Different ways of enhancing productivity in trace analysis of pesticides and PPCP in water samples using sensitive LC/MS/MS technology Jens Dahlmann and André Schreiber, Applied Biosystems
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
4 © 2009 Applied Biosystems
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
5 © 2009 Applied Biosystems
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
6 © 2009 Applied Biosystems
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
7 © 2009 Applied Biosystems
Cliquid® Software – MRM Catalogue
Cliquid™ Food & Beverage: > 500 Pesticides
8 © 2009 Applied Biosystems
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
9 © 2009 Applied Biosystems
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
10 © 2009 Applied Biosystems
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
11 © 2009 Applied Biosystems
Screening and Confirmation:
Workflow: MRM survey –MS/MS – Library Search
Collection of MS/MS…(full scan confirmation)
Library Search Results…
Quantification…
12 © 2009 Applied Biosystems
● 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
13 © 2009 Applied Biosystems
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;
14 © 2009 Applied Biosystems
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
TIC of +MRM (150 pairs): 223.2/1... Max. 1.7e5 cps.
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
XIC of +MRM (150 pairs): 305.1/1... Max. 2.9e4 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
2.5e4
2.9e4
Intensity, cps
3.0
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
TIC of +MRM (150 pairs): 223.2/1... Max. 1.7e5 cps.
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
XIC of +MRM (150 pairs): 305.1/1... Max. 2.9e4 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
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
15 © 2009 Applied Biosystems
Endocrine Disrupting Compounds (EDC), Pharmaceuticals and Personal Care Products
(PPCP)
Cliquid™ Food & Beverage: > 500 Pesticides
16 © 2009 Applied Biosystems
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
18 © 2009 Applied Biosystems
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
19 © 2009 Applied Biosystems
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
20 © 2009 Applied Biosystems
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)
21 © 2009 Applied Biosystems
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
22 © 2009 Applied Biosystems
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
23 © 2009 Applied Biosystems
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
25 © 2009 Applied Biosystems
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