5

Then, it examined the sample applied to DART separating with LC in order to perform chromatogram separation. As the suitable flow rate for DART ionization was thought to be approximately 10uL/min, the splitter

located between column and DART ionization stage. Furthermore, the closed interface was adopted for sensitivity improvement.

Figure 4 DART devices integrated with HPLC (AMR Inc.)

Analytical Condition

Column : Unison UK-C8 (2.0mmI.D. x 100mm, 3um, Imtakt Corporation, Kyoto, Japan)

Mobile phase : 1mM Ammonium formate / Acetonitrile=75/25

Flow rate : 0.2mL/min (to DART: 0.01mL/min)

DART heater temperature : 500°C

Ionization : Positive/Negative SIM mode

splitter

Pump

Injector

Column

Mobile phase






Figure 4 LC-DART mass chromatogram(a) Typical compound for DART; Quinine

(b) PAH mixture (4 compounds)

ConclusionsDART mass spectrometer coupled with HPLC was valuable for confirmation analysis of polycyclic aromatic hydrocarbons (PAHs)

As a result, by measurement of each PAHs standard reagent, each retention time was able to be confirmed and also each PAH was able to be detected in each retention time in the measurement using a PAH mixed

sample. The conclusion of this examination was understood that DART MS is effective in quick screening, and also LC-DART MS is effective in the confirmation analysis of detected PAHs in analysis of PAHs.

SIM 325(+) Quinine

SIM 202(+) pyrene

0

25000

50000

3:202.00(+)

4000

5000

6000

70004:217.00(-)

5000

7500

10000

12500 3:228.00(+)

3000

4000

5000 3:154.00(+)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

5000

10000

15000

20000

25000 3:178.00(+)

0.0 2.5 5.0 7.5 10.0 12.5 min0

10000

20000

30000

40000

50000

600002:325.00(+)

SIM 217(-) pyrene

SIM 228(+)benzo[a]anthracene

SIM 154(+) acenaphthene

SIM 178(+) anthracene

(a)

(b)

PO-CON1448E

Fast and highly sensitive analysisof multiple drugs in ground-, surface- and wastewater

ASMS 2014 TP 583

Klaus Bollig1; Sven Vedder2, Anja Grüning2

1 Shimadzu Deutschland GmbH, Duisburg, Germany; 2 Shimadzu Europe GmbH, Duisburg, Germany

2

Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

IntroductionMany pharmaceuticals from medical treatments are metabolized or partially degraded in the body. An even larger amount of these compounds is excreted intact and pollutes the aquatic environment. Relevant classes of drugs are human or veterinary antibiotics, antiepileptics, analgetics and lipid lowering drugs or radio-opaque substances. The extent of damage caused to the environment and the resulting health risk for humans or animals should not be underestimated, even though it is

not understood in detail so far. The requirement for universal, reliable and fast methods for drug determination in water is steadily increasing. Highly sensitive triple-quad-MS systems are suitable tools for the analysis of residues in ground-, surface- and wastewater, but development of a simple, rapid and robust method for simultaneous measurement of trace levels of various different classes of analytes in complex matrices is a challenge.

MethodThis study describes a fast LC-MS/MS method for the determination of trace levels of different classes of drugs in water. With online SPE no further sample pretreatment is necessary. The quaternary system with low pressure gradient eluent (LPGE) and solvent blending functionality renders addition of a third LC-Pump unnecessary. The

solvent blending function was further used for method development. High speed values for MRM recording and the fastest polarity switching time of 5 ms are essential physical parameters for a maximum of data points on various classes of analytes in different polarities during one single analysis.

One of the first steps during this automated process is the precursor ion selection, followed by m/z adjustment of the precursor. The collision energy is optimized for the most abundant fragments and finally the fragment m/z is

adjusted. Six optimization steps were performed via flow injection analysis, each taking 30 seconds (Figure 2). The result of these automated steps was the automatic generation of a final MRM method (Table 1).

LC-MS/MS Method Optimization

Figure 1. LCMS-8050 triple quadrupole mass spectrometer

3


Figure 2. Automated MRM Optimization of the drug Sulfamethazin

1st Step: m/z Precursor adjustment

5th Step: Setting Q3 Prerod Bias

2nd Step: Setting Q1 Prerod Bias

4th Step: m/z Product Ion adjustment

3rd Step: Product Ion / CE selection

6th Step: CE �ne tuning

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 min

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25

4.50

4.75

5.00

5.25

5.50

5.75

6.00

6.25(x1,000,000)

1:Sulfamethazin 279.70(+)1:Sulfamethazin 279.60(+)1:Sulfamethazin 279.50(+)1:Sulfamethazin 279.40(+)1:Sulfamethazin 279.30(+)1:Sulfamethazin 279.20(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.00(+)1:Sulfamethazin 278.90(+)1:Sulfamethazin 278.80(+)1:Sulfamethazin 278.70(+)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 min

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25

4.50

4.75

5.00

5.25

5.50

5.75

6.00

6.25

6.50

6.75

7.00

7.25(x1,000,000)

1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 min

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

1.40

1.45

(x1,000,000)

3:Sulfamethazin 279.10>65.50(+) CE: -50.03:Sulfamethazin 279.10>65.40(+) CE: -50.03:Sulfamethazin 279.10>65.30(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.10(+) CE: -50.03:Sulfamethazin 279.10>65.00(+) CE: -50.03:Sulfamethazin 279.10>64.90(+) CE: -50.03:Sulfamethazin 279.10>64.80(+) CE: -50.03:Sulfamethazin 279.10>64.70(+) CE: -50.03:Sulfamethazin 279.10>64.60(+) CE: -50.03:Sulfamethazin 279.10>64.50(+) CE: -50.02:Sulfamethazin 279.10>186.50(+) CE: -18.02:Sulfamethazin 279.10>186.40(+) CE: -18.02:Sulfamethazin 279.10>186.30(+) CE: -18.02:Sulfamethazin 279.10>186.20(+) CE: -18.02:Sulfamethazin 279.10>186.10(+) CE: -18.02:Sulfamethazin 279.10>186.00(+) CE: -18.02:Sulfamethazin 279.10>185.90(+) CE: -18.02:Sulfamethazin 279.10>185.80(+) CE: -18.02:Sulfamethazin 279.10>185.70(+) CE: -18.02:Sulfamethazin 279.10>185.60(+) CE: -18.02:Sulfamethazin 279.10>185.50(+) CE: -18.01:Sulfamethazin 279.10>92.50(+) CE: -35.01:Sulfamethazin 279.10>92.40(+) CE: -35.01:Sulfamethazin 279.10>92.30(+) CE: -35.01:Sulfamethazin 279.10>92.20(+) CE: -35.01:Sulfamethazin 279.10>92.10(+) CE: -35.01:Sulfamethazin 279.10>92.00(+) CE: -35.01:Sulfamethazin 279.10>91.90(+) CE: -35.01:Sulfamethazin 279.10>91.80(+) CE: -35.01:Sulfamethazin 279.10>91.70(+) CE: -35.01:Sulfamethazin 279.10>91.60(+) CE: -35.01:Sulfamethazin 279.10>91.50(+) CE: -35.0

0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

(x1,000,000)

3:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.0

0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7(x1,000,000)

3:Sulfamethazin 279.10>65.20(+) CE: -45.03:Sulfamethazin 279.10>65.20(+) CE: -46.03:Sulfamethazin 279.10>65.20(+) CE: -47.03:Sulfamethazin 279.10>65.20(+) CE: -48.03:Sulfamethazin 279.10>65.20(+) CE: -49.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -51.03:Sulfamethazin 279.10>65.20(+) CE: -52.03:Sulfamethazin 279.10>65.20(+) CE: -53.03:Sulfamethazin 279.10>65.20(+) CE: -54.03:Sulfamethazin 279.10>65.20(+) CE: -55.02:Sulfamethazin 279.10>186.10(+) CE: -10.02:Sulfamethazin 279.10>186.10(+) CE: -11.02:Sulfamethazin 279.10>186.10(+) CE: -12.02:Sulfamethazin 279.10>186.10(+) CE: -13.02:Sulfamethazin 279.10>186.10(+) CE: -14.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -16.02:Sulfamethazin 279.10>186.10(+) CE: -17.02:Sulfamethazin 279.10>186.10(+) CE: -18.02:Sulfamethazin 279.10>186.10(+) CE: -19.02:Sulfamethazin 279.10>186.10(+) CE: -20.01:Sulfamethazin 279.10>92.20(+) CE: -25.01:Sulfamethazin 279.10>92.20(+) CE: -26.01:Sulfamethazin 279.10>92.20(+) CE: -27.01:Sulfamethazin 279.10>92.20(+) CE: -28.01:Sulfamethazin 279.10>92.20(+) CE: -29.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -31.01:Sulfamethazin 279.10>92.20(+) CE: -32.01:Sulfamethazin 279.10>92.20(+) CE: -33.01:Sulfamethazin 279.10>92.20(+) CE: -34.01:Sulfamethazin 279.10>92.20(+) CE: -35.0

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Inten. (x100,000)

186.2

148.9

205.5124.292.3

186.2

156.1

124.2

108.2

186.1

124.2

156.1

108.2

92.2

213.2

124.2

92.2

108.2

186.1

156.1

213.3

204.1

124.2

92.2

108.2

186.1

65.2 213.2156.0

92.2

124.2108.2

65.2

149.480.0 201.2

92.2

124.265.1 108.2

80.1

92.265.2

124.3107.280.2

190.853.2 168.2

65.2

92.380.1 108.1124.253.2 197.4143.3

4


Table 1. Optimized MRM transitions of 9 drugs

Compound Mode MRM transitions Collision energy (kV)

Sulfamethazin

Sulfamethoxazol

Beza�brat

Carbamazepine

Diclofenac

Clo�bric acid

Ibuprofen

Iopamidol

Iopromid

ESI positive

ESI positive

ESI positive

ESI positive

ESI positive

ESI negative

ESI negative

ESI negative

ESI negative

279,10>186,10 / 279,10>92,20

253,90>92,20 / 253,90>156,15

362,10>139,15 / 362,10>316,25

237,10>194,20 / 237,10>179,20

296,00>214,15 / 296,00>215,15

213,00>127,00 / 213,00>85,00

205,10>161,30

775,80>126,95

790,00>127,00

-17 / -31

-26 / -15

-25 / -15

-19 / -34

-34 / -19

15 / 15

7

22

26

The solvent blending functionality entails automated mobile phase preparation on a LPGE (low pressure gradient) unit which is integrated in the binary LC pumps. The blending function eliminates the need of mobile phase pre-mixing, as necessary with ordinary binary pumps.Mobile phase composition can simply be changed in the

method without physically changing the solvents. Therefore solvent blending is a powerful tool for easy and efficient elucidation of the SPE, the gradient and the starting conditions. During this study the solvent blending function was used for optimization of the SPE conditions. A second LPGE unit was used for the analytical gradient.

Solvent Blending

Figure 3. Solvent blending functionality

1: prepare 5 mmol/L Ammonium formate (pH 8.5)

2: prepare H2O

5: prepare mobile phase A (SPE loading condition); different conditions tested !

200 mL 800 mL

6: prepare mobile phase B1 and B2 (analytical condition and gradient)

200 mL 800 mL

Traditional methodStep 1 Step 2 Step 3

Set these to system

Mobile phase blending function

Only step 1!

Set these to system

3: prepare MeOH

4: prepare 0,0025%NH4OH

LPGE Unit:Mobile phase composition for SPE loading, solvent blending allows to change conditions automatically

2nd LPGE Unit:Gradient for SPE release and separation

1: prepare 5 mmol/L Ammonium formate (pH 8.5)

2: prepare H2O

3: prepare MeOH

4: prepare 0,0025%NH4OH

5


Final methodSPE Conditions

Injection volume : 250 µl

SPE-column : Strata-X , 25 µm , 20*2 mm

SPE-�ow rate : 1 ml/min

SPE-loading buffer : 1 mmol/L ammonium formate (LPGE Pump B)

Analytical Conditions (LPGE Pump A)

Column : Kinetex C8, 2.6 µm, 100*2.1 mm

Flow rate : 0.5 ml/min

Solvent A : 0.0025% NH4OH

Solvent B : MeOH 1 min – 2.5 min analytical separation

Gradient : 0 min : 30% B

: 1 min : 30% B

: 1.5 min : 95% B

: 4.5 min : 95% B

: 4.51 min : 30% B

: 6 min : 30% B (Stop)

LCMS Conditions

Interface : ESI


Heating Gas Flow : 12 L/min


Desolvation Line Temperature : 150 ºC

Heat Block Temperature : 400 ºC

Drying Gas Flow : 6 L/min

Polaritiy Switching Time : 5 ms

Figure 4. Scheme of online-SPE extraction (A) and analytical separation (B)

HPLC/MS Work�ow

Pump 1

Autosampler

Waste

SPE-Column

Analytical-Column+ LCMS 8050

Pump 2

A Pump 1

Autosampler

Waste

SPE-Column

Analytical-Column+ LCMS 8050

Pump 2

B


ResultsIn this study we developed a fast method for direct online SPE LC-MS/MS analysis of 9 different drugs in water with a minimal LC con�guration of two binary pumps equipped with LPGE units. The solvent blending function was used for method development of the SPE extraction. The second LPGE unit was used for SPE release and analytical gradient

separation. Each compound was quanti�ed in a concentration range from 0.05 ng/ml up to 2 ng/ml. Measurements were performed on Shimadzu’s LCMS-8050 Triple Quad MS System. The calibration curves and lowest calibration point is shown in �gure 5.





Figure 5. Calibration curve and lowest calibration point at 0.05 ng/ml of each compound

PO-CON1444E

Multi-residue analysis of pyrethroidsin soil and sediment using QuEChERSby LC/MS/MS

ASMS 2014 TP 560

Yuka Fujito1, Kiyomi Arakawa1, Yoshihiro Hayakawa1

1 Shimadzu Corporation. 1, Nishinokyo-Kuwabaracho

Nakagyo-ku, Kyoto 604–8511, Japan

2

Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

IntroductionPytrethroids are one of the most widely used commercial household insecticides in agricultural or non-agricultural application sites. Synthetic pyrethroids are poorly water-soluble, but are strongly adsorbed to soil, therefore these compounds are increasingly being found in soil or sediments. Recently, soil and sediment contamination by pyrethroids has been detected in both urban and agricultural area, and it’s becoming a global concern due

to the in�uence on the insects and aquatic invertebrates. Therefore, quick, high-sensitive and universal analysis methods are required. The analysis of pyrethroids is typically performed by GC or GC/MS because of their hydrophobicity. In this study, we report the development of a simultaneous analysis technique for trace amounts of pyrethroids by LC/MS/MS.

Sample preparationSample preparation was carried out by the use of the QuEChERS method. In case of the soil samples, hydration of sample with water before acetonitrile extraction is required to improve the recovery and operability. Result of several different extraction methods that changed the

amount of the soil and water added, we �nally adopted a combination of 5 g soil (or 10 g sediment) and 5 mL water, and the following procedures were based on the original QuEChERS method.

Figure 1 Chemical structure of pyrethroids

Materials and MethodsMaterials

Sampling point

Residential garden (Kyoto, Japan)

Lake Biwa (Shiga, Japan)

Sample

Soil

Sediment

Permethrin

Pyrethrin

I : R=CH3

II : R=CO2CH3

Cyhalothrin

Te�uthrin

Esfenvalerate

3


LC/MS/MS analsisHPLC conditions (Nexera UHPLC system, Shimadzu)

Column : Phenomenex Kinetex 2.6 µm PFP 100Å (100 mm x 2.1 mm I.D.)

Mobile phase : A 5mM ammonium acetate - water

: B Methanol

Gradient program : 40 % B (0 min.) → 100 % B (10 -12 min.) → 40 % B (12.01-15 min.)

Flow rate : 0.2 mL / min.

Column temperature : 40 ºC

Injection volume : 1 μL

MS conditions (LCMS-8050, Shimadzu)

Ionization : ESI (positive / negative)

Interface temperature : 100 ºC


Heat block temperature : 400 ºC

Nebulizing gas : 3.0 L / min.

Drying gas : 15.0 L / min.

Heating gas : 3.0 L / min.

Weigh 5 g soil / 10 g sediment

(Add STDs solution)

Add 5mL water

Add 10mL acetonitrile

Add salt mixture*1 • 4g MgSO4

• 1g NaCl• 1g Trisodium citrate dehydrate• 0.5g Disodium hydrogencitrate sesquihydrate

Shake vigorously by hand 1min.

Centrifuge for 10min. (Extract 1)

Step 1 : Acetonitrile extraction

*1 : Q-sep QuEChERS Extraction Salts (RESTEK) *2 : Q-sep QuEChERS dSPE Tubes (RESTEK)

Step 2 : Clean-up

Transfer 6mL Extract 1 into dSPE tube*2

• 900 mg MgSO4

• 150 mg PSA• 45 mg GCB

Shake vigorously by hand 1min.

Centrifuge for 5min.

Transfer the supernatant into a vial

Filtration using disposable �lter

LC/MS/MS analysis

4



Table 1 MRM transitions of pyrethroids

Compounds Polarity Quantitative ion (m/z) Con�rmation ion (m/z)

pyrethrin-I

pyrethrin-II

fenpropathrin

cycloprothrin

deltamethrin

esfenvalrate

cypermethrin

cy�uthrin

ethofenprox

permethrin

cyhalothrin

bifenthrin

acrinathrin

acrinathrin

sila�uofen

+

+

+

+

+

+

+

+

+

+

+

+

+

-

+

329.20>161.20

373.20>161.20

367.20>125.20

498.90>181.10

522.80>280.90

437.10>167.30

433.10>191.10

450.90>191.00

394.20>177.30

408.10>183.30

467.10>225.10

440.00>181.20

559.00>208.20

540.10>372.20

426.20>287.10

329.20>105.20

373.20>105.20

367.20>181.20

498.90>229.20

522.80>181.10

437.10>125.30

433.10>181.20

450.90>206.10

394.20>107.20

408.10>355.20

467.10>141.10

440.00>166.10

559.00>181.10

540.10>345.30

426.20>168.20


Ultra Fast Scanning - 30,000 u / sec. Ultra Fast Polarity Switching - 5 msec. Ultra Fast MRM - Max. 555 transitions / sec

Result

In this study, we selected and evaluated 15 pyrethroids (pyrethrin, fenpropathrin, cycloprothrin, deltamethrin, esfenvarelate, cypermethrin, cyfluthrin, ethofenprox, permethrin, cyhalothrin, bifenthrin, acrinathrin, tefluthrin, silafruofen) which are the most widely used for household or

agrocultural insecticides worldwide. Except for tefluthrin, which was not ionized by LC/MS under conditions tested, all other 14 compounds were successfully detected in ESI positive mode or in both positive and negative mode.

MRM of pyrethroid standards

5


Figure 3 MRM chromatograms

Figure 4 MRM chromatograms of the LOQs of typical pyrethroids

Table 2 Calibration curves

compounds min. conc. max. conc. r2

pyrethrin I

pyrethrin II

fenpropathrin

cycloprothrin

deltamethrin

esfenvalerate

cypermethrin

cy�uthrin

ethofenprox

trans-permethrin

cis-permethrin

cyhalothrin

bifenthrin

acrinathrin (+)

acrinathrin (-)

sila�uofen

0.5

0.5

0.02

0.5

0.05

0.5

0.05

0.5

0.01

0.02

0.02

0.1

0.02

0.1

0.5

0.01

500

500

100

100

100

100

100

100

100

100

100

100

100

100

500

100

0.9996

0.9997

0.9993

0.9991

0.9992

0.9990

0.9986

0.9976

0.9993

0.9996

0.9994

0.9993

0.9995

0.9987

0.9993

0.9999

(ppb)7.0 8.0 9.0 10.0 min

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

pyrethrin-II

pyrethrin-I

fentropathrin

cycloprothrindeltamethrinesfenvalrate

cypermethrin

ethofenprox

permethrin

cyhalothrin

bifenthrin

acrinathrinsila�uofen

cy�uthrin

permethrin0.02 ppb

trans-

cis-

9.5 10.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

(x1,000)

fenpropathrin0.02 ppb

9.0 9.5

0.00

0.25

0.50

0.75

1.00

1.25(x1,000)

bifenthrin0.02 ppb

9.5 10.0 10.5

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000)

10.0 10.5 11.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

(x1,000)

sila�uofen0.01 ppb


6

Figure 5 Recovery of 14 pyrethroids from soil and sediment matrices (10 ppb spiked)

soil (residential garden)

Reco

very

(%)

ethofenprox

Figure 6 Chromatograms of prethroids in the soil

permethrin

sediment (lake)

Table 3 Result of quantitative analysis in the soil and sediment

pyrethrin-I

pyrethrin-II

fenpropathrin

cycloprothrin

deltamethrin

esfenvalrate

cypermethrin

cy�uthrin

ethofenprox

permethrin

cyhalothrin

bifenthrin

acrinathrin

sila�uofen

soil

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.01 ppb*

0.03 ppb

n.d.

n.d.

n.d.

n.d.

sediment

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

All target compounds showed good recoveries from soil and sediment matrices in the range 70-120% by the QuEChERS

method. Neither matrix effect (Ion suppression or enhancement) nor sample preparation losses were observed.

Recovery from soil and sediment matrices

The quantitative analysis of the soil and sediment sample was performed. Ethofenprox and permethrin was detected

from the soil sample at approximately 0.02 and 0.06 μg / kg, respectively.

Quantitative analysis of soil and sediment

n.d. : not detected* : <LOQ

0

20

40

60

80

100

120

140

Pyre

thrin

-2

Pyre

thrin

-1

Fenpro

pathrin

Cyclo

proth

rin

Deltam

ethrin

Esfe

nvalra

te

Cyper

met

hrin

Cy�uth

rin

Ethofe

nprox

trans-P

erm

ethrin

cis-P

erm

ethrin

Cyhalo

thrin

Bifenth

rin

Acrinat

hrin

Sila�

uofen

0

20

40

60

80

100

120

140

Pyre

thrin

-2

Pyre

thrin

-1

Fenpro

pathrin

Cyclo

proth

rin

Deltam

ethrin

Esfe

nvalra

te

Cyper

met

hrin

Cy�uth

rin

Ethofe

nprox

trans-P

erm

ethrin

cis-P

erm

ethrin

Cyhalo

thrin

Bifenth

rin

Acrinat

hrin

Sila�

uofen

STDs spiked after prep STDs spiked before prep

soil blank

solvent blank






Conclusions• A method for quanti�cation of 14 pyrethroids in soil and sediment at ppt-level concentrations was developed by

LC/MS/MS.• In this study, neither matrix effect nor sample preparation losses were observed in the recovery test, demonstrating the

applicability of QuEChERS method to sample preparation of soil and sediment.

Metabolism

• Page 197High sensitivity analysis of metabolites in serum using simultaneous SIM and MRM modes in a triple quadrupole GC/MS/MS

• Page 202Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

• Page 208Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-flight mass spectrometry

• Page 213Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta- fluorophenylpropyl column

PO-CON1443E

High Sensitivity Analysis ofMetabolites in Serum UsingSimultaneous SIM and MRM Modesin a Triple Quadrupole GC/MS/MS

ASMS 2014 ThP 641

Shuichi Kawana1, Yukihiko Kudo2, Kenichi Obayashi2,

Laura Chambers3, Haruhiko Miyagawa2

1 Shimadzu, Osaka, Japan, 2 Shimadzu, Kyoto, Japan,

3 Shimadzu Scienti�c Instruments, Columbia, MD

2

High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

IntroductionGas chromatography / mass spectrometry (GC–MS) and a gas chromatography-tandem mass spectrometry (GC-MS/MS) are highly suitable techniques for metabolomics because of the chromatographic separation, reproducible retention times and sensitive mass detection.

Sample• Human serum

MRM measurement modeSome compounds with low CID ef�ciency produce insuf�cient product ions for MRM transitions, and the MRM mode is consequently less sensitive than SIM for these compounds.

Our suggestionSIM, MRM, and simultaneous SIM/MRM modes are evaluated for analysis of metabolites in human serum.

Materials and MethodSample and Sample preparation

Sample Preparation1)

Instrumentation

Freeze-dry

Residue

Sample

Add 40 µL methoxyamine solution (20 mg/mL, pyridine)

Heat at 30 ºC for 90 min

Add 20 µL MSTFA

Heat at 37 ºC for 30 min

1) Nishiumi S et. al. Metabolomics. 2010 Nov;6(4):518-528

Supernatant 250 µL

Add 250 µL water / methanol / chloroform (1 / 2.5 / 1)

Add internal standard (2-Isopropylmalic acid)

Stir, then centrifuge

Extraction solution 225 µL

Add 200 µL Milli-Q water

Stir, then centrifuge

50uL serum

GC-MS : GCMS-TQ8040 (SHIMADZU)

Data analysis : GCMSsolution Ver.4.2

Database : GC/MS Metabolite Database Ver.2 (SHIMADZU)

Column : 30m x 0.25mm I.D., df=1.00µm (5%-Phenyl)-methylpolysiloxane

3


Simultaneous SIM and MRM modes in GC/MS/MSFigure 1 shows the theory of Simultaneous SIM and MRM modes. This analysis mode can measure SIM and MRM data in a single analysis.

Method Creation using Database and SmartMRMFigure 3 shows the GC/MS Metabolites Database Ver.2. This database involves conditions of SIM and MRM in 186 metabolites and a method creation function we call SmartMRM. SmartMRM creates MRM, SIM, SIM/MRM methods from Database automatically.

• Select the MRM, SIM and SIM/MRM conditions of 186 TMS derivatization metabolites from GC/MS Metabolites Database Ver.2.

• Select the two transitions (or ions) each metabolite.

Poor sensitivity of MRM in some compounds because of low CID ef�ciency

Figure 1 The concept of simultaneous SIM and MRM analysis mode.

Figure 3 GC/MS Metabolites Database Ver.2

Figure 2 Mass Spectrum of Precursor (or SIM) and Product ion

SIM

MRMSIM

MRM

Q1 Q3Collision Cell

SIMSIM CID

100 200 300 400 0

25

50

75

100 %

361

73

217 147 437 103 271

243 319 191

100 200 300 0

25

50

75

100

%

169

103 73

243 361

Precursor ion (or SIM) Product ion

CID

4


A number of Identi�cation metabolites in serum Table 1 shows the identi�cation results of metabolites in human serum using SIM, MRM and simultaneous SIM/MRM analysis modes in GC/MS/MS. In SIM/MRM, the metabolites, which were insuf�cient sensitivity in MRM, were measured by SIM and the other metabolites were measured by MRM.

ResultsComparison of the chromatogram between SIM and MRM in human serum

Detected the peak in MRM because of high selectivity

Peak was not detected in MRM because of low CID ef�ciency.

SIM MRM

SIM MRM

0.5

1.0

1.5

2.0

2.5

3.0

3.5

2.5

5.0

7.5

0.25

0.50

0.75

1.00

1.25

1.50

1.75

(x100,000)333.10160.10

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000)238.10218.10

(x10,000)218.10>73.00

(x100)238.10>91.00

238.10>91.00

(x10,000)333.10>143.10333.10>171.10

21.00 21.25 21.00 21.25

21.25 21.50 21.2521.00 21.50

21.2521.00 21.500.250.500.751.001.251.501.75

a) Glucuronic acid-meto-5TMS(2)

b) S-Benzyl-Cysteine-4TMS

Table 1 The number of identi�ed metabolites each analysis mode

note) A:Target and Con�rmation ions were detected.; B: Either Target or Con�rmation ion was detected. Another one was overlapped by contaminants.; C: Either Target or Con�rmation ion was detected.

Modes

SIM

MRM

SIM/MRM

A

57

131

133

B

51

14

22

C

8

1

1

Total

116

146

156






Fig.4 shows a number of metabolites in each mode can be measured. In metabolites with low CID ef�ciency, SIM are superior to MRM if there are no interfering substances to the target metabolites.

Figure 4 Detected metabolites in human serum each analysis mode.

Conclusions• Analytical results from the SIM and MRM modes identi�ed 116 and 146 metabolites, respectively.• In metabolites with poor CID ef�ciency, the sensitivity of SIM is more than 10 times higher than MRM.• Simultaneous SIM and MRM modes in a single analysis (SIM/MRM) improves the sensitivity and reproducibility for

analysis of metabolites in human serum compared to MRM alone. • A novel SIM/MRM expands the utility of a triple quadrupole GC/MS/MS

The reproducibility(n=6) in MRM and SIM/MRMTable 2 Comparison of the reproducibility results from MRM and SIM/MRM analysis. A number of detected metabolites involves A, B and C in Table 1.

%RSD

- 4.99%

5 - 9.99%

10 - 14.99%

15 - 19.99%

> 20%

MRM

73

26

8

9

30

146

SIM/MRM

76

30

10

10

30

156

Improvement

+3

+4

+2

+1

0

+10

SIM MRM

10 40 106

Metabolites with low CIDef�ciency in MRM

Metabolites withinterference in SIM

PO-CON1451E

Analysis of D- and L-amino acids usingautomated pre-column derivatizationand liquid chromatography-electrosprayionization mass spectrometry

ASMS 2014 MP739

Kenichiro Tanaka1; Hidetoshi Terada2; Yoshiko Hirao2;

Kiyomi Arakawa2; Yoshihiro Hayakawa2

1. Shimadzu Scienti�c Instruments, Inc., Columbia, MD;

2. Shimadzu Corporation, Kyoto, Japan

2

Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

IntroductionRecently, several species of D- amino acids have been found in mammals including humans and their physiological functions have been elucidated. Quantitating each enantiomer of amino acids is indispensable for such studies. In order to diagnose diseases, it is desirable that D- and L-amino acid can be separately quantitated and applied to metabolic analysis. Pre-column derivatization with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine(NAC) is widely utilized for the analysis of D- and L- amino acids since the method can be performed with a rapid reversed phase separation on a relatively simple hardware (U)HPLC con�guration with

good reliability. One of the drawbacks of pre-column derivatization is less reproducibility due to the tedious manual procedure and human errors. We have launched an autosampler for a UHPLC system equipped with an automated pretreatment function that allows overlapping injections in which the next derivatization proceeds during the current analysis for saving total analytical time. We have applied this autosampler and its function to fully automate pre-column derivatization for the determination of amino acids. In this study, we developed a methodology which enabled the automated procedure of pre-column chiral derivatization of D- and L- amino acids.

Experimental

The system used was a SHIMADZU UHPLC Nexera pre-column Amino Acids (AAs) system consisting of LC-30AD solvent delivery pump, DGU-20A5R degassing unit, SIL-30AC autosampler, CTO-30A column oven, and SHIMADZU triple quadrupole mass spectrometer LCMS-8040. The software is integrated in the LC/MS/MS

workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions can be seamlessly translated into method �les and registered to a batch queue, ready for instant analysis. A 1.9um YMC-Triart C8 column (2.0 mm x 150 mm L.) was used for the analysis.

Instruments

Derivatizing solutions: 0.1 mol/L boric acid buffer was prepared by dissolving 6.18 g of boric acid and 2.00 g of sodium hydroxide in 1 L of water. 10 mmol/L NAC solution was prepared by dissolving 16.3 mg of N-acetyl-L-cysteine in 10 mL of the 0.1 mol/L boric acid buffer. 10 mmol/L OPA solution was prepared by dissolving 6.7

mg of o-phthalaldehyde in 0.3 mL of ethanol, adding 0.7 mL of the 0.1 mol/L boric acid buffer and 4 mL of water.Fig.1 shows the schematic procedure for amino acids derivatization with the SIL-30AC.Samples, including the derivatized amino acids, were injected onto the UHPLC and separated under the conditions shown in Table 1.

Derivatization Method

3


Fig.1 Schematic procedure of automated pre-column derivatization

Table 1 UHPLC and MS analytical conditions

Mobile Phase : A : 10 mmol/L Ammonium Bicarbonate solution

B : Acetonitrile/Methanol = 1/1(v/v)

Initial B Conc. : 0%



Injection Volume : 1 μL

LC Time Program : 0 -> 5%(0.01min), 5%(0.01-1.00min), 5 ->20%(1.00 - 15.00min),

20 - 25%(15.00 - 24.00min), 25 – 90%(24.00 - 24.50min),

90%(24.50 - 27.50min), 90 - 0% (27.50 – 28.50min)

Ionization Mode : ESI

Nebulizing Gas Flow Rate : 3 L/min

Drying Gas Flow Rate : 15 L/min


Heating Block Temperature : 450 ºC

Result

A standard solution containing 27 amino acids was prepared at 1 mmol/L concentration each in 0.1 mol/L HCl solution. The MS conditions such as ESI positive and negative ionization modes were optimized in parallel with the column separation, and compound dependent parameters such as CID and pre-bias voltage were adjusted

using the function for automatic MRM optimization. The transition that provided the highest intensity was used for quanti�cation. Table 2 shows the MRM transition of each derivatized amino acid. The MRM chromatogram is illustrated in Fig.2.

Analysis of Standard Solution

(1)

Take 20 μL of 10 mmol/L NAC solution

Supply 1 μL ofsample solution to the vial for mixing

(3)

Take 20μL of 10 mmol/L OPA solution

Mix the sample solutionand derivatizing solutions

Inject 1μL of the mixed solution to the column

Supply 20 μL of NAC solution to thevial for mixing

Supply 20 μL of10 mmol/L OPA solution to the vial for mixing

(5)

Take 1 μL of sample solution

Wait for 3min untilthe derivatization ends

Take 1μL of the mixedsolution

(2) (4)

(6) (7) (8) (10)(9)

4


Fig. 2 Chromatogram of a 27 amino acid standard solution

Compound

Aspartic acid

Glutamic acid

Serine

Glutamine

Glycine

Histidine

Threonine

Arginine

Tyrosine

Valine

Tryptophan

Isoleucine

Phenylalanine

Polarity

+

+

+

+

+

+

+

+

+

+

+

+

+

Precursor m/z

395.00

409.10

367.00

408.20

337.00

417.10

381.20

436.10

443.00

379.10

466.20

393.00

427.20

Product m/z

130.00

130.05

130.00

130.05

130.00

244.05

130.05

263.10

130.05

250.05

337.10

264.05

298.05

Table 2 Compounds, Ionization polarity and MRM transition

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

12

3 45

6

78

9

10

11

12 13

14

1516

17

18 1921

22

20

23

24

2526

27

■Peaks

1. D-Aspartic acid, 2. L-Aspartic acid, 3. L-Glutamic acid, 4. D-Glutamic acid, 5. D-Serine, 6. L-Serine, 7. L-Glutamine8. D-Glutamine, 9. Glycine, 10. L-Histidine, 11. D-Histidine ,12. D-Threonine, 13. L-Threonine, 14. L-Arginine15. D-Arginine, 16. D-Alanine, 17. L-Alanine, 18. D-tyrosine, 19. L-Tyrosine, 20. L-Valine, 21. D-Valine22. L-Tryptophan, 23. D-Tryptophan, 24. L-Isoleucine, 25. D-Phenylalanine, 26. L-Phenylalanine, 27.D-Isoleucine

5


CompoundRepeatability (%RSD)

D-Aspartic acid

D-Glutamic acid

D-Serine

D-Glutamine

D-Histidine

D-Threonine

D-Arginine

D-Alanine

D-Tyrosine

D-Valine

D-Tryptophan

D-Isoleucine

D-Phenylalanine

5 μmol/L

3.5

3.7

4.8

4.1

4.3

3.8

3.4

4.0

3.2

3.3

3.9

3.1

3.5

25 μmol/L

2.5

3.1

3.0

3.4

1.8

2.6

1.7

2.3

2.9

2.2

3.2

2.9

1.8

Table 3 Reproducibility

Compound

D-Asparic acid

D-Glutamic acid

D-Serine

D-Glutamine

D-Histidine

D-Threonine

D-Arginine

D-Alanine

D-Tyrosine

D-Valine

D-Tryptophan

D-Isoleucine

D-Phenylalanine

Cali.F

Y = (44661.8)X + (1829.61)

Y = (12191.8)X + (10390.7)

Y = (22319.5)X + (-2869.30)

Y = (3458.60)X + (1521.83)

Y = (5778.33)X + (-341.182)

Y = (10800.6)X + (-1874.07)

Y = (10535.7)X + (-1298.12)

Y = (15349.1)X + (-4719.98)

Y = (17098.7)X + (-1812.69)

Y = (23707.7)X + (772.548)

Y = (18089.1)X + (-3620.41)

Y = (44017.1)X + (67903.1)

Y = (22426.0)X + (-736.090)

r2

0.998

0.999

0.999

0.999

0.998

0.999

0.998

0.999

0.999

0.999

0.998

0.999

0.999

Table 4 Linearity

Reproducibility and linearity in this analysis were evaluated with a plasma spiked standard solution. As a result, less than 5% relative standard deviation of peak areas were obtained. Table 3 shows the reproducibility of repeated analysis of spiked sample (n=6). Five different levels of

spiked sample concentration from 1 to 100 μmol/L standard solution were used for the linearity evaluation. The coef�cients of determination (r2) were approximately 0.999. Table 4 shows the summary for the linearity results.

Method Validation






Considering the frequency of amino acids analysis in physiological samples, the recovery of spiked samples were con�rmed. In addition, the results indicated that the recovery ratio of most amino acids are around 100%.Table 5 shows the summarized results for the recovery of each amino acid.

Conclusions• The combination of Shimadzu triple quadrupole mass spectrometer and Nexera UHPLC provides reliable pre-column

derivatized AAs analysis with enhanced productivity.• An established method was successfully applied to the separation of D- and L- amino acids with excellent reliability.

CompoundRecovery (100%)

D-Asparic acid

D-Glutamic acid

D-Serine

D-Glutamine

D-Histidine

D-Threonine

D-Arginine

D-Alanine

D-Tyrosine

D-Valine

D-Tryptophan

D-Isoleucine

D-Phenylalanine

5 μmol/L

100.3

92.8

97.9

103.2

104.8

101.1

102.4

93.5

98.1

101.0

97.8

98.8

104.5

25 μmol/L

107.1

97.8

100.6

104.3

100.4

98.8

99.6

99.5

101.0

99.2

100.4

102.4

100.9

Table 5 Recovery

PO-CON1476E

Characterization of metabolites in microsomal metabolism of aconitineby high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry

ASMS 2014 WP 739

Cuiping Yang1, Changkun Li2, Tianhong Zhang1,

Qian Sun2, Yueqi Li2, Guixiang Yang2, Taohong Huang2,

Shin-ichi Kawano2, Yuki Hashi2, Zhenqing Zhang1,* 1Beijing Institute of Pharmacology & Toxicology, 2Shimadzu Global COE, Shimadzu (China) Co., Ltd., China

2

Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5

0.0

2.5

5.0

7.5(x1,000,000)

Introduction

Results

Aconitine (AC) is a bioactive alkaloid from plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. AC is also well known for its high toxicity that induces severe arrhythmias leading to death. Although numerous studies have raised on its pharmacology and toxicity, data on the identi�cation

metabolites of AC in liver microsomes are limited. The study of metabolic pathways is very important for ef�cacy of therapy and evaluation of toxicity for those with narrow therapy window. The aim of our work was to obtain the metabolic pathways of AC by the human liver microsomes.

Methods and Materials

The typical reaction mixture incubation contained 10 μmol/L aconitine and was preincubated at 37 ºC for 3 min. Reactions were initiated by adding 50 μL of NADPH (20 mmol/L), then incubated at 37 ºC in a waterbath shaker for

60 min. The reactions were terminated by adding 3-volume of ice-cold acetonitrile, then vortexed and centrifuged to remove precipitated protein.

Sample Preparation

Instrument : LCMS-IT-TOF (Shimadzu Corporation, Japan);

UFLCXR system (Shimadzu Corporation, Japan);

Column : Shim-pack XR-ODS II (2.0 mmI.D. x 75 mmL.,2.2 μm)

Mobile phase : A: water (0.1% formic acid+5 mmol ammonium formate),

B: acetonitrile

Gradient program : 30%B (0-4 min)-80%B (8 min)-80%B (8-11 min)-30%B (11.01-17 min)


M11M1

M0

M2M3

M4

M5

M6

M7

M8M9

M10

M12

M13 M14M16

M15

B

Fig.1 TIC chromatogram (A) and mass chromatograms of the metabolites of AC in the microsomal incubation mixture of human (B)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.00.0

2.5

5.0

7.5(x1,000,000)

1:TIC (1.00)

A

3


Fig. 3 Proposed metabolic pro�le of AC in the human liver microsomes

Fig. 2 Proposed fragmentation pathway of AC

OH+

OH

OO

O

O

N

O

OH

H

OH

O

O

C34H48NO11+

Exact Mass: 646.3227C32H44NO9

+

Exact Mass: 586.3016

C31H40NO8+


C25H34NO8+


C29H36NO8+


C25H36NO9+

Exact Mass: 494.2390 C22H26NO4+

Exact Mass 368.1862 C21H25NO4+

Exact Mass 354.1705

O

OH

O

+

O

O

N

O

OH

H

OH

O

OH

O

+

O

O

N

O

OH

H

OH

O

OH

O

+

O

O

HN

O

OH

H

OH

O

OH

O

+

O

O

N

O

OH

H

OH

O

OH

O

+

O

O

N

O

OH

H

OH

OOH

O

O

HN

OH

OHH+

O

O

HN

HOH

OHH+

O

HO

HO

O

O

O

N

O

OH

H

OH

O

O

OH

HO

HO

O

O

O

N

O

OH

H

OH

O

O

OH

HO

O

O

O

OH

N

O

OH

H

OH

O

O

O

HO

O

O

O

O

N

HOH2C

O

OH

H

OH

O

O

O

OH

O

OH

O

O

N

O

OH

H

OH

O

O

OH

O

O

O

O

N

HOH2C

O

OH

H

OH

O

O

O

OH

O

O

O

O

N

O

OH

H

OH

O

O

O

OH

O

O

O

OH

N

O

OH

H

OH

O

O

OH

OH

O

O

O

O

N

O

OH

H

OH

O

O

O

OH

O

O

O

N

O

OH

H

OH

O O

O

O

O

N

O

OH

H

OH

O

O

O

OH

O

O

O

O

HN

O

OH

H

OH

O

OO

O

OH

O

O

N

O

OH

H

OH

O

O

OH

OH

O

O

O

O

N

O

OH

H

OH

O

O

M0

M13

M15

M11

M8M9M2

M10

M7

M16

M12

M3 M1

M5

O

HO

HO

O

O

O

N

O

OH

H

OH

O

O

M6O

HO

O

O

O

O

N

O

OH

H

OH

O

O

M4

O

HO

O

O

O

O

N

HOH2C

O

OH

H

OH

O

O

M14

4


No.

M0

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

M11

M12

M13

M14

M15

M16

RT(min)

22.3

10.5

11.2

11.3

11.8

12.2

13.3

13.5

13.7

13.8

14.1

15.0

15.1

16.0

17.3

17.6

17.9

Meas.MW(m/z)

646.3230

618.2922

616.2754

604.3140

630.2930

586.3005

616.2769

632.3035

648.3016

618.2935

618.2890

662.3179

602.2948

632.3054

662.3209

632.3068

584.2826

Pred.MW(m/z)

646.3222

618.2909

616.2752

604.3116

630.2909

586.3011

616.2752

632.3065

648.3015

618.2909

618.2909

662.3171

602.2960

632.3065

662.3171

632.3065

584.2854

mDaerror

0.8

1.3

0.2

2.4

2.1

0.6

2.3

3.0

0.1

3.0

1.5

0.8

1.6

1.1

3.8

0.3

2.8

MS2 data

586.3000, 554.2752, 526.2785, 494.2536, 476.2431, 404.2432, 368.1847, 354.1687

558.2710, 498.2469, 480.2378, 436.2093, 354.1725

556.2510, 554.2335, 494.2106, 478.2321, 434.1908, 402.1682

554.2744, 522.2398, 434.1898

570.2686, 552.2576, 510.2457, 492.2381

568.2938, 554.2705, 522.2537, 466.2168, 434.1922

584.2477, 524.2316, 434.1941

572.2866, 512.2638, 494.2468, 480.2283, 462.2214, 290.2236, 354.1652, 340.1871

588.2702, 570.2654, 528.2566, 510.2434, 406.2161

558.2714, 494.2109, 476.2400, 340.1548

558.2722, 494.2127, 476.2009, 354.1635

602.2964, 570.2654, 542.2750, 510.2434, 420.2416

584.2533, 524.2249, 510.2179, 406.1582

572.2853, 512.2661, 480.2368, 476.2445, 436.2082, 368.1812

602.2947, 570.2654, 542.2766, 510.2434, 478.2187

586.2973, 526.2738, 508.2273, 494.2490

552.2669, 492.2111, 460.2063

Formula

C34H47NO11

C32H43NO11

C32H41NO11

C32H45NO10

C33H43NO11

C32H43NO9

C32H41NO11

C33H45NO11

C33H45NO12

C32H43NO11

C32H43NO11

C34H47NO12

C32H43NO10

C33H45NO11

C34H47NO12

C33H45NO11

C32H41NO9

Biotransformation

Parent

deethylation

bidemethylation+dehydrogenation

deacetylation

demethylation+dehydrogenation

deacetylation+dehydration

bidemethylation+dehydrogenation

demethylation

oxidation+demethylation

bidemethylation

bidemethylation

oxidation

deacetylation+dehydrogenation

demethylation

oxidation

demethylation

deacetylation+dehydration+dehydrogenation

ppmerror

1.26

2.10

0.26

3.94

3.35

0.96

3.68

4.81

0.23

4.88

2.43

1.21

2.66

1.80

5.74

0.42

4.82

Table1 Mass data for characterization of metabolites in of AC in the microsomalincubation mixture of human






Conclusions In this study, totaling 16 metabolites were found and characterized in the humam liver microsomes incubation mixture, including O-demethylation, oxidation, bidemethylation, dehydrogenation, N-deethylation, deacetylation, dehydration and besides M1, M3, M4, M9, M13 and M15, all the left ten of them were �rst identi�ed and reported. Collectively, these data provide a foundation for the clinical use of AC and contributes to a wider understanding of xenobiotic metabolism and toxicity evaluation.

PO-CON1447E

Simultaneous analysis of primarymetabolites by triple quadrupole LC/MS/MSusing penta�uorophenylpropyl column

ASMS 2014 WP 613

Tsuyoshi Nakanishi1, Takako Hishiki2, Makoto Suematsu2,3

1 Shimadzu Corporation, Kyoto, Japan,

2 Department of Biochemistry, School of Medicine,

Keio University, Tokyo, Japan,

3 Japan Science and Technology Agency,

Exploratory Research for Advanced Technology,

Suematsu Gas Biology Project, Tokyo, Japan

2

Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column

IntroductionVarious metabolic pathways are controlled to keep a biological function in the cell and to monitor the rapid and slight changes of these metabolism, a simple simultaneous analysis is required for quanti�cation of primary metabolites. A typical LC/MS system with an ODS column is not effective to measure primary metabolites because of low af�nity of ODS column to hydrophilic metabolites. Here we report the

simultaneous measurement of 97 metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column. In this experiment, MRM transitions of these metabolites were optimized and this method was applied to biological samples. Furthermore, to evaluate the accuracy of developed method for quanti�cation, simultaneous analysis by PFPP column was compared to measurement of ion-paring chromatography.

Commercially available compounds were used as standards to optimize MRM transition and LC condition for separation. Mixed standard solutions were diluted to a range of 10 nM~10000 nM for a calibration curve and an aliquot of 3 µL was subjected to LC/MS/MS measurement.Mice were sacri�ced under anesthesia and the isolated heart/liver tissues were rapidly frozen in liquid nitrogen. Frozen liver or heart tissues (>50 mg) from mice were homogenized in 0.5 mL methanol including L-methionine sulfone and 2-morpholinoethanesulfonic

acid (MES) as internal standards. After a general chloroform/methanol extraction, upper aqueous layer �ltered through 5-kDa cutoff �lter. The �ltrate was dried up and dissolved in 0.1 mL puri�ed water. Further, the solution was diluted to 20-100 folds in puri�ed water. An aliquot of 3 µL was analyzed to measure primary metabolites by LC/MS instrument, Nexera UHPLC system and LCMS-8030/LCMS-8040 triple quadrupole mass spectrometer. The following is detailed conditions of LC/MS mesurement.

Methods and materials

3


UHPLC conditions (Nexera system using a PFPP column)

Column : Discovery HS F5 150 mm×2.1 mm, 3.0 µm

Mobile phase A : 0.1% Formate/water

B : 0.1% Formate/acetonitrile


Time program : B conc.0%(0-2.0 min) - 25%(5.0 min) - 35%(11.0 min)

- 95%(15.0.-20.0 min) - 0%(20.1-25.0 min)

Injection vol. : 3 µL

Column temperature : 40°C

MS conditions (LCMS-8030/LCMS-8040)

Ionization : Positive/Negative, MRM mode

DL Temp. : 250°C

HB Temp : 400°C

Drying Gas : 10 L/min

Nebulizing Gas : 2.0 L/min

Result

The MRM transitions for 97 standard compounds were optimized on both positive and negative mode by flow injection analysis (FIA). The MRM transitions of the 97 metabolites were determined as described in Table 1. Subsequently, LC condition was investigated to separate the 97 metabolites with a good resolution. As a consequence, the 97 metabolites were eluted from a PFPP column with a gradient of acetonitrile for <15 min in the

condition described in Figure 1. The linearity of this method was also confirmed by the simultaneous analysis of a serial of diluted calibration curve.

Figure 1 shows the MRM chromatogram of 97 metabolites at a concentration of 5 µM. In this figure, we can see the peak from all metabolites with a good separation.

Optimization of MRM transition

4


Table 1 MRM transition of 97 metabolites

Name Product ion Precursor ion Linearity (R2)

2-Aminobutyrate

Acetylcarnitine

Acetylcholine

Adenine

Adenosine

Adenylsuccinate

ADMA

Ala

AMP

Arg

Argininosuccinate

Asn

Asp

cAMP

Carnitine

Carnosine

cCMP

cGMP

Choline

Citicoline

Citrulline

CMP

Creatine

Creatinine

Cys

Cystathionine

Cysteamine

Cystine

Cytidine

Cytosine

Dimethylglycine

DOPA

Dopamine

Epinephrine

FAD

GABA

gamma-Glu-Cys

Gln

Glu

Gly

GMP

GSH

Guanosine

His

Histamine

Homocysteine

Homocystine

Hydroxyproline

Hypoxanthine

Ile

No.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

104.10

204.10

147.10

136.00

268.10

464.10

203.10

89.90

348.00

175.10

291.00

133.10

134.00

330.00

162.10

227.10

306.00

346.00

104.10

489.10

176.10

324.00

132.10

114.10

122.00

223.00

78.10

241.00

244.10

112.00

104.10

198.10

154.10

184.10

786.15

104.10

251.10

147.10

147.90

75.90

364.00

308.00

284.00

155.90

112.10

136.00

269.00

132.10

137.00

132.10

58.05

85.05

87.05

119.05

136.05

252.10

70.10

44.10

136.05

70.10

70.10

87.15

74.05

136.05

103.05

110.05

112.10

152.05

60.05

184.10

70.05

112.05

44.05

44.05

76.05

88.05

61.05

151.95

112.05

95.10

58.05

152.10

91.05

166.10

136.10

87.05

84.10

84.15

84.10

30.15

152.05

179.10

152.00

110.10

95.05

90.10

136.05

86.05

55.05

86.20

Polarity

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

0.99

0.99

0.99

0.98

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99*

0.99

0.99

0.99*

0.99

0.99

0.99

0.99*

0.99

0.99

0.99

0.99

0.99*

0.99

0.98*

0.99

0.99

0.99

0.99

0.99*

0.99*

0.99

0.99*

0.99

0.99*

0.99

0.99*

0.99*

0.99

0.99*

0.99

0.99

0.99*

0.99*

0.99

0.99

0.98*

0.99

Name Product ion Precursor ion Linearity (R2)

Inosine

Kynurenine

Leu

L-Norepinephrine

Lys

Met

Methionine-sulfoxide

Nicotinamide

Nicotinic acid

Ophthalmic acid

Ornitine

Pantothenate

Phe

Pro

SAH

SAM

SDMA

Ser

Serotonin

Thr/Homoserine

Thymidine

Thymine

TMP

Trp

Tyr

Uracil

Uridine

Val

2-Oxoglutarate

Allantoin

Cholate

cis-Aconitate

Citrate

FMN

Fumarate

GSSG

Guanine

Isocitrate

Lactate

Malate

NAD

Orotic acid

Pyruvate

Succinate

Taurocholate

Uric acid

Xanthine

No.

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

269.10

209.10

132.10

170.10

147.10

149.90

166.00

123.10

124.05

290.10

133.10

220.10

166.10

115.90

385.10

399.10

203.10

105.90

177.10

120.10

243.10

127.10

322.90

205.10

182.10

113.00

245.00

118.10

144.90

157.00

407.20

172.90

191.20

455.00

115.10

611.10

150.00

191.20

89.30

133.10

663.10

155.00

86.90

117.30

514.20

167.10

151.00

137.05

192.05

86.05

152.15

84.10

56.10

74.10

80.05

80.05

58.10

70.10

90.15

120.10

70.10

134.00

250.05

70.15

60.10

160.10

74.15

127.10

54.05

81.10

188.15

136.10

70.00

113.05

72.15

101.10

97.10

343.15

85.05

111.10

97.00

71.00

306.00

133.00

111.10

89.05

114.95

541.05

111.10

87.05

73.00

107.10

123.95

108.00

Polarity

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.98

0.99*

0.99

0.99*

0.99

0.99

0.99

0.99*

0.99*

0.99

0.99

0.99*

0.99

0.99

0.98*

0.98*

0.99**

0.99

0.99*

0.99

0.99**

0.99*

0.99*

0.99*

0.97*

0.99*

0.99*

0.99

0.99*

0.99*

0.99*

0.99*

0.99*

Calibration curve was obtained at a range of concentration from 10 nM to 10000 nM.* Calibration curve was obtained at a range of concentration from 100 nM to 10000 nM.** Calibration curve was obtained at a range of concentration from 1000 nM to 10000 nM.

5


Figure 1 MRM chromatogram of 97 compounds

Simultaneous analysis of 99 compounds was performed for heart / liver tissue extracts as biological samples. Figure 2 shows MRM chromatograms of 99 compounds from tissue extracts (liver/heart). In this measurement, 83/97 metabolites were detected from liver tissue extracts and 88/97 metabolites were confirmed from heart tissue extracts. These results show this method is also effective to

simultaneous analysis of biological samples. As shown in the resulting MRM chromatogram, some major peaks were derived from the metabolites which were known to be characteristic to each tissue. Furthermore, this characteristic difference in each tissue was also confirmed in some faint peaks (e.g., cholate, cystine and homocysteine).

Application to tissue extracts as biological samples

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000


6

We have previously reported simultaneous analysis of 55 metabolites which were related to central carbon metabolic pathway by using ion pairing chromatography at ASMS conference 2013. To evaluate the accuracy of this simultaneous method using PFPP column, we compared the resulting peak area of 25 metabolites, which were covered as targets in both methods. The 25 metabolites are Lysine, Arginine, Histidine, Glycine, Serine, Asparagine, Alanine, Glutamine, Threonine, Methionine, Tyrosine, Glutamate, Aspartae, Phenylalanine, Tryptophan, Cysteine, CMP, NAD, GMP, TMP, AMP, cGMP, cAMP, MES and L-Methionine sulfone as internal standards. Heart tissue extracts were prepared from mice (n=9) according to the

method described above and the aliquots were measured by the simultaneous method using either ion pairing chromatography or PFPP separation system. As a result, we could see the similar trend of elevation/decrease of peak area in metabolites of 20/25 between nine samples. The peak areas between 9 samples of representative metabolites are shown in Figure 3. This result shows that a ratio of areas between 9 samples is kept in both methods. The four metabolites (TMP, cGMP, cAMP and Cysteine) could be hardly detected on simultaneous analysis by alternately ion-paring chromatography or PFPP column. Tryptophan had a faint peak in this experiment and led to the low similarity.

Correlation between PFPP and ion pairing Methods

Figure 2 MRM chromatogram of liver/heart tissue extracts

Liver Tissue

Heart Tissue

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

0

5000000

10000000

15000000

20000000

25000000

30000000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

Acetylcarnitine

Creatine

Ophtalmic acid

GSSG

Guanosine

S-Adenosylhomocysteine

GSH

AMP






Conclusions• The 97 metabolites were separated by PFPP column with high resolution and this method was applied to biological

samples.• The utility of this simultaneous analysis using PFPP column was con�rmed by comparing between PFPP and ion paring

chromatography.

0.0E+00

5.0E+05

1.0E+06

1.5E+06

1 2 3 4 5 6 7 8 9

MES

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

1 2 3 4 5 6 7 8 9

Serine

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

1 2 3 4 5 6 7 8 9

Threonine

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

1 2 3 4 5 6 7 8 9

L-Methionine sulfone

PFPP

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1 2 3 4 5 6 7 8 9

MES

0.0E+00

5.0E+03

1.0E+04

1.5E+04

2.0E+04

2.5E+04

1 2 3 4 5 6 7 8 9

Serine

0.0E+00

1.0E+04

2.0E+04

3.0E+04

4.0E+04

1 2 3 4 5 6 7 8 9

Threonine

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1 2 3 4 5 6 7 8 9

L-Methionine sulfone

Ion pairing

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1 2 3 4 5 6 7 8 9

Aspartate

0.0E+005.0E+041.0E+051.5E+052.0E+052.5E+053.0E+05

1 2 3 4 5 6 7 8 9

GMP

0.0E+00

5.0E+06

1.0E+07

1.5E+07

1 2 3 4 5 6 7 8 9

AMP

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

1 2 3 4 5 6 7 8 9

Phenylalanine

PFPP

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

1 2 3 4 5 6 7 8 9

Aspartate

0.0E+001.0E+042.0E+043.0E+044.0E+045.0E+046.0E+04

1 2 3 4 5 6 7 8 9

GMP

0.0E+001.0E+052.0E+053.0E+054.0E+055.0E+056.0E+05

1 2 3 4 5 6 7 8 9

AMP

0.0E+00

1.0E+04

2.0E+04

3.0E+04

4.0E+04

1 2 3 4 5 6 7 8 9

Phenylalanine

Ion pairing

Figure 3 Correlation of peak areas between PFPP and ion-pairing method

Life Science

• Page 222Surface analysis of permanent wave processing hair using DART-MS

• Page 229Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromato-graph Mass Spectrometer)

PO-CON1454E

Surface analysis of permanent waveprocessing hair using DART-MS

ASMS 2014 MP 476

Shoji Takigami1, Erika Ikeda1, Yuta Takagi1,

Jun Watanabe2, Teruhisa Shiota3

1 Gunma University, Kiryu, Japan;



2

Surface analysis of permanent wave processing hair using DART-MS

IntroductionPermanent wave processing of hair is carried out at two processes as follows; (A) Reducing agent (permanent wave 1 agent) makes the bridge construction between the keratin protein molecular chains of hair, especially disul�de (S-S) bond of cystine residue cleaved to thiol (-SH) group and hear results a wave and curl.(B) Oxidizing agent (permanent wave 2 agent) makes -SH group oxidized to be reproduced S-S bond. As reducing agents used for permanent wave 1 agent, the thing of cosmetics approval, such as cysteamine hydrochloride and a butyrolactone thiol (brand name Spiera, other than quasi drugs, such as ammonium thioglycolate, acetyl cystein, and thiolactic acid, are used.

After hair is applying permanent wave processing and coloring repeatedly, the chemical structure of a keratin molecule and �ne structure in the hair have been damaged and it resulted as damage hair. It is thought that hair becomes dryness and twining if the cuticle which covers hair is damaged, so it is important to investigate the surface structure of hair and its chemical structure changing.DART (Direct Analysis in Real Time), a direct atmospheric pressure ionization source, is capable of analyzing samples directly with little or no sample preparation. Here, analysis of the ingredient which has deposited on the permanent wave processing hair surface was tried using this DART combined with a mass spectrometer.

The chemical state and property were investigated in the surface of the hair which repeated permanent wave processing with these reducing agents.

Figure 1 DART-OS ion source & LCMS-2020


Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec

TGA(thioglycolate)

CA(cysteamine hydrochloride)

BLT(butyrolactone thiol)

SH

O

HOO

O

SH

Fw 92 Fw 113 Fw 118

H2NSH

HCl

Wave ef�ciency is good in a weak alkaline (pH 8 - 9.5)

Wave ef�ciency is good in a weak alkaline (pH 8 - 9.5)

Wave ef�ciency is good in a weak acid (pH 6)

3


Methods and MaterialsThe Chinese virgin hair purchased from the market was washed with the 0.5% non-ionic surfactant containing saturated EDTA solution, and then it was considered as untreated hair sample. Permanent wave processing of hair was prepared as following; the 0.6M TGA solution and 0.6M CA solution which were adjusted to pH8.5 with aqueous ammonia and the 0.6M BLT solution adjusted to pH6.0 with arginine water, which were used as a reducing

agent. After hair sample was reduced for 15 minutes at 35°C using each solvent, it was carried out oxidation treatment at 35°C by being immersed in 8% sodium bromate solution (pH7.2) for 15 minutes. LCMS-2020 (Shimadzu) was coupled with DART-OS ion source (IonSense) and hear samples were held onto DART gas �ow directly, then their surface analyzed.



Heater Temperature (DART) : 350°C


Chinese Virgin Hair

0.5% Laureth - 9 solution - EDTA saturated 35°C 1h

Water washing and air drying

Untreated Permanent wave processing by agent 1 & 2 at 0.6M each

Analyzed by DART-MS

permanent wave 1 agent : TGA or CA (pH 8.5; aqueous ammonium) BLT (pH 6; arginine) 35°C 15min

Water washing

Water washing

Britton - Robinson buffer (pH 4.6) 35°C 15min

Water washing

permanent wave 2 agent : 8% NaBrO3 solution (pH 7.2) 35°C 15min

Air drying

Repeat6 times

4


ResultAfter repeating operation of permanent wave processing 1-6 times using TGA (thioglycollic acid), CA (cysteamine), and BLT (Butyrolactonethiol), hair was immersed for 15 minutes at 35°C and with a �ush and air-drying, then permanent wave processing hair was prepared. In order to investigate the ingredient which has deposited on the permanent wave processing hair surface, DART-MS analysis

was performed. DART-MS analysis was conducted in order of #1 Untreated (woman hair), #2 control; ammonia treatment (pH 8.5), #3 0.6M thioglycolic acid (TGA) processing, #4 0.6M butyrolactone thiol (BLT) processing, #5 0.6M cysteamine hydrochloride (CA) processing and #6 control; arginine processing (pH 6).

In the DART mass spectra of #1 untreated and #6 control, many signals considered as triglyceride and diglyceride were detected in both positive and negative spectra obtained by DART-MS. In #3 0.6M thioglycolic acid (TGA) processing spectra, the signal in particular of TGA origin was not detected. In #4 BLT processing spectra (Figure 3), the signals considered to be oxidized BLT (3, 3'-dithiobis (tetrahydrofuran2-one), molecular weight 234) were detected at m/z 235 and 252 in the positive mode. The signal m/z 235 is equivalent to [M+H]+ and m/z 252, [M+NH4]+. In the negative mode, the signals, m/z 115,

231 were detected. They were considered the signal equivalent to [M-H]- and [2M-H]- of BLT oxide compound (C4H4O2S, molecular weight 116) in which two hydrogen atoms were removed from BLT. Carrying out permanent wave processing by BLT, it was found that the dimer of BLT accumulated on the cuticle surface.In #5 CA processing spectrum (Figure 5), the signal considered to be the dimer (Fw152) origin in which CA carried out S-S bond in the positive mode was detected at m/z 153. This is equivalent to [M+H]+.

Figure 2 TIC chromatogram of each sample analyzing with DART

0

25000000

50000000

75000000 2:TIC(+)

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

5000000

10000000

150000004:TIC(-)

#1 #2 #3 #4 #5 #6




5

Figure 3 DART-MS spectra of #4 BLT processingThe BLT-related signals were detected from the positive and the negative spectra.

Figure 4 DART-MS spectra of #5 CA processingThe CA-related signal was detected from the positive spectrum

100 200 300 400 500 600 700 800 900 1000 1100 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x10,000,000)

252

486282 368 424 516

100 200 300 400 500 600 700 800 900 1000 1100 m/z0.0

1.0

2.0

3.0

4.0

5.0Inten. (x100,000)

179

115

231

321347

411 501 579

235

Positive

Negative

[M+H]+[M+NH4]+

[M-H]-

[2M-H]-

100 200 300 400 500 600 700 800 900 1000 1100 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x1,000,000)

282124

391

252

468424 563

184600102 644 691 769 851 922

153

Positive

[2M+H]+


6

In order to indicate clearly the signals specifically detected in each sample, the extraction chromatograms (XIC) were shown (Figure 5). It turned out that BLT-related signals were detected only in #4 and the CA-related signal in #5. Moreover, although the signal intensity was weak, the signal at negative m/z 325 was detected from all samples. Negative m/z 325 is equivalent to [M-H]- of 18

methyl eicosanoic acid (18MEA, molecular weight 326). 18MEA is one of lipid components which protect a cuticle. There is no significant difference of this signal in the hair between treated hair and untreated hair. We would like to inquire so that intensity difference can be found out by further verifying the detection technique in the future.

Figure 5 XIC chromatorgam of each sample analyzing with DART

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

0

500000

1000000 2:152.85(+)

0

5000000

10000000 2:234.70(+)

0

5000000

100000002:251.75(+)

0

250000

4:114.95(-)

0

500000

4:230.90(-)

0

500000

1000000

1500000 2:123.85(+)

0

50000

1000004:325.15(-)

Positive XIC m/z 153


Negative XIC m/z 231


#1


#2 #3 #4 #5 #6








ConclusionsBy direct analysis of the hair by DART-MS, the chemical structure change in the surfaces of hair, such as permanent wave processing, was able to be observed.

PO-CON1469E

Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

ASMS 2014 TP761

Sanket Chiplunkar, Prashant Hase, Dheeraj Handique,

Ankush Bhone, Durvesh Sawant, Ajit Datar,

Jitendra Kelkar, Pratap Rasam




2


IntroductionCosmetics, fragrances and toiletries (Figure 1) are used safely by millions of people worldwide. Although many people have no problems, irritant and allergic reactions may occur. Irritant and allergic skin reactions are the types of contact dermatitis. Essential oils present in fragrance contain some natural and synthetic compounds, which may cause allergic reactions to the end user after application. There are 26 potential allergens listed by

European Directive (EU) 2003/15/EC and International Fragrance Association (IFRA)[1] labeled on cosmetics. Shimadzu MDGC-GCMS technology facilitates the identi�cation and quanti�cation of these allergens to comply with the threshold limits of 100 ppm for rinse-off products.Co-eluting peaks were resolved completely with the help of MDGC-GCMS heart-cut technique.

Figure 1. Cosmetics, fragrances and toiletries

Method of Analysis

Shampoo samples were collected from local market. Standard solutions of 23 allergens were procured from ACCU Standard and dilutions were carried out in Ethanol/Acetonitrile to yield 1000 ppm concentration. Further dilutions were made in methanol.MDGC-GCMS technique was effectively used to minimize matrix effect. Co-eluting peaks were resolved with heart-cut technique using two columns of different

polarities. In MDGC-GCMS, 1st instrument was GC-2010 Plus equipped with FID as a detector and 2nd instrument was GCMS-QP2010 Ultra with MS as a detector. Columns in both the instruments were connected with Deans switch. Allergens in shampoo samples were determined by using this technique. For sample preparation, following methodology was adopted.

Extraction of allergens from shampoo sample

Part method validation was carried out by performing system precision, sample precision, linearity and recovery study. For validation, solutions of different concentrations

were prepared using 40 ppm (actual concentration) standard stock solution mixture of allergens.

1) Blank Solution : 10 mL of methanol was transferred in 20 mL centrifuge tube and vortexed for 5 minutes. The mixture was then centrifuged for 5 minutes at 3000 rpm. This solution was filtered through 0.2 µm nylon syringe filter. Initial 2 mL was discarded and remaining filtrate was collected.

2) Sample Solution : 1 g of shampoo sample was weighed in 10 mL volumetric flask and diluted up to the mark with methanol. Above mixture was transferred in 20 mL centrifuge tube. Further processing was done as mentioned in blank solution.

3) Spike Sample Solution : For recovery study, 1 g of sample was spiked with different volumes of standard stock solution. The above procedure was repeated for preparing different concentration levels of allergens in samples. These spiked samples were treated as mentioned in sample solution.

3


MDGC-GCMS Analytical ConditionsThe instrument con�guration used is shown in Figure 2. Samples were analyzed using Multi-Dimensional GC/GCMS as per the conditions given below.

Table 1. Method validation parameters

Figure 2. Multi-Dimensional GC/GCMS System by Shimadzu

Figure 3. Schematic diagram of multi-Deans switch in MDGC-GCMS

Parameter Concentration

System Precision

Sample Precision

Linearity

Accuracy / Recovery

10 ppm

10 % in Methanol

2.5, 5, 7.5, 10, 15 (ppm)

5, 10, 15 (ppm)

4


MDGC-GCMS analytical parametersChromatographic parameters (1st GC : GC-2010 Plus)

• Column : Stabilwax (30 m L x 0.25 mm I.D.; 0.25 μm)

• Injection Mode : Split




• Detector : FID

• APC Pressure : 200 kPa (For switching)

• Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min)

50.0 0.00

15.00 100.0 0.00

5.00 240.0 43.67

Chromatographic parameters (2nd GCMS : GCMS-QP2010 Ultra)

• Column : Rxi-1ms (30 m L x 0.25 mm I.D.; 0.25 μm)

• Detector : Mass spectrometer

• Ion Source Temp. : 200 ºC

• Interface Temp. : 240 ºC


• Event Time : 0.30 sec

• Mode : SIM and SCAN


80.0 13.00

3.00 180.0 0.00

10.00 260.0 20.67


Results

MDGC-GCMS technique was used to avoid matrix interference from sample. Using multi-Deans switch and heart-cut technique (Figure 3), co-eluted components from the 1st column were transferred to the 2nd column with different polarity.

Sample analysis using MDGC-GCMS

5


Figure 5. Chromatogram with 1st column (FID)

Figure 4. Chromatogram of spiked sample solution before switching

Table 2. Summary of results for precision on GC and GCMS

Figure 6. SIM chromatogram with 2nd column (MS)

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 min

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

uV (x100,000) Chromatogram

Sam

ple

- 1

Lim

onen

e

Lina

lool

Met

hyl h

eptin

e ca

rbon

ate

Sam

ple

- 2

Sam

ple

- 3 Citr

al -

1

Citr

al -

2 Citr

onel

lol

Ger

anio

lBe

nzyl

Alc

ohol

Hyd

roxy

-citr

onel

lal

Cin

nam

al

Euge

nol

Am

yl c

inna

mal

Ani

syl a

lcoh

olC

inna

myl

alc

ohol

Fern

esol

- 1

Isoe

ugen

olFe

rnes

ol -

2Fe

rnes

ol -

2H

exyl

cin

nam

ald

ehyd

e

Cou

mar

in

Am

ylci

n na

myl

alc

ohol

Sam

ple

- 5

Benz

yl b

enzo

ate

Sam

ple

- 6

Benz

yl s

alic

ylat

e

Benz

yl C

inna

mat

e

12.0 13.0 14.0 15.0 16.0 17.0 min

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

uV (x10,000) Chromatogram

Met

hyl h

eptin

e ca

rbon

ate

Sam

ple

- 2

Sam

ple

- 3

Citr

al -

1

Citr

al -

2

Citr

onel

lol

Ger

anio

l Benz

yl A

lcoh

ol

25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 min

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0uV (x10,000) Chromatogram

Am

yl c

inna

mal

Ani

syl a

lcoh

olC

inna

myl

alc

ohol

Fern

esol

- 1

Isoe

ugen

ol Fern

esol

- 2

Fern

esol

- 2

Hex

yl c

inna

m a

ldeh

yde

26.5 27.0 27.5 28.0 min0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50uV (x10,000) Chromatogram

26.2

56

26.4

91

28.1

05

Target compound - Isoeugenol

27.0 27.5 28.0 28.5 29.0 29.5-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

(x100,000)

134.00 (100.00)115.00 (100.00)92.00 (100.00)137.00 (100.00)109.00 (100.00)138.00 (100.00)103.00 (100.00)149.00 (100.00)164.00 (100.00)

Target compound - Isoeugenol

Summary of results

Result

% RSD for area (n=6) < 2.0

% RSD for area (n=6) < 2.0

Concentration

10 ppm

Unknown

Sample name

23 Allergens mixture

Shampoo

Type of sample

Standard

Cosmetic

Sr. No.

1

2


6

Figure 7. Linearity graph for linalool

Table 3. Linearity by GC

For the quantitation studies, the shampoo sample was spiked with allergens standard to achieve 5, 10 and 15 ppm concentrations. Recovery studies were performed on 13 allergens, having co-elution or matrix interference, using heart-cut technique. The quantitation of these allergens was carried out using 2nd detector (MS) in SIM mode.

In below recovery study, some allergens had recovery value out side the acceptance limit (70-130 %). Optimization can be done by means of change in sample clean up procedure and filtration study.

Quantitation of allergens in shampoo sample

Name of allergen

Linalool

Methyl heptine carbonate

Citronellol

Geraniol

Hydroxy citronellal

Cinnamal

Amyl Cinnamal

Coumarin

Amylcin namyl alcohol

Benzyl benzoate

Sr. No.

1

2

3

4

5

6

7

8

9

10

Linearity (R2)

0.9945

0.9949

0.9965

0.9962

0.9973

0.9959

0.9976

0.9971

0.9983

0.9979 0.0 2.5 5.0 7.5 10.0 12.5 Conc.0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Area (x10,000)

Figure 8. Linearity graph for benzyl cinnamate

Table 4. Linearity by GCMS

Name of allergen

Limonene

Benzyl alcohol

Citral - 1

Citral - 2

Eugenol

Anisyl alcohol

Cinnamyl alcohol

Isoeugenol

Farnesol - 1

Farnesol - 2

Hexyl cinnam aldehyde

Benzyl salicylate

Benzyl cinnamate

Sr. No.

1

2

3

4

5

6

7

8

9

10

11

12

13

Linearity (R2)

0.9945

0.9871

0.9889

0.9902

0.9894

0.9916

0.9937

0.9902

0.9919

0.9929

0.9932

0.9853

0.9927

0.0 2.5 5.0 7.5 10.0 12.5 Conc.0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Area(x10,000)






Conclusion• MDGC-GCMS method was developed for quantitation of allergens present in cosmetics. Part method validation was

performed as per ICH guidelines.[2] Results obtained for reproducibility, linearity and recovery studies were well within acceptable limits.

• Simultaneous SCAN/SIM and high-speed scan rate 20,000 u/sec are the characteristic features of GCMS-QP2010 Ultra, which enables quantitation of allergens at very low concentration level.

• Matrix effect from cosmetics was selectively eliminated using MDGC-GCMS with multi-Deans switching unit and heart-cut technique.

• MDGC-GCMS was found to be very useful technique for simultaneous identi�cation and quantitation of components from complex matrix.

Reference[1] IFRA guidelines (International Fragrance Association), GC/MS Quanti�cation of potential fragrance allergens, Version 2,

(2006), 6.[2] ICH guidelines, Validation of Analytical Procedures: Text And Methodology Q2(R1), Version 4, (2005).

Figure 9. Overlay SIM chromatogram of unspiked and spiked sample

Table 5. Quantitation of allergens – Recovery Study

Name of allergen

Limonene

Benzyl alcohol

Citral - 1

Citral - 2

Eugenol

Anisyl alcohol

Cinnamyl alcohol

Isoeugenol

Farnesol - 1

Farnesol - 2

Hexyl cinnam aldehyde

Benzyl salicylate

Benzyl cinnamate

Sr. No.

1

2

3

4

5

6

7

8

9

10

11

12

13

Level -15 ppm

127

114

101

97

96

94

98

103

83

84

121

63

66

Level -210 ppm

% Recovery

126

114

106

103

105

105

106

108

95

95

122

47

61

Level -315 ppm

129

123

114

112

116

116

115

118

107

106

130

32

5625.0 27.5 30.0 32.5

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

(x1,000)

Farnesol-1

min

Farnesol-2

Spiked

Unspiked

m/z : 69.00

Technical Applications

�•�Page�238Applications�of�desorption�corona�beam� ionization-mass�spectrometry

�•�Page�243Rapid�analysis�of�carbon�fiber�reinforced�plastic�using�DART-MS

•�Page�249Analysis�of�styrene�leached�from�polystyrene�cups�using�GCMS�coupled�with�Headspace�(HS)�sampler

PO-CON1474E

Applications of Desorption Corona Beam Ionization-Mass Spectrometry

ASMS 2014 WP 393

Yuki Hashi1, Shin-ichi Kawano1, Changkun Li1, Qian Sun1,

Taohong Huang1, Tomoomi Hoshi2, Wenjian Sun3

1Shimadzu (China) Co., Ltd., Shanghai, China 2Shimadzu Corporation, Kyoto, Japan 3Shimadzu Research Laboratory (Shanghai) Co., Ltd.,

Shanghai, China

2


IntroductionNumerous ambient ionization mass spectrometric techniques have been developed for high throughput analysis of various compounds with minimum sample pretreatment.(1) Desorption corona beam ionization (DCBI) is a more recent technique.(2) In DCBI, helium is used as discharge gas and heating of the gas is required for sample

desorption. A visible thin corona beam is formed by using hollow needle/ring electrode structure. This feature facilitates localizing sampling areas and obtaining good reproducibility of data. Details of DCBI hardware are shown in Figs. 1 and 2. In this study, DCBI was applied for analysis of various samples.

Figure 2 DCBI interface

Figure 1 Schematic diagram of DCBI

+

-

HVDC

LVDC

MS inlet

Counter electrode

Heated thin wall tubing

Helium �ow

Sample and stage

Dischargeneedle

Sampling capillary

Corona beam

MS Inlet

DCBI probe

Manual liquidsampler

3


Results and DiscussionIn this experiment, all compounds with variety of polarity from non- to high-polar gave protonated molecules (Figs. 3-8). Methomyl gave also fragment ions (m/z 106) by

cleavage at methylcarbamoyl group, while fragment ions with signi�cant intensity were not observed for other compounds. Analysis time was less than 1 minute.

Method

Samples (melamine, saturated hydrocarbon mixture, polyaromatic hydrocarbon mixture, testosterone, pirimicarb, and methomyl) were dissolved in methanol or acetonitrile.

Sample Preparation

Samples were analyzed using a DCBI system coupled to a LCMS-2020 quadrupole mass spectrometer (Shimadzu Corporation, Japan). The system was operated with the DCBI control software and LabSolutions for LCMS version 5.42.

DCBI-MS Analysis


Figure 3 Mass spectrum of melamine (0.5 mg/mL)

DCBI

Flow rate : 0.6 L/min

HV discharge : +2.0-3.0 kV

He gas temperature : 350 ºC

Sample volume : 1, or 2 µL

MS (LCMS-2020 quadrupole mass spectrometer)

Polarity : Positive


BH temperature : 400 ºC

Mass range : m/z 100-500

100.0 105.0 110.0 115.0 120.0 125.0 130.0 135.0 140.0 145.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

Inten. (x1,000)

127.1

136.0 148.6

4


Figure 4 Mass spectrum of saturated hydrocarbon mixture (1 mg/mL)

Figure 5 Mass spectrum of polyaromatic hydrocarbon mixture (2 mg/mL)

Figure 6 Mass spectrum of testosterone (1 mg/mL)

Compound MWC10H22 142C11H24 156C12H26 170C13H28 184C14H30 198C15H32 212C16H34 226C17H36 240C18H38 254C19H40 268C20H42 282C21H44 296C22H46 310C23H48 324C24H50 338C25H52 352

100 150 200 250 300 350 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50Inten. (x100,000)

241.3213.2

255.3269.3

199.2

283.3

297.3185.2

311.3

171.2 325.3

339.3157.2

115.1 143.2 367.4

100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5Inten. (x10,000)

153.1

155.2

179.1

203.1167.2

209.1195.1129.1

141.2 235.1115.1 276.2

Compound MWNaphthalene 128Acenaphthylene 152Acenaphthene 154Fluorene 166Anthracene 178Phenanthrene 178Pyrene 202Fluoranthene 202Chrysene 228Benzo[a]anthracene 228

150 200 250 300 350 400 450 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Inten. (x10,000)

289.2

331.2 461.4112.1

424.5






ConclusionThe DCBI system was successfully applied for analysis of samples with various polarity.Mass spectra were quickly obtained after sample introduction to the DCBI probe.The method is useful for fast identi�cation of various compounds.

References(1) Monge ME et al, Chem. Rev. 113 (2013), 2269-2308(2) Hua W et al, Analyst 135 (2010), 688-695

Figure 7 Mass spectrum of pirimicarb (0.5 mg/mL) Figure 8 Mass spectrum of methomyl (0.5 mg/mL)

100 150 200 250 300 350 400 450 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Inten. (x100,000)

239.2

182.2

100 150 200 250 300 350 400 450 m/z0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Inten. (x100,000)

163.0

105.9

194.0121.9 252.0 354.1 394.3208.0

PO-CON1456E

Rapid analysis of carbon �berreinforced plastic using DART-MS

ASMS 2014 TP 782

Hideaki Kusano1, Jun Watanabe1, Yuki Kudo2,

Teruhisa Shiota3


2 Bio Chromato, Inc., Fujisawa, Japan;


2

Rapid analysis of carbon �ber reinforced plastic using DART-MS

IntroductionDART (Direct Analysis in Real Time) can ionize and analyze samples directly under atmospheric pressure, independent of the sample forms. Then it is also possible to measure in form as it is, without sample preparation. Qualitative analysis of target compounds can be conducted very fast and easily by combining DART with LCMS-2020/8030 which have ultra high-speed scanning and ultra high-speed polarity switching. Carbon-�ber-reinforced plastics, CFRP is the �ber-reinforced plastic which used carbon �ber for the reinforced material, which is only called carbon resin or

carbon in many cases. An epoxy resin is mainly used for a base material in CFRP. While CFRP is widely used taking advantage of strength and lightness, most approaches which measure CFRP with analytical instruments were not tried, triggered by the dif�culty of the preparation.DART (Direct Analysis in Real Time), a direct atmospheric pressure ionization source, is capable of analyzing samples with little or no sample preparation. Here, rapid analysis of carbon �ber reinforced plastic was carried out using DART combined with a mass spectrometer.

Methods and MaterialsThermosetting polyimide (carbon-�ber-reinforced plastics) and thermoplastic polyimide (control sample) were privately manufactured. After cutting a sample in a suitable size, it applied DART-MS analysis. They were introduced to the DART gas using tweezers. The DART-OS ion source (IonSense, MA, USA) was interfaced onto the single quadrupole mass spectrometer LCMS-8030 (Shimadzu,

Kyoto Japan). Ultra-fast polarity switching was utilized on the mass spectrometer to collect full scan data. LCMS-8030 can achieve the polarity switching time of 15msec and the scanning speed of up to 15,000u/sec, therefore the loop time can be set at less than 1 second despite the relatively large scanning range of 50-1,000u.

Figure 1 CFRP：carbon-�ber-reinforced plastic



3


Result3 CFRP samples were analyzed by DART-MS. Mass chromatograms of each sample were shown in Figure 3 and mass spectra in Figure 4.

Figure 2 DART-OS ion source (IonSense) & triple quadrupole LCMS (Shimadzu)

Figure 3 TIC chromatogram of CFRP samples #1, #2, #3


UFswitching　High-Speed Polarity Switching 15msec UFscanning　High-Speed Scanning 15,000u/sec

Sample

#1 thermoplastic polyimide (control)

#2 thermosetting polyimide (molded; dried)

#3 thermosetting polyimide (immediately after molded; wet state with solvent)


Heater Temperature (DART) : 300ºC


0

25000000

50000000

1:MIC1(+)

7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 min

0

1000000

2000000

3000000

4000000

5000000

6000000 2:MIC1(-)

#1 #2 #3



4


Since the thermosetting polyimide used for this measurement was molded using the organic solvent (N-methyl pyrrolidone, C5H9NO, molecular weight 99), molecular related ions of N-methyl pyrrolidone, [M+H]+ (m/z 100) and [2M+H]+ (m/z 199), were detected very strongly in the mass spectrum of #1. The mass spectrum of #2 also showed the same ions that intensity was intentionally detected strongly compared with #3 although intensity was weak compared with #1. Even if

it raised the heating gas temperature of DART to high temperature (up to 500°C), MS signal considered to originate in the structural information of CFRP was not able to be obtained.Then, the optional heating mechanism, ionRocket (Bio Chromato, Inc.; Figure 5), in which a sample could be heated directly was developed to the sample stage of DART, and analysis of CFRP was verified by heating the sample directly up to 600°C.

Figure 4 DART-MS spectra of each sample

Sample

#4 thermosetting polyimide (molded; dried)

#5 thermoplastic polyimide (control)


Heater Temperature (DART) : 400°C

Temperature control (ionRocket) : 0-1min room temp., 4min 600°C

Measuring mode (MS) : Positive scanning

50 100 150 200 250 m/z0.0

2.5

5.0

7.5

Inten. (x1,000,000)

199.1100.1 282.2228.3172.1

Positive, m/z 50-300#1

50 100 150 200 250 m/z0.0

2.5

5.0

7.5

Inten. (x1,000,000)

199.1100.1

Positive, m/z 50-300 #3

50 100 150 200 250 m/z0.0

2.5

5.0

7.5

Inten. (x1,000,000)

199.1100.1172.2 282.3

[M+H]+ [2M+H]+

Positive, m/z 50-300#2 N-methyl pyrrolidoneC5H9NOMw 99


5

When heating temperature was set to 600ºC, the rudder shape signals of 28u (C2H4) interval was appeared around m/z 900. This signal was more notably detected with the thermosetting polyimide sample than the thermoplastic sample. Since the sample was heated at

high temperature, it was considered that the thermal decomposition of resin started, the thermal decomposition ingredient of polyimide clustered, and possibly the structures of the rudder signals of equal interval were generated.

Figure 5 DART-MS system integrated with ionRocket

r.t.

600°C

time[min]1 4

excitation helium

DART ion source

evaporated ingredient

small heating furnace

sample pot

heater

MSspectrometer






Figure 6 DART-MS with ionRocket spectra of each sample

AcknowledgmentWe are deeply grateful to Mr. Yuichi Ishida, Japan Aerospace Exploration Agency (JAXA), offered the CFRP sample used for this experiment.

ConclusionsThe result of having analyzed the carbon fiber plastic CFRP (thermosetting polyimide and thermoplastic polyimide) using DART-MS,

a. residue of the solvent used in fabrication was able to be checked by direct analysis of CFRP by DART. b. analyzing CFRP by DART and the heating option ionRocket, the difference between thermosetting polyimide and thermoplastic polyimide was able to be found out.

Zoom

#4

#4 thermosetting polyimide

#5 thermoplastic polyimide

#5

PO-CON1464E

Analysis of styrene leached from polystyrene cups using GCMS coupledwith Headspace (HS) sampler

ASMS 2014 TP763

Ankush Bhone(1), Dheeraj Handique(1), Prashant Hase(1),

Sanket Chiplunkar(1), Durvesh Sawant(1), Ajit Datar(1),

Jitendra Kelkar(1), Pratap Rasam(1), Nivedita Subhedar(2)

(1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh



(2) Ramnarain Ruia College, L. Nappo Road,

Matunga (E), Mumbai-400019, Maharashtra, India.

2

Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

IntroductionWorldwide studies have revealed the negative impacts of household disposable polystyrene cups (Figure 1) on human health and environment.Molecular structure of styrene is shown in Figure 2. Styrene is considered as a possible human carcinogen by the WHO and International Agency for Research on Cancer (IARC).[1] Migration of styrene from polystyrene cups containing beverages has been observed.[2] Styrene enters into our body through the food we take, mimics estrogens in the

body and can therefore disrupt normal hormonal functions. This could also lead to breast and prostate cancer.The objective of this study is to develop a sensitive, selective, accurate and reliable method for styrene determination using low carryover headspace sampler, HS-20 coupled with Ultra Fast Scan Speed 20,000 u/sec, GCMS-QP2010 Ultra to assess the risk involved in using polystyrene cups.

Figure 1. Polystyrene cup Figure 2. Structure of styrene

Method of Analysis

This study was carried out by extracting styrene from commercially available polystyrene cups and recoveries were established by spiking polystyrene cups with standard solution of styrene. Solutions were prepared as follows,

Extraction of styrene from polystyrene cups

Method was partly validated to support the findings by performing reproducibility, linearity, LOD, LOQ and recovery studies. For validation, solutions of different concentrations were prepared using standard stock solution of styrene (1000 ppm) as mentioned in Table 1.

1) Standard Stock Solution: 1000 ppm of styrene standard stock solution in DMF: Water-50:50 (v/v) was prepared. It was further diluted with water to make 100 ppm and 1 ppm of standard styrene solutions.

2) Calibration Curve: Calibration curve was plotted using standard styrene solutions in the concentration range of 1 to 50 ppb with water as a diluent. 5 mL of each standard styrene solution was transferred in separate 20 mL headspace vials and crimped with automated crimper.

3) Sample Preparation: 150 mL of boiling water (around 100 ºC)[1] was poured into polystyrene cups. The cup was covered with aluminium foil and kept at room temperature for 1 hour. After an hour, 5 mL of sample from the cup was transferred into the 20 mL headspace vial and crimped with automated crimper.

3


HS-GCMS analytical parametersHeadspace parameters

• Sampling Mode : Loop

• Oven Temp. : 80.0 ºC

• Sample Line Temp. : 130.0 ºC

• Transfer Line Temp. : 140.0 ºC

• Equilibrating Time : 20.00 min

• Pressurizing Time : 0.50 min

• Pressure Equilib. Time : 0.10 min

• Load Time : 0.50 min

• Load Equilib. Time : 0.10 min

• Injection Time : 1.00 min

• Needle Flush Time : 10.00 min

• GC Cycle Time : 23.00 min

HS-GCMS Analytical ConditionsFigure 3 shows the analytical instrument, HS-20 coupled with GCMS-QP2010 Ultra on which samples were analyzed with following instrument parameter.

Table 1. Method validation parameters

Figure 3. HS-20 coupled with GCMS-QP2010 Ultra by Shimadzu

Parameter Concentration (ppb)

Linearity

Accuracy / Recovery

Precision at LOQ level

Reproducibility

1, 2.5, 5, 10, 20, 50

2.5, 10, 50

1

50

4


Chromatographic parameters

• Column : Rxi-5Sil MS (30 m L x 0.25 mm I.D., 0.25 μm)

• Injection Mode : Split



• Flow Control Mode : Linear Velocity

• Linear Velocity : 36.3 cm/sec

• Pressure : 53.5 kPa


• Total Flow : 14.0 mL/min



50.0 0.00

40.00 200.0 1.00

30.00 280.0 5.00

Mass Spectrometry parameters

• Ion Source Temp. : 200 ºC

• Interface Temp. : 230 ºC


• Event Time : 0.20 sec

• Mode : SIM

• m/z : 104,103 and 78

• Start Time : 1.00 min

• End Time : 5.00 min

Results

Mass spectrum of styrene is shown in Figure 4. From the mass spectrum, base peak of m/z 104 was used for quantitation where as m/z 103 and 78 were used as reference ions. SIM chromatogram of 50 ppb standard styrene solution

with m/z 104, 103 and 78 is shown in Figure 5.Method validation data is summarized in Table 2. Figures 6 and 7 show overlay of SIM chromatograms for m/z 104 at linearity levels and calibration curve respectively.

Fragmentation of styrene

5


Figure 4. Mass spectrum of styrene

Figure 5. SIM chromatogram of 50 ppb standard styrene solution

Table 2. Validation summary

Summary of validation results

Result

% RSD : 1.74 (n=6)

R2 : 0.9996

LOD : 0.2 ppb

LOQ : 1 ppb

S/N ratio : 38 (n=6)

% RSD : 3.2 (n=6)

Concentration in ppb

50

1 – 50

1 – 50

1

Parameter

Reproducibility (% RSD)

Linearity* (R2)

LOD

LOQ

Precision at LOQ

Compound Name

Styrene

Sr. No.

1

2

3

4

5

45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 105.0 m/z

0

25

50

75

100

Inten.104

103

78

51

52 63 74 8965 985844

85

2.325 2.350 2.375 2.400 2.425 2.450 2.475 2.500 2.525

0.0

2.5

5.0

7.5

(x1,000,000)

78.00 (10.00)103.00 (10.00)104.00 (10.00)

min

* Linearity levels – 1, 2.5, 5, 10, 20 and 50 ppb.


6

Figure 7. Calibration curve for StyreneFigure 6. Overlay of SIM chromatograms for m/z 104 at linearity levels

Figure 8. Overlay SIM chromatograms of spiked and unspiked samples

Table 3. Summary of results for sample analysis

Analysis of leachable styrene from polystyrene cups was done as per method described earlier. Recovery studies were carried out by spiking 2.5, 10 and 50 ppb of standard

styrene solutions in polystyrene cups. Figure 8 shows overlay SIM chromatogram of spiked and unspiked samples. Table 3 shows the summary of results.

Quantitation of styrene in polystyrene cup sample

Sample Name

Unspiked sample

Spiked polystyrene cups

Sr. No.

1

2

Observed Concentration

in ppb

9.8

12.0

18.5

55.9

Parameter

Precision

Recovery

Spiked Concentration

in ppb

NA

2.5

10

50

% Recovery

NA

88.0

87.0

92.2

0 10 20 30 40 Conc.0

250000

500000

750000

1000000

1250000

Area

R2 = 0.9996

2.2 2.3 2.4 2.5 2.6

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x1,000,000)

1 ppb

2.5 ppb

5 ppb

10 ppb

20 ppb

50 ppb

min

m/z : 104.00

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

0.0

2.5

5.0

7.5

(x100,000)

Spiked

Unspiked

m/z : 104.00

min






Conclusion• HS-GCMS method was developed for quantitation of styrene leached from polystyrene cup. Part method validation was

performed. Results obtained for reproducibility, linearity, LOQ and recovery studies were within acceptable criteria.• With low carryover, the characteristic feature of HS-20 headspace, reproducibility even at very low concentration level

could be achieved easily.• Ultra Fast Scan Speed 20,000 u/sec is the characteristic feature of GCMS-QP2010 Ultra mass spectrometer, useful for

quantitation of styrene at very low level (ppb level) with high sensitivity.

References[1] Maqbool Ahmad, Ahmad S. Bajahlan, Journal of Environmental Sciences, Volume 19, (2007), 422, 424.[2] M. S. Taw�ka; A. Huyghebaerta, Journal of Food Additives and Contaminants, Volume 15, (1998), 595.

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