chapter clinical, forensic and pharmaceutical applications

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

• 3Simultaneous 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

• Page 36Single 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 1a,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

Flow rate : 0.474 mL/min

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)

Column temperature : 50 °C

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.

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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.

Materials and Methods

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

Column temperature : 40ºC

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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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

Analytical Conditions

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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ivud

ine

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Telb

ivud

ine-

D3

0 2500 5000 7500 Conc. Ratio0.0

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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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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

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N

O

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O

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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

Nebulizing Gas Flow : 3.0 L/min

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

• Column : Shim-pack XR-ODS (100 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 – 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 (%)


• MS interface : ESI

• 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,


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

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

Mumbai-400059, Maharashtra, India.

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.

chapter clinical, forensic and pharmaceutical applications

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