Different ways of enhancing productivity in trace analysis of

Different ways of enhancing productivity in trace analysis of pesticides and PPCP inwater samples using sensitive LC/MS/MS technology

Jens Dahlmann and André Schreiber, Applied Biosystems

2 © 2009 Applied Biosystems

Outline

● Analysis of environmental contaminants such as pesticides, PPCP in drinking and surface water– Many compounds– Fast and accurate results– As easy as possible– Robust, selective, sensitive…1. Screening with Multiple Reaction Monitoring (MRM)2. Confirmation using MS/MS library search

● Automation and Ease of Use of acquisition and reporting


The instrumentation LC/MS/MS


Experimental

● Direct injection of water ● HPLC

– Phenomenex Synergi 4μ Fusion-RP 80A 50x2mm (25°C)– Eluent A: water + 5mM ammonium formate– Eluent B: acetonitrile + 5mM ammonium formate– 8min gradient: 80/20 – 10/90 (A/B), total run time 20min– Injection of 20µL

● MS/MS– 3200 QTRAP® LC/MS/MS system (used in triple quadrupole ‘QqQ’

and linear ion trap ‘LIT’ mode)– TurboV™ source with Electrospray Ionization


Turbo V™ Source: ESI

The softest ionization for polar and ionic substances

LC

High Voltage

Nebulizer Gas

Atmospheric pressure Vacuum

Formation of charged droplets

Evaporation (TurboV heaters)

Coulomb-Explosion

Exhaust

Curtain Gas

Curtain Gas

Mass Analyzer

Orifice

TurboV heaters


Screening Strategy Using LC/MS/MS

● Multiple Reaction Monitoring– Standard quantitation experiment

● Two or more MRM transitions– Quantifier and qualifier MRM– Retention time and– MRM ratio used for confirmation– Tolerance of +/-20%

● Limitation in number of MRM transitions per experiment (LINAC® collision cell ~ 300 MRM with 5ms dwell time)

XIC of +MRM (300 pairs): Exp 1, 180.1/77.0 amu from Sample 2 (Std with 2mM and 0.2%) of ... Max. 3.8e4 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5


Cliquid® Software – MRM Catalogue

Cliquid™ Food & Beverage: > 500 Pesticides


10 ppb MRM sensitivity (API 3200™ system)

5 6 7 8Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

5.9e4 6.4

4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

Intensity, cps

5.7

6 8T im e, m in

0 .0

1 00 0.0

2 00 0.0

3 00 0.0

4 00 0.0

5 00 0.0

6 00 0.0

7 00 0.0

8 00 0.0

9 00 0.0

1 .0 e4

1 .1 e4

1 .2 e4

1 .3 e4

1 .4 e4

1 .5 e4

1 .6 e47.5

8 1 0T im e , m in

0.0

50 00 .0

1.0 e4

1.5 e4

2.0 e4

2.5 e4

3.0 e4

3.5 e4

4.0 e4

Intensity, cps

8 .3

Thiabendazole Thiacloprid

202/175 253/126202/131 253/99

Atrazine Azoxystrobin

216/174 404/372216/104 404/344


Screening Strategy Using LC/MS/MS - QTRAP®

● QTRAP® LC/MS/MS systems: Information Dependent Acquisition (IDA)

● Multi Target Screening (MTS)● Multiple Reaction Monitoring

– Selective and sensitive survey scan

● Dependent Enhanced Product Ion (EPI)– Sensitivity in EPI is comparable to MRM– Less acquisition time in comparison to many 2nd

MRM transitions (100 MRM at 5 ms dwell + 5 ms pause ~ 1s) vs. 2 EPI of 500 amu at 4000 amu/s with 100 ms fill ~ 1s)

– Complete spectral information– Mass spectral library searching

DynamicExclusion

Survey Scan

Dependant Scans

Threshold


XIC of +MRM (10 pairs): Exp 1, 216.1/174.0 amu from Sample 4 (0.5) of Data EPI vs MS2 - EPI dynFT 216.wiff (Turbo Spray) Max. 660.0 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0

100

200

300

400

500

600660 7.21

+EPI (216.10) CE (35): Exp 2, 7.121 to 7.301 min from Sample 4 (0.5) of Data EPI vs MS2 - EPI dynFT 216.wiff (Turbo Spray) Max. 3.5e4 cps.

70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230m/z, amu

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4 174.0

96.1 216.079.0

145.9104.0

+MS2 (216.10) CE (35): Exp 2, 6.628 to 7.861 min from Sample 4 (0.5) of Data EPI vs MS2 - MS2 216.wiff (Turbo Spray) Max. 89.2 cps.

70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230m/z, amu

0

20

40

60

8089 174.0

Confirmation by Q TRAP® or QqQ (0.5 ng/mL Atrazine)

MRM

Enhanced Product Ion scan (Q TRAP® system)Purity Fit 76.5%

Product Ion scan (QqQ)no library match


Screening and Confirmation:

Workflow: MRM survey –MS/MS – Library Search

Collection of MS/MS…(full scan confirmation)

Library Search Results…

Quantification…


● River water and fruit samples– Direct injection of water– QuEChERS extraction of fruits (disperse SPE) and 10x dilution to

minimize possible matrix effects ● UHPLC

– Column: Phenomenex Synergi Fusion-RP 2.5μm (50x2mm)– Ultra fast gradient water/methanol + 5mM ammonium format– Total run time of less than 5min

● MS/MS– AB SCIEX Triple Quad™ 5500 LC/MS/MS System– Turbo V™ source with ESI probe– 150 MRM transitions (dwell time = 2ms and pause time = 3ms)– Quantifier-qualifier ratio for confirmation

Simultaneous Analysis for more than 500 Pesticides


Detection of Diazinon in an Urban Water Sample

Control of sucking and chewing insects and mites on a very wide range ofcrops, including deciduous fruit trees, citrus fruit, vines, olives, bananas,pineapples, vegetables, potatoes, beet, sugar cane, coffee, cocoa, tea,tobacco, maize, sorghum, alfalfa, flax, cotton, rice, ornamentals,glasshouse crops, forestry, etc., at 300-600 g/ha;


TIC of +MRM (150 pairs): 223.2/1... Max. 5.5e5 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e55.5e5

Intensity, cps

2.9

2.62.5

1.23.31.7

0.80.4 2.1

XIC of +MRM (150 pairs): 305.1/1... Max. 2.1e4 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

Intensity, cps

3.0


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Intensity, cps

0.40.6

3.0

1.3 1.6


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

2.9e4

Intensity, cps

3.0


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e55.5e5

Intensity, cps

2.9

2.62.5

1.23.31.7

0.80.4 2.1


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

Intensity, cps

3.0


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Intensity, cps

0.40.6

3.0

1.3 1.6


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

2.9e4

Intensity, cps

3.0

Detection of Diazinon in an Urban Water Sample

TIC of XIC of1μg/L standard 1μg/L Diazinon

MRM ratio = 0.46

(20%: 0.37-0.55)

TIC of water sample 1.3μg/L DiazinonMRM ratio = 0.42


Endocrine Disrupting Compounds (EDC), Pharmaceuticals and Personal Care Products

(PPCP)

Cliquid™ Food & Beverage: > 500 Pesticides


EDC and PPCP studied by LC/MS/MS

● Analgesics● Antibiotics● Antidepressants● Antiarthritics● Lipid Regulator● Androgens● Estrogens● Pesticides● Insect Repellents● Sunscreen● Stimulants● …

Compound Type Compound Type Compound TypeAcetaminophen Analgesic Amoxicillin Antibiotic Chloridazon Herbicide

Ketoprofen Analgesic Lincomycin Antibiotic Propazine HerbicideCodine Analgesic Oxytetracycline Antibiotic Diuron Herbicide

Hydrocodone Analgesic Chlortetracycline Antibiotic Hexazinone HerbicideAcetylsalicylic Acid Analgesic Viginiamycin Antibiotic Bromacil Herbicide

Ibuprofen Analgesic Erythromycin Antibiotic Metazachlor HerbicideAndorostenedione Androgen Roxithromycin Antibiotic Metolachlor Herbicide

Testosterone Androgen Tylosone Tartrate Antibiotic 2,4-D HerbicideWarfarin Anti coagulant Carbadox Antibiotic DEET Insect Repellant

Diclofenac Anti-arthritic Triclosan (Irgasan) Antibiotic Bezafibrate Lipid Regulator

Meprobamate Anti-axiety Chloamphenicol Antibiotic Clofibric AcidMetabolite of Lipid

RegulatorSulfadiazine Antibiotic Fluoxetine Anti-depresant Diazepam Muscle-relaxant

Sulfamethoxazole Antibiotic Carbamazepine Anti-seizure Norethisterone Ovulation Inhibitor

Sulfathiazole Antibiotic PentoxifyllineBlood Viscosity Reducing Agent Theophylline Stimulant

Sulfamerazine Antibiotic Estrone Estrogen Theobromine StimulantSulfamethizole Antibiotic Estradiol 17a Estrogen Caffeine StimulantSulfamethazine Antibiotic Ethynylestradiol Estrogen Oxybenzone Sunscreen

Sulfachloropyridazine Antibiotic Progesterone Estrogen Sildenafil Virility regulatorTrimethoprim Antibiotic Equilin Estrogen replacement Vardenafil Virility regulator

Sulfadimethoxine Antibiotic Diethylstilbestrol Estrogen replacement CotinineCiprofolxacin Antibiotic Simazine Herbicide KetorolacPenicillin G Antibiotic Isoproturon Herbicide Meclofenamic Acid

Chlorotoluron Herbicide NifedipineAtrazine Herbicide Indomethacin

Diatrizoate2,4-Dichlorobenzoic Acid


100ng/mL EDC and PPCP standard (MRM detection)


Fast and Comprehensive Screening for Pharmaceuticals and Personal Care Products● Environmental samples

– Direct injection of water or SPE cleanup ● UHPLC

– Column: Zorbax SB-C18 1.8μm (50x4.6mm)– Fast gradient water/methanol + 0.1% formic acid

● MS/MS– AB SCIEX QTRAP® 5500 LC/MS/MS System– Turbo V™ source with ESI probe– 111 MRM transitions (dwell time = 3ms and pause time = 3ms)– Polarity switching in 50ms by eQ™ Electronics– IDA of EPI spectra (CE = 35V and CES = 15V) for confirmation – Linear Accelerator™ trap scans with 20000Da/s


XIC of +MRM (78 pairs): Exp 1, 235.2/86.1 Da ID: Lidocaine_1 from Sample 1 of Pos Neg... Max. 1.1e6 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.00.002.0e54.0e56.0e58.0e51.0e61.2e6

Intensity, cps

6.5

XIC of -MRM (33 pairs): Exp 2, 307.0/160.9 Da ID: Warfarin_1 from Sample 1 of Pos Neg... Max. 2.7e6 cps.

2 4 6 8 10 12 14 160.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e62.7e6

Intensity, cps

11.8

XIC of +MRM (78 pairs): Exp 1, 235.2/86.1 Da ID: Lidocaine_1 from Sample 1 of Pos Neg... Max. 1.1e6 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.00.002.0e54.0e56.0e58.0e51.0e61.2e6

Intensity, cps

6.5

XIC of -MRM (33 pairs): Exp 2, 307.0/160.9 Da ID: Warfarin_1 from Sample 1 of Pos Neg... Max. 2.7e6 cps.

2 4 6 8 10 12 14 160.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e62.7e6

Intensity, cps

11.8

Fast and Comprehensive Screening for Pharmaceuticals and Personal Care Products

Detection 111 PPCP in positive and negative polarity in a single LC/MS/MS run

78 PPCPPositive polarity

33 PPCPNegative polarity


XIC of +MRM (78 pairs): Exp 1, 24... Max. 2.2e5 cps.

5 10 15Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.2e5

Intensity, cps

4.8

XIC of -MRM (33 pairs): Exp 2, 28... Max. 6.8e4 cps.

5 10 15Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

6.8e4

Intensity, cps

13.4

+EPI (240.16) Charge (+0) CE (3... Max. 3.5e4 cps.

50 100 150 200 250 300m/z, Da

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

Intensity, cps

148.1

130.0120.1 222.291.0 240.1166.1

-EPI (286.79) Charge (+0) CE (3... Max. 1.0e4 cps.

50 100 150 200 250 300m/z, Da

0.0

2000.0

4000.0

6000.0

8000.0

1.00e4

Intensity, cps

160.9

142.0

287.0

175.9 241.2124.9223.1

88.9269.4

197.3107.0

(+/-) Salbutamol (+/-) TriclosanMRM: 240/148 MRM: 287/35

(+) EPI (-) EPI

XIC of +MRM (78 pairs): Exp 1, 24... Max. 2.2e5 cps.

5 10 15Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.2e5

Intensity, cps

4.8

XIC of -MRM (33 pairs): Exp 2, 28... Max. 6.8e4 cps.

5 10 15Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

6.8e4

Intensity, cps

13.4

+EPI (240.16) Charge (+0) CE (3... Max. 3.5e4 cps.

50 100 150 200 250 300m/z, Da

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

Intensity, cps

148.1

130.0120.1 222.291.0 240.1166.1

-EPI (286.79) Charge (+0) CE (3... Max. 1.0e4 cps.

50 100 150 200 250 300m/z, Da

0.0

2000.0

4000.0

6000.0

8000.0

1.00e4

Intensity, cps

160.9

142.0

287.0

175.9 241.2124.9223.1

88.9269.4

197.3107.0

(+/-) Salbutamol (+/-) TriclosanMRM: 240/148 MRM: 287/35

(+) EPI (-) EPI

PPCP Spiked into Water Sample (MRM-EPI Screen)


XIC of +MRM (53 pairs): Exp 1, 237.1/194.0 amu from Sample 7 (EDC River 1 Influent) of Samples POS mode_1EPI.wiff (Turbo Spray) Max. 7.3e4 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

9.3e4

7.6

6.2

XIC of +MRM (53 pairs): Exp 1, 237.1/194.0 amu from Sample 7 (EDC River 1 Influent) of Samples POS mode_1EPI.wiff (Turbo Spray), ... Max. 5.4e4 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

7.6

6.2

+EPI (237.08) Charge (+0) CE (35) CES (15) FT (200): ... Max. 4.9e4 cps.

80 100 120 140 160 180 200 220 240 260 280 300m/z, amu

0.0

1.0e4

2.0e4

3.0e4

4.0e4

194.0237.1

219.1135.091.1

201.2175.2165.2103.181.1 161.397.2 181.4123.2 137.0

+EPI (237.08) Charge (+0) CE (35) CES (15) FT (174.2... Max. 4.9e5 cps.

80 100 120 140 160 180 200 220 240 260 280 300m/z, amu

1.0e5

2.0e5

3.0e5

4.0e5

4.9e5

Inte

ns

ity, c

ps

194.0

237.1

165.1 179.2219.191.1 105.1 145.0

River 2 – Carbamazepine (MRM and EPI)

Peak 1?

Peak 2Carbamazepine (12.1ng/L)

Influent

EPI24.6%

EPI89.2%

MRM


Results River 2 (suburban upstream and downstream)Compound River 2 - downstream (ng/L) River 2 - upstream (ng/L)Acetaminophen 3190.0 >1Bromacil 30.8 25.8 0.8Caffeine 4930.0 31000.0 6.3Carbamazepine 64.5 179.0 2.8Codeine 63.1 >1Cotinine 44.4 467.0 10.5DEET 171.0 470.0 2.7Diazepam 8.4 8.4 1.0Diuron 133.0 29.9 0.2Erythromycin 65.7 63.4 1.0Isoproturon 1.8 0.6 0.4Ketorolac 4.2 >1Lincomycin 3.8 >1Meprobamate 146.0 86.0 0.6Monensin 4.1 <1Pentoxifylline 5.6 2.1 0.4Prometon 24.5 4.6 0.2Propazine 1.4 >1Sulfadiazine 20.5 90.5 4.4Sulfamethazine 14.9 115.0 7.7Sulfamethoxazole 61.1 106.0 1.7Sulfathiazole 9.1 >1Trimethoprim 137.0 711.0 5.2


Conclusion

● LC/MS/MS: ideal technology for quantification and conformation of e.g. pesticides and contaminates in water samples

● MRM triggered Enhanced Product Ion scan of QTRAP®

systems with higher degree of confirmation by mass spectral library search

● API 3200™ / 3200 QTRAP® LC/MS/MS systems give sufficient sensitivity to analyze food at 10 ppb levels

● QTRAP 5500™ LC/MS/MS system gives higher sensitivity (allows direct injections of water samples at 0.1 μg/L and dilution of extracts to reduce matrix effects) in ultra fast LC methods up to 1000 MRMs


Trademarks/Licensing

For research use only. Not for use in diagnostic procedures.

AB (Design) and Applied Biosystems are registered trademarks of Applied Biosystems or its subsidiaries in the US and/or certain other countries.

AcQurate, DiscoveryQuant, eQ, iMethod, LightSight, Linear Accelerator, MarkerView, MIDAS, MRMPilot, MultiQuant, ProteinPilot, Scheduled MRM, Triple Quad, and Turbo V are trademarks and Analyst, Cliquid, QJet, QTRAP, and Qurved LINAC are registered trademarks of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applied Biosystems and MDS Inc. All other trademarks are the sole property of their respective owners.

© 2008 Applied Biosystems and MDS Inc. Joint Owners. All rights reserved.

Different ways of enhancing productivity in trace analysis of

Documents

Transcript of Different ways of enhancing productivity in trace analysis of