livre blanc de la spectrométrie de masse introduction methods vulcanization is a cross-linking...

Livre blanc de la

Spectrométrie de MasseSpécial ASMS 2012

«Plus de 30 applications LC-MS/MS, GC-MS/MS, MALDI-TOF...»

©Shimadzu Corporation 2012

Fondé en 1875, Shimadzu est un groupe multinational japo-nais de 3 milliards de dollars côté à la bourse de Tokyo. Avec près de 10000 employé s dans le monde Shimadzu Corpora -tion regroupe trois activités principales : l’instrumentation ana-lytique et physique, le diagnostic médical et l’aéronautique. Présent dans plus de 100 pays, Shimadzu est un fabricant d’ins-trumentation analytique et d’équipement de contrôle environne-mental. En ef fet Shimadzu dispose d’une large gamme d’ins-truments analytiques : Chromatographie liquide et gazeuse, spectrométrie de masse, une gamme complète de Maldi, des robots de préparation, spectrométrie UV-Vis, FTIR, analyse élé-mentaire.

A propos de Shimadzu

Shimadzu Corporation confirme ses capacités d’innovation dans le domaine de la spectrométrie de masse Ultra Rapide en commercialisant simultanément un nouveau GCMS-TQ8030 Triple Quad et deux sys-tèmes LCMS Triple Quad (LCMS-8040 et LCMS-8080).Le lancement de ces trois systèmes pendant l’ASMS-2012 renforce le rôle joué par Shimadzu sur le marché mondial de la spectrométrie de masse. Appelée UFMS (Ultra Fast Mass Spectrometry), cette gamme conjugue haute sensibilité, excellente qua-lité des données et cycles ultra rapides.

Shimadzu, acteur mondial majeur en spectrométrie de masse

Shimadzu est aussi un fabricant mondial de machines de caractérisation de matériaux et d’essais méca-niques. En effet Shimadzu offre une large gamme de machines électromécaniques ou hydrauliques, sta-tiques ou dynamiques, de traction, compression, flexion, pelage, cisaillement fatigue…

Fondé en 1968 en Allemagne, Shimadzu Europe fourni des solutions analytiques aux scientifiques euro-péens. Aujourd’hui Shimadzu Europe représente plus de 9 filiales et 13 distributeurs répartis à travers l’Eu-rope dont Shimadzu France, filiale de plus de 65 personnes en forte croissance depuis sa création en 2002.

GCMS-TQ8030 : Système Triple Quadrupôle

• Mode SCAN / MRM pour éviter les faux positifs/négatifs• Gain x10 en sensibilité par rapport à la GCMS SQ• Permet de suivre 600 transitions MRM/s

LCMS-8030: Triple quadripôle ultra rapide

• Ultra rapide (jusqu’à 500 transitions MRM par seconde)• Cycles d’acquisition du signal ultra rapide (<2 ms)• Pas de compromis entre vitesse, sensibilité et robustesse

LCMS-8040: Encore plus de sensibilité

• Evolution du LCMS-8030 pour plus de sensibilité• Plateforme idéale pour les applications « Qual/Quant »,• Ultra rapide (jusqu’à 555 transitions MRM par seconde)

Spectromètres de masse Shimadzu utilisés dans ce livre

LCMS-IT-TOF: MSn à haute résolution

• Couplage unique Ion Trap - TOF• Haute résolution & précision de masse < 5 ppm• Changement de polarité 100 ms

LCMS-8080: Système LC-MS/MS le plus sensible

• Nouvelle source d’ionisation • Nouvelle interface qui réduit le bruit de fond chimique• Robustesse exceptionnelle

http://www.shimadzu-france.com/fr/?q=GCMS-Triple-Quad

http://www.shimadzu-france.com/fr/?q=LCMS-Triple-Quad



http://www.shimadzu-france.com/fr/?q=LCMS-IT-TOF

PAGE TECHN. APPLICATION5 LCMS-IT-

TOFDifferential Analysis in vulcanizing accelerators for rubber products by High mass Accuracy MSn and Multivariate Statistical Technique

10 LC-MS/MS Multi-component quantitative analysis of pharmaceuticals and personal care products in the environment by LC-MS/MS with fast polarity switching

16 LC-MS/MS Evaluation of the higher sensitive LC/MS/MS incorporates novel desolvation technologies to achieve low femto-gram LOQ

21 LC-MS/MS Multi-class pesticides analysis in challenging vegetable matrices using fast 5 msec MRM with 15 msec polarity switching

26 LC-MS/MS Exploring the application of a universal method for pesticide screening in foods using a high data acquisition speed MS/MS

31 LC-MS/MS Rapid and Highly Sensitive Quantitative Analysis and Screening of Aflatoxins in Foods Using Liquid Chromatography Triple Quadrupole Mass Spectrometry

39 LC-MS/MS High throughput analysis of anion surfactant using ultra-high speed LC-MS/MS and 1 mm inside diameter column

45 LC-MS/MS Simultaneous analysis of anionic, amphoteric and non-ionic surfactants using ultra-high speed LC-MS/MS

51 LC-MS/MS Screening analysis for drugs of abuse by LC-MS/MS enables fast polarity switching MRM triggered product ion scanning on the fly

57 GC-MS/MS Comprehensive Two-dimensional Gas Chromatograph Quadruple Mass Spectrometer for PlantMetabolite Analysis

62 LCMS-IT-TOF

Analysis of degradation products in electrolyte for rechargeable lithium-ion battery through high mass accuracy MSn and multivariate statistical technique

67 LCMS/MS Sensitive detection and quantification of hydrogen sulfide as a gasotransmitter by combi-ning monobromobimane-based derivatization to triple quadrupole LC/MS/MS

72 MALDI-TOF Metabolite maps reconstructed using a quantitative data by capillary electrophoresis-mass spectrometry (CE-MS)

77 LC-MS/MS Quantitative analysis of hydrophilic metabolite using ion-paring chromatography with a high-speed triple quadrupole mass spectrometer

83 LC-MS-IT-TOF

Analysis of trace amount of 17-β-Estradiol and its metabolites in aqueous samples using online-SPE and accurate MSn analysis

88 LCMS/MS Analysis of phthalate esters in environmental water samples by online-SPE-LC coupled with high-speed triple quadruple mass spectrometer

93 LC-MS/MS Development of an ion transmission enhanced tandem ion guide system for triple quadruple mass spectrometer

98 LC-MS/MS High Throughput Quantitative Analysis of Multi-mycotoxin in Beer-based Drinks using UH-PLC-MS/MS

104 DART/MS High throughput molecular weight confirmation of Pharmaceutical Compounds using DART MS analysis with ultra-fast polarity switching

110 DART/MS Componential analysis of pepper of various origins using DART-MS using ultra-fast polarity switching

116 LC-MS/MS Identification of the modified amino acid residue in the modified heme protein using LC/MS/MS

121 LC-MS/MS Identification of triazolam, etizolam and their metabolites in biological samples by liquid chromatography tandem mass spectrometry

127 LCMS-IT-TOF

Development of an LC-Based Metabolomic Approach for Polar Compounds in Brewage Samples using Fast Polarity Switching TOFMS Acquisition

134 MALDI-TOF Differentiation of isobaric residues in SPITC-derivatized tryptic peptides using MS/MS tech-nique in a Curved Field Reflectron.

Takahiro Goda, Hiroki Nakajima, Satoshi Yamaki,

Tsutomu Nishine, Masaru Furuta, Naoki Hamada

Shimadzu Corporation, Kyoto, JAPAN

Differential Analysis in vulcanizing accelerators for rubber products by High mass Accuracy MSn and Multivariate Statistical Technique

ASMS 2012 ThP21 - 455

2

Introduction

Methods

Vulcanization is a cross-linking reaction for forming bridges between individual polymer chains via addition of sulfur (Fig. 1). The purpose of vulcanization is to convert the rubber into a more durable material. In general, a reaction rate of vulcanization is increased by adding vulcanizing accelerator to mixture of rubber and sulfur.Each tire manufacturer employs one particular accelerator from among many commercially available reagents in order to meet set standards .Therefore, analyzing vulcanizing

accelerator compounds is a key stage in the tire manufacturing process, however, it is difficult to detect differences in similar structures when produced by different manufacturers. In this study we show differential analysis of similar structured sulfenamide-based vulcanizing accelerators (Fig. 2) produced by different manufacturers using high mass accuracy MSn and multivariate statistical technique.

Five N-(tert-butyl)-2-benzothiazole sulfenamide (NS; NS-1,

NS-2, NS-3, NS-4, NS-5) and five

N-cyclohexyl-2-benzothiazole sulfenamide (CZ; CZ-1, CZ-2,

CZ-3, CZ-4, CZ-5) were used in this study. Each NS and

each CZ were produced by different manufacturers.

Sample solutions were prepared at 100 mg/L dissolved in

tetrahydrofuran and acetonitrile. Equal amount of NS

solutions were mixed and used as a quality control (QC)

sample for NS analysis to identify robust and reproducible

ion signals. The QC sample for CZ analysis also was

prepared by the same method. LCMS measurement was

performed by LCMS-IT-TOF (Shimadzu Corporation, Kyoto,

Japan). SIMCA-P+ (Umetrics) and MetID Solution (Shimadzu

Corporation) were used for multivariate statistical analysis

and for searching structural analogues using MSn data

acquired by LCMS-IT-TOF measurement, respectively.

Formula Predictor (Shimadzu Corporation) was used for

predicting the formulae of characteristic compounds (Fig. 3).

Fig. 3 Work flow of the analysis of vulcanizing accelerators.

Fig. 2 Typical sulfenamide-based vulcanizing accelerators.


Profiling Solution (SHIMADZU)

Automatic construction of peak matrixHigh mass accuracy MSn data

LCMS-IT-TOF (SHIMADZU)

Searching out statistical analogues

MetID Solution (SHIMADZU)

Formula prediction

Structural analogues of main compound which are characteristic to each sample

Multivariate analysis

SIMCA-P+ (Umetrics)

Formula Predictor(SHIMADZU)

cross-linking point

N-(tert-butyl)-2-benzothiazole sulfenamide

N, N'-Dicyclohexyl-2-benzothiazolyl sulfenamide

N-cyclohexyl-2-benzothiazole sulfenamide

N-Oxydiethylene-2- benzothiazole sulfenamide

polymer chains

vulcanizationvulcanization

N

S

S NH

N

S

S NH CH3

CH3

CH3

N

S

S NN

S

S N O

3


Results and discussionAs a result of principal component analysis (PCA) for NS, the groups of each sample type were located at the different sites on the score plot (Fig. 4a) showing that they were comprised of different components. The unique peaks of each sample were observed on the loading plot (Fig. 4b). Candidates of the structural analogues of NS were

identified using unique peaks based on fragment ions and neutral losses (Fig. 5).The extracted ion chromatograms (EICs) suggested that these were characteristic components of each sample (Fig. 6). By the same method, each sample of CZ was identified as containing characteristic components.

Table 1 LCMS analytical conditions.

Fig. 4 Result of PCA for NS (a: score plot, b: loading plot). Fig. 6 EICs of characteristic peaks of each samples.

Fig. 5 Analysis window of MetID Solution.

Column Flow rate Column temperature Mobile phaseA Mobile phaseB Time program Injection volume

Ionization modeProbe voltage CDL temperature BH temperature Nebulizing gas flow Drying gas pressureScan range

: Shim-pack XR-ODS (2.0 mmI.D.x75 mmL、 2.2 mm) : 0.45 mL/min : 40°C: water containing 5 mmol ammonium acetate: acetonitrile : 0%B(0 min) – 100%B(9 – 12 min) – 0%B(12.01 – 15 min) :1 mL

: ESI(+) : 4.5kV : 200°C : 200°C : 1.5 L/min : 0.1 MPa : m/z 100 - 1000

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 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000,000)

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 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000,000)

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 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000,000)

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 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000,000)

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 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000,000)

507.0859 (25.00) 372.0657 (50.00) 404.0403 (25.00) 253.0828 (25.00)

358.1076 (10.00) 300.9922 (25.00) 183.9885 (50.00) 271.0569 (50.00) TIC (1.00)

NS -1

NS -2

NS -3

NS -4

NS -5

No. 1

No. 7

No. 3

No. 5

No. 6

507.0859 (25.00) 372.0657 (50.00) 404.0403 (25.00) 253.0828 (25.00)

358.1076 (10.00) 300.9922 (25.00) 183.9885 (50.00) 271.0569 (50.00) TIC (1.00)

507.0859 (25.00) 372.0657 (50.00) 404.0403 (25.00) 253.0828 (25.00)

358.1076 (10.00) 300.9922 (25.00) 183.9885 (50.00) 271.0569 (50.00) TIC (1.00)

507.0859 (25.00) 372.0657 (50.00) 404.0403 (25.00) 253.0828 (25.00)

358.1076 (10.00) 300.9922 (25.00) 183.9885 (50.00) 271.0569 (50.00) TIC (1.00)

507.0859 (25.00) 372.0657 (50.00) 404.0403 (25.00) 253.0828 (25.00)

358.1076 (10.00) 300.9922 (25.00) 183.9885 (50.00) 271.0569 (50.00) TIC (1.00)

-18000000

-16000000

-14000000

-12000000

-10000000

-8000000

-6000000

-4000000

-2000000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

-25000000 -20000000 -15000000 -10000000 -5000000 0 5000000 10000000 15000000 20000000 25000000

t[2]

t[1]

NS_1000ppm_20110901_01.M1 (PCA-X)t[Comp. 1]/t[Comp. 2]

R2X[1] = 0.566677 R2X[2] = 0.270169 Ellipse: Hotelling T2 (0.95)

nsa01nsa02

nsa03

nsb04nsb05nsb06 nsc07

nsc08

nsc09nsd10nsd11

nsd12

nse13nse14nse15

nsmix16nsmix17nsmix18

NS-2

NS-1

NS-3 NS-4

NS-5

QA/QC

a

-0.2

-0.1

-0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

-0.2 -0.1 -0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

p[2]

p[1]

NS_1000 ppm_20110901_01.M1 (PCA-X)p[Comp. 1]/p[Comp. 2]Colored according to model terms

R2X[1] = 0.566677 R2X[2] = 0.270169

100.063100.071100.081100.101100.09100.091100.108100.11100.119100.114100.11100.998101.049101.069101.052101.069101.073101.109102.038105.035107.056107.059107.061107.074107.968107.998108.007108.002108.009108.009108.014108.014108.051108.077108.071108.076108.993109.003109.009109.101110.012111.055111.061111.114111.116112.038112.044112.049112.053112.05112.049112.056112.089112.113113.045113.097113.137114.079114.082114.086115.023115.043115.057115.061115.064115.074115.103115.114115.122115.117116.044116.054116.053116.061116.058116.067117.08117.099118.046118.057118.085118.088119.055119.061119.085119.082119.098120.074120.075121.021121.059121.089121.099121.097121.995121.996122.01122.026122.059122.097123.001123.017123.061123.08124.028124.027124.036124.038124.046124.072124.077124.081124.085124.093124.093125.088125.087125.112126.062126.069127.039127.047127.047127.069127.058127.062127.072127.07127.078128.046128.045128.051128.058128.064128.076128.077128.094129.046129.054129.059129.062129.086129.1130.036130.046130.049130.061130.063130.076130.083130.098130.093130.093130.106130.135130.157130.151130.16130.166131.053131.059131.089131.102132.037132.055132.055132.102132.101132.103132.114132.987132.993132.998133.053133.052133.056133.066133.061133.062133.063133.071133.07133.076133.081133.085133.095133.104134.006134.075135.039135.097135.122136.021136.02

136.021136.021136.031136.044136.062136.065136.057136.067136.08136.109136.097137.018137.058137.057137.064137.086137.096138.065138.055138.061138.097138.11138.123138.141138.144139.025139.048139.049139.047139.085139.075139.08139.108139.122139.118139.115139.141139.137140.001140.005140.014140.016140.035140.101140.963141.072141.07141.089141.079141.1141.09141.109141.123141.121141.124141.126141.983141.986142.087142.099142.101142.108143.048143.06143.048143.059143.063143.061143.069143.072143.077143.077143.082143.125143.127144.001143.996144.068144.068144.07144.069144.1144.956144.979144.981144.98144.984145.075145.059145.079145.082145.08145.077145.073145.083145.085145.083145.12145.108145.111145.113145.12145.12145.131145.122145.132146.067146.08146.099146.101146.101146.974146.985146.984146.987147.096147.077147.073147.101147.1147.111147.112148.044148.094148.113148.124149.019149.017149.018149.028149.027149.033149.028149.025149.082149.075149.072149.086149.098149.109149.109149.12149.123149.145150.053150.072150.087150.092150.089150.118150.118150.128150.14151.03151.026151.026151.046151.058151.081151.096151.096151.113151.117152.013152.034152.045152.05152.051152.052152.06152.05152.057152.119152.131153.008152.998152.999153.019153.021153.086153.095153.133153.132153.142153.995154.001154.006154.067154.078154.091154.08154.08154.083154.093155.082155.07155.083155.083155.101155.113155.967155.982156.041156.058156.074156.073156.085156.1156.099156.112156.118157.084157.079157.066157.074157.074157.09157.093157.088157.096157.093157.117157.098157.127157.133157.136157.133157.143157.138157.144157.993158.078158.097158.101158.105158.114158.118158.125158.154158.189159.047159.097159.091159.109159.107159.132159.969161.111161.112162.06162.079162.097162.114162.124163.043163.047163.08163.077163.093163.1163.112163.123163.117163.133163.136163.132163.135163.134164.089164.095164.115164.119165.049165.05165.05165.058165.054165.068165.077165.079165.1165.099165.091165.104165.119165.115165.119165.124165.133165.979165.97165.978166.067166.083166.066166.103166.112166.116166.115166.98166.988166.983166.986167.03167.043167.064167.067167.077167.087167.09167.105167.109167.13167.13167.155167.986167.979167.995167.987167.989167.991168.053168.084168.096168.092168.122168.111168.127168.14168.148168.97168.984169.077169.084169.09169.099169.112169.104169.11169.11169.142170.072170.093170.085170.115170.125170.129171.078171.085171.084171.106171.122171.111171.11171.112171.156171.151171.15171.148171.14171.151171.156171.152171.147171.166171.173172.086172.089172.08172.092172.105172.109172.119172.111172.114172.131172.134172.128172.152172.151172.149172.161172.171173.071173.074173.105173.095173.122173.126173.152173.149173.984173.995174.036174.046174.053174.102174.095174.095174.133175.035175.04175.078175.09175.084175.111175.111175.111175.116175.122175.135176.026176.024176.056176.105176.125176.127176.115176.13176.144176.942177.056177.074177.061177.068177.114177.097177.121177.117177.107177.129177.124177.145178.028178.031178.068178.101178.122178.126178.128178.132178.154178.156178.168178.177178.17179.051179.074179.083179.095179.124179.111179.124179.117179.124179.127179.121179.142179.146179.161180180.041180.084180.083180.076180.086180.095180.107180.096180.096180.108180.121180.12180.158180.134180.158180.158180.171180.174180.176180.984180.983180.993181.027181.039181.036181.096181.099181.128181.125181.125181.15181.165181.166181.217181.229181.971181.973181.978181.998182.02182.044182.081182.093182.082182.095182.124182.123182.133182.147182.143182.19182.192182.742182.991183.008183.011183.077183.079183.091183.12183.092183.119183.12183.134183.151183.128183.144183.196183.198183.203183.259183.803

183.989183.98183.995184.012184.012184.064184.07184.111184.131184.34184.994185.001185.008185.003185.012185.028185.011185.02185.045185.083185.097185.109185.123185.131185.133185.135186.054186.075186.076186.094186.133186.129186.143186.979187.061187.074187.109187.085187.122187.139187.138188.039188.055188.044188.059188.089188.097188.104188.129188.13188.137188.14188.148188.149188.193189.045189.053189.054189.055189.115189.125189.124189.137189.135189.144189.153190.055190.054190.079190.075190.122190.119190.126190.126190.117190.13190.16190.951191.055191.08191.092191.084191.115191.096191.123191.111191.108191.136191.149191.164191.176191.991192.095192.107192.127192.12192.116192.133192.132192.137192.177192.173192.17193193.089193.087193.092193.104193.103193.122193.111193.132193.137193.131193.142193.146193.147193.149193.152193.169193.158194.023194.04194.107194.092194.106194.104194.119194.116194.123194.119194.124194.138194.135194.132194.173195.035195.074195.081195.098195.09195.087195.12195.12195.119195.122195.135195.146195.151196.005196.019196.018196.057196.08196.089196.099196.125196.132196.14196.151196.147196.136196.147196.159196.161196.983197.023197.082197.094197.097197.11197.123197.129197.134197.161197.199198.007198.038198.065198.065198.105198.109198.123198.12198.193198.952198.999198.997199.005199.003199.002199.002199.006199.008199.017199.019199.035

199.094199.094199.104199.122199.105199.178199.184199.178199.175199.182199.206199.631199.985200.005200.012200.009200.029200.05200.08200.083200.074200.127200.13200.118200.105200.12200.159200.16200.135200.147200.157200.158200.166200.193201.012201.063201.058201.131201.165202.072202.072202.071202.085202.109202.134202.135202.148202.169202.193202.992203.029203.059203.079203.108203.111203.107203.139203.132203.158203.162204.001204.065204.093204.08204.089204.085204.089204.111204.118204.098204.128204.154204.167204.172204.99205.087205.067205.085205.096205.085205.077205.087205.101205.103205.115205.142205.154205.164206.051206.062206.066206.067206.121206.148206.146206.141206.15206.151206.162206.175206.175206.191206.987207.007206.998207.006207.008207.011207.087207.095207.097207.115207.123207.12207.129207.146207.164207.154207.171207.153207.17207.184207.194207.198207.982208.002208.008208.014208.014208.085208.086208.122208.113208.126208.134208.132208.15208.124208.152208.182209208.984208.987208.996209.012209.025209.111209.116209.12209.143209.142209.142209.149209.167209.166209.193210.075210.103210.107210.104210.132210.107210.139210.161210.146210.149210.181210.166210.169210.177210.199210.942210.988211.053211.111211.124211.113211.129211.123211.131211.15211.148211.188212.083212.114212.134212.142212.144212.157212.928212.94212.957213.085213.096213.089213.091213.101213.093213.107213.108213.107213.12213.128213.121213.14213.153213.191213.191214.064214.119214.111214.137214.168214.177214.992214.992214.995215.091215.093215.101215.097215.104215.116215.13215.136215.16215.181

215.977215.976216.059216.071216.09216.105216.105216.122216.157217.104217.083217.12217.104217.15217.127217.17217.15217.156217.144217.158217.186217.182217.187217.974218.003218.104218.11218.106218.106218.142218.149218.162218.172218.193218.163218.183218.177218.177218.198218.191219.028219.098219.121219.102219.123219.12219.132219.129219.158219.156219.174219.191219.191219.198219.212220.066220.08220.109220.108220.094220.097220.108220.122220.127220.152220.155220.162220.155220.179221.016221.024221.055221.063221.085221.077221.113221.078221.117221.164221.158221.162222.047222.087222.072222.108222.105222.137222.121222.137222.118222.132222.153222.162222.178222.217223.036223.048223.064223.091223.098223.108223.083223.096223.095223.148223.147223.16223.17224.024224.042224.064224.065224.07224.086224.122224.136224.132224.173225.02225.037225.101225.084225.093225.09225.094225.14225.132225.128225.118225.149225.164225.177225.179

225.194

225.195225.203225.214226.004226.037226.043226.092226.141226.156226.169226.178226.183226.219226.221227.052

227.064

227.094227.063227.071227.091227.118227.124227.14227.146227.956228.062228.089228.113228.112228.105228.143228.16228.166228.184228.183228.192228.187228.19228.232228.227228.266228.956

229.019229.06229.062229.089229.093229.124229.142229.148229.172229.194230.068230.089230.109230.132230.123230.154230.149230.138230.139230.179230.167230.196230.21230.956231.073231.07231.091231.113231.1231.113231.142231.117231.157231.158232.057232.077232.132232.142232.144232.137232.163232.178232.199232.969 233.004233.015233.028233.086

233.111

233.127233.123233.144233.145233.995234.026234.068234.085234.138234.132234.135234.131234.176234.176234.187234.949235.001235.114235.125235.124235.114235.126235.156235.147235.174235.155235.167235.173235.195235.185235.21235.229236.014236.016236.021236.004236.012236.008236.024

236.02

236.02

236.024236.009236.023236.032236.026236.026236.03236.062236.098236.11236.126236.15236.163236.171236.995237.002237.027237.024237.052237.056237.109237.124237.127237.142237.136237.15237.18237.192238.034238.021238.019238.023238.059238.082238.077238.077238.109238.112238.148238.139238.186238.637238.775238.841238.961238.983239.146239.193239.216239.962239.96240.004240.017240.068240.069240.073240.073240.169240.168240.167240.174240.174240.192240.207240.218240.303240.372240.917240.983240.981240.986241.06241.06241.069241.131241.13241.159241.165241.186241.176241.182241.193241.182241.187241.194241.209241.26241.343242.063242.07242.076242.086242.101242.102242.164242.202242.18242.195243.067243.063243.084243.083243.095243.09243.11243.122243.133243.149243.135243.158243.163243.165243.195243.207243.202244.065244.095244.111244.126244.123244.159244.172244.183244.199244.215244.238245.08245.099245.101245.117245.122245.161245.174245.187245.208245.21245.234245.233246.063246.079246.096246.087246.101246.108246.106246.129246.137246.141246.167246.179246.165246.181246.186246.201246.21247.019247.021247.089247.114247.152247.144247.18247.178247.176247.186247.193247.203247.204248.065248.101248.144248.14248.159248.157248.171248.19248.187248.212249.083249.107249.081249.1249.129249.139249.148249.168249.201249.201249.211249.208249.209249.213249.502250.097250.12250.123250.174250.202250.17250.178250.179250.191250.195251.128251.135251.128251.155251.159251.175251.185251.174251.181251.186251.277251.968252.013252.026252.092252.084252.084252.105252.101252.12252.126252.161252.16252.191252.202252.204252.206252.956252.999253.044253.029253.057253.065253.07

253.082

253.088253.1253.105253.112253.111253.13253.133253.134253.15253.148253.161253.178253.169253.166253.926253.942253.995

254.031

254.029253.994254.012254.032254.018254.01254.052254.048254.037254.059254.083254.171254.174254.205254.225254.224254.905254.933254.951255.031255.029255.035

255.062

255.049255.054255.062255.072255.087255.076255.149255.189255.195255.2255.236255.946255.953255.967255.986255.994256.01256.063256.079256.097256.177256.191256.189256.267256.268256.258257.069257.077257.075257.084257.107257.114257.119257.155257.162257.155257.16257.179257.189257.191257.196257.258257.281258.027258.035258.077258.102258.101258.107258.146258.155258.161258.224258.224258.216258.236258.983259.008259.037

259.036259.034259.043259.071259.12259.156259.198259.935259.963260.04260.063260.075260.102260.171260.173260.186260.95260.962261.026261.019261.037261.081261.092261.122261.128261.117261.133261.148261.148261.166261.169261.195261.211261.214261.229262.048262.084262.146262.146262.165262.172262.188262.192262.206263.022263.018263.066263.069263.068263.09263.09263.113263.109263.139263.136263.152263.157263.203263.229263.247264.072264.074264.087264.088264.133264.145264.186264.172264.224264.197264.227264.229264.226264.242264.992265265.083

265.083

265.082265.163265.204265.233265.234265.233265.233265.369265.951266.028266.092266.187266.227266.206266.236266.234266.231266.237266.472266.998267.095267.083267.111267.089267.136267.143267.154267.15267.186267.165267.167267.228267.184267.21267.209267.242267.252267.252267.267267.246267.244267.26267.262267.997268.055268.06268.093268.065268.075268.1268.1268.106268.109268.095268.105268.13268.132268.178268.182268.17268.198268.217268.974268.995269.017269.018

269.028269.02269.019269.099269.088269.082269.152269.167269.132269.166269.199269.192269.217269.216269.26269.255269.257269.953270.001270.018269.998270.023270.027270.029270.032270.053270.136270.168270.174270.188270.188270.2270.207270.202270.233270.248270.977270.977271.005271.01

271.038271.029

271.056

271.057

271.057271.058

271.099271.117271.131271.148271.159271.147271.146271.161271.167271.228271.214271.225271.254271.934271.963271.97272.103272.105272.129272.142272.161272.151272.157272.184272.19272.188272.237272.269272.959273.01273.051273.094273.125273.112273.185273.199273.238273.977274.035274.041274.092274.204274.187274.938275.06275.133275.175275.155275.16275.181275.193275.191275.236275.254275.268275.929275.938275.953276.031276.035276.077276.147276.24276.24276.272276.922277.078277.053277.083277.1277.085277.161277.197278.07278.12278.108278.124278.155278.194278.188278.2278.206278.224278.237278.239278.252278.252279.074279.095279.094279.099279.092279.096279.137279.124279.13279.159279.138279.147279.161279.26279.421279.942280.005280.049280.091280.091280.095280.158280.157280.168280.169280.17280.265280.255280.275280.966281.064

281.079

281.151281.114281.11281.118281.118281.127281.151281.224281.232281.254281.233281.232281.245281.26281.28282.111282.105282.121282.169282.223282.224282.219282.224282.231282.234282.25282.247282.28282.257282.287282.298283.035283.035283.082283.141283.164283.173283.187283.183283.198283.196283.211283.261283.263283.268283.266284.041284.063284.09284.134284.122284.156284.192284.212284.219284.245284.243284.245284.289284.286284.293284.307284.931284.963

285.02

285.036285.054

285.13285.077285.124285.156285.125285.142285.168285.165285.179285.204285.197285.188285.188285.215285.223285.225285.24285.275286.018

286.016

286.019

286.051286.061286.065286.098286.124286.13286.179286.159286.147286.185286.168286.179286.229286.246286.244286.988286.977287.077287.082287.085287.127287.143287.163287.192287.204287.222287.236287.247287.256287.251288.051288.097288.113288.127288.132288.146288.161288.16288.164288.19288.252288.221288.25288.249288.263288.276288.981288.996289.081289.104289.1289.14289.159289.169289.189289.211289.205289.253289.241289.245289.242289.261289.251289.254290.103290.154290.16290.183290.168290.176290.199290.25290.252290.908291.008291.027291.031291.079291.105291.105291.126291.152291.195291.228291.217291.918291.917291.99291.992292.007292.03292.105292.134292.124292.159292.18292.202292.242292.249292.247292.263292.289292.904292.903292.923292.915293.069293.083293.103293.123293.133293.133293.149293.16293.176293.18293.192293.227293.215293.249293.901293.971293.975293.963294.02294.102294.119294.18294.168294.217294.204294.222294.221294.242294.265294.277294.91294.912294.967294.979295.076295.091295.127295.123295.166295.181295.156295.207295.181295.221295.185295.215295.189295.229295.239295.226295.236295.241295.238295.982296.074296.096296.097296.115296.158296.185296.205296.198296.214296.213296.228296.228

297.063

297.081297.146297.159297.126297.168297.181297.158297.198297.238297.225297.236297.245297.239297.252297.265297.963297.997298.063298.104298.117298.126298.144298.197298.179298.217298.251298.259298.971299.06299.097299.102299.149299.17299.156299.189299.176299.18299.205299.197299.239300.109300.182300.186300.22300.253300.269300.304300.303300.99

300.991

300.995 300.99

300.993

300.993300.996301.049301.102301.146301.135301.156301.149301.158301.168301.188301.226301.244301.24301.295301.995302.045302.023302.039302.034302.164302.133302.165302.167302.228302.279302.986303.024303.029303.062303.104303.168303.202303.228303.238303.245303.995303.99303.989304.044304.034304.071304.066304.087304.121304.145304.142304.18304.175304.174304.171304.171304.192304.203304.224304.239304.248304.258305.009305.019305.025305.103305.109305.113305.142305.159305.242305.243305.242305.247305.271305.277305.276305.293306.131306.168306.162

306.271

306.263306.265306.267306.265306.534307.082307.142307.155307.21307.26307.259307.243307.26307.271307.262307.269307.277307.295307.999

308.002307.993308.092308.084308.12308.162308.193308.182308.206308.205308.232308.238308.263308.262308.261308.268308.957309.119309.092309.109309.145309.184309.176309.188309.209309.193309.226309.211309.229309.234309.239309.243309.255309.275310.016310.018310.026310.089310.126310.134310.158310.147310.181310.172310.203310.198310.211310.225310.249310.268310.279310.276310.307311.149311.118311.152311.148311.169311.192311.239311.238311.275311.972311.996311.999312.161312.203312.207312.235312.238312.237312.242313.093313.133313.128313.137313.169313.205313.187313.215313.236313.254313.258313.899313.973314.1314.131314.136314.185314.201314.201314.215314.205314.222314.245314.263314.258314.266314.239314.285314.307314.902314.972315.018315.07315.085315.098315.104315.159315.126315.15315.158315.158315.172315.178315.193315.209315.961316.061316.118316.108316.163316.182316.189316.214316.249316.269316.283316.987316.995317.063317.076317.109317.146317.138317.176317.183317.241317.922317.947318.076318.096318.12318.158318.145318.177318.187318.167318.193318.179318.205318.215318.261318.264318.284319.005319.002319.026319.017319.034319.075319.086319.117319.122319.138319.15319.207319.227319.247319.26319.997320.081320.161320.16320.169320.184320.274320.232320.252320.269321.094321.192321.182321.18321.197321.262321.236321.248321.258321.247321.268321.272321.95322.096322.149322.173322.23322.267322.303322.995322.991323.073323.125323.152323.179323.169323.211323.195323.245323.242323.261323.292323.28323.278323.269323.279324.07324.079324.126324.126324.126324.159324.227324.231324.254324.246324.253324.269324.277324.286324.298325.074325.102325.145325.145325.172325.163325.177325.183325.202325.214325.232325.232325.29325.286325.291325.294325.315325.914326.085326.133326.131326.176326.202326.223326.255326.237326.259326.274326.276326.301327.008327.038327.061327.088327.075327.077327.117327.09327.099327.147327.194327.154327.172327.231327.222327.238327.291327.283328.112328.165328.153328.212328.214328.245328.248328.248328.295328.296328.322328.932328.998328.992329.005329.08329.1329.104329.132329.152329.151329.16329.19329.26329.274329.944330.151330.159330.177330.167330.184330.289330.937330.946330.954330.978331.076331.095331.16331.186331.213331.247331.261331.273331.987332.14332.199332.187332.233332.276332.301332.82332.857332.873332.961

332.964332.967332.967333.099333.275333.973334.214334.92334.936334.951334.974334.967335.124335.163335.165335.183335.197335.213335.241335.223335.24335.243335.905335.934335.933335.963336.093336.111336.218336.224336.205336.22336.957337.135337.207337.236337.25337.224337.274337.288337.932337.946338.051338.139338.213338.255338.257338.266338.257338.279338.921338.898338.924338.934339.009339.146339.147339.154339.154339.19339.214339.263339.279339.273339.587340.032340.025340.067340.086340.102340.118340.148340.153340.164340.176340.274340.286340.279340.9340.942341.029341.073341.098341.14341.193341.201341.184341.214341.241341.261341.286341.292341.286341.288341.304341.303341.931342.043342.026342.031342.099

342.111342.143342.142342.156342.226342.286342.261342.295342.275342.29342.315343.037343.115343.126343.121343.133343.131343.169343.203343.211343.225343.232343.253343.235343.288343.312343.314343.301343.305343.309343.647344.026344.02344.043344.083344.077344.107344.109344.109344.169344.214344.22344.296344.306344.303344.318344.322345.109345.173345.172345.19345.208345.223345.239345.257345.249345.322346.098346.154346.146346.167346.176346.191346.196346.214346.212346.236346.229346.254346.229346.274346.285346.299346.976346.98346.977346.98347.104347.127347.145

347.158

347.172347.194347.247347.247347.265347.311347.953347.979348.109348.134348.165348.174348.227348.278348.267348.303348.957348.936348.955349.073349.111349.159349.163349.196349.212349.205349.216349.229349.267349.297349.949349.954350.168350.203350.221350.221350.272350.239350.25350.246350.257350.28350.324350.293350.296350.31350.32350.877350.948351.116351.162351.137351.137351.157351.2351.229351.235351.242351.256351.263351.301351.319351.941351.948351.96352.122352.144352.162352.198352.246352.262352.253352.278352.298352.27352.341352.345352.335352.624352.879352.911352.908352.931353.135353.139353.167353.249353.255353.259353.248353.278353.259353.282353.264353.326353.338353.346353.34353.365353.909353.96353.951354.08354.097354.126354.126354.219354.248354.25354.252354.251354.299354.307354.288354.289354.938355.059355.072355.082355.086355.099355.178355.227355.179355.196355.269355.27355.278355.277355.275355.297355.934355.94355.966355.964355.959356.169356.179356.196356.22356.22356.269356.254356.259356.259356.29356.328356.953357.094357.095357.083357.128357.146357.192357.187357.188357.237357.305357.938357.927357.936357.947

358.107

358.104358.092358.108358.145358.157358.21358.201358.283358.255358.287359.121359.181359.183359.203359.184359.207359.239359.293359.281359.301359.314359.307359.327360.071360.135360.248360.251360.256360.273360.301360.904361.105361.103361.131361.159361.196361.224361.211361.245361.243361.266361.244361.251361.311361.304361.312361.273361.318361.313361.31362.081362.099362.24362.272362.3362.261362.289362.338362.92362.927362.934363.09363.147363.281363.273363.267363.294363.259363.286363.317363.281363.906364.119364.159364.214364.264364.279364.283364.281364.269364.29364.296364.342364.348365.115365.178365.168365.192365.185365.225365.205365.201365.208365.269365.305365.298365.29366.097366.114366.129366.167366.177366.201366.216366.211366.233366.255366.267366.308366.299366.314366.983367.085367.116367.184367.198367.219367.232367.241367.232367.29367.324367.323367.965368.134368.145368.16368.172368.177368.202368.213368.207368.271368.253368.278368.286368.322368.886369.145369.206369.184369.245369.221369.238369.259369.289369.269369.314369.351369.906369.961370.08370.143370.206370.221370.215370.247370.297370.316370.307370.321370.306370.89370.915370.972370.975371.001371.061371.102371.102371.102371.106371.205371.209371.212371.254371.247371.274371.253371.333371.288371.309371.251371.317371.298371.317371.329371.323371.949371.945371.961371.984372.064372.067372.095372.091372.112372.192372.245372.222372.884372.958372.942373.086373.09373.116373.15373.152373.175373.211373.231373.226373.223373.22373.266373.279373.277373.303373.952373.943374.073374.1374.103374.11374.119374.19374.18374.212374.24374.254374.31374.862375.063375.09375.09375.117375.178375.167375.196375.211375.24375.263375.242375.304375.337375.896375.951376.061376.101376.083376.155376.232376.256376.271376.284376.339377.085377.13377.167377.2377.235377.307377.308377.31377.899377.902377.93378.167378.237378.3378.255378.318378.315378.333378.336378.333378.914378.941378.981379.167379.235379.275379.305379.313379.271379.331379.316379.312379.886379.941380.191380.249380.949381.08381.136381.115381.169381.177381.163381.213381.214381.229381.288381.306381.302381.332381.352382.064382.195382.233382.232382.281382.317382.949383.137383.166383.218383.301383.989384.128384.129384.175384.181384.182384.288384.294384.305385.117385.141385.227385.226385.229385.321385.34385.331385.993386.091386.161386.197386.241386.272386.238386.261386.267386.332386.348386.991387.086387.172387.171387.208387.24387.278387.309388.061388.133388.134388.19388.199388.25388.281388.29388.282388.286388.34388.88388.886388.943388.963388.962388.994389.176389.196389.216389.218389.256389.255389.273389.283389.294389.321389.306389.9389.92389.978390.018390.064390.179390.246390.303390.282390.35390.926390.916390.944390.969391.092391.181391.171391.203391.22391.283391.283391.308391.874391.939391.954391.941391.978392.023392.14392.285392.287392.293392.299392.298392.314392.302392.299392.332392.362392.549392.578393.106393.119393.166393.27393.292393.285393.251393.251393.25393.296393.315393.324393.301393.319393.912393.947394.218394.213394.237394.256394.301394.328394.277394.345394.341394.341394.357394.364394.356394.887394.899395.18395.184395.218395.242395.292395.284395.283395.341395.35396.199396.266396.221396.293396.313396.882396.894396.906397.056397.275397.277397.21397.289397.288397.29397.298397.307397.321397.894397.899398.073398.079398.08

398.073398.157398.277398.264398.293398.298398.318398.378398.375398.894398.895399.079399.165399.207399.227399.275399.289399.263399.292399.247399.322399.35399.358399.359400.075400.121400.204400.215400.247400.287400.3400.325400.964401.002401.158401.248401.211401.254401.21401.263401.235401.27401.26401.284401.275401.31402.015402.058402.169402.185402.249402.264402.276402.33403.216403.166403.242403.271403.269403.232403.268403.3403.349403.613403.955403.968403.979404.036

404.039

404.037404.036

404.191404.197404.258404.275404.286404.27404.274404.324404.908404.953404.958404.969405.035

404.969405.026405.027405.043405.185405.275405.271405.36405.938405.962406.034405.987406.026406.038

406.179406.181406.223406.287406.941407.036407.036407.077407.119407.165407.255407.33407.928407.931408.287408.286408.309408.334408.361409.219409.237409.294409.289409.329410.134410.247410.252411.186411.224411.26411.301411.966411.976412.149412.142412.178412.194412.21412.225412.224412.223412.243412.261412.261412.263412.283412.326412.921413.115413.182413.265413.274413.277413.312413.919413.963414.063414.22414.221414.269414.262414.279414.323

414.352414.94414.945415.151415.187415.243415.22415.249415.287415.313415.296415.312415.939416.162416.169416.164416.239416.28416.304416.318416.916416.945416.94417.036417.128417.178417.196417.257417.272417.278417.399418.138418.231418.242418.236418.238418.259418.278418.284418.996418.999

419.174419.158419.264419.261419.261419.265419.267419.264419.294419.319419.709419.898419.983420.007420.033420.035420.276420.264420.276420.312420.304420.35420.993421.176421.162421.251421.268421.275421.306421.316421.344421.994422.01422.151422.227422.28422.284422.298422.285422.327422.263422.328422.344422.97423.031423.243423.216423.215423.272423.271423.32423.346423.961424.011424.198424.201424.243424.308424.286424.319424.31424.356424.921424.951425.198425.213425.236425.211425.24425.272425.269425.272425.29425.337425.358425.379425.929425.936425.94425.932425.986425.957426.014426.044426.145426.212426.204426.223426.211426.245426.25426.282426.275426.31426.949427.175427.254427.237427.232427.276427.286427.291427.312427.31427.945428.16428.205428.236428.281428.242428.294428.283428.33428.389428.878428.967428.98428.99428.997429.011429.216429.246429.256429.265429.281429.264429.285429.96429.982429.961430.052430.056

430.055430.056430.232430.238430.884430.967430.943

430.947

430.943431.046431.22431.235431.235431.233431.317431.315431.958432.212432.222432.22432.266432.284432.269432.293432.315432.852432.985433.238433.258433.265433.304433.904433.946433.998434.208434.251434.229434.253434.304434.339434.358435.012435.131435.138435.139435.183435.259435.248435.27435.272435.384

436.006

436.025436.024436.335436.301436.253436.249436.261436.333436.347436.302436.341436.358436.368436.359436.361436.404436.885436.897437.027437.181437.263437.223437.257437.282437.346437.308437.356437.381437.878438.128438.269438.307438.341438.388439.002439.209439.29439.307439.322439.329440.234440.268440.311440.316441.124441.266441.317441.309441.326441.322442.025442.168442.189442.253442.318442.346443.012443.274443.178443.249443.246443.287444.005444.067444.285444.305444.325445.057445.117445.119445.116445.207445.275445.24445.98445.999445.995446.052446.119446.198446.249446.283446.275446.3446.323446.859446.993447.116447.234447.237447.245447.272

447.976

448.3448.29448.306448.348449.003449.225449.241449.25449.34449.875449.999450.269450.333450.947451.019451.219451.25451.27451.379452.008452.188452.327452.273452.317452.4453.179453.261453.93454.284454.286454.303454.295455.274455.304455.979456.302456.317456.967457.281457.299457.309458.262

458.344

458.326458.821459.194459.209459.264459.263459.294460.296460.315461.193461.267461.26461.265461.286461.292461.999462.217462.274462.28462.381462.956463.128463.27463.262463.316464.128464.371464.372464.384465.137465.269465.303465.958465.961465.964465.96465.989466.321466.244466.266466.337466.297466.265466.35466.346466.411467.244467.996467.957468.003468.293468.338468.936469.169470.285470.345470.345470.935471.226471.245472.217472.357472.366472.905473.278473.355474.076474.232474.232474.272474.287474.301474.337475.085475.241475.236475.297475.366475.862475.859476.244476.283476.306477.23477.283478.213478.241478.302479.262479.359479.864479.965479.98480.158480.32480.328480.832480.84481.162481.222481.232481.221481.271481.314481.334481.949482.224482.274482.318482.401484.233484.279484.269485.276485.329485.347485.356486.357487.106487.226487.202487.218487.292487.979488.203488.232488.244488.233489.131489.128489.226489.305490.854491.231491.205491.209491.216492.062492.838492.863493.138493.227493.27493.301494.048

494.319494.443496.038496.056496.21496.243496.254496.413496.444496.98497.255497.928497.939

497.93

497.934

497.95498.069498.085498.096498.248498.275498.27498.936499.156499.928500.243500.935500.942501.215501.928501.947502.214

502.372

502.35502.851502.905503.189503.268503.918503.971505.033505.189505.267505.397506.07506.286

507.083

507.069507.086507.246507.3507.379507.872508.258508.854508.872509.873510.083510.439510.885510.856511.075512.119512.363512.362512.404512.834514.372514.421514.859514.863515.357515.848515.858516.281516.378516.348516.412517.395519.148519.305520.306522.245523.076523.089523.223523.348524.071524.352524.863525.27526.271526.871527.042527.052527.407527.902529.368529.382529.903529.911531.324532.072

532.1

532.106532.391533.108533.392534.05535.108535.471535.699536.071536.294537.052537.145537.21537.267538.319539.053539.061539.412540.056540.448540.484541.043542.323542.323542.844544.394546.105546.274

546.393

546.367546.391546.395546.42546.868547.921548.287548.731548.922549.41551.012552.875553.036553.239553.859554.175

554.276554.468554.849554.866555.18556.173556.406556.436557.947558.128559.22560.41563.07563.352563.356563.367563.41564.363564.337565.102568.038568.392568.882569.305571.294571.454571.918572.28573.178573.265573.302573.376573.389574.323574.337575.346575.794576.407576.424579.426581.034581.88582.315582.828583.039583.808584.475585.006588.327588.338588.869589.313589.411589.723590.323590.296

590.417

590.419590.432591.323591.323591.457593.289593.444593.855594.825598.016600.978

601.446601.85601.968602.841602.844602.977603.25603.383603.97604.43604.455604.843604.856605.007605.443605.828606.014609.275611.402612.424615.453619.115619.753620.453623.462625.084625.893626.362628.197628.489629.197630.196633.421634.069634.308

634.447

634.454637.505638.233638.783639.31641.331642.244642.769645.464646.331647.273648.451651.562651.594652.311652.324652.356654.348657.284662.949

663.09665.921667.449667.484667.765667.903670.38677.449677.953

678.477

678.476679.454680.308680.816682.264691.325691.598692.49693.494697.069702.212703.219704.214705.223706.344706.361712.299721.613721.802

722.503

722.527724.221724.37724.373725.335725.537726.25727.357730.3735.652738.371758.432758.455766.535

785.332785.35796.424797.395807.542808.752810.558

810.572812.614812.6812.62812.625812.626812.667813.62813.661814.594814.609814.63826.612826.61836.761854.58855.583855.607856.366856.376856.407876.374898.609899.616942.638944.641962.639962.652962.663963.652963.656963.637964.655964.648964.675964.656964.676

SIMCA-P+ 12.0.1 - 2011-09-07 06: 13:54 (UTC+9)

SIMCA-P+ 12.0.1 - 2011-09-07 06:15:37 (UTC+9)

NS-2

NS-1

b

4

The formula of peak No. 7 (Fig.6) of NS-2 was predicted as C12H16N2S2. Considering MSn spectra and neutral loss ions, it was determined that peak No. 7 has the structure of NS with addition of –CH2 to the phenyl group (Fig. 7). We also

determined the structures of other characteristic compounds and thought to be either impurities or by-products in the process of synthesis.

Fig. 7 MSn spectra of NS (left) and peak No. 7 (right) detected from NS-2 sample (see Fig. 6).

Table 2 Predicted formulae and structures of characteristic components of NS (left) and CZ (right).

ConclusionDifferences in similar structured sulfenamide-based vulcanizing accelerators were identified from different manufacturers with characteristic components of each sample detected using high mass accuracy MSn and multivariate statistical techniques. The structural analogues of main compounds were

detected using MSn data analysis software (MetID Solution).Formulae and structures of the analogues were assigned using Formula Predictor software.The impurities detected from each sulfenamide-based vulcanizing accelerator differed from manufacture to manufacture.

AcknowledgementWe wish to thank Dr. Fumito Yatsuyanagi and Ms.Yuko Sekine of THE YOKOHAMA RUBBER CO.,LTD. for supplying the samples and their constructive comments.


No. R.T. (min)

Predicted formula and structure

ions

Sample Measured value (m/z )

Theoretical value (m/z)

Error (ppm)

1 5.62

C11H14N2O2S 2

271.0567 271.0569 -0.74 1

2 6.35

C7H5NOS 2

183.9887 183.9885 1.09 4

3 6.48

C14H8N2S 3

300.9923 300.9922 0.33 3

4 6.50 C15H23N3OS3 358.1062 358.1076 -3.91 2

5 6.72

C11H14N2O2S 2

271.0582 271.0569 4.80 4

6 7.52 C14H8N2S 3

300.9921 300.9922 -0.33 5

7 7.58

C12H16N2S 2

253.0831 253.0828 1.24 2

8 8.48 C14H17N3O5S 2 404.0399 404.0403 -0.99 4 9 8.85 C18H17N3S 3 372.0647 372.0657 -2.69 4

10 9.17 C18H26N4O5S 4 507.0850 507.0859 -1.77 4

No. R.T. (min)

Predicted formula and structure

ions

Sample Measured value (m/z)

Theoretical value (m/z)

Error (ppm)

1 3.62 C10H17NO3

200.1280 200.1281 -0.50 4

2 4.78 C11H11NO2S 2 254.0308 254.0304 1.57 4

3 5.07 C11H11NO4S 2

286.0207 286.0202 1.75 4

4 6.25 C13H10N2S 227.0628 227.0637 -3.96 4

5 6.83

C11H14N2S 2(NS)

239.0671 239.0671 0.00 1

6 7.30

C14H8N2S 4

332.9648 332.9643 1.50 4

7 8.25 C30H49N3O 468.3956 468.3948 1.71 1

8 8.31

C12H12N2O5S

297.0533 297.0540 -2.36 4

9 8.36

C14H18N2S 2

279.0985 279.0984 0.36 1

10 8.90 C24H25N3OS 4 500.0950 500.0953 -0.3 4

50 100 150 200 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0Inten. (x1,000,000)

239.0666(1)

183.00 332.9620(1) 459.2364(1)

50 100 15 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.0

1.0

2.0

3.0

4.0Inten. (x1,000,000)

183.0042

9.0664(1)

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.00

0.25

0.50

0.75

1.00

1.25

Inten. (x1,000,000)

165.9778

7.0053

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.0

0.5

1.0

1.5

Inten. (x100,000)

122.0045

90.0369

50 100 150 200 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.0

1.0

2.0

3.0

4.0

5.0

Inten. (x1,000,000)

341.2860(1)

239.0663(1)420.0319(1)253.0830(1)

118.0851 569.2763(1)

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.00

0.25

0.50

0.75

1.00

Inten. (x1,000,000)

17 . 3

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.0

0.5

1.0

1.5

Inten. (x100,000)

136.0212

147.0128

92.4451

50 100 150 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z0.0

0.5

1.0

1.5

2.0Inten. (x1,000,000)

197.021

17

MS1

MS2

MS3

MS4

C11H15N2S 2+

NL :-56

NL :-17

NL :-44

:-56

NL :-17

NL :-44

N

S

S NH CH3

CH3

CH3

CH3

150000000000 0 20

212 NN

C7H7N2S 2+

C7H4NS 2+

C6H4NS +

N

S

S NH CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

CH3

CH3

CH3

MS1

MS2

MS3

MS4

OH× 2

N

S

S NH CH3

CH3

CH3

OH× 2 N

S

S NH CH3

CH3

CH3

CH3

N

S

S NH CH3

CH3

CH3

+O

N

S

S H

+C7H3NS

N

S

S H

+C7H3NS

N

S

S H

N

S

S NH CH3

CH3

CH3

+C4H6O4S

N

S

+C7H3NS 2

N

S

S H

+C7H7NO5

N

S

N

S

S NH

CH3

+C4H5NO3

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, 2012

First Edition: May, 2012

www.shimadzu.com/an/

Natsuyo Asano, Kiyomi Arakawa, Shinjiro Fujita,

Kazuo Mukaibatake, Ichiro Hirano SHIMADZU

CORPORATION, Kyoto, Japan

Multi-component quantitative analysis of pharmaceuticals and personal care products in the environment by LC-MS/MS with fast polarity switching

ASMS 2012 MP22-532

2

IntroductionPharmaceuticals and personal care products (PPCPs) constitute a group of emerging contaminants which have received considerable attention in recent years. Monitoring of PPCPs in the environment is vital as many of these compounds are ubiquitous, persistent and biologically active with recognised endocrine-disruption functions. Given the hazardous nature of these compounds, there is a need to provide fast and sensitive multi-residue methods

that are able to analyze multiple classes of compound within one analytical procedure. Here we report a new multi-residue UHPLC-ESI-QqQ method that utilizes fast polarity switching with an optimized chromatographic gradient that removes matrix effects and results in excellent ng/L detection levels. Furthermore, we have evaluated the performance of polarity switching in comparison to dedicated single polarity experiments.

Materials and MethodsNatural river and lake water was collected from the Shiga region (Japan) and spiked, without any sample

pre-treatment, at a range of concentration levels (1 – 10000 ng/L) with 15 PPCPs.

A higher sensitivity triple quadruple mass spectrometer (LCMS-8080, Shimadzu, Japan) operating in SRM mode

with fast polarity switching (20 msec) was used for the detection of positively and negatively charged analytes.

Table 1. Analytical conditions.

UHPLC

LC system:

Analysis Column:

Mobile Phase A:

Mobile Phase B:

Gradient Program:

Flow rate:

Column Temperature:

Injection Volume:

MS

MS system:

Ionization:

Nebulizing Gas Flow:

Curtain Gas Flow:

Heating Gas Flow:

Probe Temperature :

HSID Temperature :

Nexera (Shimadzu, Japan)

Shim-pack XR-ODSIII (2.0 mmI.D. x 50 mmL., 1.6 µm)

0.1% Formic acid - Water

Acetonitrile

0%B (0-6 min) – 80%B (16 min) – 100%B (16.01-18 min) – 0%B (18.01-21 min)

0.4 mL/min

40°C

40 µL

LCMS-8080 (Shimadzu, Japan)

ESI (positive/negative)

3.00

3.50

12.00

450°C

300°C


3

ResultsAnalysis of PPCPs spiked in environmental water

Fig. 1 Gradient program of LC.

Table 2 MRM mode parameters and analysis results for each PPCPs.

As a result of the complex matrix in which PPCPs are present, the occurrence of ion suppression/enhancement can reduce MS/MS detection limits. For this reason, an optimized gradient was developed that focused target

analytes at the head of the chromatographic column while allowing the interfering environmental matrix to be eluted (Fig. 1).

All compounds were measured by SRM with fast polarity switching (20 msec) for multi-component analysis. Excellent limits of quantification were achieved in the range 1 – 50 ng/L for nearly all studied compounds, with outstanding

linearity (R2 > 0.999). The analysis results of 15 PPCPs are shown in Table 2, Fig. 2 shows calibration curves for three selected PPCPs: Carbamazepine, Albeterol and Ibprofen.

Before the organic phase was increased, the aqueous mobile phase held at 100% for 6min.

Albuterol

Acetaminophen

Trimethoprim

Sulfamethoxazole

Carbamazepine

Dehydronifedipine

Naproxen

Antipyrine

Doxycycline

Isopropylantipyrine

Warfarin

Ibuprofen

Gemfibrozil

Triclocarban

Triclosan

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

pos

neg

neg

neg

neg

neg

240.20>148.20

152.10>110.30

291.20>230.20

254.00>92.30

237.10>194.20

345.20>284.10

231.10>185.20

189.00>56.20

445.00>428.00

231.00>189.00

309.00>163.00

307.00>161.20

205.30>161.40

249.30>121.30

313.10>160.20

287.00>34.90

5

50

5

25

1

5

10

10

100

2.5

5

25

50

25

25

50

LOQ (ng/L)TransitionPolarityCompound

River Lake River Lake

%Recoery (100 ng/L)

5

50

25

50

2.5

25

25

25

50

5

10

50

50

50

25

100

148

80

143

104

94

97

99

106

79

103

86

91

106

114

120

105

112

87

118

76

68

75

76

82

56

82

60

103

87

77

98

74


4

(a) Carbamazepine (b) Albuterol

(a) Polarity Switching: 20 ms (b) Non-polarity Switching

(c) Ibprofen

Fig. 2 Calibration curves for Carbamazepine (a), Albeterol (b) and Ibprofen (c); (a) was spiked in river water, (b) and (c) were spiked in lake water.

Fig. 3 Comparison of polarity switched analysis (a) and non-polarity switched analysis (positive only or negative only analysis) (b).


Polarity SwitchingTo evaluate the capability of polarity switching the data quality obtained was compared to dedicated positive or negative analysis. Data quality obtained during polarity switching experiments was directly comparable to that achieved during dedicated

positive or negative analysis (Fig. 3). Long term stability was investigated by making 100 injections over 10 hours. Polarity switching data indicates excellent stability over the analysis time (Fig. 4, Table 3).

Fig. 4 Results for area variation across the 100 serial analyses.

0.0 25.0 50.0 75.0 Conc. (ng/L) 0

2.0

4.0

6.0

8.0 1 – 100 ng/L

R2 = 0.9996

0 250 500 750 Conc. (ng/L) 0

0.5

1.0

1.5

2.0

2.5

3.0

Area (×100,000) Area (×100,000) Area (×100,000)

5 – 1000 ng/L

R2 = 0.9994

0 1000 2000 3000 4000 Conc. (ng/L) 0

0.25

0.50

0.75

1.00

1.25

1.50

1.75 50 – 5000 ng/L

R2 = 0.9999

1.50 2.00 2.50 3.00 min 0

5.0

10.0

15.0 ×104 ×104 ×104

Inte

nsi

ty

1: Carbamazepine (pos)

2: Dehydronifedipine (pos)

3: Gemfibrozil (neg)

4: Triclocarban (neg)

1.50 2.00 min 0

5.0

10.0

15.0

Inte

nsi

ty

1: Carbamazepine (pos) 2: Dehydronifedipine (pos)

2.75 3.00 min 0

5.0

10.0

15.0

Inte

nsi

ty

3: Gemfibrozil (neg)

4: Triclocarban (neg)

0

0.5

1.0

1.5

2.0

2.5

3.0

0 20 40 60 80 100 Injection

Are

a

Carbamazepine

Dehydronifedipine

Gemfibrozil

Triclocarban

100 injections (10hours)

5

Table 3 Analysis results for respective compounds.

CarbamazepineDehydronifedipine

GemfibrozilTriclocarban

posposnegneg

237.10 > 194.20345.20 > 284.10249.30 > 121.30313.10 > 160.20

2.703.943.103.52

Transition %RSDPolarityCompound

ConclusionOptimization of the LC gradient program resulted in the reduction of matrix effect and the recoveries of 70 – 120 % for almost all studied compounds.Using LCMS-8080, excellent sensitivity and linearity were obtained for PPCPs spiked in environmental water samples.

Fast polarity switching results were shown to be comparable to dedicated single polarity experiments for the analysis of PPCPs in environmental samples.


Shinjiro Fujita, Natsuyo Asano, Kazuo Mukaibatake

SHIMADZU CORPORATION, Kyoto, JAPAN

Evaluation of the higher sensitive LC/MS/MS incorporates novel desolvation technologies to achieve low femto-gram LOQ

ASMS 2012 ThP26-566

2

IntroductionThe triple quadrupole mass spectrometer is widely used in various application fields to quantify the trace amount of compounds because of excellent sensitivity and selectivity. In order to exceed the low femto-gram barrier, many researchers have been developing new desolvation technologies at either ESI sprayer or MS inlet. In this paper we present the development novel

desolvation devices coupled to a highly sensitive triple quadrupole mass spectrometer utilizing a coaxial hot gas ion source and the heated multi-orthogonal interface (Hot Source Induced Desolvation: HSID) mounted at the inlet of mass spectrometer. The combination of the coaxial hot gas and the HSID enhanced desolvation efficiency which resulted in low femto-gram limit of quantitation.

Methods7 commercially available drug samples (Verapamil, Alprazolam, Carbamazepine, Cilostazol, Lidocaine, Fluticasone and Testosterone) were prepared for the sensitivity evaluation of LCMS-8080 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) equipped with coaxial hot gas and HSID interface. All samples were analyzed by the Multiple Reaction Monitoring (MRM). MRM parameters including MRM transitions and collision energy as well as compound dependent ion transfer voltages were optimized through automatic MRM optimization functionality incorporated in LabSolution software (Shimadzu Corporation, Japan). The temperatures of

coaxial hot gas and HSID were optimized for each sample. Chromatographic separations were carried out on a Nexera MP system (Shimadzu Corporation, Japan) using a Shim-pack XR-ODSIII (50 mmL.× 2.0 mmI.D., 1.6 mm).

functionality incorporated in LabSolution software (Shimadzu Corporation, Japan). The temperatures of

High temperature gas blowing around the electro spray coaxially achieves highly efficient desolvation and accelerates ionization, resulting in larger volumes of ions introduced into the mass spectrometer.

Noise derived from neutral species or unwanted ions are strongly reduced in the multi orthogonal region which is heated to a high temperature and achieves excellent signal to noise ratio.


Fig. 1 LCMS-8080 triple quadrupole mass spectrometer. Fig. 2 Coaxial hot gas and HSID.

Coaxial Hot Gas

HSID (Hot Source Induced Desolvation)

Coaxial hot gas (<500 degrees C)

Ionization Probe

HSID

Multi Orthogonal

Heated atHigh Temperature(<300 degrees C)

3

ResultsMRM chromatogram for verapamil at 0.5 fg/ µL. Extremely high sensitivity has been achieved with the lower limit of detection in the region of below femto-gram level.

Coaxial Hot GasHSIDMobile phase AMobile phase BFlow rate

: 500 degrees C: 280 degrees C: 5mM Annmonium acetate-water: Acetonitrile: 0.5 mL/min

MRM chromatogram for testosterone, a type of steroid. At 2 fg/ µL, at the LOQ region, the RSD is 4.34% for 6 repeated analysis.



: 450 degrees C: 240 degrees C: 0.1 % formic acid - water: Acetonitrile: 0.4 mL/min

MRM chromatogram for 3 drugs at 5 fg/ µL. In each case, the area repeatability (RSD) was less than 6%, a low noise level has been achieved, and with excellent selectivity, the LOQ is several femto-gram or less.

: 500 degrees C: 280 degrees C: 5mM Annmonium acetate-water: Acetonitrile: 0.4 mL/min

5 fg/uL

Verapamil 0.5 fg/ μL m/z 455.50 > 165.10 S/N = 16

Testosterone 2fg/ μL m/z 289.10 > 97.00 4.34 %RSD (Area, n=6)

Lidocaine m/z 235.10>86.20 Carbamazepine m/z 237.10>194.00 Cilostazol m/z 370.10>288.00

5 fg/ μL

Fig. 3 MRM chromatograms at lower concentration.

Table 2 ynamic range, linearity (R2) and Area %RSD for each compound

Compounds Dynamic Range (fg/ µL) R2 Area %RSD

Verapamil

Alprazolam

Carbamazepine

Cilostazol

Lidocaine

Fluticasone

Testosterone

0.5-200,000

2-100,000

2-20,000

2-50,000

5-50,000

5-200,000

2-20,000

0.9995

0.9998

0.9999

0.9999

0.9996

0.9999

0.9997

0.08-10.70

1.78-7.92

0.93-6.54

0.42-12.38

0.36-8.10

0.39-18.63

0.40-4.34


4

With cilostazol calibration curves, an extremely good linearity value of R2 = 0.9999 was obtained across a dynamic range from 2 fg/ µL to 50,000 fg/ µL. In addition,

even at low concentrations, a relative error under 10 % and RSD under 15 % were achieved.

Using LCMS-8080 equipped with coaxial hot gas and HSID, low femto-gram LOQs were achieved for all 7 drugs (Verapamil, Alprazolam, Carbamazepine, Cilostazol,

Lidocaine, Fluticasone and Testosterone) delivering excellent linearity of R2 > 0.999 across wide range from low to high concentrations.

Conclusions


Concentration, ×103 fg/ µL 0 10 20 30 40 50

0

2.0

4.0

6.0

8.0

10.0 Cilostazol

m/z 370.10>288.00

0.002 - 50 ng/ mL

R2 = 0.9999

Table 3 Relative Error (RE) and Area %RSD for each concentration of Cilostazol.

Fig. 4 Calibration curves for Cilostazol.

Perared Concentration x103 fg/ µL

Measured Concentration x103 fg/ µL

Rerative Error (RE) %

Area %RSD (N=5)

0.002

0.005

0.05

0.2

0.5

5

20

50

0.0021

0.0048

0.049

0.20

0.50

4.99

20.09

49.93

5.89

-3.76

-1.62

-1.35

-0.20

-0.24

0.43

-0.14

12.38

5.03

2.78

1.47

1.46

0.42

0.93

0.57

Yuka Fujito1, Yusuke Inohana2, Kiyomi Arakawa2,

Ichiro Hirano2 1Shimadzu Analytical & Measuring Center, Inc.,

Kyoto, Japan ; 2Shimadzu Corporation, Kyoto, Japan

Multi-class pesticides analysis in challenging vegetable matrices using fast 5 msec MRM with 15 msec polarity switching

ASMS 2012 WP27-575

2

IntroductionMany regulatory authorities have established multi-class residual pesticides methods for the analysis of vegetables, fruits and other food stuffs. There is, however no global agreement on the provision of a target list of pesticides and this presents a risk with products moving between different regulatory requirements. In order to eliminate this risk, food safety laboratories need to ideally screen as many compounds as possible in a single run which may reach

maximum residual limits (MRL); typically 10 ppb in food matrices. In this study, we report the application of ultra-fast 5 msec MRM with 15 msec polarity switching for the analysis of 138 pesticides in vegetable matrices (72 and 66 compounds measured by LC-QqQ and GC-QqQ in the European Union Reference Laboratory (EURL) method) .

Materials and Methods


Sample Preparation (QuEChERS EU method) LC/MS/MS analysis

Vegetables

Paprika (Sweet pepper)Leek (Garlic chives)

Origin

New ZealandJapan

ColumnMobile phase

Gradient program

Flow rateColumn temperature

IonizationIon spray voltageMRM

: Shim-pack XR-ODSII (75 mm x 2 mmI.D., 2.2 um): A ; 2 mM ammonium formate containing 0.1% formic acid – water B ; Methanol: 5% B (0-2.5 min.)→55% B (2.51-6 min.)→80% B (6.01-12 min.) →100% (12-15 min.)→5% (15.01-20 min.): 0.2 mL / min.: 40°C

: ESI (Positive / Negative): +4.5 kV / -3.5 kV: 276 MRM transitions (2 MRMs / compound) Dwell time 5 msec. / Pause time 1 msec.

Food sample Analytical Conditions

Features of LCMS-8040

HPLC : Nexera UHPLC system

MS : LCMS-8040 Triple quadrupole mass spectrometer

Step 1 : Sample Extraction

1. Homoginize vegetables with food processor and homoginizer

2. Weigh 10 g homoginized sample

Add 10 mL acetonitrile

Add Salt-mixture*1

・4 g MgSO4

・1 g NaCl ・0.5 g Na2H citrate ・1.5H2O ・1 g Na3 citrate ・2H2O

Extract 13. Shake vigorously by hand 1 min.

4. Centrifuge for 5 min. at 4000 rpm (Extract 1)

*1 : Citrate Extraction Tube (SIGMA ALDRICH)

Add 200 uL acetonitrile

1

3

4

Step 2 : Sample Clean up

5. Transfer 6 mL Extract 1 into Dispersive SPE tube*2

6. Shake vigorously by hand 2 min.

YellowPaprika

RedPaprika

containing ・ 900 mg MgSO4

・ 150 mg PSA ・ 45 mg ENVI-Carb

Leek

Extract 2

*2 : PSA/ENVI-Carb SPE Clean Up Tube 2 (SIGMA ALDRICH) Fig. 1 LCMS-8040 Triple Quadrupole Mass Spectrometer

7. Centrifuge for 5 min. at 3000 rpm (Extract 2)

8. Transfer 1 mL Extract 2 into a vial

9. Vortex sample to mix it

10. Filtrate sample with disposable filter

6

7

8

5 times higher sensitivity compared to LCMS-8030An ultra fast scan speed of 15000 u / sec.An ultra fast polarity switching of 15 msec.An ultra fast MRM transition speed of 555 ch./ sec.

LCMS-8040Ultra Fast Mass Spectrometer

3

ResultsMRM of pesticide standards

Compounds for LC-QqQ Compounds for GC-QqQ


Technique(on the EURL method)

by LC-QqQby GC-QqQ

LOQs < 10 ppb LOQs > 10 ppb Not Ionization

72 (100%)47 (71%)

0 (0%)6 (9%)

0 (0%)13 (20%)

Table 1 LOQs of 138 pesticides in the EURL method by LCMS-8040

Fig. 2 Calibration curves and MRM chromatograms of typical pesticides

・ 71 % of pesticides measured by GC-QqQ in the EURL method could achieved excellent LOQs (0.08-10 ppb) .

1-1000 ppbr2=0.9999

S/N 38 (1ppb)

S/N 82 (1ppb)

S/N 73 (1ppb)

S/N 73 (1ppb)

1-1000 ppbr2=0.9992

1-1000ppbr2=0.9999

1-1000ppbr2=0.9996

0 500 Conc. 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Area (x10,000,000)

Azoxystrobin Carbofuran Ethion Methidathion

0 500 Conc. 0.00

0.25

0.50

0.75

1.00 Area (x100,000,000)

0 500 Conc. 0.0

2.5

5.0

7.5

Area (x10,000,000)

0 500 Conc. 0.0

2.5

5.0

7.5

Area (x10,000,000)

5.0 6.0

0.0

0.5

1.0

1.5

2.0 (x100,000)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00 (x100,000)

9.0 10.0 6.0 7.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

(x100,000)

6.0 7.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00 (x100,000)

1 ppb

5 ppb

10 ppb

4

Fig. 3 Recovery of pesticides in the vegetable matrices (5 ppb spiked)

Recovery of pesticides in vegetable matrices


Approximately 90% of pesticides represented good recoveries in the range of 70-120% in all studied matrices.Some pesticides indicating up to 120% of recovery were also detected in the matrix blank (Fig. 4）.

Azoxystrobin

5 ppb standards spikedMatrix blank

Imidacloprid Clothianidin

Azoxystrobin8.19 ppb3.77 ppb

5 ppm

Imidacloprid8.99 ppb3.19 ppb

3 ppm

Clothianidin21.39 ppb25.78 ppb

15 ppm

Fludioxonil556.58 ppb550.92 ppb

10 ppm

Fig. 4 MRM Chromatograms and quantitative results of 4 pesticides

5ppb standards spikedMatrix blankMRL (Japan)

ConclusionMRL of pesticide in vegetable matrices pre-treatment by QuEChERS method could be measured successfully using fast 5 msec MRM with 15 msec polarity switching.

Majority of pesticides being suggested to GC-QqQ technology on the EURL method was successfully covered by LC-QqQ.

Azoxytrobin

Imidacloprid

Kresoxim -methyl

Trichlorfon

Dichlorvos Flutriafol

Triacloprid

Recovery (%)

0

20

40

60

80

100

120

140

160

180

200

220

240

▲

(out of range)

Fludioxonil Clothianidin

▲ (622 %)

(11439 %)

■ Red paprika

◆ Yellow paprika

▲ Leek

Fludioxonil6.5 7.0 7.5

0

50000

100000

150000

200000

250000

300000

3.5 4.0 4.5

0

10000

20000

30000

40000

50000

60000

70000

80000

3.5 4.0 4.5

0

2500

5000

7500

10000

12500

15000

17500

20000

6.0 6.5 7.0

0

25000

50000

75000

100000

125000

150000

Ichiro Hirano1 Yuka Fujito2, Kiyomi Arakawa1,

Yusuke Inohana1 1Shimadzu Corporation, Kyoto, Japan, 2Shimadzu Analytical & Measuring Center, Inc.,

Kyoto, Japan

Exploring the application of a universal method for pesticide screening in foods using a high data acquisition speed MS/MS　　

ASMS 2012 WP27-574

2

IntroductionEffective management, use, and disposal of agrochemicals, particularly pesticides, is an increasingly important health and environment issue in developing countries where economies may be heavily reliant on agriculture. The conventional approach to monitor these pesticides is to develop highly optimized triple quadrupole MRM methods to achieve the required levels of sensitivity, selectivity and

speed of analysis whilst still providing confidence in pesticide identification. In this study LCMS technology, developed for ultra-fast scanning MRM analysis, allows the possibility of a single generic ‘universal’ method. High speed MRM analysis and a generic parameters were used for screening 172 pesticides (344 MRM transitions) with 5 msec dwell and 1 msec pause times in food matrices.


Exploring the application of a universal method for pesticide screening in foods using a high data acquisition speed MS/MS

Sample Preparation

Analytical Conditions

Setting of MRM analysis & integration parameter

ColumnMobile phase

Gradient program

Flow rateColumn temperature

IonizationIon spray voltageMRM

: Shim-pack XR-ODSII (75 mm x 2 mmI.D., 2.2 um): A ; 5 mM ammonium acetate – water B ; 5 mM ammonium acetate – methanol: 30% B (0 min.) → 80% B (4 min.) → 95% B (10-15 min.) → 30% B (15.01-20 min.): 0.2 mL / min.: 40°C

: ESI (Positive / Negative): +4.5 kV / -3.5 kV: 344 MRM transitions (2 MRMs / compound)

HPLC : Nexera UHPLC system

Positive

Negative

Features of LCMS-8040

MS : LCMS-8040 Triple quadrupole mass spectrometer

Sample: 5 g

Homogenize

Extraction

Suction Filtration

Salting-out

Dehydration

Concentration

Purification by ENVI-Carb/LC-NH2 column

Concentration

Analyte

LC/MS analysis

Addition of 20 mL of water and then left to stand for 15 min.

Addition of 50 mL of acetonitrile

Preparation of 100 mL solution of supernatant with acetonitlie.

Partitioned 20 mL of extracted solution (equivalent to 1 g of sample)

Addition of 10 g of NaCl and 20 mL of 0.5 mol/L phosphate buffer (pH 7.0)

Shaking for 10 min after adding adequate amount of anhydrous sodium sulfate.

Filtration

FiltrInstrument Parameters View (Realtime analysis)

167 compounds from ‘Residual pesticide

method package’+

5 compounds

Acquisition time set to the entire chromatographic run (0-20 min.).

‘Largest peak’ selected for identification.

Dwell 5 msec.Pause 1 msec.Loop time 2.058 sec.

MS Data Processing View (Postrun analysis)

Residue dissolved in 2 mL of 25% toluene / acetonitrile

Conditioned with 10 mL of 25% toluene / acetonitrile

Elution with 20 mL of 25% toluene / acetonitrile

4 mL of solution was prepared by dissolving in methanol

(Method reported by Japan’ s Ministry of Health, Labour and Welfare)

Fig. 1 LCMS-8040 Triple Quadrupole Mass Spectrometer

5 times higher sensitivity compared to LCMS-8030An ultra fast scan speed of 15000 u / sec.An ultra fast polarity switching of 15 msec.An ultra fast MRM transition speed of 555 ch./ sec.

In this study, no scheduling of MRM transitions was applied; thereby creating a universal generic method.


3

ResultsScreening of 10 pesticides in food matrices

10 ppb of pesticides spiked in the leek extract

Mevinphos

5.0(×100,000)

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

0.0 2.5 5.0 7.5 10.0 min

Tricyclazole

Propoxur

Carbofuran

Fosthiazate

Indanofan

Lufenuron

Pyrimidifen

Hexythiazox

Chlorfluazuron

Fig. 2 MRM chromatogram of 10 pesticides

Table 2 Result of 10 pesticides screening (10 ppb spiked in each matrices)

・ All peaks were automatically selected as the target compound to permit automatic identification of target analytes without retention time information. (* Number of false positives out of 172 screened pesticides.)


LeeK Paprika Green tea leaves

8 7 10

Compounds

False positives*

Carbofuran　

Chlorfluazuron　

Fosthiazate　

Hexythiazox　

Indanofan　

Lufenuron　

Mevinphos

Propoxur

Pyrimidifen

Tricyclazole

4

Fig. 3 Result of automatic identification (10 ppb spiked in the green tea leaves)

ConclusionPesticides spiked in all matrices at 10 ppb (10 compounds) could be automatically detected using fast 5 msec MRM with 15 msec polarity switching without retention time information.


Mevinphos

Tricyclazole

Propoxur

Carbofuran

Fosthiazate

Indanofan

Lufenuron

Pyrimidifen

Hexythiazox

Chlorfluazuron

Manami Kobayashi, Satoshi Yamaki, Ayako Nomura,

Kyoko Watanabe, Yoshihiro Hayakawa, Tsutomu

Nishine Shimadzu Corporation, Kyoto, Japan

Rapid and Highly Sensitive Quantitative Analysis and Screening of Aflatoxins in Foods Using Liquid Chromatography Triple Quadrupole Mass Spectrometry

ASMS 2012 WP27-585

2

IntroductionAflatoxins (AFs) are the most harmful mycotoxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus and can contaminate foods such as cereals and nuts. To reduce the risk of the ingestion from foods, analyses of the AFs are carried out in many countries. It is necessary to quantitate the total aflatoxin (B1, B2, G1, G2) in foods by the regulation in JAPAN. The conventional LC/MS method proposed by the Ministry of Health, Labour and Welfare of Japan has a total analysis time of 30 minutes. In this study, we examined two alternative high-throughput LC-MS/MS methods. The first optimized for sensitivity & quantification; the second, a rapid screening method, using UHPLC for the purpose of increasing work flow.

Materials and MethodsThe system consisted of a SHIMADZU “Nexera” HPLC system, and “LCMS-8030” triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) used in the positive ion MS/MS mode. AFs standard solution were obtained from Biopure, MYCOTOXIN MIX5(AFLATOXINS) and Wako Chemicals, Aflatoxins Mixture Solution 1. Sample preparation work flow (Fig. 2) shows how AFs in roast peanut was prepared by an immunoaffinity column (AFLAKING, HORIBA, JAPAN). Based on starting material 50g (roast peanut powder) spiked Aflatoxin B1 and G1(4 µg/Kg), B2 and G2 (1 µg/Kg) standard solution, final concentration of the sample solutions became: 2 µg/L B1 and G1, 0.5 µg/L B2 and G2.

Rapid and Highly Sensitive Quantitative Analysis and Screening of Aflatoxins in Foods Using Liquid ChromatographyTriple Quadrupole Mass Spectrometry

Fig. 2 Sample preparation work flow.

Fig. 1 Structure of Aflatoxins.

Aflatoxin G1

Aflatoxin B1

Aflatoxin B2

Aflatoxin G2

O O

O

O O

OCH3

O O

O

O O

OCH3

O O

O

O

OCH3

O

O

O O

O

O

OCH3

O

O

5 g NaCl

Mixing for 5 min

Centrifugation

Roast Peanut 50.0 g

10 mL up to 50 mL with water

Clean -up by immunoaffinity column “ AFLAKING”

Filtration with glass micro filter

10 mL

Sample solution

Evaporation by N2 gas

1.0 mL water / acetonitrile=9/1 (v/v)

Elution with acetonitrile (3 mL)

200 mL water / methanol= 2 / 8 (v / v)

Add AFs standard solution

3

ExperimentAnalysis Standard Solution



The chromatogram and analytical conditions of the conventional method (Fig. 3, Table 1) were compared to and the high-speed method with using ultra-fast liquid chromatography (Fig. 4, Table 3). The flow rate was raised

to 0.45 mL/min accelerating AFs elution to 4 minutes (operating back pressure 44-50 Mpa). MRM transitions summarized in Table 2.

Column:

Mobile Phase A:Mobile Phase B: Gradient Program:

Flow Rate: Column Temp. :Injection Vol.:

Probe Voltage: Nebulizing Gas Flow:Drying Gas Flow:DL Temp.:Heat Block Temp.:

Table 1 Analytical conditions (typical 30 minute method).

HPLC

MS

Shim-pack FC-ODS （150 mm L. × 2.0 mm i.d., 3 µm）10 mmol/L Ammonium acetate-water Methanol 40%B(0-15 min) → 100%B(15.01-20 min) → 40%B (20.01-30 min)0.2 mL/min 40°C6 µL

+4.5 kV ESI-Positive mode3 L/min15 L/min 250°C 400°C

Column:


Flow Rate: Column Temp.:Injection Vol.:


Table 3 Analytical conditions (optimized fast method).

HPLC

MS

Shim-pack XR-ODS II (100 mm L. ×2.0 mm i.d., 2.2 µm)10 mmol/L Ammonium acetate-water Methanol 40%B (0-4.5 min)→ 100%B(4.51-6.5 min)→ 40%B (6.51-12 min)0.45 mL/min 50°C6 µL

+4.5 kV ESI-Positive mode3 L/min15 L/min 250°C 400°C

Fig. 3 Chromatograms of AFs (0.5 µg/L each).

Fig. 4 Chromatograms of AFs (0.5 µg/L each).

1.0 2.0 3.0 4.0 min

- 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

(x1,000)

■Peaks 1. Aflatoxin G2 :331.10>245.00(+) 2. Aflatoxin G1 :328.90>242.95(+) 3. Aflatoxin B 2 :315.10>258.95(+) 4. Aflatoxin B 1 :313.10>240.95(+)

1

2

3

4

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

(x100)

Peaks 1. Aflatoxin G2 :331.10>245.00(+) 2. Aflatoxin G1 :328.90>242.95(+) 3. Aflatoxin B 2 :315.10>258.95(+) 4. Aflatoxin B1 :313.10>240.95(+)

4

3

2

1

Table 2 MRM Parameter.

Compound Transition Pause time

Dwell time CE

(V) Resolution (Q1,Q3) (ms) (ms)

Aflatoxin B1 313.10 > 240.95 3 100 -40 Unit Aflatoxin B2 315.10 > 258.95 3 100 -33 Unit Aflatoxin G1 328.90 > 242.95 3 100 -30 Unit Aflatoxin G2 331.10 > 245.00 3 100 -32 Unit

4

Fig. 5 Callibration curves AFs (linearity beyond R2=0.999 was acquired).

Fig. 6 Chromatographic optimization of column temperature.


B1 B2 G1 G2

0.0 2.5 5.0 7.5 Conc. 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 Area (x10,000)

0.0 2.5 5.0 7.5 Conc. 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 Area (x10,000)

0.0 2.5 5.0 7.5 Conc. 0.0

1.0

2.0

3.0

4.0

5.0

6.0 Area (x10,000)

0.0 2.5 5.0 7.5 Conc. 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00 Area (x10,000)

µg/L µg/L µg/L µg/L

Table 4 L.O.Q. and Linearity (n=6). Compound L.O.Q. (µg/L) Linearity R^2

Aflatoxin B1 0.1 0.1-10 µg/L 0.9995372

Aflatoxin B2 0.1 0.1-10 µg/L 0.9997556

Aflatoxin G1 0.05 0.05-10 µg/L 0.9994336

Aflatoxin G2 0.1 0.1-10 µg/L 0.9992275

Effect of Column TemperatureColumn temperature was optimized in order to accelerate compound elution without compromising peak shape or intensity. Finally a column temperature of 50°C was chosen (Fig. 6).

0.0 1.0 2.0 3.0 4.0 5.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

(x1,000)

40°C

50°C

60°C

0.0 1.0 2.0 3.0 4.0 5.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

(x1,000)

0.0 1.0 2.0 3.0 4.0 5.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

(x1,000)

Peaks 1. Aflatoxin G2 :331.10>245.00(+) 2. Aflatoxin G1 :328.90>242.95(+) 3. Aflatoxin B2 :315.10>258.95(+) 4. Aflatoxin B1 :313.10>240.95(+)

1

2

3

4

5

Effect of ESI Probe Position



Further optimisation was achieved through optimization of ESI probe position ranging from -2 mm to +3 mm from the central position (Fig. 7). Chromatographic comparison (Fig. 8) illustrates both peak intensity and level of noise are

influenced by probe position. Optimization required highest S/N and minimum noise at +1 mm (Fig. 9 & 10). [Noise was calculated by ASTM method with 3 blocks of 0.5 min around each

Fig. 8 Chromatograms comparison of a ESI probe position difference.

Fig. 7 ESI probe of LCMS-8030.

0

10

20

30

40

50

60

70

central +0.5 +1 +1.5 +2

probe position (mm)

S/N

AF G2AF G1AF B2AF B1

0

50

100

150

200

250

central +0.5 +1 +1.5 +2

Noi

se

AF G2AF G1AF B2AF B1

G2 G1

Central

+0.5 mm

+1.0 mm

+1.5 mm

+2.0mm

Fig. 9 S/N of probe position difference.

Fig. 10 Noise of probe position difference.

Probe Position

1.0 2.0 min

(x1,000)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

2.0 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

(x1,000)

6

1.0 2.0 3.0

0.0

0.5

1.0

1.5

2.0

2.5

(x1,000)

2.0 3.0 4.0

0.0

1.0

2.0

3.0

4.0

(x100)

3.0 4.0

0.00

0.25

0.50

0.75

1.00

1.25

(x1,000)

1.0 2.0 3.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 (x100)

G2 G1 B2 B1

0.43 µg/L 1.64 µg/L 1.54 µg/L 0.34 µg/L

Fig. 11 Chromatograms of AFs in roast peanut matrix.

Table 5. Results of quantity analysis. Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2

Conc. (µg/L) (µg/L) (µg/L) (µg/L)

Recovery (%)

Conc.

Recovery (%)

Conc.

Recovery (%)

Conc.

Recovery (%)

Sample1 1.54 75 0.34 69 1.64 82 0.43 86

Sample2 1.44 70 0.34 69 1.64 82 0.37 74


ResultsAnalysis of Roast Peanut Matrix The recovery test of AFs spiked into the roast peanut powder was performed in duplicate experiments. Overlaid chromatograms of spiked AFs in roast peanut matrix to un-spiked were shown (Fig. 11). Solid line - spiked compared to dotted line - un-spiked. Interference peaks were not detected in un-spiked samples. Quantitation results using external standard method show the recovery rate was in the range of 69-86% (Table 5). This relatively low recovery rate is a known problem when extracting AFs with a solvent from the powder of a roast peanut. Further method development is underway to increase recovery rate.

7


Ultra High-speed Method For Screening Analysis

Further improvements to speed of analysis were made using a reduced column particle size and length (1.6 µm, Shim-pack XR-ODS III 50 mm × 2.0 mm). With these

conditions, AFs eluted within 2 minutes with L.O.Q. of 0.5 µg/L. This ultra high-speed analysis could prove useful when screening many samples.

Conclusion Analysis of AFs in the roast peanut was studied.

Accelerated method was developed due to capacity of ultra high pressure liquid chromatography (Nexera).New choice of Shim-pack column enabled faster elution times. Two high-speed methods were developed eluting AFs within 4 and 2 min.

Column temperature and ESI probe position were important conditions of AFs analysis.Immunoaffinity column was useful for cleanup from the roast peanut matrix.The results of recovery test was 69-86%.

Column:


Flow Rate: Column Temp. :Injection Vol.:


Table 6. Analytical conditions.

HPLC

MS

Shim-pack XR-ODS III （50 mm L. ×2.0 mm i.d., 1.6 µm）10 mmol/L Ammonium acetate-water Methanol 40%B(0-2.25 min)→ 100%B(2.26-3.25 min)→ 40%B(3.26-6.00 min)0.45 mL/min 50°C 6 µL

+4.5 kV ESI-Positive mode3 L/min15 L/min 250°C400°C

0.5 1.0 1.5 2.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

(x100)

■Peaks 1. Aflatoxin G2 :331.10>245.00(+) 2. Aflatoxin G1 :328.90>242.95(+) 3. Aflatoxin B 2 :315.10>258.95(+) 4. Aflatoxin B 1 :313.10>240.95(+)

4

3

1

2

Table 7 MRM Parameters.

Fig. 12 Chromatograms of AFs (0.5 mg/L each).

Compound Transition Pause time

Dwelltime CE

(V) Resolution (Q1,Q3)

(ms) (ms)

Aflatoxin B1 313.10 > 240.95 1 50 -40 Unit

Aflatoxin B2 315.10 > 258.95 1 50 -33 Unit

Aflatoxin G1 328.90 > 242.95 1 50 -30 Unit

Aflatoxin G2 331.10 > 245.00 1 50 -32 Unit

Keiko Matsumoto1, Jun Watanabe1, Manabu

Yukiyama1, Junko Iida1, Itatu Yazawa2　 1SHIMADZU CORPORATION; 2Imtakt Corporation

High throughput analysis of anion surfactant using ultra-high speed LC-MS/MS and 1 mm inside diameter column

ASMS 2012 TP31-611

2

IntroductionHigh resolution separations with high speed MS/MS data acquisition have created new opportunities in academic and applied research. High resolution separations can be achieved by ultra-high pressure columns with a particle diameter > 2 um or as an alternative technology using particles with a diameter of 3 um and 1 mmID. Both

LAS surfactants (C10 to C14) were obtained from Wako Pure Chemical Ind., Ltd(Osaka,Japan). Several levels of calibrators were made from the stock solution.Commercial LAS products consist of more than 20 individual components. The ratio of the various homologues and isomers representing different alkyl chain lengths and aromatic ring positions along the linear alkyl chains is relatively constant across the various household applications. The analysis of LAS surfactants (C10 to C14) share a common fragment ion at m/z 183 independent of linear carbon chain length and a fragment ion at m/z 119.

technologies are designed for higher resolution but they approach the problem in a different way. In this paper we have applied a 1 mm inside diameter column phase to the analysis of linear alkylbenzene sulfonate anion surfactants using a high speed MS/MS detection system.

As high resolution analysis, various isomers of LAS surfactant can be separated and be detected severally. Chromatographic separations were carried out using Unison UK-C18 HT (1 mmI.D. , 50 mmL., 3 um) , which has high pressure resistance and large sample capacity1). The

column temperature was maintained at 40°C . Flow rate was 0.15 mL/min with a binary gradient system. Components were detected in electrospray negative MRM mode for quantitative analysis.

Nexera MP UHPLC system was connected to LCMS-8030 triple quadrupole mass spectrometer.

Methods and MaterialsAutosampler SIL-30ACMP

・Utrafast injection performance exceeding that of current models ・Ultralow carryover ・6 microtiter plates can be loaded, enabling a maximum of 2304 samples to be analyzed

LCMS-8030 High Speed Mass Spectrometer Polarity Switching 15 msec Scanning Speed Max. 15000 u/sec


Fig. 1 UHPLC Nexera MP ＆LCMS-8030

Fig. 2 Structure of LAS (C11)

CH3

SO 3H

CH3

m/z 183

ResultsConventional HPLC Analysis

3


UHPLC conditions (Nexera MP system) Column: Mobile phase A: Flow rate: Time program: Injection vol.: Column temperature:

Unison UK-C18 HT 1 mmI.D.x 50 mmL., 3 um10 mM Ammonium acetate, B: Acetonitrile0.15 mL/minB conc.40%(0 min) 60%(5 min) 95%(5.01-7 min) 40%(7.01-10 min)5 µL40°C

MS conditions (LCMS-8030) Ionization: ESI, Negative MRM mode MRM transition are shown in Table 1.

Fig. 3 Mass chromatograms of conventional conditions (LAS C10 - C14 each 20 ppb)

Table 1 MRM transition of LAS

Table 2 Recovery data spiked in tap water sample at 20 ppb (n=5)

C10 C11 C12 C13 C14

20 ppb Standard sample 263865 218485 240309 255308 205600

Spiked tap water sample 282372 227065 244975 265646 222209

Recovery (%) 107.0 103.9 101.9 104.0 108.1

C10 C11 C12 C13

C14

C10 C11 C12 C13 C14

Quantitative ion Q1/Q3 297/183 311/183 325/183 339/183 353/183

297/119 311/119 325/119 339/119 353/119 Qualitative ion Q1/Q3

Quantitative ion

Qualitative ion

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

2500

5000

7500

10000

12500

15000

17500

20000

5:353.20>119.10(- ) 5:353.20>183.10(- ) 4:339.20>119.10(- ) 4:339.20>183.10(- ) 3:325.20>119.10(- ) 3:325.20>183.10(- ) 2:311.20>119.10(- ) 2:311.20>183.10(- ) 1:297.15>119.10(- ) 1:297.15>183.10(- )

The above counts are peak area

Several Peaks for each LAS carbon chain were detected. The result shows that excellent　separation was achieved with these conditions in spite of comparatively high speed analysis. Spiked tap water sample (20ppb LAS) showed good recovery

at almost 100% (Table 2). These results indicate that the LC-MS/MS method was not influenced by sample matrix in tap water.

4

Fig. 5 Mass chromatograms of high throughput condition　　 (LAS C10～ C14 each 20 ppb)

Fig. 4 HPLC path of SIL-30AC

High throughput analysisAlthough several peaks were detected for each LAS as mentioned above, it does however make quantification more complex. In high throughput analysis, the condition of which each LAS is detected as one peak was re-examined

The Nexera UHPLC system has a unique wash process in which the needle and sample loop can be washed, separating it from the HPLC line after injection. Washing of the needle seal and shortened the HPLC line result from this function (Fig. 4). Using this function, chromatographic separations were carried out on LC-MS/MS condition as follows.

Each LAS was separated as one peak within 1 minute. Total analysis cycle was within 2 minutes.　In this condition, the linearity of calibration curve and repeatability for each LAS was excellent and all LAS can be

detected from 0.1ppb (Fig. 6, Table 3). Spiked Tap water sample (200 ppb LAS) showed good recoveries with almost 100%. It indicates that this LC-MS/MS method was not influenced by sample matrix in tap water.

Modified UHPLC conditions: Flow rate: 0.3 mL/min(0-1 min) 0.5 mL/min(1.01-1.30 min) 0.3 mL/min(1.31-1.5 min) Time program: B conc.55%(0 min) 90%(0.7 min) 95%(0.71-0.75 min) 55%(0.76-1.5 min)

MS conditions (LCMS-8030): same as previously used.

C10 C11 C12 C13 C14

min 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0

10000

20000

30000

40000

50000

60000

5:353.20>119.10(-)5:353.20>183.10(-)4:339.20>119.10(-)4:339.20>183.10(-)3:325.20>119.10(-)3:325.20>183.10(-)2:311.20>119.10(-)2:311.20>183.10(-)1:297.15>119.10(-)1:297.15>183.10(-)

0.7

Fig. 6 Representative calibration curve in high thoughput conditions (LAS C10, C12, C14)

C10

R 2=0.998

C12

R 2=0.996

C14

R 2=0.998

％RSD

C10 3.09

C11 4.11

C12 5.77

C13 7.50

C14 3.43

Table 3 Area repeatability of 0.5 ppb (n=5)

0 10 20 30 40 ppb 0

50000

100000

150000

200000

250000

300000

350000

400000

Area

0 10 20 30 40 ppb 0

100000

200000

300000

400000

Area

0 10 20 30 40 ppb 0

100000

200000

300000

400000

500000

600000 Area


5

ConclusionsUsing 1 mm inside diameter column, 5 LAS were separated with high resolution within 1 minute and were detected with high sensitivity. The excellent linearity was obtained in the calibration curves of all LAS.

1) J. Watanabe et al., 56th ASMS Conference, LCMS I (WP)-295 (2008).

LCMS-8030 ultra fast precursor ion scanning is useful and reliable even for such a high thoughput analysis where extremely narrow peaks were obtained.

References

Simultaneous screening analysis of LAS using precursor ion scanning Simultaneous screening analysis of LAS using high speed precursor ion scanning was also conducted. M/z 183 was applied as a common fragment ion of LAS. It is commonly known that increasing the scanning speed in a conventional triple-quadrupole mass spectrometer results in mass errors. Increasing a scan speed, the detected m/z of LAS was examined using LCMS-8030.UHPLC conditions: same as previous high thoughput analysis conditionMS conditions (LCMS-8030) Ionization: ESI, Negative, precursor ion scan mode Prec of 183 (Scan range: m/z 180-500) Scan time: 1 sec (326 µ/sec), 0.33 sec (1000 µ/sec), 0.1 sec (3750 µ/sec).

Fig. 7 Measurement results of precursor ion scans; LAS standard solution left：TIC chromatogram, right：C12 (third peak) peaktop mass spectrum

LAS was analyzed at various scan speeds. The TICs and mass spectra are shown above for measurements at 326, 1000 and 3750 u/sec. At 326 u/sec, a proper peak shape was not obtained due to insufficient data points. The mass chromatogram at 3750 u/sec shows sharper peaks. The

results indicate that sufficient data points are obtained at a scan rate of over 3000 u/s.In addition, no precursor ion mass error was apparent at any scanning speed in the results.

326 u/sec

1000 u/sec

3750 u/sec

0.25 0.50 0.75 min 0

250000

500000

750000 1:TIC(- )

0.25 0.50 0.75 min 0

250000

500000

750000 1:TIC(- )

0.25 0.50 0.75 min 0

250000

500000

750000 1:TIC(- )

300 310 320 330 340 350 m/z 0.0

1.0

2.0

3.0

4.0

5.0 Inten. (x100,000)

325.20

300 310 320 330 340 350 m/z 0.0

1.0

2.0

3.0

4.0

5.0 Inten. (x100,000)

325.15

300 310 320 330 340 350 m/z 0.0

1.0

2.0

3.0

4.0

5.0 Inten. (x100,000)

325.15


Keiko Matsumoto1, Jun Watanabe1, Junko Iida1

1Shimadzu Corporation.1, Nishinokyo-Kuwabaracho

Nakagyo-ku, Kyoto 604–8511, Japan

Simultaneous analysis of anionic, amphoteric and non-ionic surfactants using ultra-high speed LC-MS/MS

ASMS 2012 TP31-612

2

IntroductionSurfactant chemistry has made a considerable impact on a number of household products including detergents, shampoos and toothpaste. Products are generally classified by the type of each hydrophilic substructure into anionic, cationic, amphoteric and non-ionic surfactants. Either anionic or non-ionic surfactants are typically used as synthetic detergents, however, to better elucidate the

potential risk in environmental samples, mainly in agricultural soils and sediments, methods need to take into account a range of surfactant chemistries. Current surfactant monitoring methodologies tend to focus on a specific surfactant. Here, we have developed the simultaneous analysis method for typical anionic, amphoteric and non-ionic surfactant using LC-MS/MS.

Commercially available surfactants were used for this experiment. Standards of surfactants were diluted with water / acetonitrile =3/7 to an appropriate concentration and then analyzed by LC-MS/MS. As an LC-MS/MS system, UHPLC was coupled to triple quadrupole mass spectrometer (Nexera MP with LCMS-8040, Shimadzu Corporation, Kyoto, Japan). Separation was achieved using a YMC-Triart C8 column (100 mmL., 2.0 mmI.D., 1.9 um particles) and column oven temperature was maintained at

40°C. Samples were eluted at flow rate 300 uL/min with a binary gradient system; the mobile phase consisted of (A) 10 mM ammonium acetate buffer and (B) mixture of 10 mM ammonium acetate / acetonitrile / isopropanol (1/4/5). LC-MS/MS with electrospray ionization was operated in multiple-reaction-monitoring (MRM) mode with ultra-fast polarity switching.

Methods and Materials


Fig. 2 LCMS-8040 triple quadrupole mass spectrometer

Fig. 1 Structure of anionic, amphoteric and non-ionic surfactant

High Speed Mass Spectrometer Ultra Fast Polarity Switching 15 msecUltra Fast MRM Max. 555 transition /sec

CH3 (CH 2)nCH 2 N+

CH3

O-

O

CH3

Betain (n=10,12,14)

CnH(2n+1)

SO 3H

LAS (n=10 -14)

Heptaethyleneglycoldodecylether

(CH 2)11OH (OCH 2CH 2)7 CH3

3

ResultsMethod development for surfactants

UHPLC conditions (Nexera MP system)Column:Mobile phase A: B:Flow rate:Time program:Injection vol.:Column temperature:

MS conditions (LCMS-8040)Ionization: ESI, Positive/Negative MRM mode

YMC TriartC8 100 mm×2.0 mm, 1.9 um10 mM Ammonium acetate10 mM Ammonium acetate / Acetonitrile/ isopropanol (1/4/5)0.3 mL/minB conc.75%(0 min) -95%(1.5-3 min) - 75%(3.01-5 min)10 uL40°C

The following standard surfactants were selected and analyzed; anionic surfactant: linear alkylbenzene sulfonate (LAS) C10-C14 mixture, amphoteric surfactant: EMPIGEN BB Detergent (Betaine) C10, C12, C14 mixture (Sigma-Aldrich, St.Louis, MO), and non-ionic surfactant: heptaethyleneglycoldodecylether (HEDE). Full scan measurement by flow injection analysis (FIA) was conducted to determine the optimum ionization polarity of target compounds followed by MRM transition optimization by FIA. As a consequence, all LASs were detected as the de-protonated ions (M-H) in negative ion

mode and m/z 183 was selected as the product ion of MRM transitions for all LASs (C10-C14). All Betaine were detected as protonated ions (M+H) in positive ion and m/z 104 was selected as the product ions of MRM transitions for all Betaines (C10, C12 and C14). HEDE yielded the protonated ion (M+H) in positive ion as the precursor and m/z 133 was selected as product ion for MRM transition. As compounds selected in this experiment formed either positive or negative ion, high-speed polarity switching is an important element to consider in developing an optimized LC-MS/MS method.

Table 1 MRM transition of LAS

Compound

LAS C10

LAS C11

LAS C12

LAS C13

LAS C14

Betain C10

Betain C12

Betain C14

HEDE

Polarity

-

-

-

-

-

+

+

+

+

MRM transition

297.15 > 182.60

311.20 > 182.60

325.20 > 182.70

339.20 > 182.60

353.40 > 182.60

271.95 > 103.80

300.00 > 103.70

328.20 > 103.70

495.30 > 133.15


4

Fig. 3 shows MRM chromatograms of the nine surfactants. It took 5 minutes per one LC-MS/MS analysis, including column rinsing, and excellent separation and high sensitive detection were obtained.

Fig. 3 Mass Chromatgrams of typical anionic, amphoteric and non-ionic surfactant 　　（concentration of each compound : 5ppb）

Fig. 4 Representative calibration curve （Betain C10, HEDE, LAS C13）


1.00 1.25 1.50 1.75 2.00 2.25 2.50 min 0

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

Betain C10 （+）

LAS C10 （-） LAS C11 （-）

LAS C12 （-）

Betain C12 （+）

LAS C13 （-）

HEDE （+）

LAS C14 （-）

Betain C14 （+）

The dilution series of these compounds were analyzed. All compounds were detected at ppb level with excellent linearity　(Table 2, Fig. 4).

Table 2 Linearity 9 sufactants

Compound

LAS C10

LAS C11

LAS C12

LAS C13

LAS C14

Betain C10

Betain C12

Betain C14

HEDE

Polarity

1-500 ppb

1-500 ppb

1-500 ppb

1-500 ppb

0.5-100 ppb

1-100 ppb

1-100 ppb

1-100 ppb

1-100 ppb

MRM transition

0.998

0.999

0.997

0.999

0.998

0.999

0.999

0.999

0.999

LAS C13

1-500 ppb R2=0.999

Betain C10

1-100 ppb R2=0.999

1-100 ppb R2=0.999

HEDE

ppb

Area

0.0 25.0 50.0 75.0 ppb 0

25000

50000

75000

100000

125000

150000

175000 Area

0 100 200 300 400 ppb 0

2500000

5000000

7500000

10000000

Area

0.0 25.0 50.0 75.0 0

500000

1000000

1500000

2000000

5


Quantitative Analysis of real world sampleThe quantitative analysis of real world sample using the kitchen detergents and liquid soap was achieved using this method. Kitchen detergents and liquid soap was diluted 1000 to 1 with water / acetonitril = 3/7. Finally, it was filtered through a 0.2 um filter and analyzed by LC-MS/MS. MRM chromatograms of each surfactants in the kitchen detergents and liquid soap is shown Fig. 5,6.

The 1000 to 1 dilution of liquid soap contained approximately 5 to 6 ppb LAS C12 and C13. Therefore, it was determined that undiluted liquid soap contains 5 to 6 ppm LAS C12 and C13. On the other hand, undiluted kitchen detergent contains approximately 40 ppm LAS C10, C11, C12 and C13, and approximately 75 ppm HEDE.

Fig. 5 Measurement results of liquid soap (dilution 1000 times)

Fig. 6 Measurement results of Kitchen detergent (dilution 1000 times)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 min

0

25000

50000

liquid soap （× 1000）

LAS C10 (-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

LAS C11

LAS C12

LAS C13

0.0 0.5 1.0 1.5 2.0 2.5 3.0 min 0

100000

200000

300000

400000

500000

HEDE(+)

LAS C13

LAS C12

LAS C11

LAS C10

Kitchen detargent (×1000)

matrix

ConclusionsTypical anionic, amphoteric and non-ionic surfactant were separated with high resolution within 2.5 minute. Even though selected compounds formed either positive or negative ion, all surfactant were detected with high sensitivity. High-speed polarity switching is an important element for simultaneous analysis of various surfactants.

This method was able to be applied to the quantification of surfactants in kitchen detergents and liquid soap.

Concentration (ppb)75.2 ppb

no detectionno detectionno detectionno detection

35.7 ppb31.0 ppb40.4 ppb44.3 ppb

LAS C10LAS C11LAS C12LAS C13LAS C14

HEDEBetain C10Betain C12BetainC14

Concentration (ppb)< 1ppb< 1ppb4.9ppb6.1ppb

no detectionno detectionno detectionno detectionno detection

LAS C10LAS C11LAS C12LAS C13LAS C14

HEDEBetain C10Betain C12BetainC14

Keiko Kudo1; Toshikazu Minohata2; Junko Iida2;

Hitoshi Tsuchihashi3; Kiochi Suzuki3; Kei Zaitsu4;

Noriaki Shima4; Munehiro Katagi4; Noriaki Ikeda1 1Kyushu University, Fukuoka, Japan; 2Shimadzu Corporation, Kyoto, Japan; 3Osaka Medical Collage, Takatsuki, Japan; 4Osaka Prefectural Police, Osaka, Japan

Screening analysis for drugs of abuse by LC-MS/MS enables fast polarity switching MRM triggered product ion scanning on the fly

ASMS 2012 MP24-591

2

IntroductionIn recent years the need for forensic, toxicological and clinical analyses have increased, and as a consequence of sample complexity, analysis has become increasingly challenging due to a growing trend in the use of illicit drugs and non-medicinal prescription drugs. Screening applications requires rapid and unambiguous results that can be achieved using a generic analysis method designed

for a large number of target compounds. To meet this need, a universal high speed MRM triggered product ion scanning method with fast polarity switching was applied to simultaneously screen, quantitate and confirm by reference to an MS/MS data base containing the majority of drugs of abuse available in Japan.



[Abused Drugs]　Amphetamine　Benzoyl ecgonine　Cocaine　Codeine　Dihydrocodeine　Ecgonine methyl ester　Ephedrine　Ketamine　MDA　MDMA　Methamphetamine　Methylephedrine　Methylphenidate　Morphine　Sildenafil　THC　THC-COOH

[Psychotropic Drugs]　Amitriptyline　Amoxapine　Aripiprazole　Chlorpromazine　Clomipramine　Dosulepin　Fluvoxamine　Haloperidol　Imipramine　Levomepromazine　Maprotiline　Mianserin　Mirtazapine　Nortriptyline　Olanzapine　Paroxetine　Promethazine　Quetiapine　Risperidone　Sertraline　Sulpiride　Trazodone　Zotepine

[Hypnotic Drugs]　7-Aminoflunitrazepam　7-Aminonimetazepam　7-Aminonitrazepam　8-Hydroxyetizolam　Allylisopropylacetylurea　alpha-Hydroxybrotizolam　alpha-Hydroxytriazolam　Alprazolam　Amobarbital (neg)　Barbital (neg)　Bromazepam　Bromovalerylurea　Brotizolam　Diazepam　desmethyldiazepam　Estazolam　Ethyl loflazepate　Etizolam　Flunitrazepam　Flurazepam　Hydroxyzine　Lorazepam　Lormetazepam　Midazolam　Nimetazepam　Nitrazepam　Oxazepam　Pentobarbital (neg)　Phenobarbital (neg)　Quazepam　Temazepam　Thiamylal (neg)　Triazolam　Zolpidem　Zopiclone

[Medical Drugs]　Acetaminophen　Acetylpheneturide　Atropine　Biperiden　Bupivacaine　Carbamazepin　Chlorpheniramine　Clonazepam　Dextromethorphan　Diclofenac　Diltiazem　Diphenhydramine　Diprophyline　Ethenzamide　Glibenclamide　Glimepiride　Ibuprofen (neg)　Lidocaine　Loxoprofen (neg)　Mepivacaine　Mexiletine　Pancuronium　Pentazocine　Salicylic_acid (neg)　Trihexyphenidyl　Vecuronium　Warfarin

[Pesticides]　Diquat　Fenitrothion (MEP)　Glufosinate　Malathion　Methomyl　Paraquat

[Natural Toxines]　Aconitine　Colchicine　Tetrodotoxin

Table 1 List of compounds for Forensic method.

3

Analysis of several drugs was performed using fast polarity switching and high speed data acquisition LC/MS/MS. This was achieved using Synchronized Survey Scan® which refers to the execution of MS/MS scanning triggered by survey

scan signals (in this case, MRM). Therefore, during the elution of a peak in MRM analysis, a full-product ion mass spectrum can also be obtained.

Samples were analyzed using a Nexera UHPLC system coupled to a LCMS-8030 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) with LC/MS/MS Method Package for Forensic Toxicology. Database contains product ion scan spectra for 286 forensic and toxicology-related compounds such as 87 Abused drugs, 105 Psychotropic drugs, 70 Hypnotic drugs and others. This library provides Synchronized Survey Scan parameters (product ion spectral data

acquisition parameters based on the MRM intensity as threshold) optimized for screening analysis. The simple quantitative method included the most frequently analyzed 111 components of Abused drugs, Psychotropic drugs and Hypnotic drugs for method validation (Table 1).Samples were separated using a Shim-pack FC-ODS using a gradient elution with ammonium formate and methanol.

MRM parameter Product Ion Scan parameter

Positive

Negative

Fig. 1 User Interface of MRM-Product Ion Scan setting at LabSolutions software.


HPLC (Nexera UHPLC system) Column

Mobile Phase A

Mobile Phase B

Gradient Program

Flow Rate

Column Temperature

Injection Volume

: Shim-pack FC-ODS (2.0 mmI.D. x 150 mmL., 3 um)

: 10 mM ammonium formate

: Methanol

: 5%B (0 min) - 95%B (15-20 min) - 5%B (20.01 - 30 min)

: 0.3 mL / min

: 40ºC

: 5 uL

Mass (LCMS-8030 triple quadrupole mass spectrometry)

Ionization

Polarity

Probe Voltage

Nebulizing Gas Flow

Drying Gas Pressure

DL Temperature

BH Temperature

: ESI

: Positive & Negative

: +4.5 kV (ESI-Positive mode);

-3.5 kV (ESI-Negative mode)

: 1.5 L / min

: 10 L / min

: 250ºC

: 400ºC


4

Fig. 2 MRM - Product Ion Scan screening data about 4 compounds.

ResultsMS/MS Library MatchingMRM chromatograms of four compounds (each 1000 ng/mL) spiked into urine and analyzed by Nexera coupled to LCMS-8030 following sample preparation (Fig. 2). These product ion scans were searched against the MS/MS library and the four previously identified peaks were assigned a high hit score. The assay generates both MRM and Product Ion Scan data (MS/MS) due to the speed of data acquisition

from the LC/MS/MS system. This results in quantitative data and library searching / product matching data to help with product ion confirmation. Fast polarity switching helps to provide information rich product ion spectra resulting in better detection and identificationfor each compound

Allylisopropylacetylurea

Diclofenac

Amobarbital (neg)

Thiamylal (neg)


10.0 12.4

0.0

1.0

2.0

3.0

4.0

(x100,000)

185.00>55.05(+)

12.5 10.1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

(x100,000)

296.00>214.00(+)

296.00>215.05(+)

10.0 12.5

0.0

1.0

2.0

3.0

4.0

5.0

6.0

(x1,000)

225.15>42.00(-)

12.5 10.3

0.00

0.25

0.50

0.75

1.00

(x10,000)

253.00>101.00(-)

253.00>58.10(-)

5

Fig. 3 Registration of 1st coefficient and intersection by calibration curve.

Table 2 The calculated results of 12 compounds in whole blood using LC-MS/MS (n=2 average).

ConclusionA high speed LC/MS/MS data acquisition system was applied to drug screening in forensic, toxicological and clinicalanalysis. To achieve a highly specific and sensitive detection method in screening and quantitation, an MRM triggered production scanning method using a polarity switching speed of 15msec and a scan speed of 15,000u/sec was applied to 111components including illicit drugs, psychotropics, hypnotics, pesticides and other substances. As the MRM acquisition time was very fast, this enabled product ion spectra to be generated in both positive andnegative ionization mode which could be matched against a user library of compounds as an automated aid toscreening and compound identification.

Simple Quantitative Method for Forensic analysesBased on the chromatogram obtained by injection of a fixed volume of individual reference standard solutions, the ratio of peak area of the reference standard was calculated and compared to that of the internal standard (Diazepam-d5). The resulting calibration curve was prepared by plotting the ratios of the amount of the reference standard to that of the internal standard. 1st coefficient and intersection were calculated from the calibration curve and were registered to the LCMS method(Fig. 3).

The method was validated using 12 of 111 compounds, between 0,05 ng/mL and 5 ng/mL, spiked into whole blood and treated with solid phase extraction (Table 2). The results from this method indicated a high quantitative performance and could prove useful as rapid confirmationand simple quantitative analysis.


0.05 ng/uL

Compounds

Diazepam-d5 (IS)AlprazolamAripiprazoleAtropineBrotizolamColchicineEstazolamEthyl loflazepateEtizolamFlunitrazepamHaloperidolRisperidoneTriazolam

12.98711.85715.592

7.22511.987

9.79411.46413.06812.09211.22912.01111.77811.728

396,803 114,210

59,975 327,992

42,175 21,970

128,563 85,673 73,746 77,218

616,938 783,134

34,424

[0.500]0.0380.0250.0840.0440.0150.0480.0280.0320.0600.0480.0380.042

342,441 918,575 700,323

3,105,470 325,945 159,050

1,078,497 489,250 575,984 545,933

5,378,666 6,811,884

283,935

[0.500]0.525 0.205 0.935 0.502 0.270 0.639 0.272 0.421 0.649 0.610 0.510 0.550

77,460 2,497,911 7,120,340

17,445,635 1,043,787

696,217 5,580,490 1,012,405 2,519,896 1,816,819

28,654,837 30,675,213

746,810

[0.500]6.72 3.07

10.92 7.49 3.72 5.49 2.68 5.67 7.12 7.10 6.72 6.78

R.T Area Conc.Area Conc.Area Conc.

0.5 ng/uL 5 ng/uL

60th ASMS Conference on Mass Spectrometry 　May 20 - 24, 2012 　Vancouver, BC, Canada

Shuichi Kawana1; Katsuhiro Nakagawa1; Riki Kitano1;

Haruhiko Miyagawa1; Richard Whitney2; Mark

Taylor2; Ling Gee Siang3

1Shimadzu Corp., Kyoto, JAPAN; 2Shimadzu Scientific Instruments, Columbia, MD; 3SHIMADZU (Asia Pacific) Pte. Ltd., Singapore,

Singapore

Comprehensive Two-dimensional Gas Chromatograph Quadruple Mass Spectrometer for Plant Metabolite Analysis

ASMS 2012 ThP27-592

2

Introduction

Experimental

- Identification of metabolic engineering targets- Understanding of stress response- Genetically modified food certification - Human nutrition

Several thousands of target compounds such as organic acids, amino acids, sugars etc. Different concentration levels for each compound

GCxGC　Column:　　Injection:　Injection volume:　　　Injection temp.:　Column temp.: 　Carrier gas:　Modulation time:

MS　Interface temp.:　Ion-source temp.:　Ionization mode:　MS mode:　Event time:

1st DB-5 30 m x 0.25 mm I.D., df = 1.0 µm2nd BPX-50 2.5 m x 0.1 mm I.D., df = 0.1 µmsplit (1:50)1 µL　　　　　280°C100°C (4min)→2°C /min→320°C (10 min)He (Constant = 150 kPa)6 sec

280°C200°CEIscan (m/z 50-500)0.03 sec20,000 u/sec

Ultra-high separation efficiency and wider dynamic range required


Applications of plant metabolomics

Characteristics of plant metabolite analysis 　

GC×GC-QMS

System Configuration of GCxGC-QMS

Sample Preparation

Analysis Conditions

Injector

Modulator

QMS GC Oven

1st columnnon-polar stationary phase

2nd Column Polar stationary phase

1st Column

2nd

Col

umn

GC-MS: GCMS-QP2010 Ultra(Shimadzu Corp.)GC*GC modulator: ZX1-GCxGC modulator (Zoex Corp.)

Oryza sativa subsp. japonica

(rice)

30 mg of Samples

900 µL of Extraction Solution

400 µL of Supernatant Solution

Residue

GCXGC-QMS

Echinochloa utilis (Japanese

barnyard millet)

Panicum miliaceum (millet)

Setaria italica(Pearl millet)

Add internal standard (10 µL of 0.1 mg/mL TA, 005 mg/mL HDA, and C24)

Stir, then centrifuge

Add 400 µL Milli-Q water

Stir, then centrifuge; separation into two phase

Dryness under nitrogen gas flow at 35°C

Add 100 µL of BSTFA

Heat at 80OC for 60 min

(TMS derivatization)

Add 1 mL water / methanol / chloroform (1 / 2.5 / 1)

3

ResultsScan Interval and Chromatographic Peak Shape

Analysis Results of oryza sativa subsp. japonica

GCxGC image

50 Hz (20,000 u/sec) 　25 Hz (10,000 u/sec) 10 Hz (4,000 u/sec)

oryza sativa subsp. japonica (rice)

Measured spectrum

Standard spectrum（Glycerol-3TMS)

Although this component was not be separated by 1st column, it was separated by GCxGC.

Similarity index：800

TIC

2147 peaks

~100 msec 2nd column

44.54 44.55 44.56 44.57 44.58 44.59 44.60 44.61 44.62 44.63

1000000

2000000

3000000

4000000

5000000

6000000

7000000 TIC

Methyl oleate

Methyl linolelaidate

Methyl linolenate

47.70 47.71 47.72 47.73 47.74 47.75 47.76 47.77

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000 TIC

47.70 47.71 47.72 47.73 47.74 47.75 47.76 47.77

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000 TIC


4

SummaryA fast scanning quadruple mass spectrometer (QMS) was shown to have a sufficient scan speed to fully characterize the ultra narrow peaks generated by the GC X GC system.Using this system, cereal samples (oryza sativa subsp. japonica (rice), echinochloa utilis (Japanese barnyard millet), panicum miliaceum (millet), and setaria italica (pearl millet)) were analyzed.　The number of detected chromatographic peaks ranged from1950 to 2333.These results demonstrated the GCxGC-QMS can be effectively used to detect several thousand plant metabolites in a wide concentration range.

Oryza sativa subsp. japonica (rice) Echinochloa utilis (Japanese barnyard millet)

Setaria italica (pearl millet)Panicum miliaceum (millet)

A B

C D

> 800700 - 799600 - 699 500 - 599

< 499Total

>600 (%)

506091329158

1214792.6

73771704245

0233389.4

Number of Chromatographic PeaksSimilarity Index

193681377186

0195090.4

806491101128

3196193.3

A B C D


Hiroki Nakajima, Satoshi Yamaki, Tsutomu Nishine,

Masaru Furuta　　SHIMADZU CORPORATION, Kyoto, Japan

Analysis of degradation products in electrolyte for rechargeable lithium-ion battery throughhigh mass accuracy MSn and multivariate statistical technique

ASMS 2012 WP26-541

2

Introduction


Experiment

Rechargeable lithium-ion batteries (LiB) are one of the major power sources for portable electronic devices and electric vehicles because of their high voltage and high energy density (Fig. 1-(a)). The electrolyte of a LiB is consisting of a lithium salt in an aprotic organic solvent. The typical operational potential of a LiB is between 0 and 5 V. Therefore, solvent can be reduced or oxidized at the

negative and positive electrodes during the battery charging process. As a result, various degradation products are generated in the electrolyte and cause some problems such as a decrease in the capacitance of battery (Fig. 1-(b)). Here, we present the analysis method of degradation products generated in electrolyte using high mass accuracy MSn and multivariate statistical technique.

The electrolyte was a mixture of ethylene carbonate (EC) and diethyl carbonate (DEC) (EC : DEC = 1 : 1 vol%) containing 1M lithium hexafluorophosphate (LiPF6). The electrolyte A taken from unused lithium-ion battery and the electrolyte B taken from lithium-ion battery repeated charge and discharge cycles (60°C, 30 times) were used as samples. Those samples were prepared 1/10 dilution with methanol for LCMS-IT-TOF (Shimadzu Corporation) measurement. Orthogonal Partial Least Squares

Discriminant Analysis (OPLS-DA) was performed using data acquired by LCMS-IT-TOF measurement of electrolyte A and electrolyte B (n=3) to find the compounds generated in electrolyte B. Then, those compounds were identified chemical formula using software “Formula Predictor” (Shimadzu Corporation). SIMCA-P+ (Umetrics) and Profiling Solution (Shimadzu Corporation) were used for OPLS-DA (Scheme).

e- e-

Li xC6 Li -xMO2

Li + Anode Cathode

Separator

Electrolyte

M=Mn. Co, Ni

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35 Cycle Number

Cap

acity

/ m

Ah Charge Discharge

(a) (b)

Electrolyte

Fig. 1 Rechargeable lithium-ion battery component of lithium-ion battery (a), a decrease in the capacitance of battery (b).

1. Acquisition of the high mass accuracy MSn data (LCMS-IT-TOF)

3. Searching of degradation products (SIMCA-P+)

2. Peak alignment and generation of peak list (Profiling Solution)

Scheme Work flow of the analysis of degradation product in electrolyte for LiB

4. Prediction of chemical formula (Formula Predictor)

Structual estimation

3

Results and discussion


MS data of electrolyte A and electrolyte B were acquired using LCMS-IT-TOF under the analytical conditions shown Table 1. On the score plot of OPLS-DA, the group of electrolyte A and electrolyte B were located at left side and right side, respectively (Figure 2-(a)). 15 unique ions of electrolyte B were observed at right side on S-plot (Figure 2-(b)). And, those ions were not detected on the extracted ion chromatogram (EIC) of electrolyte A (Fig. 3). These results suggested that those ions were degradation products generated in the electrolyte of lithium-ion battery repeated charge and discharge 30 cycles.

: Shim-pack FC-ODS (2.0 mmI.D.x150 mm, 3 mm) : water: 0.2 mL/min: 40°C : methanol : 5%B (0 min) → 55%B (30 min) → 5%B (30.01 min) : 1 µL : ESI(+) : 4.5 kV : 200°C : 200°C : 1.5 L/min : 0.1 MPa : m/z 80 - 1000

Column Flow rate Column temp. Mobile phaseA Mobile phaseB Time prog.Injection volume Ionization mode Probe voltage CDL temperature BH temperature Nebulizing gas Drying gasScan range

Table 1 LCMS analytical conditions

(M+H)+

(M+NH4)+

(M+Na) +

(M+K) +

255.0 260.0 265.0 270.0 275.0 280.0 285.0 290.0 295.0 300.0 305.0 310.0 315.0 m/z 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Inten. (x10,000,000)

289.0529

284.0982

305.0251

267.0707

17.0275

21.9822

37.9544

C H 3 O

O

O O O

O O

O

O C H 3

-20000

-15000

-10000

-5000

0

5000

10000

15000

20000

-40000 -30000 -20000 -10000 0 10000 20000 30000 40000

to[1

]

t[1] R2X[1] = 0.674304

a1

a2 a3

b4

b5 b6

Electrolyte A Electrolyte B

-1.0

-0.8

-0.6

-0.4

-0.2

-0.0

0.2

0.4

0.6

0.8

1.0

-0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28

p(co

rr)[1

]

w[1] R2X[1] = 0.674304

1

2

3

4

5 15

6 7 8

9

10 11

12

13 14

Electrolyte B

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

(x10,000,000)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

(x10,000,000)

Electrolyte A

Fig. 2 The result of OPLS-DA, score plot (a), S-plot (b)

(a)

(b)

(a)

(b)

Fig. 3 EICs of ions detected in Electrolyte B (b) These ions were not detected on EIC of electrolyte A (a).

Fig. 4 MS data and predicted structure of peak number 2 (m/z 284.0982) being one of 15 unique ions of electrolyte B

4

ConclusionIt was clear that the chemical species of degradation products generated in the electrolyte with increasing charge and discharge cycles were carbonate and phosphate from result of this study. The formulae of 15 degradation products detected in Electrolyte B were identified as below.


The formula of peak number 2 (m/z 284.0982) being one of 15 unique ions of electrolyte B was predicted as C9H14O9 (polycarbonate) using high mass accuracy MS data and formula predictor. Indeed, the structure of C9H14O9 was predicted as H3C-(OCO2-C2H4)2-OCO-CH3 referring to some articles on degradation products in electrolyte. MSn

of the ion (m/z 284.0982) also was measured to determine the validity of the predicted chemical structure. Each product ions and neutral loss in MS2 and MS3 data showed that the predicted structure of C9H14O9 was correct.

By the same method, formula of peak number 13 (m/z 283.0336 ) was identified as C3H7O4P. Structure of it was determined as phosphate shown in Fig. 6.

C9H15O9+

NL: C 5H8O6

Precursor ion : m/z 284.0982 (C9H14O9 +NH4)+

100 200 300 400 500 600 700 800 900 m/z 0.0

1.0

2.0

3.0

4.0 Inten. (x100,000)

267.0701

100 200 300 400 500 600 700 800 900 m/z 0.0

1.0

2.0

3.0

4.0

5.0 Inten. (x10,000)

103.0374

MS

MS2

MS3

100 200 300 400 500 600 700 800 900 m/z 0.0

1.0

2.0

3.0

4.0 Inten. (x10,000,000)

289.0534

284.0983

226.9505 103.0067

C H 3 O

O

O O O

O O

O

O C H 3

C H 3 O

O

O O O

O O

O

O C H 3

C H 3 O

O

O O O

O O

O

O C H 3

NH4+

H+

C4H7O3+

C4H7O3+

H+

-NH3

Fig. 5 MSn data of peak number 2 (m/z 284.0982) detected in electrolyte B

Fig. 6 of Structure peak number 13

Peak No. m/z R.T.(min) Ion species M.W Predicted formula Mass accuracy (ppm)

123456789101112131415

C H 3 O P O

O O

C H 3

C H 3

229.0678284.0982295.1032177.0512400.1458295.056 262.0853185.0577251.1111 269.0162350.1003283.0336381.0938293.0777245.0641

26.64520.401 29.482 6.178 31.29415.83325.37410.05427.41619.87928.4264.385 30.857 28.487 15.713

(M+Na)+(M+NH4)+

(M+H)+

(M+Li)+

(M+NH4)+

(M+Na)+

(M+NH4)+

(M+H)+

(M+H)+

(M+H)+

(M+NH4)+

(2M+Li)+

(M+Na)+

(M+Na)+

(M+Na)+

206294170382272244184250268332138358270222266

C8H14O6

C9H18O9

C11H18O9

C4H11O5PC14H22O12

C8H17O8P C10H13O5P C5H13O5P C8H17O5F3

C7H7O2F6P C8H17O7F4PC3H7O4P C12H23O10P C9H19O7P C8H14O7

-2.62-0.35+1.69+2.92+2.50-1.36+1.53+4.32+2.39-2.60+1.14+4.32-1.57+1.36-0.41

Takako Hishiki 1, Tsuyoshi Nakanishi 2 and Makoto

Suematsu 1, 3

1 Department of Biochemistry, School of Medicine,

Keio University, Tokyo, Japan

2 MS Business Unit, Shimadzu Corporation, Kyoto,

Japan

3 Japan Science and Technology Agency, Exploratory

Research for Advanced Technology, Suematsu Gas

Biology Project, Tokyo, Japan

Sensitive detection and quantification of hydrogen sulfide as a gasotransmitter by combining monobromobimane-based derivatization to triple quadrupole LC/MS/MS

ASMS 2012 WP25-512

2

OverviewHydrogen sulfide (H2S) regulates various cellular functions as a gasotransmitter and reliable and accurate quantification of H2S concentration in biological samples is required. Here the quantitative analysis of endogenous H2S level in tissue samples was performed by coupling the monobromobimane-based assay to triple quadrupole

LC/MS/MS. Furthermore, this method has an advantage of the measurement at neutral pH to quantify biologically free-gaseous H2S not acid-labile H2S and this approach is expected to be a useful method to quantify free H2S level in tissue samples.

IntroductionHydrogen sulfide (H2S) is known to be a third gaseous mediator and also to have important roles on a vasodilation in both the peripheral and cerebral circulation. To elucidate various signal transduction pathways elicited by H2S, reliable and accurate measurement of H2S concentration in biological samples becomes increasingly significant. Until now, head space gas analysis using gas chromatography and measurement by polarographic sensor have been

reported to quantify H2S concentration in biological samples. However these approaches often yielded a higher value resulting from acid-labile H2S not from biologically free-gaseous H2S. Here we developed sensitive quantitative analysis of H2S in biological samples, mouse brain tissues by combining a monobromobimane-based derivatization at a neutral pH to SRM (selected reaction monitoring) using triple quadrupole LC/MS/MS.

Methods Thiol-specific derivatization agent monobromobimane was used to quantify the endogenous H2S in brain tissues. Frozen brains from 12-d-old mice were placed in two volumes of ice-cold 5 mM monobromobimane (mBBr) in 10 mM Tris-HCl (pH 7.5) and then homogenized. Methanol was added to precipitate proteins. The mixture was vortexed and centrifuged. A supernatant was desalted by a solid-phase extraction and filtrated through a 5-kDa cutoff filter. An aliquot of 10 µL was analyzed to quantify sulfide-dibimane (SDB), monobromobimane-based derivatives of H2S by LC/MS/MS instrument, Nexera UHPLC system and LCMS-8030 triple quadrupole mass spectrometer. SDB was eluted from an ODS column (150 mmL. X 2.1 mmI.D. 1.7 µm particle size) with a gradient of acetonitrile, detected in negative mode ESI / SRM mode.

Fig. 1 Analytical condition of SRM to quantify endogenous hydrogen sulfide


Column : commercially available ODS column (150 mmL. X 2.1 mmI.D. 1.7 mm particle size) Flow rate : 0.2 mL/minColumn temp. : 40°CMobile phase : A) water containing 0.1% formic acid : B) acetonitrile containing 0.1% formic acidTime prog. : 2%B (0 → 5min) → 50%B (23 min) → 100%B (25 → 27 min) → 2%B (28 → 30 min)Ionization mode : ESI negativeDL temp. : 250°CHB temp. : 400°CNebulizing gas flow : 3.0 L/minDrying gas flow : 15 L/minTransition : m/z 413.10 → m/z 191.15 (Q1 : 30 V, CE : 20 V, Q3 : 20 V)

3

Results

Fig. 2 Scheme of the derivatization of hydrogen sulfide with monobromobimane

Fig. 3 MS/MS spectra of SDB standard on various collision energy (CE)

Fig. 4 Calibration curves of SDB and NaHS standard

Fig. 5 MS chromatogram and MS spectrum on SRM of SDB derived from H2S in murine brain tissues

+ HS-

+

+ 2 (Br-)


Hydrogen sulfide is derivatized with monobromobimane reagent and forms sulfide-dibimane (SDB). Reacted SDB is stable at room temperature and is measured on SRM analysis. In this study, the transition of SDB was determined at m/z 413 → 191 at CE of 20 V in ESI/negative mode.

(A) A calibration curve of SDB standard. A calibration curve was plotted by dilution series of SDB standard and were linear at a range of 1-5000 nM (R2>0.99).

(B) A calibration curve of NaHS solution forming H2S A calibration curve was also plotted by using the derivatization with mBBr to H2S resulting from NaHS solution. Various concentrations of NaHS were reacted with excess monobromobimane (1 mM) for 10 min on ice.

N N

O

O Br

N N

O

O

N N

O

O S

0 1 2 3 4 5

0

200

400

600

800

1000A

SDB (µm)

Peak

are

a x

103

B

0 1 2 3 4 5

0

200

400

600

800

1000

NaHS (µm)

Peak

are

a x

103

1

2

3

0

10 20 0 5 15 25 100 200 300 400

5

10

0

15

m/z

A

MRM

Retention time (min)

19.2 min

B

SDB m/z 413.1

m/z 191.2 m/z 191.2

MS spectrum on SRM

Inte

nsity

(cps

) x 1

04

Inte

nsity

(cps

) x 1

03

MS chromatogram on SRM

m/z 413.1→191.2

4

Endogenous H2S concentration in neonatal mouse brain was determined by SRM analysis using a monobromobimane-based assay. To quantify H2S level in brain tissues, a calibration curves was plotted by a SDB standard and applied as a external calibration. Fig. 6 shows H2S concentration in WT mouse increased under 6% O2 fraction (hypoxia). On the contrary, the alteration of H2S concentration from heme oxygenase (HO)-2-null mouse brains could not be found between 21% and 6% fraction. Under normoxia, H2S levels were similar in WT and HO-2-null mice, presumably due to compensation from other sources of H2S in the transsulfuration pathway.

Error bar show a standard error (n=9 or 6).*P<0.05 compared with WT at 21% O2.


3

2

1

021

9 6 9 6

O2 fraction (%)

Normoxia : 21% O2 fractionHypoxia : 6% O2 fraction

Fig. 6 Endogenous H2S concentration in neonatal brain of both WT and HO-2-null under normoxia/hypoxia

WT*

HO-2-null

6 21 6

SDB

(pm

ol/m

g br

ain)

Hydrogen sulfide is known to be as the gasotransmitter to control multiple biological functions. Therefore reliable and accurate quantitative analysis of H2S level in tissues is required. In this study we performed the quantitative analysis of hydrogen sulfide in mouse brain tissue by coupling the monobromobimane-based assay to triple quadrupole LC/MS/MS.

First analytical condition of SDB was determined on SRM by a SDB standard and then the linearity of calibration curves was validated by using a SDB standard and NaHS reagent forming H2S. Correlation coefficient of these calibration

curve was confirmed to be >0.99 and

The endogenous H2S level in mouse brain tissues was quantified as the derivatized SDB by triple quadrupole LC/MS/MS. This result shows the of the endogenous H2S is at the level of pmol/mg tissue. And the increase of the H2S level in WT mouse was confirmed under the hypoxia. On the other hand, the change of H2S level could not be found in the HO-2-null mouse. These results suggests this quantitative analysis by using triple quadrupole LC/MS/MS coupled with monobromobimane-based assay is helpful to quantify the endogenous H2S level in tissue samples.

Conclusions

(1) Morikawa T, et al. (2012) Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway. Proc. Natl. Acad. Sci. U.S.A. 109:1293-1298

References

Tsuyoshi Nakanishi 1, Takako Hishiki 2, Shigeki

Kajihara 3, Kiyoshi Ogawa 3 and Makoto Suematsu 2, 4

1 MS Business Unit, Shimadzu Corporation, Kyoto,

Japan

2 Department of Biochemistry, School of Medicine,

Keio University, Tokyo, Japan

3 Technology Research Laboratory, Shimadzu

Corporation, Kyoto, Japan

4 Japan Science and Technology Agency, Exploratory

Research for Advanced Technology, Suematsu Gas

Biology Project, Tokyo, Japan

Metabolite maps reconstructed using a quantitative data by capillary electrophoresis-mass spectrometry (CE-MS)

ASMS 2012 MP15-346

2

OverviewImaging mass spectrometry is a powerful tool to display the unique distribution of endogenous metabolites in tissue slices and this technique is currently used to the wide variety of animal and plant systems. Here to reflect the content of metabolites in tissues to the MS images, the

quantitative data by CE-MS was to tried to couple to the results of MALDI imaging experiment. Metabolite maps were reconstructed by the information of each metabolite quantified from the CE-MS measurement.

IntroductionImaging mass spectrometry (IMS) provides information of metabolite localization or distribution of a dosed drug in tissue sections. In general IMS technique requires a homogeneous matrix coating for a good reproducibility. This procedure also influences variable quantitative results on IMS technique. To improve these drawbacks, we achieved a reproducible matrix deposition by usage of a robotic spotter and coupled quantitative results of

metabolites by CE-MS with ion images obtained by IMS to reconstruct metabolite maps from different slices. Here we quantified ATP and its degradation metabolites, ADP and AMP, using CE-MS in brains from both wild type or transgenic mice deficient with a target enzyme. Furthermore, we examined a regional variation of metabolites in energy metabolism under normoxia/hypoxia by this method.

MethodsImaging mass spectrometry (IMS)Murine brain tissue (10 µm thick) was prepared using in situ freezing method(1). A 9-aminoacridine was selected as a MALDI-matrix and microdispensed onto frozen brain slices as a spatial resolution at 200 µm by a chemical inkjet printer (CHIP-1000, Shimadzu Corp.). Imaging mass spectrometry was performed in negative mode by MALDI-TOF/TOF MS instrument (AXIMA Performance) on the basis of positional information of each matrix deposit. Ion images were reconstructed by BioMap software after TIC normalization.

Quantification of metabolites by CE-MSFrozen brain sections from mice were plunged into ice-cold methanol containing internal standards and homogenized with a polytron homogenizer. After chloroform/methanol extraction, the upper aqueous layer was centrifugally filtered through a 5-kDa cutoff filter. Quantification of metabolites by CE-MSD was performed as described previously(1).

Coupling of quantitative data to mass images of metabolites To construct metabolite mapping between different slices, the quantitative data by CE-MS was linked to the IMS data of each metabolite. Here we estimated the apparent concentration (Ci) of each metabolite at the ith spot (corresponding to the ith pixel on a mass image) of tissue as follows: Ci = C ’ x Inti/Intave, where C ’, means the metabolite concentration of tissue determined by CE-MS quantification, Inti is the intensity of a target metabolite on a mass spectrum at the ith spot, and Intave is the median of intensities of the metabolite from all of the spots. Thus metabolite maps (AMP, ADP and ATP) were reconstructed to evaluate the fluctuation of energy metabolism under the normoxia/hypoxia using transgenic mice deficient with a target enzyme. Furthermore, energy charge (EC) was also calculated as:

EC=([ATP]+1/2[ADP])/([ATP]+[ADP]+[AMP])


3

Results

Fig. 1 Typical mass spectrum from wild-type murine brain tissue using 9-aminoacridine as a MALDI-matrix

Fig. 2 Typical mass image of metabolites using 9-aminoacridine as a MALDI-matrix

Fig. 3 Metabolite maps reconstructed from the quantitative data by CE-MS

A mouse coronal brain tissue was prepared according to in situ freezing method to maintain a level of ATP and ADP. Tissue slices were placed onto a conductive slide glass and then dried up. A 9-aminoacridine matrix was microdispensed onto tissue slices at a spatial resolution at 200 mm by a robotic spotter. A mass spectrum was acquired from each matrix spot onto tissue slices by MALDI-TOF/TOF instrument (AXIMA Performance, Shimadzu Corp.). Fig. 1 shows a typical mass spectrum from a murine brain tissue when using 9-aminoacridine as a MALDI-matrix. Metabolites with phosphate group and phospholipids were observed on this mass spectrum.

Typical mass image of metabolites were displayed from obtained mass spectra by BioMap software (Fig. 2). To evaluate variation of metabolites between multiple samples from different individuals, we performed TIC normalization with in-house designed software. Furthermore, to display metabolite maps corresponding to a content of metabolites in tissue, we tried to couple the quantitative data by CE-MS to mass images of each metabolite (see Fig. 3).

The concentration of metabolites quantified by CE-MS was coupled to the IMS data and apparent concentration (on tissue) of each metabolites was estimated on MS images. Fig. 3 shows mass images of AMP, ADP, ATP and EC map which was calculated from the three metabolites (AMP, ADP and ATP). First metabolite levels were quantified by CE-MS and the information about the metabolite concentration in tissue was used to reconstruct the metabolite maps. Here the apparent concentration (Ci) of each metabolite at the ith spot was calculated as Ci=C’ x Inti/Intave (see method). We could reproducibly compare both the distribution and abundance of metabolites between multiple tissues from the different individuals.

Metabolite concentration determined by CE-MS was coupled to MS images

Energy Charge =([ATP]+1/2[ADP])([ATP]+[ADP]+[AMP])

0

1

2

3

8 9 8 9

ATP

wh

ole

(µm

ol/g

)

21 10O2 fraction (%)

21 10


4

(A) Alterations in AMP (AMPwhole), ADP (ADPwhole), ATP (ATPwhole), and energy charge (ECwhole) in the whole brain. The concentrations of adenylates were determined by CE-MS. *P < 0.05 compared with WT normoxia; †P <0.05 compared with HO-2-null normoxia. HO-2, heme oxygenase 2(B) Representative IMS showing spatial distribution of apparent ATP concentration (ATPapp) and energy charge (ECreg). Note the basal increase in ATP in HO-2-null mice. (Bottom) H&E staining after IMS. cx, cortex; hp, hippocampus.(C) Quantitative analysis of regional ATP concentration and energy charge in WT and HO-2-null mice. *P < 0.05 compared with WT normoxia; †P < 0.05 compared with HO-2-null normoxia.

From results of quantitative analysis by CE-MS, remarkable decrease of ATP level in HO-2-null mice under the hypoxia was shown in Fig. 4 Furthermore, a value of EC under the hypoxia remained unchanged in WT mice but dropped to <0.5 in the HO-2-null mice.IMS data coupled by CE-MS quantitative results also displayed the characteristic decrease of EC value on the cortex region.

MS images of typical metabolites were displayed on mouse brain tissue (10 µm thick) using 9-aminoacridine as a MALDI-matrix. Then chemical inkjet printer was used for homogenous matrix deposition onto tissue slices and resulted to reproducible MS images between multiple samples.By using the IMS data coupled to the quantitative data of CE-MS measurement, metabolite maps were reconstructed using brain tissue slices from the different individuals. The metabolite maps which were reconstructed on the basis of

the quantitative results by CE-MS, reflect the endogenous concentration of each metabolite. In fact, AMP, ADP and ATP which related to energy metabolism were detected from tissue slices under the normoxia/hypoxia and these MS images displayed an unique alteration on the distribution and the content in WT and a transgenic mouse. These results suggests this developed approach is useful to semiquantitatively evaluate the distribution of metabolites on the IMS experiment.

Conclusions

(1) Hattori K, et al. (2010) Paradoxical ATP elevation in ischemic penumbra revealed by quantitative imaging mass spectrometry. Antioxid Redox Signal 13:1157-1167.

(2) Morikawa T, et al. (2012) Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway. Proc. Natl. Acad. Sci. U.S.A. 109:1293-1298.

References

Fig. 4 Impaired ability of HO-2-null mice to maintain ATP levels on exposure to 10% O2 for 1 min


Zanariah Hashim1; Yudai Dempo1; Tairo Ogura2;

Ichiro Hirano2; Junko Iida2; Takeshi Bamba1; Eiichiro

Fukusaki1 1Dept.Biotech., Grad. Sch. Eng., Osaka Univ., Suita,

JAPAN; 2Shimadzu corporation, Kyoto , JAPAN

Quantitative analysis of hydrophilic metabolite using ion-paring chromatography with a high-speed triple quadrupole mass spectrometer

ASMS 2012 ThP11-251

2

IntroductionMetabolomics, the comprehensive study of metabolites, allows for detailed phenotypic analysis through a combination of metabolite information. Among the many kinds of metabolic reactions, central pathways to energy metabolism are of the most biologically important pathways, and includes more than a hundred of hydrophilic compounds such as sugar phosphates, organic acids, and nucleotides. Ion-pare chromatography coupled to a triple

quadrupole mass spectrometer is one of the techniques to analyze these hydrophilic metabolites. In order to improve throughput of analysis, high-speed with a quantitative capability is required from the mass spectrometer. In this study, we report a developed analytical system for hydrophilic metabolite using ion-pare chromatography with a high-speed triple quadrupole mass spectrometer.



Nexera series UHPLCLCMS-8040 triple-quadrupole mass spectrometer10 mM tri-butyl ammonium and 15 mM acetic acid in water/methanol (97/3)methanol0.3 mL/min0%B(0-0.5 min) - 25%B(7.5 min) -90%B(11-11.5 min) - 0%B(11.6-15 min)L-Column ODSII (2 mm I.D. x 150 mmL., 3 µm)40°C3 µL

LCMS-8040 triple-quadrupole mass spectrometerESI negative mode250°C 400°C10 L/min2 L/min

Apparatus:

Mobile phase A:

Mobile phase B:Flow rate:Time program :

Analytical column:Oven temp.:Injection Vol.:

LC-MSApparatus:Ionization: DL Temp.:HB Temp:Drying Gas:Nebulizing Gas:

UHPLC

Fig. 1 LCMS-8040 Triple Quadrupole Mass Spectrometer

Fig. 2 Typical chromatograms of 96 standard compounds.


ResultsQuantitative analysis of standard samplesBy using ion-paring chromatography coupled to high speed triple-quadrupole mass spectrometry, hydrophilic metabolites including sugar phosphates, organic acids, coenzymes, and nucleotides were analyzed successfully within 15 minutes (Fig. 2). In comparison of several brands of ODS column, we chose L-Column ODS in order to favor separation of sugar phosphates. The qualitative capability of the chosen separation method in terms of limit of detection, dynamic range, carry over, and stability are summarised (Table 1).

0.0 2.5 5.0 7.5 10.0 min 0.0

1.0

2.0

3.0

4.0

5.0

6.0 (x100,000)

3

Fig. 3 Typical chromatograms of yeast extract. Figu. 4 Linearity between sample dilution to peak area.

Analysis of yeast extractYeast extracts were analyzed as an experimental model to confirm applicability of the developed method to biological samples. 75 components were detected successfully between concentration range 0.4 – 100,000 nM (compound dependant).

Linearity was also tested through serial dilution to confirm the range of linearity in which metabolites could be quantified (Fig. 4). These results show that this method can detect change of a metabolite quantitatively.

ConclusionsAccelerated quantitative analysis of hydrophilic metabolites was developed that was able to analyse 96 components in 15 minutes. The result of analysis of yeast extract showed

applicability of this method to future studies involving biological samples.

y = 4474.7x-21556R² = 0.9992

peak

are

ayeast extract (%)

G6P

0.0 2.5 5.0 7.5 10.0 min 0.0

0.5

1.0

1.5

2.0

2.5 (×1,000,000)　


4

ArginineHistidine

4-AminobutyrateSerine

AsparagineGlutamine

HydroxyprolineHomoserineThreonineLeucineRibitol

TrehaloseProline

CytidineMethionineTheanineGuanineIsoleucineTyrosine

Amino adipic acidGlutamate

UridineAspartateThymineInosine

GuanosinePhenylalanine

ShikimateGlycerateThymidineGlycolateGlyoxylate

InositolD-glucose/galactose

LactatePyroglutamate

Glucose-6-phosphatePIPES

Ribose-5-phosphateSedoheptulose-7-phosphate

Fructose-6-phosphateTryptophan

α-GlycerophosphateGlucose-1-phosphate

Glyceraldehyde-3-phosphateErythrose-4-phosphateRibulose-5-phosphateβ-Glycerophosphate

Orotate

173.1>131.2154>93.15

102>84104>74.15131>113.15145>127.05130>84.15118>100

118>74.05130.1>84151>89.1341>89

114>68.1242>109.15148>47.05173>155.25150>133.1130.1>45

180>163.05160>116.2146>102.2243>110.15132>88.05125>42.05267>135.15282.1>150.2164>103.15173>93.15105>75.15

241.1>42.0575>47.0573>45.05179>87

179>89.0589>43.1128>84.1

258.9>97.05301>193.25229.1>96.95288.9>97.1258.9>97.1

203.1>116.15171.1>79.1258.9>79.05168.90>97.10

198.9>97.2229>97.1

170.9>79.05155>111.15

12345678910111213141516171819202122232425262728293031323335343637383940414243444546474849

7.8 - 50023.6 - 500

144.6 - 5000095.3 - 200062.6 - 200006.2 - 1000028.8 - 10004.8 - 100029.2 - 200055.1 - 1000198.1 - 500092.6 - 20000184.3 - 50004.2 - 500001.7 - 500010 - 200007.5 - 5000

447.6 - 5000029.8 - 20000024 - 100000

148.8 - 2000002.2 - 10000

160.6 - 20000058.5 - 500003.9 - 200003.5 - 10000

190.9 - 2000031.8 - 50000147.2 - 2000063.7 - 20000

1526.1 - 200002297.5 - 1000002481.4 - 50000

64.2 - 50000238.6 - 10000151.8 - 10000270.2 - 10000

2.1 - 1000072.7 - 1000014.3 - 10000293.7 - 10000

1.2 - 1000025.3 - 10000497.9 - 10000

1543.8 - 1000001191.9 - 100000

118.3 - 5000032 - 20000

28.3 - 10000

0.9924 0.9979 0.9884 0.9824 0.9832 0.9842 0.9800 0.9895 0.9846 0.9935 0.9868 0.9897 0.9957 0.9901 0.9938 0.9850 0.9879 0.9902 0.9980 0.9880 0.9894 0.9911 0.9906 0.9888 0.9894 0.9976 0.9880 0.9865 0.9889 0.9872 0.9975 0.9988 0.9962 0.9979 0.9744 0.9819 0.9922 0.9958 0.9993 0.9875 0.9895 0.9852 0.9912 0.9925 0.9919 0.9885 0.9969 0.9868 0.9930

LinearityLOD (nM)*

m/z

0.9050.9071.0371.1381.1511.161.1791.1821.1861.2011.2431.3021.3311.8451.9872.0062.4992.5782.8343.3453.4773.5483.6854.0034.5594.7064.8544.9055.0885.2495.3365.5776.346.3466.3486.4386.4446.6376.7596.8676.9276.9787.0747.113

7.37.5

7.6227.7537.84

2.4 7.1 43.4 28.6 18.8 1.9 8.6 1.4 8.8 16.5 59.4 27.8 55.3 1.3 0.5 3.0 2.3

134.3 8.9 7.2 44.6 0.7 48.2 17.6 1.2 1.0 57.3 9.5 44.1 19.1 457.8 689.3 744.4 19.3 71.6 45.5 81.1 0.6 21.8 4.3 88.1 0.4 7.6

149.4 463.1 357.6 35.5 9.6 8.5

R.T.CompoundLinear Range R2 R.T. Peak area

0.14 0.25 0.63 0.15 0.05 0.11 0.21 0.17 0.11 0.31 0.08 0.42 0.66 0.03 0.05 0.09 0.07 0.35 0.16 0.26 0.18 0.25 0.31 0.18 0.16 0.15 0.14 0.07 0.14 0.10 0.12 1.24 0.16 0.09 0.09 0.07 0.06 0.07 0.10 0.14 0.17 0.07 0.19 0.21 0.54 0.75 0.15 0.13 0.05

8.1 3.6 39.9 6.4 7.1 5.8 14.0 1.4 5.4 15.4 5.1 17.4 31.9 1.8 4.2 2.7 7.9 32.8 8.1 5.0 9.2 3.6 12.8 4.2 2.4 1.9 5.7 5.8 7.4 10.5 9.8 30.1 14.7 4.3 3.3 6.8 2.0 1.1 5.0 2.9 4.2 3.8 3.7 3.2 21.0 8.8 6.0 1.2 2.0


RSD % at 1µM** (n=4)

5

Fructose-1-phosphateCMPNAD

PyruvateDihydroxy acetone-3-phosphate

UMPGMP

OxalacetateTMPAMP

NicotinatePantothenate

SuccinateFumarate

cAMPMalate

UDP-glucose2-Oxoglutarate

CDP6-Phosphogluconate

ADP-glucoseGDPUDP

ADP-riboseNADPKDPG

3-PhosphoglycerateFructose-2,6-bisphosphateFructose-1,6-bisphosphate

NADHRibulose-1,5-bisphosphate

IsocitrateCitrateADP

PhosphoenolpyruvateFMN

2-IsopropylmalateFADCTPGTP

NADPHUTPATP

Coenzyme AMalonyl coenzyme AAcetyl coenzyme A

Succinyl coenzyme A　　

258.9>97.05322>79.1

662.1>540.187>43.05

168.9>97.05322.9>97.1362>79.1131>87

321>79.1346>79.05122>78.15

218>88117>73.2115>71.1328>134.1

132.9>115.2564.8>323.1145>101.2

401.8>79.05275>79

588>346.15442>79.1

402.9>79.05558>346.15741.8>620.1256.9>97.05184.9>97.05338.9>241.15

338.9>97.1664>78.95

308.9>97.05190.9>117190.9>87

425.9>79.1167>78.95455>97.1175>115.2783.9>97.1481.9>159.1521.9>159.05

744>159482.9>159.1505.9>159.1

766.5>79852.1>408.1

808>408866>408

5051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596

260.1 - 1000059.5 - 10000

5 - 10000427.7 - 2000018.7 - 2000037.9 - 20000102.3 - 50000

27944.7 - 50000030.9 - 50000

101.6 - 10000019.1 - 50003.4 - 5000

20.5 - 10000897.7 - 500000

2.3 - 5000019.8 - 10000

2 - 500082 - 1000094 - 5000

29.2 - 2000160.8 - 5000

3.5 - 50044.9 - 1000

6.1 - 50016.1 - 200040.4 - 1000026.2 - 2000089.4 - 500043.8 - 200012.7 - 200051.6 - 1000015.8 - 5000151.3 - 200077.1 - 200021.4 - 1000067.4 - 500017.6 - 500010.4 - 1000147 - 50000

254.1 - 5000015 - 2000

175.4 - 100000119.5 - 5000056.9 - 200002.1 - 100005.5 - 100001.1 - 10000

0.9968 0.9925 0.9844 0.9871 0.9873 0.9917 0.9909 0.9961 0.9966 0.9945 0.9914 0.9934 0.9881 0.9923 0.9900 0.9959 0.9919 0.9825 0.9875 0.9836 0.9870 0.9913 0.9820 0.9969 0.9722 0.9906 0.9802 0.9728 0.9865 0.9744 0.9785 0.9789 0.9915 0.9938 0.9764 0.9774 0.9976 0.9909 0.9840 0.9963 0.9960 0.9914 0.9851 0.9863 0.9923 0.9894 0.9832

LinearityLOD (nM)*

m/z

7.9257.9648.2438.2758.3918.6618.9959.319.7199.8119.98310.02210.15510.27810.46510.57810.71210.74510.75310.77110.80610.80610.80710.8110.81110.82810.82910.83410.83810.87610.88710.89110.89210.91310.92810.98510.99811.15511.17111.18511.20111.20611.22611.34311.36711.38211.391

78.0 17.8 1.5

128.3 5.6 11.4 30.7

8383.4 9.3 30.5 5.7 1.0 6.1

269.3 0.7 5.9 0.6 24.6 28.2 8.8 48.2 1.0 13.5 1.8 4.8 12.1 7.8 26.8 13.1 3.8 15.5 4.8 45.4 23.1 6.4 20.2 5.3 3.1 44.1 76.2 4.5 52.6 35.8 17.1 0.6 1.6 0.3

R.T.CompoundLinear Range R2 R.T. Peak area

RSD % at 1µM** (n=4)

0.10 0.09 0.06 0.05 0.09 0.05 0.08 0.02 0.04 0.03 0.05 0.04 0.05 0.05 0.03 0.04 0.03 0.04 0.04 0.07 0.06 0.02 0.03 0.03 0.03 0.04 0.03 0.03 0.04 0.03 0.02 0.03 0.09 0.02 0.03 0.01 0.02 0.01 0.10 0.15 0.02 0.03 0.24 0.02 0.01 0.02 0.02

3.0 3.4 2.1 9.9 1.1 5.0 9.9 4.6 3.2 10.5 4.0 8.0 4.7 15.3 3.7 4.8 5.5 4.1 7.5 20.4 8.0 4.7 5.7 7.2 3.9 8.1 6.4 10.2 0.5 7.6 7.7 12.9 11.7 7.2 6.4 1.9 2.9 3.3 14.3 11.0 5.2 6.7 12.1 11.3 7.0 7.2 9.5


Tetsuo Tanigawa, Tairo Ogura, Ichiro Hirano, Junko

Iida

Shimadzu corporation, Kyoto , JAPAN


ASMS 2012 MP21 - 527

2

IntroductionIt has been suggested that many of hormone active compounds, such as naturally occurring estrogen, may be present in environmental water. These may pose a potential health risk as these compounds can act as endocrine disrupting chemicals. It is known that most estrogens, such as 17-β-estradiol (E2), can be metabolized to conjugated forms which have low activity and subsequently excreted, however, it is also known that some conjugated estrogens can return to a highly active free form through

de-conjugation. Moreover, treated wastewater has been suggested to contain unknown estrogenic metabolites that as yet have not been fully characterized.For these reasons the qualitative detection of estrogen-related compounds, such as conjugates in aqueous samples, has great importance. In this study we have developed a qualitative analytical system with online solid phase extraction (online-SPE) and accurate MSn analysis.


Fig. 1 Flow Diagram of online-SPE LCMS-IT-TOF system.

17β-estradiol (E2)C18H24O2

17β-estradiol 3-sulfate-17-(beta-D-glucuronide) (E2-3S-17G)

C24H32O11S

17β-Estradiol 17-(β-D-glucuronide) (E2-17G)

C24H32O8

17beta-Estradiol-3,17-disulfate(E2-3S-17S)C18H24O8S2

17β-Estradiol 3-(β-D-glucuronide)(E2-3G)

C24H32O

Fig. 2 Structure of 17β-Estradiol and its conjugates.

1. Reservoir for online-SPE2. Pump for online-SPE3. SIL-10AP with 5 mL syringe4. Preparative SPE column 5. Flow Change Valve (SPE column)6. Reservoirs for analysis7. Pumps for analysis8. Analytical column9. LCMS-IT-TOF

Materials & MethodsSamples were measured by an electrospray ion-trap time-of-flight mass spectrometer (LCMS-IT-TOF, Shimadzu Corporation, Kyoto, Japan) coupled to online-SPE LC system (Nexera series, Shimadzu Co.). Analytical conditions were as follows, analytical column: Shimpack XR-ODS II, C18, 75 × 2 mm, 2.2 µm (Shimadzu Co.); preparative column: MAYI-ODS, 10 × 2 mm (Shimadzu Co.); flow rate:

0.25 mL/min for analytical pump, 2 mL/min for sample loading pump; column temperature: 40°C; mobile phase A and sample loading solvent: water containing 10 mM ammonium acetate; mobile phase B: methanol. 5mL of water sample, filtered by 0.45 µm filter prior to analysis, was injected by SIL-10AP (Shimadzu Co.).

6

7

5

8

1

2

3

9

4 Drain

OH

OH

OO

O

OH

OH

OHOH

OH

OH

OO

O

OH

OH

OH

OH

O

O

S OH

O

O

SOH

O

O

OO

O

OH

OH

OHOH

OS

OH

O

O

Fig. 3 Chromatogram of river water containing E2 and its conjugates (2 ppt each)

Results

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0 (x100,000,000)

E2 -3S -17G E2 -3S -17S

E2 -17G E2 -3G

E2 (n.d.)

17β-Estradiol 17-(β-D-glucuronide) (E2 -17G) C24H32O8

100 200 300 400 500 m/z0.0

2.5

Inten. (x1,000,000)

351. 1264

N.D.

100 200 300 400 500 m/z0.0

1.0

2.0

Inten. (x1,000,000)

175. 0272

100 200 300 400 500 m/z0.0

2.5

5.0Inten. (x1,000,000)

447. 2026

299. 0942

100 200 300 400 500 m/z0.0

0.5

1.0

Inten. (x100,000)

113. 0259

m/zcalc. 351.1272 error: 0.8 mDa

OO

O

OH

OH

OHOH

OS

OH

O

O

OO

O

OH

OH

OHOH

OH



100 200 300 400 500 m/z0.0

1.0

2.0

3.0

Inten. (x1,000,000)

527. 1575

414. 2134

17β-estradiol 3-sulfate-17-(beta-D-glucuronide) (E2 -3S -17G)

C24H32O11S

(a)

MS2

MS3

MS

(b)

MS2

MS3

MS


3

4

3

17β-Estradiol 3-(β-D-glucuronide) (E2 -3G) C24H32O8

100 200 300 400 500 m/z0.0

2.5

5.0

Inten. (x10,000)

183. 0823269. 1530

100 200 300 400 500 m/z0.0

2.5

5.0

7.5

Inten. (x1,000,000)

215. 0409431. 0824

199. 0453

100 200 300 400 500 m/z0.0

2.5

5.0Inten. (x1,000,000)

351. 1256

100 200 300 400 500 m/z0.0

1.0

2.0

Inten. (x1,000,000)

271. 1709

100 200 300 400 500 m/z0.0

2.5

5.0

Inten. (x1,000,000)

447. 2023

299. 0946 401. 0768

100 200 300 400 500 m/z0.00

0.25

0.50

0.75

1.00Inten. (x1,000,000)

271. 1693157. 0166

429. 1905

O

O

S OH

O

O

SOH

O

O



OH

OO

O

OH

OH

OH

OH




145. 0675

m/zcalc. 145.0659error: 1.6 mDa

(d)

MS2

MS3

MS

(c)

MS2

MS3

MS

17β-Estradiol-3,17-disulfate (E2 -3S -17S) C18H24O8S 2

ConclusionsTrace amounts of E2 conjugates E2-3G, E2-17G, E2-3S-17S, and E2-3S-17G were detected in aqueous samples by using online-SPE and accurate MSn analysis.

Excellent mass accuracy and comprehensive MSn data enabled confident assignment to conjugate structures.

Through techniques developed in this study it is expected to apply these methods to identify other compounds and respective metabolites caused by reduction or oxidation formed by microbes in the natural environment.

Fig. 4 Accurate MSn analysis of 17β-estradiol conjugates (a-d).

Table 1 Mass accuracy and peak intensity (S/N) of 17β-estradiol conjugates.

Compound R.T. m/z obs. m/z calc. Error(mDa)

S/N at 2ppt

E2 11.18* 271.1702* 271.1704 0.2* N.D

E2-3S,17G 7.57 527.1575 527.1593 2.9 11.2

E2-3S,17S 8.80 431.0824 431.0840 1.6 9.8

E2-17G 9.86 447.2026 447.2024 0.2 18.6

E2-3G 9.98 447.2023 447.2024 0.1 37.9

*at 10 ppt


Yoshiyuki Watabe1; Tairo Ogura1; Ichiro Hirano1;

Shoji F. Nakayama2

1Shimadzu corporation, Kyoto , JAPAN; 2National Institute for Environmental Studies,

Tsukuba, JAPAN

Analysis of phthalate esters in environmental water samples by online-SPE-LC coupled with high-speed triple quadruple mass spectrometer

ASMS 2012 MP21 - 528

2

IntroductionPhthalate esters are produced in large quantities throughout the world and used as primary plasticizers. These compounds, however, are of environmental concern due to their suspected endocrine disrupting potential. Furthermore, phthalate di-esters can be bio-transformed into mono-esters. Subsequently these compounds may be transported from wastewater to environmental water primarily due to insufficient wastewater treatment. Since

both phthalate di- and mono-esters are likely to exist as a mixture in the environment, it is important to develop a simultaneous quantification method for both forms. In this study we developed a rapid online-SPE-LC system coupled with a high-speed triple quadrupole mass spectrometer for the simultaneous determination of phthalate di- and mono-esters to trace levels.


Fig. 2 Flow Diagram of online-SPE LCMS-8030 system.

Materials & Methods

Sample was injected into SPE column by mobile phase C. Phthalate esters from the analytical system were trapped by scrubber column

Phthalate esters in samples was eluted by mobile phase for analysis.

Analysis of phthalate esters were performed and SPE column was washed simultaneously.

Equilibration of SPE column was performed by mobile phase C.

At the starting of equilibration, the mobile phase was purged to avoid accumulation of phthalate esters on the SPE column.

1: Reservoirs for analysis2: Pumps for analysis3: Scrubber column 4: Manual Injector5: Analytical column6: LCMS-80307: Reservoirs for online-SPE8: Pumps for online-SPE9: Scrubber column10: SIL-10AP with 5 mL syringe11: Flow Change Valve (Purge)12: Flow Change Valve (SPE)13: SPE column

3) Analysis and wash SPE column

12

5

7

8

10

3 4 6 11

9 13

1) Sample loading

C D

2) Elution

12

5

7

8

10

3 4 6 11

9 13

1

2

A B 1

2

12

5

10

3 4 6 11

9 13

4) Purge preparative mobile phase

1

2

12

5

10

3 4 6 11

9 13

A B

5) Equilibration of SPE column

1

2

12

5

10

3 4 6 11

9 13

A B

C D

A B 1

2

A B

7

8

C

7

8

C D

7

8

C D

D

3

MMPMEP

MEHHPMnBPMBzPMEHPMOPMNPDMPDEPDBP

DEHPDOPBzBP

2.853.655.325.575.737.878.128.144.956.428.9212.0412.308.71

100-1000020-1000010-1000010-100005-100005-100005-100005-100002-100005-1000050-1000020-1000020-100005-10000

0.99970.99950.99960.99910.99740.99940.99780.99870.99970.99910.99980.99560.99660.9994

R2Linear range (ng/L)RT (min)

69.5 ± 11.6103.6 ± 14.6

72.8 ± 0.1133.9 ± 16.1

89.2 ± 3.568.3 ± 12.7

53.3 ± 255.6 ± 12.697.4 ± 5.1

100.8 ± 17.8101.1 ± 15.5105.3 ± 19.6105.4 ± 4.7115.5 ± 9.8

Recovery (%) at 500 ng/L*Compound RSD (%) at LOQ

9.314.213.011.112.78.514.217.817.48.44.28.47.219.7

Method performance characteristics in river water.

Development of analytical system.

*Recovery(%) = peak area of each components loaded by online - SPE (500 ng/L x 1 mL)

peak area of each components loaded by manual injector (100 µg/L x 5 µL)


Fig. 2 Typical chromatogram of standard sample and blank samples.

ResultsHigh speed simultaneous analysis of phthalate esters was achieved by using high speed polarity switching (10msec) technology. Background contaminations were minimized with a scrubber column inserted after a mixing chamber.

Background peaks were however still detected with the scrubber column in place suggested that auto-sampler and valves can also a source of contamination for DBP, DEHP, DOP.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min 0

50000

100000

150000

200000

250000

300000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min 0

50000

100000

150000

200000

250000

300000

Load

Elution from SPE column

Separation by gradient elution Equilibration Analysis

SPE Wash SPE column Purge flow line & Equilibration

0.0 2.5 5.0 7.5 10.0 12.5 min 0

50000

100000

150000

200000

250000

STD (500 ng/L each, with Scrubber) x50 magnify for monoesters

Blank water (with Scrubber) x50 magnify for monoesters

Blank water (without Scrubber) x50 magnify for monoesters

MMP MEP

DMP

MnBP

MBzP

DEP

MEHP MBzP

MNP BzBP

DBP

DEHP

DOP

DMP (<2ng/L)

DEP (<5ng/L)

BzBP (n.d.)

DBP (137ng/L)

DEHP (659ng/L)

DOP (739ng/L)

Phthalate mono-esters were not detected

Phthalate mono-esters were not detected

DMP (85ng/L)

DEP (40ng/L)

BzBP (20ng/L)

DBP (761ng/L)

DEHP (3002ng/L)

DOP (3782ng/L)

4

ConclusionsA novel simultaneous analysis method of phthalate di- and mono-esters combined with online-SPE was developed.The cycle time including online-SPE and column separation was 15 minutes.Background contamination from online-SPE and LCMS system were successfully minimized.Further method validation, including assessment of column life of SPE and recovery at several concentrations is underway.

Monomethylphthalate

Monomethylphthalate

monoethylphthalate

monoethylphthalate

monoethylhydroxyhexylphthalate

monoethylhydroxyhexylphthalate

monobutylphthalate

monobutylphthalate

monobenzylphthalate

monobenzylphthalate

monoethylhexylphthalate

monoethylhexylphthalate

Compound name

MMP-N

MMP-IS

MEP-N

MEP-IS

MEHHP-N

MEHHP-IS

MnBP-N

MnBP-IS

MBzP-N

MBzP-IS

MEHP-N

MEHP-IS

Abbreviation

200

200

50

50

30

30

30

30

30

30

30

30

Dwell

Time

77.3

79.3

77.3

79.3

121.2

124.2

77.3

79.3

77.3

79.3

134.3

137.3

Q3

m/z

11

11

19

19

18

18

20

20

23

23

14

14

Q1

m/z

Q1

m/z

179

183

193

197

293

297

221

225

255

259

277

281

+/-

-

-

-

-

-

-

-

-

-

-

-

-

Compound name Abbreviation Dwell

Time

Q3

m/z

Q1

m/z

Q1

m/z+/-

monooctylphthalate

monooctylphthalate

monononylphthalate

monononylphthalate

dimethylphthalate

diethylphthalate

dibutylphthalate

dioctylphthalate

diethylhexylphthalate

benzylbutylphthalate

MOP-N

MOP-IS

MNP-N

MNP-IS

DMP-N

DEP-N

DBP-N

DOP-N

DEHP-N

BzBP-N

30

30

30

30

3

3

3

50

50

3

77.3

79.3

141.3

141.3

163.1

149.05

149.05

149.05

149.05

91.1

26

26

19

19

-11

-20

-17

-23

-23

-25

277

281

291

295

195

223.1

279.2

391.3

391.3

313.2

-

-

-

-

+

+

+

+

+

+


Mobile phase A:Mobile phase B:Scrubber column:

Analytical column:

Time program :

Flow rate:Oven temp.:

Ionization:

DL Temp.: Drying Gas:Pause time:Monitoring ion, Collision energy, Dwelltime:

0.1% formic acid in waterAcetonitrileShim-pack XR-ODSII (2 mm I.D. x 50 mmL., 2.2 µm)Shim-pack XR-ODSII (2 mm I.D. x 75 mmL., 2.2 µm)30%B(0-2.5 min)→98%B(10-12.5 min)→30%B(12.1-15 min)0.25 mL/min40°C

ESI pos./neg. polarity switching (pos. for di-esters, neg. for mono-esters)250°C BH Temp: 400°C10 L/min Nebulize Gas: 2 L/min3 msec Polarity switching: 10msec See below

LC conditions

Mobile phase A:Mobile phase B:Scrubber column:

Preparative column:

Flow rate:Injection vol.:

0.1% formic acid in waterAcetonitrileShim-pack XR-ODS (4.6 mm I.D. x 50 mmL., 2.2 µm)

ENV-MASK (Purchased from Chemco Inc.) (2.0 mm I.D. x 10 mmL., 8 µm)2 mL/min1000 µL

online-SPE condition

MS conditions

MRM parameters for target analytes

Daisuke Okumura; Manabu Ueda; Tomohito Nakano

Shimadzu Corporation, Nishinokyo-kuwabaracho,

Nakagyo-ku, Kyoto 604-8511, Japan

Development of an ion transmission enhanced tandem ion guide system for triple quadruple mass spectrometer

ASMS 2012 ThP26-584

2

IntroductionThe application of atmospheric pressure ionization sources in mass spectrometry necessitates the presence of a differential pumping system in order to maintain high vacuum in the analyzer. Commercial triple quadrupole mass spectrometers typically have from 3 to 4 differential pumping stages and an RF ion guide is generally utilized as a ion focusing device in the higher pressure region of the ion path. The ions generated under atmospheric pressure

need to be efficiently focused to minimize the loss of ions prior to introduction into quadrupole analyzer by use of an RF lens system. Using a QqQ with 4 differential pumping stages, we have investigated the application of a fringing field in the RF ion guides installed in the second and third differential pumping stages with a resultant improvement in sensitivity by a factor of five.

MethodsThree distinct designs of the tandem quadrupole RF ion

guide in differential pumping stages have been

investigated. In order to examine the characteristics and

performance of these configurations, each RF ion guide

was installed in a triple quadrupole mass spectrometer

(LCMS-8040, Shimadzu Corporation, Kyoto, Japan). Each

tandem RF ion guide system had inscribed radii of a) 1.5

mm, b) 2.0 mm and c) 2.8 mm. Each system has one DC

lens between RF ion guides, and its diameter was 4 mm.

Fig. 2 The optimum operating RF voltage of quadrupole ion

guides was acquired experimentally. The voltages

were 27 V, 50 V and 100V for R= 1.5, 2.0 and 2.8

respectively.

Fig. 1 LCMS-8040 triple quadrupole mass spectrometer.

Fig. 3 Pseudo-potential of each tandem RF ion guide systems.


2R

a) R=1.5 mm b) R=2.0 mm c) R=2.8 mm

0 50 100 150

RF Voltage

Inte

nsity

0 50 100 150

RF Voltage

Inte

nsity

0 50 100 150

RF Voltage

Inte

nsity

-1 -0.5 0 0.5 1

r (mm)

V*

(eV

) R 1.5

R 2.0

R 2.8

3

Fig. 4 Simulated fringing focusing field estimates of smaller R ion guides was found to be more effective than those of larger R. Therefore we have achieved higher sensitivity using smaller R ion guides.

ResultsWe acquired Q1 scan spectra of pesticides at three different scan speeds with flow injection analysis (5000, 10000 and 15000 u/sec). The benefit of the quadrupole ion guide, with it’ s narrower mass range transmission relative

to hexapole and octopole ion guides, was an increase in the absolute intensity of ion species in the Q1 scan at all three scan speeds by a factor of 2-5 times.


LCMS-8040

LCMS-8030

Fig. 5 Q1 scan spectra of pesticides at three different scan speed.

15000 u/sec 10000 u/sec 5000 u/sec

200 300 400 500 600 m/z

0.5

1.0

1.5

2.0 Inten. (x10,000,000)

x 2

x3x 3

x4

x 5

200 300 400 500 600 m/z

0.5

1.0

1.5

2.0 Inten. (x10,000,000)

x 2

x 3 x 3

x 3

x5

200 300 400 500 600 m/z

0.5

1.0

1.5

2.0 Inten. (x10,000,000)

x2

x 3 x 3

x 3

x 5

15000 u/sec 10000 u/sec 5000 u/sec

200 300 400 500 600 m/z

0.5

1.0

1.5

2.0 Inten. (x10,000,000)

200 300 400 500 600 m/z

0.5

1.0

1.5

2.0 Inten. (x10,000,000)

200 300 400 500 600 m/z

0.5

1.0

1.5

2.0 Inten. (x10,000,000)

4

In case of typical triple quadrupole mass spectrometer

In some instruments a mass displacement effect can occur with linked scans, such as precursor or neutral loss scans, when performed at higher scan speeds.

Precursor ion scans were performed at two scan speeds, 2727 u/sec and 6000 u/sec. These scans showed no mass displacement at either speed for either the LCMS-8030 or LCMS-8040. LCMS-8040 sensitivity was about two times higher than for the LCMS-8030.

300.5

298.0 299.0 300.0 301.0 302.0 303.0 m/z

301.0

300.0

6000u/sec

0

0.5

1.0

1.5

3.0 4.0 5.0 min

2.0

2727u/sec

Precursor: ion of m/z 149

250.0 240.0 m/z 0.0

2.5

5.0

Inten. (x10 5)

251.25

m/z 149

Dipropyl phthalate

250.0 240.0 0.0

2.5

5.0

7.5

Inten. (x 10 5)

251.25

m/z

0

0.5

1.0

Inten. (x107) Inten. (x107)

3.0 4.0 5.0 min

8040

8030

Fig. 6 Precursor ion scan of standard mixture sample oｆ 8 phthalate esters.

ConclusionThe development of a tandem ion guide system with an enhanced fringing field between two ion guides has

enabled increases in sensitivity for the LCMS-8040 up to a maximum of 5-fold.


Masayoshi Tamura1; Keiko Matsumoto2; Jun

Watanabe2; Junko Iida2; Yasushi Nagatomi1; Naoki

Mochizuki1 1Asahi Group Holdings, Ibaraki, JAPAN; 2SHIMADZU CORPORATION, Kyoto, JAPAN

High Throughput Quantitative Analysis of Multi-mycotoxin in Beer-based Drinks using UHPLC-MS/MS

ASMS 2012 WP27-580

2

IntroductionMycotoxins often exist as contaminants in grains. To ensure consumer food safety, manufactures of food and beverages have to strictly manage risks from such contaminants. To maintain the high-quality of food standards it is therefore essential to rapidly determine the concentrations of hazardous mycotoxins in foods or beverages. UHPLC-MS/MS offers the best combination of selectivity, sensitivity, and speed for detection of these compounds in

complex matrices. In this study, a high throughput method for the quantification of 14 mycotoxins in beers was developed. Highest sensitivity of analysis is crucial to food safety, additionally, autosampler and system carry over need to be monitored to ensure these factors do not become a problem. In these experiments elimination of carry over was investigated through novel rinse condition cycles of the UHPLC autosampler.

14 mycotoxins (patulin(PAT), nivalenol(NIV), deoxynivalenol(DON), aflatoxin(AF) B1, B2, G1, G2, T-2 toxin(T-2), HT-2 toxin(HT-2), zearalenone(ZON), fumonisin(FM) B1, B2, B3 and ochratoxin A(OTA)) were determined by LC-MS/MS using a UFLC HPLC system coupled to a LCMS-8030 triple quadrupole mass spectrometer. The MRM method of 14 mycotoxins was optimized on

each compound-dependent parameter and MRM transition (Q1/Q3). As a result, all compounds were detected with high sensitivity by ESI. AFB1, B2, G1, G2, T-2, HT-2, FMB1, B2, B3 and OTA were detected in positive mode. While PAT, NIV, DON, ZON were detected in negative mode. Ultra Fast Polarity Switching of 15 msec enabled simultaneous determination of the compounds in both modes.

Methods and Materials



Fig. 1 Structure of mycotoxins

High Speed Mass Spectrometer Polarity Switching 15 msecScanning Speed Max. 15000 u/sec

3

ResultsMethod development for 14 mycotoxins

Analytical Conditions for LC-MS/MSHPLC: UFLC systemColumn:Mobile phase A: B:Flow rate:Gradient program:Column temperature:

MS: LCMS-8030 triple quadrupole mass spectrometer Ionization: ESI, Positive/Negative MRM mode Ion spray voltage: -3.5 kV

TriartC18 100 mm×2.0 mm, 1.9 um10 mM Ammonium acetate - Water, 2% Acetic acid - Methanol0.4 mL/minB conc.2%(0 min) - 55%(3 min) - 85%(7.0-8.0 min) - 2%(8.01-11 min)Column temperature: 40 C

For UHPLC separation, various LC mobile phase conditions were examined. Tailing of fumonisins peaks were observed when only ammonium acetate was added in mobile phase. It was found that pH of a mobile phase effected peak shape of fumonisins. In order to reduce tailing of fumonisins, acetic acid was added in mobile phase B and

the gradient program was controlled to maintain high concentration of acetic acid when fumonisins were eluted. By controlling the concentration of acetic acid and ammonium acetate with gradient program, 14 mycotoxins were separated and detected excellently in 11 minutes (Fig. 3).

Each mycotoxin standard was analyzed at six concentration levels. Good linearity was observed in the calibration curves, and excellent sensitivity was achieved.

MRM Transition

　AF G1（＋）

　AF G2 （＋）

　AF B1 （＋）

　AF B2 （＋）

　HT-2 （＋）

　T-2 （＋）

　OTA （＋）

　ZON（－）

　NIV（－）

　DON（－）

　PAT（－）

　FM B1 （＋）

　FM B2 （＋）

　FM B3 （＋）

329.05 > 243.05

331.00 > 245.00

313.00 > 241.05

315.00 > 259.00

442.00 > 263.05（[M+NH4]+）

483.95 > 305.00 （[M+NH4]+）

404.10 > 238.90

317.15 > 273.00

371.10 > 281.25（[M+CH3COO]-）

355.10 > 295.15

（[M+CH3COO]-）

153.10 > 109.20

722.45 > 334.30

706.45 > 336.25

706.45 > 336.25

2.0 3.0 4.0 5.0 6.0 7.0 min

0

10000

20000

30000

40000

50000 HT-2 (+)

PAT （-）

NIV （-）

DON （-）

AF G2 (+) AF G1 (+)

AF B2 (+)

AF B1 (+)

FMB1 (+)

FMB3 (+)

FMB2 (+)

T-2 (+)

OTA (+)

ZON ( -)

Ultra Fast Polarity Switching 　Mycotoxin MRM transition

Fig. 3 14 mycotoxins analysis by LC-MS/MS (PAT, NIV, DON, HT-2, T-2, OTA, ZON, FM B1/B2/B3 50ppb, AF B1/B2/G1/G2 10 ppb)


4

Table 1 Linearity 14 mycotoxins

Fig. 4 Possible coordination interaction with metal ion

Mycotoxin

　AF G1

　AF G2

　AF B1

　AF B2

　HT-2

　T-2

　OTA

Range

0.4-20 ppb

0.4-20 ppb

0.4-20 ppb

0.4-20 ppb

2-100 ppb

2-100 ppb

2-100 ppb

Coefficient(r2)

0.999

0.999

0.999

0.999

0.998

0.999

0.999

Mycotoxin

ZON

NIV

DON

PAT

FM B1

FM B2

FM B3

Range

2-100ppb

2-100ppb

2-100ppb

10-100ppb

2-100ppb

2-100ppb

2-100ppb

Coefficient(r2)

0.999

0.999

0.997

0.999

0.995

0.994

0.997


Rinse condition for eliminating carry over Carry over of fumonisins was initially observed using the general rinse condition, because fumonisins formed complexs with trace metal ions in the sample’ s flow path.

Probably, several carboxyl groups of fumonisins coordinated with metal ion (Fig. 4).

For eliminating carry over, rinse solvent and rinse method were examined. The performance of Nexera autosampler SIL-30AC, which can wash both inner and outer needle surfaces with 4 different solvents, was used. It was thought that carboxyl groups of fumonisins may preferentially pair with hydrogen ions in the presence of low pH, therefore formic acid was added to rinse solvent. When investigating rinse methods, it was discovered the inner and outer rinse of needle reduced carry over more than the outer rinse of needle. Finally the modified rinse

solvent consisted of:1% formic acid aq./methanol / acetonitrile / isopropanol (1/1/1/1). To test the modified rinse cycle method, one injection of the 100ppb fumonisins standard solution was followed by one blank injection to check for carryover. Figure 5 shows chromatograms of the standards of FMB2 and B3, and the following blank injection. Low carry over was observed in the blank injection. It resulted from washing fumonisins adsorbed inside needle with the needle's inner and outer rinse method and the effective rinse solvent.

5

Quantitative Analysis of 14 mycotoxins in beer-based drinksMycotoxins were extracted from samples and were purified with a solid phase extraction (SPE) cartridge.20 commercial beers were analyzed by using this method. The calibration curves were assessed using beer samples spiked with mycotoxins. PAT, AFB1, B2, G1, G2, NIV, T-2

and ZON were not detected in any of the beer samples. Some of the tested samples were found to be contaminated with DON, HT-2, OTA, FMB1, B2, and B3 at concentrations of less than their respective LOQs (each 5 ppb).

Table 2 Mycotoxins detected in analyzed samples

ConclusionsHigh throughput LC-MS/MS method for 14 mycotoxins was developed, and could be applied to the quantification of these compounds in beers.Carry over of fumonisins was eliminated by using both the needle's inner and outer rinse method with effective rinse solvent. Results from these experiments indicate that the health risk to consumers posed by intake mycotoxins in commercial beers is relatively low.

AcknowledgemetsReagents were provided from Wako Pure Chemical Ind.,(Osaka,Japan) with substantial cooperation.

5.5 6.0 6.5 7.0 min

0

500

1000

1500

2000

2500

FMB3

FMB2

100ppb

Blank

Fig. 5 Carry over evaluation of fumonisins Fig. 6 HPLC path of SIL-30AC

DON HT- 2 F MB1 F MB2 F MB3 OTA DON HT-2 FMB1 FMB2 FMB3 OTAMexico < 5 < 5 Holｌand < 5 < 5（1sample） (1/1) (1/1) （2samples） (1/2) (1/2)

USA < 5 Ireland（1sample） (1/1) （2sample）

China < 5 England < 5 < 5 < 5（1sample） (1/1) （1sample） (1/1) (1/1) (1/1)Philippine < 5 Germany（1sample） (1/1) （1sample）

Australia Czech（1sample）（1sample）

Japan < 5 < 5 Belguin 6.7 < 5（5sample） (3/5) (2/5) （2samples） (1/2) (1/2)

< 5 < 5 < 5(1/2) (1/2) (1/2)

< 5 (less than 5ppb)

Concentration of mycotoxin/ppb(detected rate)Producing

country

Concentration of mycotoxin/ppb(detected rate) Producing

country


Manabu Ueda1; Tsubasa Ibushi1; Teruhisa Shiota2;

Hirotaka Honda2; Jun Watanabe1; Kazuo

Mukaibatake1; Junko Iida1 1Shimadzu Corporation, Kyoto, JAPAN 2AMR, Inc., Tokyo, JAPAN

High throughput molecular weight confirmation of Pharmaceutical Compounds using DART MS analysis with ultra-fast polarity switching

ASMS 2012 ThP25-554

2

IntroductionDART, a direct atmospheric pressure ionization source, is known to have little or no negative effect from the solvent used to dissolve the sample. Even with problematic solvents for LC/MS operation such as non-volatile solvents or solvents containing salt, the target compounds can be instantly identified using DART-MS without any sample preparation. High-throughput molecular-weight (MW) confirmation of synthesized compounds is difficult to

achieve using time of flight mass spectrometers, which have been a common choice to interface DART, due to its limitation in switching between positive and negative ion modes. By combining an automated DART ion source with a quadrupole mass spectrometer with ultra-fast polarity switching capability, 11 different pharmaceutical compounds with various polarities were successfully determined in approximately 10 sec/sample.

Materials and MethodsDART-MS analyses of 10ppm pharmaceutical compounds dissolved in 100% DMSO were carried out. The DART-SVP ion source (IonSense Inc., MA, USA) was coupled to the single quadrupole LCMS-2020 (Shimadzu Corporation, Kyoto, Japan), and DART-ID CUBE (IonSense), the new type of DART, was also coupled to the said mass spectrometer. Introduction of samples for DART-SVP was automated by

using the X-Z scanner, which is capable of automating up to 96 samples/run. Ultra-fast polarity switching was utilized on the mass spectrometer to collect full scan data. LCMS-2020 can achieve the polarity switching time of 15msec and the scanning speed of up to 15000u/sec, therefore the loop time can be set at less than 1 second despite the relatively large scanning range of 100-1000u.


Fig. 1 DART-SVP ion source & LCMS-2020 (a) DART-SVP & LCMS-2020 Overview; (b) DART-SVP interface enlarged view; (c) DART-SVP interface using the X-Z scanner

(a) (b)

(c)

3

Fig. 2 DART-ID CUBE ion source & LCMS-2020 (a) The OpenSpot Sample Card; Spot solution with pipettor, or place droplet of liquid containing analyte on center spot, then card is ready to analyze (b) DART-ID CUBE & LCMS-2020 Overview; arrow shows the guide slot which the sample card is inserted into.


DART-ID CUBEFirst, the new DART-ID CUBE ion source was tested and successfully interfaced to LCMS-2020. As ID CUBE required external high voltage (HV) from LC/MS instruments, HV supply of LCMS-2020 easily connected to ID CUBE. Mass spectrum was identified for both the positive standard sample of 100 ppm quinine and the negative standard sample of 100 ppm methylparaben. 5 uL of samples were

spotted on a specialized sampling card and inserted into the ID CUBE source. Vaporization of sample is more rapid with ID CUBE than it is with DART-SVP since the sample is directly heated with electric current running through the metal mesh on which the sample is applied, while the DART-SVP heats the sample with heated gas.

Results

(a)

(b)

4

Fig. 3 Standard sample MS spectra using DART-ID CUBE

Fig. 4a Pharmaceutical compounds DART-MS analysis results

Quinine Methylparaben


50 100 150 200 250 300 350 400 450 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0Inten. (x1,000,000)

151.1

303.1167.1 241.189.2 136.1

496.0479.4417.9375.8291.3

53.1

50 100 150 200 250 300 350 400 450 m/z0.0

1.0

2.0

3.0

Inten. (x1,000,000)

324.9

325.9

135.8 374.9294.9211.9175.8499.9

96.860.9

453.3

Positive MS Negative MS M+H M-H

DART-SVPNext, the DART gas (helium) heater temperature was raised to 350C and commercially available pharmaceutical compounds such as Atenolol, Warfarin, Yohimbine, Cilostazol, Nifedipine, Diazepam, Nitrendipine, and

Diphenhydramine were applied on a metal mesh of the X-Z tool, which has the same size and configuration as the 96 well microtiter plate, to automatically introduce them into the DART ionization gas.

100 200 300 400 m/z0.0

1.0

2.0

3.0

Inten.(x1,000,000)

267

345

93 139 175 352225 476283 423249 313 39061

100 200 300 400 m/z0.0

1.0

2.0

3.0

4.0

Inten.(x100,000)

89

169

145259 294 34537497 207 437125 405

49346564

100 200 300 400 m/z0.0

0.5

1.0

1.5

2.0Inten.(x1,000,000)

157

41697 338175139 309202 48538027868 235100 200 300 400 m/z

0.0

2.5

5.0

7.5Inten.(x100,000)

307

1697997 283157 255 35064 220121 432399 456

480

100 200 300 400 m/z0.0

0.5

1.0

1.5

2.0Inten.(x1,000,000)

433

355

15793 175 449415235 333

493202 387292

56

100 200 300 400 m/z0.0

1.0

2.0

3.0

4.0Inten.(x100,000)89 353

389

169400 443

14597 335242125 207 30352

483

100 200 300 400 m/z0.0

1.0

2.0

3.0

4.0Inten.(x1,000,000)

370

338207 288102 41979 386 447471125149176499

100 200 300 400 m/z0.0

2.5

5.0

7.5

Inten.(x100,000)

368415 458

384 428281 489255 303 337

5123320916975 119145

100 200 300 400 m/z0.00

0.25

0.50

0.75

1.00

Inten.(x100,000)

157

13993

407175 338235107 315283 393 440 48268100 200 300 400 m/z

0.00

0.25

0.50

0.75

1.00

Inten.(x1,000,000)

345 425

381491

89 408222169 441 477313145 35925964

100 200 300 400 m/z0.0

1.0

2.0

3.0

Inten.(x100,000)

285

157

36313993 175 224 338 416373 47468 278

Positive MS

Negative MS

Atenolol

Warfarin

Yohimbine

Cilostazol

Nifedipine

Diazepam

DMSO 2M+H

M+H

M-H

100 200 300 400 m/z0.0

2.5

5.0

7.5Inten.(x100,000)

89

17997

259145 393339205 29552

427 465493

5

Fig. 4b Pharmaceutical compounds DART-MS analysis results


11 different pharmaceutical compounds were analyzed by DART & LCMS-2020.With DART, the choice of solvent for the preparation of sample rarely poses a problem. Here the samples were dissolved in concentrated DMSO.

The capability of LCMS-2020 to do ultra high-speed polarity switching was fully utilized.Target ions were detected in positive and/or negative mode for all samples.

Relatively large DMSO peak was observed in positive mode scan, but the target compounds were successfully identified as well. Positive ion spectrum was observed for Atenolol, Diazepam, Diphenhydramine, Verapamil, Triazolam and Alplazolam. Negative ion spectrum was observed for Warfarin, Nifedipine and Nitrendipine. Both positive and

negative ion spectra were observed for Yohimbine and Cilostazol. DART-MS with the ultra-fast polarity switching and ultra-fast scanning demonstrated its ability to perform high throughput analysis of pharmaceutical compounds dissolved in DMSO as solvent at analysis speed @ approximately 10 sec/sample.

Nitrendipine

100 200 300 400 m/z0.0

1.0

2.0

Inten. (x100,000)

157

139 33893 175 235 41637797 278202 45768

100 200 300 400 m/z0.0

0.5

1.0

1.5

Inten. (x1,000,000)

359

439

8939516997 259 473145 391327283209

51

100 200 300 400 m/z0.0

2.5

5.0Inten. (x100,000)

256

167

334

16593 139 258 354 416320224 390493

190 47328672

100 200 300 400 m/z0.00

0.25

0.50

0.75

1.00Inten. (x100,000)

169

2499714595 259 339283211127 189 31564 433385409359

496479

100 200 300 400 m/z0.0

0.5

1.0

Inten. (x1,000,000)

455

93 441139 291 368 471175 235202 41149553

100 200 300 400 m/z0.0

0.5

1.0

1.5

2.0

Inten. (x100,000)

16989

145249 33997 315

489125 357 391281205 43953

Diphenhydramine

Verapamil

100 200 300 400 m/z0.0

0.5

1.0

Inten. (x1,000,000)

343 421

40393 139 358235 455175 309499

20251

100 200 300 400 m/z0.0

0.5

1.0

Inten. (x100,000)

169

357

97145

281242 32379 373125 267223 395 43752

473

100 200 300 400 m/z0.0

1.0

2.0

3.0

4.0

Inten. (x100,000)

309

387

93 139 352175 421235 278 443 48269

100 200 300 400 m/z0.0

1.0

2.0

3.0

Inten. (x100,000)

89 169

289

388323

404242 339145 45936920597 42153

Triazolam

Alplazolam

Positive MS Negative MS

Conclusions

Teruhisa Shiota1; Manabu Ueda2; Tsubasa Ibushi2;

Jun Watanabe2; Kazuo Mukaibatake2; Junko Iida2;

Yasuhiko Bando1 1AMR, Inc., Tokyo, JAPAN 2Shimadzu Corporation, Kyoto, JAPAN

Componential analysis of pepper of various origins using DART-MS using ultra-fast polarity switching

ASMS 2012 Thp25-548

2

IntroductionDART (Direct Analysis in Real Time), a direct atmospheric pressure ionization source, is capable of analyzing food samples with little or no sample preparation. Time of flight mass spectrometers, which have been a common choice to interface DART, were not suitable in carrying out simultaneous detection of compounds with different polarities from the same sample due to its limitation in

switching between positive and negative ion modes in terms of time required to do so. Analysis of different parts of pepper (exodermis, endodermis and seeds) along with olive oil and soy sauce of different types and origins were carried out using the DART combined with a mass spectrometer with ultra-fast polarity switching capability to conduct chemometric analysis.

Materials and MethodsCommercially available peppers from different origins were introduced to the DART gas using tweezers. Glass capillary was used to dip and transfer olive oil to the ionization area, and soy sauce was spotted on a metal mesh to scan with the DART beam. The DART-SVP ion source (IonSense Inc., MA, USA) was interfaced onto the single quadrupole mass spectrometer LCMS-2020 (Shimadzu Corporation, Kyoto,

Japan). Ultra-fast polarity switching was utilized on the mass spectrometer to collect full scan data. LCMS-2020 can achieve the polarity switching time of 15 msec and the scanning speed of up to 15000 u/sec, therefore the loop time can be set at less than 1 second despite the relatively large scanning range of 100-1000u.


Fig. 1 DART-SVP ion source LCMS-2020

PeppersCapsaicin (C18H27NO3, MW 305), which is responsible for the spicy flavor of pepper, is usually most abundant in placenta. Commercially available peppers from Korea,

China and several areas in Japan were cut and separated to each part of the exodermis, endodermis and the seed and each sample was analyzed by DART-SVP LCMS-2020.

Results

3

Fig. 2 Commercially available peppers from different origins and sample preparation for DART-MS

Fig. 3 Positive/Negative mass spectrum of pepper seed @350°C

Fig. 4 DART gas heater temperature comparison; 200°C, 350°C, 500°C Mass spectra in positive ion mode of product M, N pepper seeds

The signal indicating capsaicin was detected in both polarities; at m/z 306 in positive ion mode, m/z 304 in negative mode. The signal in positive ion mode was more intense than in negative ion mode. After careful

optimization of analytical conditions using different DART gas heater temperatures at 200°C, 350°C and 500°C, it was determined that 350C was the suitable temperature setting to analyze capsaicin.

250 500 750 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0Inten. (x1,000,000)

305.9

613.1

136.8 270.0 355.9 577.2421.0151.8 241.993.7 933.7250 500 750 m/z

0.00

0.25

0.50

0.75

1.00

Inten. (x1,000,000)

304.2

367.4

279.3395.4241.3 586.4170.2 451.3 979.5

920.6

[M+H]+ [M-H]-

Positive Scan Negative Scan

200 250 300 350 400 450 m/z0.0

0.5

1.0

1.5

2.0Inten. (x1,000,000)

305.90269.95

321.90271.00 441.15353.90181.80

200 250 300 350 400 450 m/z0.0

0.5

1.0

1.5

2.0Inten. (x1,000,000)

305.90

269.95321.90

355.90 469.10181.80

200 250 300 350 400 450 m/z0.0

0.5

1.0

1.5

2.0Inten. (x1,000,000)

305.90376.95321.85269.95 437.90

200 250 300 350 400 450 m/z0.0

1.0

2.0

Inten. (x1,000,000)

305.90269.95 321.85 386.00241.90 473.10

200 250 300 350 400 450 m/z0.0

1.0

2.0

Inten. (x1,000,000)

305.90

269.95 321.85 355.90 420.95291.85253.95181.80

471.00

200 250 300 350 400 450 m/z0.0

1.0

2.0

Inten. (x1,000,000)

305.90 376.95308.85269.95

181.75 489.90460.05

Product N Product M

200C 200C

350C 350C

500C 500C


4

Fig. 5 Capsaicin localization comparison; seeds, exodermis, endodermis Mass spectra in positive ion mode of product M, S pepper seeds

Fig. 6 Various soy sauces and fish sauces DART-MS analysis results There found different spectrum pattern between soy sources and fish sources.

In most of the samples, the signal of capsaicin was most intense in seeds, which implicates that there were placental fragments on the surface of the seeds, which, even in small quantity, was giving out the intense signal for capsaicin.

250.0 275.0 300.0 325.0 m/z0.0

0.5

1.0

1.5

2.0

2.5Inten.(x1,000,000)

305.85

293.90 321.90269.90 303.90 322.85291.80 339.05281.90267.90 311.95

253.85256.90

250.0 275.0 300.0 325.0 m/z0.0

0.5

1.0

1.5

2.0

2.5Inten.(x1,000,000)

305.85251.80

291.85 331.85277.85267.85

250.0 275.0 300.0 325.0 m/z0.0

0.5

1.0

1.5

2.0

2.5 Inten. (x1,000,000)

305.80251.75291.85 323.80265.80 277.85 341.85

Product M

seed

endodermis

exodermis

250.0 275.0 300.0 325.0 m/z0.00

0.25

0.50

0.75

1.00

Inten.(x1,000,000)

305.90

321.90303.90251.80

269.95 322.90293.90 339.95283.95

250.0 275.0 300.0 325.0 m/z0.00

0.25

0.50

0.75

1.00

Inten.(x1,000,000)

305.85251.80291.85 323.80269.90

345.85311.90

346.95

250.0 275.0 300.0 325.0 m/z0.00

0.25

0.50

0.75

1.00

Inten.(x1,000,000)

305.85

291.80251.80

323.85275.85261.85 341.90

Product S

seed

endodermis

exodermis

Soy SauceNext, soy sauces and fish sauces from different origins were analyzed. Low molecular weight spectra were observed in different profiles depending on the sample ingredients, indicating the different balances in amino acid contents.

100 200 300 400 m/z0.0

1.0

2.0

3.0

Inten.(x100,000)

115.75

228.80137.80 252.8069.80

100 200 300 400 m/z0.0

2.5

Inten.(x100,000)

187.10

217.10128.10189.10

117.10164.10238.15 384.1089.15

51.30475.10

100 200 300 400 m/z0.0

1.0

2.0Inten.(x100,000)

115.75

230.80

198.75146.7569.85

100 200 300 400 m/z0.0

1.0

2.0

3.0

Inten.(x100,000)

217.10187.10

128.15 189.10238.10 289.10164.05 384.20

51.25117.15

100 200 300 400 m/z0.0

0.5

1.0

Inten.(x100,000)

115.75

226.80146.80 196.80

69.75

100 200 300 400 m/z0.0

1.0

2.0Inten.(x100,000)

217.15

202.15128.15

236.10146.15

384.2051.55

117.10

100 200 300 400 m/z0.0

0.5

1.0

Inten.(x100,000)

111.75226.80

88.80

71.85 196.85240.85

100 200 300 400 m/z0.0

1.0

2.0

3.0Inten.(x100,000)

217.10

202.20128.15 418.10

51.95 480.15

100 200 300 400 m/z0.0

0.5

1.0

1.5

Inten.(x100,000)

113.75 226.80

172.8059.85

259.7583.80 199.80 449.95

100 200 300 400 m/z0.0

1.0

2.0

3.0Inten.(x100,000)

128.15

164.10117.15

166.1089.15 257.1551.40

209.10 369.95

Soy sauce A

Soy sauce B

Soy sauce C

Fish sauce K

Fish sauce N



5

Fig. 7Commercially available olive oils and grape seed oils prepared for DART-MS

Fig. 8 Mass spectra in positive/negative ion mode of olive oil, Filiproberio Extravirgin Olive Oil; DART gas heater temperature comparison (200 - 500C)

Ultra-fast polarity switching is useful for DART analysis of food samples.Some compounds have optimum DART gas heater temperature.

Rapid DART analysis of food samples using ultra-fast polarity switching and its chemometric analysis is available to distinguish their origins.

For olive oil and grape seeds oil samples, high molecular weight spectra were more dominant in both polarity at temperature setting of 500°C than 200°C and 350°C, suggesting the possibility to analyze the differences in triglycerides contents.Multivariable analyses were conducted using these spectral

data to distinguish different food samples from different origins and/or deferent ingredients. This indicates that DART-MS can be a powerful tool for origin determination/ authentication of agricultural produce and processed goods. (data not shown)

Conclusions

Olive Oil

250 500 750 m/z0.0

0.5

1.0

Inten.(x100,000)

242.75224.75

168.80411.10106.75 444.10296.90

94.75 509.10 683.95 824.55 934.60250 500 750 m/z

0.0

2.5

5.0

7.5Inten.(x100,000)

281.30

241.10183.15 313.25

563.50344.1589.15

250 500 750 m/z0.0

2.5

5.0

Inten.(x100,000)

411.05

120.75 321.85 444.10 603.25210.75 902.50807.4594.75

250 500 750 m/z0.0

2.5

5.0

7.5Inten.(x100,000)

281.30

319.15

585.35393.15639.1589.20 183.10 467.10 901.60

250 500 750 m/z0.0

2.5

5.0

Inten.(x100,000)

603.25 902.50

577.20430.05

638.30 850.50447.00312.95120.75

250 500 750 m/z0.0

2.5

Inten.(x100,000)

319.15395.40

377.15471.35

639.2589.20697.20183.15 791.40 901.70487.30


200C

350C

500C

200C

350C

500C

Squalene

Triolein Diolein

Oleic acid


Yuki Ando1; Keiko Matsumoto2; Jun Watanabe2;

Junko Iida2; Shun Hirota1 1Nara Institute of Science and Technology, Nara,

JAPAN 2Shimadzu Corporation, Kyoto, JAPAN

Identification of the modified amino acid residue in the modified heme protein using LC/MS/MS

ASMS 2012 TP09-180

2

IntroductionAn amino acid in a heme protein can be modified by a reaction with hydrogen peroxide, and the protein may lose its physiological activity. Moreover, it has been reported that the tryptophan which was inserted near the heme in myoglobin can be oxidized by an organic acid peroxide. In this study, we found that a methionine residue is modified in a mutant of another heme protein. However, among

the peptide fragments obtained by the peptidase digestion of the modified protein, the two peptides which contain methionine was constructed with the same set of amino acids and exhibited the same molecular weight. We identified the modified amino acid in this mutant protein by using LC/MS/MS.

Materials and MethodsThe mutant of the heme protein used in this study was a mixture of non-modified and modified proteins, where about 50% of the native protein was oxidized. After digesting the mutant of the heme protein with trypsin, the obtained peptide fragments were analysed on the mass spectrometer. Preliminary analysis was first by ESI-TOF mass spectrometer. Secondary analysis was then performed by LC/MS/MS using the following analytical conditions; Nexera

UHPLC system was connected to a LCMS-8030 triple quadrupole mass spectrometer (Fig. 1). Chromatographic separations were carried out using an ODS column, Shim-pack XR-ODS II (150 mm x 2.0 mm, 2.2 um). The sample was eluted at 0.2 mL/min with a binary gradient system and applied to MS/MS with an ESI source, and then analyzed with positive polarity.



HPLC: Nexera UHPLC system (Shimadzu Corporation, Kyoto, Japan)

Mass spectrometer: LCMS-8030 (Shimadzu Corporation, Kyoto, Japan)

Column:

Mobile phase A:

Flow rate:

Time program:

Injection volume:

Column temperature:

Shim-pack XR-ODS II 150 mm x 2.0 mm, 2.2 um

0.1% Formic Acid B: Acetonitrile

0.2 mL/min

B conc.2%(0 min)-50%(40.0 min)-90%

(41.0-45.0 min) -2%(45.01-55 min)

10 uL

40°C

Ionization:

DL temperature:

Nebulizer gas:

temperature:

Drying gas:

ESI, Positive

250°C

2.0 L /min

400°C

15 L/min


High Speed Mass Spectrometer Polarity Switching ・15 msec

Scanning Speed ・Max. 15000 u/sec

3

ResultsESI-MS ananysisThe tryptic digested sample of the heme protein was analyzed by ESI-TOF MS, JMS-T100LC AccuTOF (JEOL Ltd., Tokyo, Japan). Many peaks were detected in the solution of the peptide fragments obtained by the tryptic digestion (Fig. 2). Peptide F2 (sequence: IFIMK) and peptide F8 (sequence: MIFIK) exhibit a same molecular weight at m/z 651 (M+H), but differ in sequence. Another peak was detected at m/z 667, which was 16u-larger than the original peak. The peptide equivalent to m/z 667 (M+H) was not obtained from the solution of the peptide fragments obtained by tryptic digestion of the original heme protein, suggesting that this additional peak was due to the oxidized species of peptide F2 or F8. However, since MS/MS was not available in the ESI-TOF MS analysis, it was not able to identify the modified peptide.

LC/MS analysisWe analyzed the sample was by LC/MS/MS. Full scan MS measurement was performed, and all the peptide fragments obtained by tryptic digestion and longer than five amino acids were detected (Fig. 3).

Two peaks at m/z 326 (M+2H) correspond to the mass of the peptides F2 or F8, and the peak at m/z 334 (M+2H) to the modified peptide.

Fig. 2 ESI-MS spectra of the tryptic digested sample

Fig. 3 LC/MS results of the peptide sample

(F2 or F8 + 16)

(F2 or F8)

(F2 or F8)

(F1)

(F7) (F4) (F9)

(F3)

(F6)

(F5)

500 1000 1500 m/z0.0

1.0

2.0

3.0Inten.(x100,000)

547.30

434.20315.25

500 1000 1500 m/z0.0

1.0

2.0

3.0Inten.(x1,000,000)

339.85[F7 + 2H] 2+ (677)

500 1000 1500 m/z0.0

2.5

5.0

Inten.(x100,000)714.95

477.05

[F5 + 2H] 2+ (1429)

500 1000 1500 m/z0.0

0.5

1.0

1.5Inten.(x1,000,000)

390.45

584.90

[F4 + 2H] 2+ (1168)

500 1000 1500 m/z0.0

0.5

1.0

1.5Inten.(x1,000,000)

453.95

474.50906.60

[F9 + 2H] 2+ (907)

[F1 + H] + (547)

500 1000 1500 m/z0.0

2.5

5.0

7.5Inten.(x100,000)

550.75564.45

825.40

[F3 + 2H] 2+ (1649)

500 1000 1500 m/z0.0

2.5

5.0

7.5Inten.(x100,000)1004.65

670.25

555.60

[F6 + 2H] 2+ (2008)

500 1000 1500 m/z0.0

0.5

1.0

1.5

2.0

2.5Inten.(x1,000,000)334.35

667.45

500 1000 1500 m/z0.0

1.0

2.0

3.0

4.0

Inten.(x1,000,000)346.90

651.45

500 1000 1500 m/z0.0

0.5

1.0

1.5

2.0

2.5Inten.(x1,000,000)

346.90

651.45

RT 11.6min

RT 13.7min RT 14.5min [F2 or F8 +16+ 2H] 2+ （667）

[F2 or F8 + 2H] 2+ （651）

[F2 or F8 + 2H] 2+ （651）

8 )

or F8


4

Fig. 4 Method settings for multiple product ion scan

Fig. 5 MS/MS analysis: (a) MS/MS of m/z 326.4, peak 13.7 min (b) MS/MS of m/z 326.4, peak 14.5 min (c) MS/MS of m/z 334.4, peak 11.6 min Data indicates the peptide sequences were: (a) was IFIMK (F2), (b) was MIFIK (F8), (c) was MIF(O)IK (modified F8).

LC/MS/MS analysisIn order to analyze the sequence of the peptide exhibiting these peaks, product ion scan (MS/MS) measurements were performed. For acquisition of optimal MS/MS data by one injection, the product ion scan was set to have multiple scans with three collision energies (CE=15, 30, 45 v).

With the ultra high-speed scanning capacity (up to 15000u/sec), the LCMS-8030 mass spectrometer was able to acquire sufficient data points from each peak, even when acquiring data in full scanning mode.

The results of product ion scan data showed that MS/MS spectra @ collision energy 15 V had good information for peptide sequencing and that the two peaks of m/z 326

were F2 and F8, respectively, and that the peak of m/z 334 was oxidized F8 (MIFIK).

Peptide mapping of the heme protein was carried out by LC/MS/MSThe modified amino acid in this mutant protein was identified by product ion scan spectrum using LC/MS/MS.

Ultra high-speed scanning capacity of LCMS-8030 mass spectrometer was very usefull for multiple product ion scan.

Conclusions

Precursor ion :326.40 Precursor ion :326.40 Precursor ion :334.40 (a) (b) (c)

100 200 300 400 500 600 m/z 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Inten. (x100,000) 86.2

326.3 233.2 391.3 278.2 538.3 147.2

M

538.3 147. 2 261.2 391.3

I F

[I (Immonium ion) ]+ 86.2

100 200 300 400 500 600 m/z 0.0

2.5

5.0

7.5

Inten. (x10,000) 104.2

217.2

147.2 326.3

407.3 56.3

261.2 520.3 130.3 302.2

520.3 147.2 261.2 407.3

[M (Immonium ion) ]+ )104.2

I F I

100 200 300 400 500 600 m/z 0.00

0.25

0.50

0.75

1.00

1.25 Inten. (x100,000)

302.3

334.3

147.2 74.2

194.6 130.2 407.4 261.1 84.3

520.3 .3407.4 261.1 147.2

130.15

120.2

I F I

[M (Immonium ion) +O] +


Noriko Shoji1; Toshikazu Minohata2; Naohiro

Kuriyama1; Chie Yokoyama1; Keiko Matsumoto2; Jun

Watanabe2; Junko Iida2 1YMC Co., LTD., Komatsu, JAPAN 2Shimadzu Corporation, Kyoto, JAPAN

Identification of triazolam, etizolam and their metabolites in biological samples by liquid chromatography tandem mass spectrometry

ASMS 2012 WP28-611

2

IntroductionBenzodiazepines are one of the mostly widely prescribed groups of drugs because of their sedative, hypnotic, anxiolytic, antiepileptic and muscle relaxant properties. This class of compounds and their associated metabolites are also frequently present in clinical and forensic samples. For this reason, the analysis of benzodiazepines in biological fluids is of great importance to clinicians and forensic toxicologists. A key analytical challenge in the analysis of benzodiazepines is to identify etizolam, triazolam, and their

metabolites (alpha-hydroxyetizolam, 8-ethylhydroxyetizolam, alpha-hydroxytriazolam and 4-Hydroxytriazolam) as a mixture, because of their very similar chemical structure, molecular weight and fragmentation during mass spectrometry. In this study we report a new high resolution separating method for the simultaneous analysis of etizolam, triazolam and their metabolites.


Fig. 1 Structure of etizolam, triazolam and their metabolites


N

NN

NCH3

Cl

S

CH3 N

NN

NCH3

Cl

S

OH

N

NN

NCH3

Cl

Cl N

NN

NCH3

Cl

Cl

OH

N

NN

N

Cl

Cl

OH

EtizolamM.W. 342.07059

8-Ethylhydroxye tizolamM.W. 358.06550

Tria zolamM.W. 342.04389

4-Hydroxytria zolamM.W. 358.03881

alpha-Hydroxytria zolamM.W. 358.03881

N

NN

NCH3

Cl

S

CH3

OH

alpha-Hydroxye tizolamM.W. 358.06550

3

Materials and MethodsThree samples were prepared: A) mixture of all standards (alpha-hydroxytriazolam, 4-hydroxytriazolam, triazolam and etizolam), B) metabolised matrix of triazolam and etizolam and C) blank metabolized matrix using human liver S9.

Triazolam, etizolam, and NADPH regeneration system solution A (NADP+, Glucose-6-phosphate, MgCl2 in H2O), NADPH regeneration system solution B (Glucose-6-phosphate dehydrogenase in sodium citrate buffer), human liver S9 were mixed in 100 mM phosphate buffer (pH 7.4). The mixture was incubated at 37 deg C

overnight (approx. 17 hrs). The control sample was prepared without triazolam and etizolam added. [Final concentration in incubation mixture; 100 mM triazolam and etizolam, 1.6 mM NADP, 3.3 mM Glucose-6-phosphate, 0.4 U/mL Glucose-6-phosphate dehydrogenase, 3.3 mM MgCl2, 2 mg/mL S9 protein

Solid phase extraction (SPE) of incubation mixture:

In vitro metabolism of triazolam and etizolam in human liver S9:

The incubation mixture was extracted on YMC Dispo SPE C18 column (100 mg/mL) as follows and injected onto LC/MS/MS. 1. Condition with 2 mL MeOH 2. Equilibrate with 2 mL 0.1% CH3COOH 3. Load 250 mL sample 4. Elute with 1 mL MeOH 5. Dry extract and resolve in 500 mL H2O

Samples were analyzed with UHPLC and a triple quadruple mass spectrometer using following conditions


HPLC: Nexera UHPLC system (Shimadzu Corporation, Japan)

Column: YMC-Triart C18 column, 1.9 µm, 12 nm (150 × 2 mm) Mobile phase: (A) 10 mM formic acid (B) 10 mM formic acid / acetonitrile (1/1) Flow rate: 0.3 mL/min Time program: B conc. 40%(0 min)-65%(40 min)-40%(40.01-60 min) Injection volume: 1 uL Column temperature: 40°C

Mass spectrometer: LCMS-8030 (Shimadzu Corporation, Japan)

Ionization: Electrospray ionization, Positive Scan type: multiple-reaction-monitoring mode (MRM) MRM triggered automatic MS/MS data acquisition


4


ResultsThe simultaneous analysis of drugs of abuse in clinical and forensic laboratories requires highly specific methods. The developed method in this study contained not only optimized MRM transition parameters and chromatographic conditions, but also product ion scanning which is automatically triggered once an MRM exceeds a specified threshold. The method was applied to the analysis of benzodiazepines; including etizolam, triazolam, and their known metabolites. In this experiment three samples were prepared (as described above). Sample (A) was a matrix-free mixture of etizolam, triazolam, and their known metabolites, sample (B) was a

metabolized matrix of triazolam and etizolam and, sample (C) was a blank metabolism matrix of human liver S9. Firstly, sample (A) was analyzed with the method of 12 MRM transitions, which were quantitative and qualitative transitions for etizolam, triazolam, and their known metabolites and it resulted in excellent separation for all four compounds. Next, sample (C) (blank matrix) was analyzed and no peaks were observed; therefore highlighting the excellent selectivity of the method. Sample (B) (metabolized drug) was then analyzed and three new peaks were found (in addition to the four peaks in sample (A)).

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.00

0.25

0.50

0.75

1.00 (x100,000)

6:TIC(+) 5:TIC(+) 4:TIC(+) 3:TIC(+) 2:TIC(+) 1:TIC(+)

(C)

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.0

2.5

5.0

7.5

(x10,000)

6:TIC(+) 5:TIC(+) 4:TIC(+) 3:TIC(+) 2:TIC(+) 1:TIC(+)

(A)

Etizolam

Triazolam

4-Hydroxytriazolam

alpha-Hydroxytriazolam

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.0

0.5

1.0

1.5

(x100,000)

6:TIC(+) 5:TIC(+) 4:TIC(+)(5.00) 3:TIC(+)(5.00) 2:TIC(+)(5.00) 1:TIC(+)(5.00)

(B) Peak 1 8-Ethylhydroxyetizolam

Peak 3 alpha-Hydroxyetizolam

Peak 5

CEQualitativeCEQunatitativecompounds

-39359.05>111.20-22359.05>341.104-Hydroxytriazolam

-18359.05>341.15-27359.05>176.20alpha-Hydroxytriazolam

-27359.05>287.20-28359.05>286.20alpha-Hydroxyetizolam (M-VI)

-20359.05>315.25-24359.05>305.058-Hydroxyetizolam (M -III)

-27343.05>315.00-24343.05>308.20Triazolam

-37343.05>138.15-28343.05>314.10Etizolam

CEQualitativeCEQunatitativecompounds

-39359.05>111.20-22359.05>341.104-Hydroxytriazolam

-18359.05>341.15-27359.05>176.20alpha-Hydroxytriazolam

-27359.05>287.20-28359.05>286.20alpha-Hydroxyetizolam (M-VI)

-20359.05>315.25-24359.05>305.058-Hydroxyetizolam (M -III)

-27343.05>315.00-24343.05>308.20Triazolam

-37343.05>138.15-28343.05>314.10Etizolam

Peak 2

Peak 4

Peak 6

Peak 7

Fig. 3 12 MRM transitions for 6 drugs and metabolites (above) and MRM chromatograms for sample (A), (B), (C).

5


Two of the three unknown peaks were identified as 8-Ethylhydroxyetizolam and alpha-Hydroxyetizolam as they are known metabolites. However the third unknown peak, which was detected the same MRM transition as that of metabolites of these two compounds, was not identified.Next, sample (B) was re-acquired with MRM triggered

automatic MS/MS and product ion scans. These product ion scan spectra were searched against a hypnotics MS/MS library and the six previously identified peaks were assigned a high hit score. In the same manner as described here, this method is highly applicable to the screening of drugs of abuse in biological samples.

Loop time < 1 sec.

50 100 150 200 250 300 350 m/z0.0

0.5

1.0

1.5

2.0

Inten.(x10,000)

341.0

314.0272.9

111.1249.8183.1 358.4

Peak 4: 4 -Hydroxytriazolam

50 100 150 200 250 300 350 m/z0.0

2.5

5.0

7.5

Inten.(x10,000)

176.1

359.0313.0

277.0149.1 341.0266.0243.0204.1

Peak 2: alpha -Hydroxytriazolam

50 100 150 200 250 300 350 m/z0.0

2.5

5.0

Inten.(x10,000)

282.1 341.1315.1 359.1

247.1179.1154.1 204.057.2

331.1258.1232.096.0 291.1

Peak 1: 8 -Ethylhydroxyetizolam

50 100 150 200 250 300 350 m/z0.0

1.0

2.0

3.0

4.0

Inten.(x10,000)

286.1

287.1341.1305.1269.173.2 236.0137.3 330.1176.3209.4

Peak 3: alpha -Hydroxyetizolam

50 100 150 200 250 300 m/z0.0

1.0

2.0

3.0Inten.(x100,000)314.1

138.1 259.1

224.1 295.1245.1171.1123.157.2148.1 343.1

Peak 7: Etizolam

50 100 150 200 250 300 m/z0.0

2.5

5.0

Inten.(x100,000)

308.1

343.1

239.1165.1138.1 275.1

Peak 6: Triazolam

50 100 150 200 250 300 350 m/z0.0

0.5

1.0

Inten.(x10,000)

341.1

238.0273.0300.0314.0258.1

223.062.4 135.5110.5 331.3

Peak 5: ?

Fig. 4 MRM triggered MS/MS method and MS/MS spectra of Peak 1 to Peak 7

ConclusionsMetabolite analysis using LC/MS/MS with small particle size column achieved high resolution separation for the simultaneous analysis of etizolam, triazolam and their metabolites.

The metabolites were detected and confirmed with MRM triggered automatic MS/MS data acquisition.

Satoshi Yamaki, Manami Kobayashi, Tsutomu

Nishine

Shimadzu Corporation, Kyoto, JAPAN


ASMS 2012 WP13-300

2

IntroductionThe metabolomics technique can rapidly bring information about the similarities and differences within a chromatographic dataset. A metabolomic based approach has been established for metabolite profiling and biomarker discovery, however, it is equally applicable to other research fields including industrial chemical product characterization, food analysis and natural product research. Brewing and fermentation product of beer etc contains several polar metabolites such as amino acids, nucleic acids and organic acids, which provide important contribution on the product‘s quality, flavor and being useful index of a fermentation performance. Profiling studies for several beers using 1H NMR were performed for the requirement of quality control

and index compounds were determined 1, 2. The identification of carbohydrates is easily indicated by NMR but its method may need to be improved in respect of sensitivity. Liquid chromatography-mass spectrometry is one of a widely used tool in the field of metabolomics and metabolite profiling by high sensitivity and selectivity. To obtain the complete profile from a sample, it is necessary to run the LC/MS both positive and negative modes 3.In this study, we developed an LC-based approach to determine metabolite profiles including polar metabolites and to identify specific endogenous components, aiming at high throughput and comprehensive methods using TOFMS acquisition.

1) “Nexera” ※1 Ultra High Performance Liquid chromatograph2) “LCMS-IT-TOF” ※1 Hybrid Mass Spectrometer Fast scanning, fast polarity switching and formula prediction with high accuracy MSn analysis.

3) “Profiling Solution ver. 1.1” ※1 ,Create an aligned data array.

“SIMCA-P+ ver. 12” ※2 ,Data mining tool using multivariate statistical analysis.

“Formula Predictor ver. 1.2” ※1 ,Predicting the molecular formula of target compounds.

※1 Shimadzu, ※2 Umetrics　

Analytical equipment

Strategy of differential analysis using MS-based methods

Approach of this study


Fig. 2 Work flow of the analysis of metabolites in fermentation products.

Fig. 1 Nexera UHPLC and LCMS-IT-TOF.

Analytical Instrument A large number of gata One array of data Software of Statistical Analysis

3

ResultsIdentification of the isolated compounds From beer sample, about 60 peaks were detected by peak integration function in positive ion total ion current chromatogram (TICC) and about 30 peaks were detected in negative ion TICC within a 20 min HPLC separation (Fig. 3). When mass accuracy was checked with the known compound such as malic acid and adenosine, it turned out that MS measurement was performed in the accuracy of less than 3 ppm (using external calibration) acquired with fast polarity switching. These detected peaks were verified using formula prediction software that takes into account MSn information, mass accuracy and isotope modeling. Furthermore, tyrosine, phenylalanine, proline, pyroglutamic acid, fumaric acid and hypoxanthine were tentatively assigned by reference to published literature, and identified using authentic standards.

Table 2 Analytical conditions of LC/MS

Fig. 3 MS chromatogram of a lager beer No. 20 in Table 1. The important components include amino acids, organic acids and nucleic acids were detected.


Table 1 Some Characteristics of the Beers analyzed

*1: Tax category of Japanese liquor. Beer: Malt content, 67% or higher.Low-malt beer: Less than 67% malt (analyzed product contains less than 25% malt).

The third beer: Use malt alternatives, or mix of low -malt beer and another type of spirits.(High-malt beer: The manufacturer sells the beer of 100% of malt use as high premium beer of added value.)

sample no. classification *1 type % alcohol origin1 the third beer (no malt) 5.0 Japan2 the third beer 5.0 Japan3 the third beer 5.0 Japan4 the third beer 5.0 Japan5 the third beer 5.0 Japan6 the third beer 5.0 Japan7 low-malt beer lager 5.5 Japan8 low-malt beer lager 5.5 Japan9 low-malt beer lager 5.5 Japan10 beer lager 5.0 Japan11 beer lager 5.0 Japan12 beer ale 6.5 Japan13 beer lager 5.0 Japan14 beer lager 6.0 Japan15 beer lager 5.0 Japan16 beer lager 4.5 Mexico17 beer ale 7.0 Belgium18 beer ale 9.0 Belgium19 beer (high-malt beer) lager 5.5 Japan20 beer (high-malt beer) lager 5.0 Japan21 beer (high-malt beer) lager 5.5 Japan22 beer (high-malt beer) lager 5.5 Japan23 beer (high-malt beer) lager 5.0 Japan24 beer (high-malt beer) lager 5.0 Korea25 beer (high-malt beer) ale 5.0 Japan

Column:

Flow rate:

Column temp.:

Mobile phase:

Time prog.:

Injection vol.:

Mixer vol.:

Ionization mode:

Probe voltage:

CDL temperature:

BH temperature:

Nebulizing gas flow:

Drying gas flow:

CDL,Q-array voltage:

Scan range:

Phenomenex Synergi Hydro-RP 80A

(150 mm L. x 2.0 mmI.D., 4.0 um)

0.2 mL/min

40°C

A) Water containing 0.1% formic acid

B) 80% Acetonitrile containing 0.1% formic acid

0%B (0-8 min) → 100%B (18-22 min) →

0%B (22.01 min) → STOP 37 min

2 uL

0.5 mL

ESI positive and negative

+4.5 kV/-3.5 kV

200°C

200°C

1.5 L/min

0.1 MPa

Default value

m/z 85-1000

Positive TICC

Negative TICC

4

Fig. 4 Multivariate statistics were performed on the aligned data set of positive and negative ions using Umetrics SIMCA-P+ software: (a) Score plot from 25 beer samples (groups are highlighted by the circles manually drawn). (b) Loading plot, that indicate each metabolites as m/z retention time pairs.

Principal Component AnalysisPooled QC sample analysis was used to assess the performance of the system by repeatedly injecting it. PCA result of QC samples showed its to be tightly clustered together for both positive and negative ESI data (data not shown). Profiling software produced a data array of m/z and retention time pairs. About fifteen hundred ions (1072 ions detected in positive ion, 480 ions detected in negative ion) were detected in common with twenty five beer samples.PCA of LC/MS data resulted in the separation of samples into six groups: two groups of the third beer, one group of low-malt beer, two groups of beer, and one group of high-malt beer (Fig. 4a). Six groups may be suggested based on the distribution of samples along PC1, which

explain most of the variability (41%). These groups of beers roughly are separated according to the malt content: low-malt beers in negative PC1, high-malt beers in positive PC1, and beers characterized by PC1 close to zero. This results suggest that LC/MS approach could be applied to understand the characteristics of sample, for example, label, malt contents and type of beer drinks. The ion of m/z 205.0976 in positive mode and m/z 191.0199 in negative mode was extracted as a characteristic peak of high-malt beer. These detected peaks were tentatively assigned as tryptophan (C11H12N2O2) and citric acid (C6H8O7) respectively, using formula prediction software, and were further identified by comparison with authentic standards.

Differential analysis using degradation model sampleIn addition, this system was applied to the confirmation of quality deterioration of beer sample. Beers were heated at sixty degrees centigrade for 30 min, 1 hour, 4 hours, 10 hours, 1 day, 3 days, 6 days, and analyzed in the same system. Fig. 5 shows the PCA score and loading plot of one high-malt lager beer (No. 20 in Table 1) of no heat, 4hours,

1 day, 3 days and 6days heated treatment. Sample of heat deterioration were not clearly showed tendency on score plot, since it is considered an imperfect degradation examination. However, several constituents were found as the differentiating components between short term and 6 days treatment (Fig. 6).

(a) Score plot of positive ion mode

1 17

18

24

9

7 8

4

6

3

16

13

11

25 15

10 2

5

12

22

20 23

21 19

14

8

2515

10

5

12

224

2

20 23

2119

142

22

6

3 1110

2

17

18

・The third beer ・Low-malt beer ・Beer ・High-malt beer

(No malt)

(Belgian ale)

(b) Loading plot

PC1(41%)

PC2(

10%

)

1

m/z 268.102_R.T.10.29: adenosine

m/z 205.097_R.T.14.91: tryptohan

m/z 116.071_R.T.2.08: proline

m/z 118.086_R.T.2.063: valine


5

This result suggests that LC/MS approach could be applied to evaluate the content of degradation of several fields such as industrial products. These detected components were tentatively assigned using Formula Predictor software

(Fig. 7) and MSn spectra. One of the decreasing intensity of ions by heat treatment was tentatively assigned as deoxyadenosine (C10H13N5O3).

Fig. 5 PCA score plot and loading plot of a lager beer obtained from degradation model data on positive ion mode.

Fig. 6 XVat plot of m/z 252.109 eluted at 12.198 minutes. Y axis shows the intensity of detected ion and X axis shows the sample name.

Fig. 7 The formula prediction software results on the m/z 252.1091 ion are displayed. The highest score calculated corresponds to the molecular formula C10H13N5O3.

3 days

6 days 1 day

No heat

4 hours

PC1(33%)

PC2(

28%

)

Peaks which decrease intensity by heating

(a) Score plot (b) Loading plot

No heat 4 hours 1 day 3 days 6 days

N

N

N

N

NH2

O

OH

OH

DeoxyadenosineMolecular Formula = C10H13N5O3Formula Weight = 251.24192Monoisotopic Mass = 251.101839 Da[M+H]+ = 252.109116 Da[M-H]- = 250.094563 Da

m/z 136.06


6

ConclusionsMass spectrometry-based metabolite profiling was used to identify changes in chemical component levels in fermentation product model using fast polarity switching TOFMS analysis.Bioactive marker compounds that belong to the polar metabolites were measured and identified using a combination of high accuracy MSn data and verified by reference to authentic standards and to internal and external databases.

Mass spectrometry in combination with multivariate analysis is useful for the rapid determination of subtle differences and exploring the potential markers for quality control not only within the beer products but also in other beverages and biofluids.

1) I. Duarte et. al., J Agric. Food Chem., 2002, 50, 2475-24812) C. Almeida et. al., J Agric. Food Chem., 2006, 54, 700-706 3) S. Yamaki et. Al., 59th ASMS Conference in Denver, WP 354 (2011)

References


Yuzo Yamazaki, Keisuke Shima　　SHIMADZU CORPORATION, Kyoto, Japan

Differentiation of isobaric residues in SPITC-derivatized tryptic peptides using MS/MS technique in a Curved Field Reflectron.

ASMS 2012 TP09-189

2

OverviewSpecific fragment ions obtained by CFR can discriminate isobaric residues in SPITC-derivatized peptides without impairing interpretable sequence information.Differentiations of Ile/Leu and αα- /βAsp are performed successfully.Switching PSD to HE-CID rapidly is valuable for de novo sequencing.

IntroductionFixing a strong negative charge at N-terminus of tryptic peptide is a quite effective chemical derivertization for de novo sequencing by using post-source decay (PSD) on MALDI-TOFMS. However, whereas the chemical derivertization causes interpretable y-ions mainly, one

cannot differentiate isobaric amino acid residues, for instance, Ile/Leu and α- /βAsp. We report our study of differentiation of these residues by using PSD and a high energy CID.

MethodsMALDI-TOFMS Instrument: AXIMA-Performance (Shimadzu Biotech/Kratos) Measurement: PSD and high-energy CID-MS/MS in positive ion mode. Collision gas: helium Collision energy: 20 keV (laboratory frame of reference).

Curved Field Reflectron(1),(2)

Mix-mode of PSD and CID. All fragment ions generated in both PSD and CID are detected simultaneously. No precursor suppression system. No stitch, seamless PSD. Only 30 sec. to switch PSD to CID. High energy collision at 20 keV. No deceleration, no re-acceleration. Easy operation.

Samples Tryptic digests of BSA and β-casein Synthesized peptides including α-and βAsp, originated from human α-crystallin

Derivatization of 4-sulphophenyl isothiocyanate(SPITC) on a ZipTip(3). Guanidination: 3 hours at 37°C Sulfonation: 2 hours at 50°C


Gas on / CIDGas off / PSD

N2 Laser337 nm, 3ns

Gas on / CID

N2 Laser337 nm, 3ns

Gas on / CIDGas off / PSD

N2 Laser337 nm, 3ns

- O3S-AA1-AA2-AA3------------ AAn- Arg - COOH - +

PSD y-ions

y1

y2

y3

yn-2

yn-1

SO

O

O

N C S NH2 peptide COOH

SO

O

O

HN C

SHN peptide COOH

Fig.1 Description of CFR Fig.2 SPITC derivatization for de novo sequencing

3

ResultsDifferentiation of Ile/Leu


Fig.1 Description of CFR Fig.1 Description of CFR Fig.1 Description of CFR

0

10

20

30

40

50

60

70

80

90

100

%Int.

900 950 1000 1050 1100 1150 1200m/z

1[c].G3

927.6

1121.6

1142.5

904.5 997.7 1113.5 1163.7

0

20

40

60

80

100

%Int.

100 200 300 400 500 600 700 800 900 1000 1100m/z

1[c].G3

2[c].G3

X10

1142.6

1142.6

927.4

927.3

764.2

764.2

969.5

969.5

245.9

1139.3

136.0

358.8175.0

245.9

651.3488.2405.8

358.8651.3488.2

276.9

910.3705.3412.8

988.0918.0

988.185.9

174.9

543.0774.5

747.0

341.8 482.3 634.7

378.6

146.9 200.9

277.0

0

20

40

60

80

100

%Int.

240 260 280 300 320 340 360 380 400 420m/z

1[c].G3

2[c].G3

245.9

358.8

245.9

405.8

358.8

378.6276.9

405.8

313.9 412.8248.9 292.9

341.8

378.6277.0

45

58

Ile HE -CID

PSD

300.8

Xle

Xle

HE -CID

PSD

+215

SPITC - NH- Y- L - Y- E- I - A- R- COOH

Xle

Xle

y3 y2 y6 y5

y3

y2

0

10

20

30

40

50

60

70

80

90

100

%Int.

1450 1500 1550 1600 1650 1700m/z

1[c].G7

1567.7

1480.8

1724.9

1479.8

1640.0

1676.9

1537.8 1694.91573.7

0

20

40

60

80

100

%Int.

200 400 600 800 1000 1200 1400 1600m/z

1[c].H3

2[c].H3

X10

1481.5

1696.2

1679.2

1696.3

1368.3 1633.31523.6

1679.21368.3

1549.6

1523.61649.0

571.3

1415.8

571.3

686.61018.9

686.5

1182.1814.3

1018.8814.3 1277.3387.2

387.3

274.2 500.3

500.3

1350.7

274.2

868.8 1140.3

992.0

175.2 992.1740.4640.6441.4342.2

669.0

526.3

175.1

257.4136.1

0

20

40

60

80

100

%Int.

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

1[c].H3

2[c].H3387.3

274.2500.3

500.3274.2

441.4342.2

512.3526.3329.1 407.5243.3 299.9 370.3 427.9257.4 314.2 355.9333.0

510.4 539.9410.8 461.8428.1370.2322.7257.3 300.2

SPITC - NH- LGEYGFQNALI VR- COOH

+215

Xle

HE -CID

PSD Xle

Xle Xle

45

Ile HE -CID

PSD 300.8

y3 y2 y4

Leu

59

y2

y3 y4

y12

y13

Xle

Xle

0

10

20

30

40

50

60

70

80

90

100

%Int.

2200 2250 2300 2350 2400m/z

1[c].E20

X5

2186.2

2243.3

2191.3 2229.32211.2 2353.22247.3

2401.3

2171.2 2285.2

SPITC - NH- DMPIQAFLL YQEPVL GPVR- COOH

0

20

40

60

80

100

%Int.

400 600 800 1000 1200 1400 1600 1800 2000 2200 2400m/z

1[c].E20

2[c].E20

X5

2187.9

2402.7

2072.7

2230.0

2188.0

2388.32135.2

2072.9

736.9

2402.9

1157.91731.0

2230.0

1531.71384.5

736.6

1941.2

2262.5

1157.8

994.9

2388.1

1271.2

866.1

1602.7 1730.9540.9 995.0 1940.3427.8 866.1

541.0427.9

1325.6790.9 920.5410.7

719.6

0

20

40

60

80

100

%Int.

1100 1150 1200 1250 1300 1350 1400 1450m/z

1[c].E20

2[c].E201157.9

1271.3

1384.5

1157.8

1271.21384.6

1325.6

1434.9

1211.9

+215

Xle HE -CID

PSD

Xle Xle Xle?

Xle Xle Xle Xle?

HE -CID

PSD

y12 Leu

59

Leu

59

y4

y5

y16

y17

y10

y11 y12

y11 y10

MS MS MS

Fig. 3 Analysis of tryptic peptide of BSA #1

Fig. 4 Analysis of tryptic peptide of BSA #2

Fig. 5 Analysis of tryptic peptide of β-casein

113 gap

No

Xle 113

CID gas on

y-59(w)

Leu 113 Yes

y-45(w)

Ile 113

HE -CID PSD

Fig. 6 A possible flow, where de novo sequencing incorporates the differentiation.

4

ConclusionsIle and Leu in SPITC-derivatized peptides were differentiated successfully by high energy CID-MS/MS in a CFR.y-59 and y-45 between two y-ions, a mass difference of which is 113, were specific side chain fragmentations that indicate Leu and Ile respectively.Since PSD of the derivatized-peptide is still useful to read out an amino acid sequence easily, taking spectrum in both PSD and HE-CID could be more preferable to obtain a

(1)Cordero MM, et al; Rapid Commun Mass Spectrom., 1995, 9, pp1356-61.(2)Cornish TJ, et al; Rapid Commun Mass Spectrom., 1993, 7, pp1037-40.(3)Chen, P., et al; Rapid Commun. Mass Spectrom., 2004, 18, pp191-198.(4)Yamazaki, Y.,et al; Anal. Chem., 2010, 82 (15), pp 6384–6394.

complete amino acid sequence. The SPITC derivatization induced more cleavable N-terminal side of the Asp residue than the one in a underivertized counterpart. Notably, the specific y-ion ratios of Asp isomers were still retained after the derivertization, which is thought to be useful to perform further quantitative analysis.


0

10

20

30

40

50

60

70

80

90

100

%Int.

1150 1200 1250 1300 1350 1400 1450m/z

1[c].G1

1175.5

1177.7

1390.51180.7

1231.71188.81157.6

T6α

+215

0

10

20

30

40

50

60

70

80

90

100

%Int.

1150 1200 1250 1300 1350 1400 1450m/z

1[c].G2

1175.6

1390.61180.8

1157.6 1231.7

T6β

+215

SPITC - NH- TVL D(α/β) SGISEV R- COOH

y7

y8

0

20

40

60

80

100

%Int.

200 400 600 800 1000 1200 1400m/z

1[c].G1

2[c].G2

X5

1390.9

1390.8

1175.7

1175.6

1074.5

1074.4

747.3 862.3

1217.7

1217.7

489.9 747.4

489.8

1373.0

660.2

660.3 862.5

975.4

274.0 975.4402.9

274.0 402.9

175.1 1131.7

844.7 1131.5175.1 603.3

0

10

20

30

40

50

60

70

80

90

100

%Int.

650 700 750 800 850 900 950 1000m/z

747.3 862.3

660.2

862.5

975.4

844.7

SPITC -derivatized

PSD PSD

T6α

T6β

T6α T6β y7 y8

0

10

20

30

40

50

60

70

80

90

100

%Int.

650 700 750 800 850 900 950 1000m/z

746.9

861.8

844.3815.9

0

20

40

60

80

100

%Int.

200 400 600 800 1000 1200m/z

1[c].G1

2[c].G2

X10 X10

1174.8

1175.1

746.9 1181.4

746.9

861.8

273.7

861.9

844.3

174.8 572.7 659.6 1074.1974.9

273.7

472.9372.2 729.1

772.4

659.9174.8 975.0

256.7

572.7472.9 1074.1

544.6 602.7

372.3

315.3 668.1

256.6 315.4

underivatized

PSD

T6α

T6β

T6α

T6β

y7

y8

T6 peptide;

(A)

(C)

(B)

Fig. 7 Effect of the derivatization on a differentiation of Asp isomers. MS spectra after the derivatization (A), PSD spectra (B), and an amino acid sequence of T6 peptide and a proposed fragmentation of Asp isomers (C).

Differentiation of α-and βAsp(4)

References

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Noisiel 77448 Marne la Vallée Cedex 2

Tél : 01 60 95 10 10Fax : 01 60 06 51 66

Email : [email protected] : www.shimadzu.fr

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