Using LC-MS/MS and Advanced Software Tools to Screen for Unknown and Non-targeted PPCP in Environmental Samples Stacy Tremintin and André Schreiber AB SCIEX
Outline Pharmaceuticals and Personal Care Products (PPCP), Endocrine Disrupting Compounds (EDC), Pesticides Screening for PPCP in water samples Comprehensive screening using Multi-MRM methods Direct injection of water with LOD < 10ppt Hybrid triple quadrupole linear ion trap systems (QTRAP ) Compound Identification MRM ratio MS/MS library searching Multi-Target Screening (MTS) and Quantitation General Unknown Screening (GUS) Non-targeted peak finding algorithm Statistical data analysis (Principal Components Analysis PCA) Automatic data reporting 2 2010 AB SCIEX
LC-MS/MS analysis of EDC and PPCP Screening for hundreds or possibly thousands of compounds covering a wide range of chemical properties in environmental samples (water, soil, biota ) Minimum sample cleanup without time consuming derivatization and generic LC setup LC-MS/MS ideal for analysis of medium polar, polar and ionic compounds in trace levels Positive and negative ionization with different ion sources (ESI, APCI, APPI) Quantitation and identification at ng/l (ppt) levels Screening for unknown environmental pollutants 3 2010 AB SCIEX
Screening strategies using LC-MS/MS Screening using full scan (high resolution) spectra lacks in selectivity and sensitivity causing a high risk of false positive and false negative results Fragment ions are needed for identification 2 Multiple Reaction Monitoring transitions per compound MRM ratio for identification [1] Scheduled MRM for extended screening [3-4] Information Dependent Acquisition (IDA) of MS/MS Multi Target Screening: MRM to trigger MS/MS [2] Unknown Screening: Full scan MS to trigger MS/MS [5] [1] Christopher Borton et al.: at ASMS conference (2006) Seattle, WA [2] Jim Krol et al.: at AOAC annual meeting (2007) Anaheim, CA [3] André Schreiber et al.: at ASMS conference (2008) Denver, CO [4] Rolf Kern et al.: at Pittcon (2009) Chicago, IL [5] André Schreiber et al.: at LC/MS/MS workshop (2010) Barcelona, Spain 4 2010 AB SCIEX
Experimental Sample preparation Direct injection of drinking water samples SPE cleanup of waste water and sea water HPLC separation Shimadzu Prominence UFLC XR system Different RP columns with fast gradients of water/methanol and volatile buffer, such as (ammonium acetate, formate, formic acid) MS/MS detection New QTRAP 5500 LC-MS/MS system with Turbo V source and Electrospray Ionization (ESI) probe MRM with Scheduled MRM algorithm for targeted quantitation Enhanced MS (EMS) for non-target screening Enhanced Product Ion (EPI) CE=35 with CES=15V for compound identification using MS/MS library searching 5 2010 AB SCIEX
New Technology in the 5500 QTRAP Systems Curtain Plate Orifice QJet Q0 Q3L Q1 Q3/LIT Q2/Q2L TurboV source for highest ionization efficiency, ruggedness and robustness with low carry-over Ceramic interface and QJet 2 ion guide for best ion transmission Space saving design of collision cell with Linear Accelerator fields Faster electronics for fast MRM (2ms dwell time) and polarity switching in 50ms without loss in S/N Linear Accelerator Q3 for fastest QTRAP scanning (20,000Da/s) 6 2010 AB SCIEX
Multi Target Screening and Quantitation 7 2010 AB SCIEX
MRM offers highest Selectivity and Sensitivity Ion production Ion filtering Ion filtering Ion transport Fragmentation 8 2010 AB SCIEX
MRM in Multi-Target Analysis 1.7e6 1.5e6 Many MRM transitions with long cycle time insufficient number of data points 9.1e4 8.0e4 3.1 1.0e6 5.0e5 0.58 6.0e4 4.0e4 2.0e4 cycle time 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 Time, min 2.9 3.0 3.1 3.2 3 Time min cycle time 1.7e6 1.5e6 1.0e6 Many MRM transitions with short dwell time for better qualitative data 1.11e5 1.00e5 8.00e4 6.00e4 3.1 cycle time 5.0e5 0.58 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 Time, min cycle time 4.00e4 2.00e4 0 2.9 3.0 3.1 3.2 3. Time, min 9 2010 AB SCIEX
The Key for Multi-Target Analysis Smart Software 1.7e6 1.5e6 Scheduled MRM Algorithm 1.0e6 5.0e5 0.58 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 Time, min Adjusts detection windows automatically depending on retention time Optimizes dwell times for each analyte and cycle time Allows detecting many more MRM transitions Allows using UHPLC 10 2010 AB SCIEX
Maintaining Quantitative Accuracy Maximizing Effective Duty Cycle Poor peak sampling makes robust peak integration very difficult (left) Proper cycle time provides good peak sampling and increases assay reproducibility 300 Normal MRMs 300 Scheduled MRMs 12% CV 8.4% CV 7.8% CV 5.3% CV 11 2010 AB SCIEX
LC-MS/MS Screening Direct Injection of Water XIC of +MRM (158 pairs): 415.1/178.1 amu Expected RT: 5.2 ID: Diltiazem 1 from Samp... Max. 8.2e6 cps 8.0e6 7.0e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e6 1μg/L (100μL injected) 5.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 Time, min 158 Scheduled MRM transitions to screen for PPCP (EPA 1694) Injection of 100µL into QTRAP 5500 LC/MS/MS system Phenomenex Luna C18 HST (100x3mm 2.5µm) and gradient of water/acetonitrile with 0.1% formic acid 12 2010 AB SCIEX
LC-MS/MS Screening Farmland Drain Sample XIC of +MRM (158 pairs): 216.1/96.1 amu Expected RT: 6.9 ID: Atrazine 2 from Sample 37 (Farmland Drai... Max. 1.1e5 cps. 1.13e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 LOD of PPCP <10ppt 6.9 70ppt Atrazine 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e4 5000 2ppt Sulfadimethoxazine 6ppt Metolachlor 2ppt Propazine 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 13 Time, min 2010 AB SCIEX
LC-MS/MS Screening Urban Water Sample XIC of +MRM (158 pairs): 216.1/96.1 amu Expected RT: 6.9 ID: Atrazine 2 from Sample 36 (Urban) of 200... Max. 1.9e4 cps. 1.15e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 200ppt Cotinine 100ppt Metformin LOD of PPCP <10ppt 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 420ppt Caffeine 10ppt Atrazine 5ppt Metolachlor 1ppt 2ppt Simazine Thiabendazole 20ppt Diuron 6.9 1.50e4 1.00e4 5000 6.6 6.9 7.2 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 14 Time, min 2010 AB SCIEX
Multi-Target Screening with MS/MS Library Searching 15 2010 AB SCIEX
An Alternative to MRM Ratios Full Scan MS/MS Q3 of a QTRAP System can be used as quadrupole or as linear ion trap Ion trapping Ion production Ion filtering Ion filtering Ion transport Fragmentation 16 2010 AB SCIEX
Trapping and Scanning in a Linear Ion Trap (Q3) (1) Trapping Exit lens (2) Scanning Entrance lens 17 2010 AB SCIEX
Multi-Target Screening with MS/MS Library Search Scheduled MRM Algorithm intensity threshold Threshold EPI 297 159 69 173 81 141 109 115 176 201 255 Library Search 18 2010 AB SCIEX
LC-MS/MS Screening Farmland Drain Sample XIC of +MRM (79 pairs): Exp 1, 130.1/71.1 amu Expected RT: 0.6 ID: Metformin from S... Max. 9.2e4 cps 9.2e4 8.0e4 6.0e4 4.0e4 0.5 Scheduled MRM EPI 2.0e4 0.8 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 Time, min +EPI (311.30) Charge (+0) CE... Max. 1.2e5 cps. +EPI (216.10) Charge (+0) CE... Max. 1.1e6 cps 1.16e5 1.00e5 5.00e4 108.0 156.0 92.0 Sulfadimethoxazine FIT 83.7% 292.9 311.0 0 100 150 200 250 300 350 400 m/z, Da +EPI (230.20) Charge (+0) CE... Max. 1.9e5 cps. In te n s ity, c p s 1.15e6 1.00e6 5.00e5 67.9 103.7 146.0 78.6 174.1 216.1 Atrazine FIT 98.3% 0 100 150 200 250 300 350 400 m/z, Da +EPI (284.20) Charge (+0) CE... Max. 7.9e4 cps 1.9e5 1.5e5 1.0e5 Propazine FIT = 98.8% 230.2 In te n s ity, c p s 5.0e4 146.0 2.0e4 79.0 195.1 212.2 73.0 133.9 146.3 184.0 266.5 100 150 200 250 300 350 400 100 150 200 250 300 350 400 19 m/z, Da m/z, Da 2010 AB SCIEX 7.9e4 6.0e4 4.0e4 Metolachlor FIT=91.8% 176.2 252.1 284.2
LC-MS/MS Screening Urban Water Sample XIC of +MRM (79 pairs): Exp 1, 216.1/174.1 amu Expected RT: 6.9 ID: Atrazine from Sample 15 (Urban)... Max. 3174.0 cps. 5.7e4 5.0e4 4.0e4 3.0e4 Scheduled MRM EPI 2.0e4 1.0e4 7.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 Time, min +EPI (130.10) Cha... Max. 2.0e5 cps. +EPI (177.20) Cha... Max. 3.1e5 cps. +EPI (202.10) Cha... Max. 1.4e5 cps. 130.1 2.0e5 1.5e5 1.0e5 Metformin Fit 57.8% 71.0? Intensity, cps 5.0e4 88.8 112.1 60.8 102.0 100 150 200 m/z, Da +EPI (202.10) Cha... Max. 5.7e4 cps. 3.0e5 Cotinine Fit 76.8% 2.5e5 2.0e5 1.5e5 8 1.0e5 5.0e4 72.8 118.1 97.9 177.1 149.3 159.0 In ten s ity, cp s 100 150 200 m/z, Da +EPI (216.10) Cha... Max. 7.4e4 cps. 1.4e5 1.2e5 1.0e5 202.1 Thiabendazole Fit 94.2% 8.0e4 184.1 6.0e4 175.0 131.0 4.0e4 139.1 2.0e4 70.9 90.8 156.0 100 150 200 250 m/z, Da +EPI (284.20) Cha... Max. 8.2e4 cps. 5.7e4 5.0e4 4.0e4 Simazine Fit 83.8% 202.1 3.0e4 2.0e4 114.8 130.1 1.0e4 73.8 105.0 127.9 167.2 Intensity, cps 7.4e4 6.0e4 4.0e4 2.0e4 76.9 96.1 Atrazine Fit 94.9% 100 150 200 250 100 150 200 250 300 100 150 200 250 300 350 20 m/z, Da m/z, Da m/z, Da 2010 AB SCIEX 216.2 17 133.9 188.2 129.1 154.0 198.2 In ten sity, cp s 8.0e4 6.0e4 4.0e4 Metolachlor Fit 87.9% 176.0 252.0 284.3 2.0e4 134.0 105.0 163.1 266.2
LC-MS/MS Screening MRM vs. EPI Identification XIC of +MRM (158 pairs): 130.1... Max. 1.8e5 cps. +EPI (130.10) Charge (+0) CE... Max. 1.2e6 cps 0.6 1.8e5 1.5e5 1.0e5 5.0e4 Standard 1μg/L MRM ratio 83 2 4 6 8 10 Time, min XIC of +MRM (158 pairs): 130.1... Max. 2.7e4 cps. Intensity, cps 88.9 1.20e6 1.00e6 8.00e5 70.9 6.00e5 4.00e5 68.9 83.9 2.00e5 75.8 Library spectrum (Metformin) 112.0 130.2 0 80 100 120 140 160 180 200 m/z, Da +EPI (130) CE (20): Exp 1,... Max. 1.1e6 cps 5.0e4 4.0e4 3.0e4 0.7 2.0e4 1.0e4 Water sample 1μg/L MRM ratio 82? Intensity, cps 71.0 1.14e6 1.00e6 8.00e5 6.00e5 68.0 4.00e5 2.00e5 Unknown spectrum 130.2 85.1 113.0 66.4 132.0 0 2 4 6 8 10 80 100 120 140 160 180 200 21 Time, min m/z, Da 2010 AB SCIEX
General Unknown Screening and Quantitation 22 2010 AB SCIEX
0. 8 1. 8 2. 2 2. 9 3. 9 4. 1 4. 7 5. 2 5. 8 6. 2 6. 6 7. 2 8. 2 8. 5 9. 2 9. 7 9. 9 1 0. 5 1 1. 1 1 2. 5 1 3. 1 1 3. 9 1 4. 4 General Unknown Screening using a Non-Target Peak Finding Algorithm in Cliquid Software EMS survey 0.5 7. 4 intensity threshold, DBS EPI Threshold 297 159 69 173 81 141 109 115 176 201 255 Library Search 23 2010 AB SCIEX
Comparison of chromatograms and mass spectra 2 unknown samples Chromatograms (TIC) Mass spectra TIC of +MRM (53 pairs): Exp 1, from Sample 9 (EDC River 2 Influent) of Samples POS mode_1epi.wiff (Turbo Spray) 8.17 5.0e5 4.5e5 +EMS: Max. Exp 5.2e5 1, 10 cps. to 19.238 min from Sample 1 (GUS Testmix in urine extract (150ng/mL)) of Data Forensic samples ST Boniface_08... 195.1 8.0e5 149.2 7.0e5 Max. 8.1e5 cps. 4.0e5 6.0e5 114.1 3.5e5 153.1 3.0e5 6.24 6.42 5.0e5 7.62 304.2 432.2 2.5e5 4.0e5 138.1 374.3 211.2 416.3 470.2 2.0e5 3.0e5 171.1 235.2 287.2 309.1 120.1 335.2 1.5e5 2.0e5 156.1 177.1 251.2 346.3 1.74 5.46 123.1 227.2 301.2 311.2 429.2 1.0e5 16 379.2 448.2 7.14 144.1 185.2 193.2 273.1 355.2 365.2 475.1 8.39 5.0e4 10.58 1.0e5 130.1 298.2 315.2 413.3 242.3 385.2 449.3 445.2 267.2 467.2 494.4 540.2 563.3 573.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 10019 120 20140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 Time, min m/z, amu TIC of +MRM (53 pairs): Exp 1, from Sample 10 (EDC River 2 Effluent) of Samples POS mode_1epi.wiff (Turbo Spray) +EMS: Max. Exp 9.9e5 1, 8.024 cps. to 29.929 min from Sample 1 (GUS Testmix in urine extract (150ng/mL)) of Data Forensic samples ST Boniface_08... Max. 1.6e6 cps. 9.9e5 8.18 1.6e6 149.0 9.0e5 1.4e6 8.0e5 1.2e6 7.0e5 6.0e5 5.0e5 5.45 7.64 1.0e6 8.0e5 4.0e5 3.0e5 2.0e5 1.0e5 1.73 5.27 6.25 4.58 17 1.90 6.45 2.36 4.13 7.17 6.0e5 4.0e5 2.0e5 332.4 360.4 432.2 153.1 305.2 195.1 287.2 374.3 416.3 470.2 124.1 330.2 338.4 363.2 167.2 211.2 251.2284.3 311.2 391.3 429.2 201.1 155.2 369.2 427.3 475.1 105.1 357.2 423.2 485.3 522.5 536.2 583.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 10019 120 20140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 Time, min m/z, amu 24 2010 AB SCIEX
PCA Explanation 1 y 2 dimensional plot x 25 2010 AB SCIEX
PCA Explanation 2 z y x 3 dimensional plot 26 2010 AB SCIEX
PCA Explanation 3 PC1 z (0, 0, 0) PC1 Score y x PC1 PC1 = 0.1 x + 0.3 y +0.95 z 27 2010 AB SCIEX
Analysis of Street Cocaine Samples Procaine Sample 2 Cocaine Sample 1 Benzoylecgonine Sample 3 Diltiazem Principal Components Analysis (PCA) scores and loadings plot 28 2010 AB SCIEX
Screening for Unexpected PPCP Samples from wilderness areas with extensive recreational use DEET Drinking water produced from river bank filtrate during flood Increasing urbanity of samples 2.5µg/L 3.6µg/L Distribution of the insect repellent DEET 2.6µg/L 2.4µg/L 3.3µg/L 29 2010 AB SCIEX
Quantitation of Benzoylecgonine (Metabolite of Cocaine) in 70 Water Samples Berlin BE Concentration (µg/ 0.200 0.175 0.150 0.125 0.100 75 50 25 Std. 0.1µg/L Zermatt Creeks Drinking water Canoe Lake (Algonquin) Lake Zürich Lakes Dresden Philadelphia Rio de Janeiro Santa Fe Rome Vienna Toronto 00 Sample Rivers 30 2010 AB SCIEX
Cliquid General Unknown Screening 31 2010 AB SCIEX
Summary LC-MS/MS is a powerful tool for the analysis of Pharmaceuticals and Personal Care Products in environmental samples The combination of high resolution LC separation and high sensitivity MS/MS is the most powerful tool to screen and quantify targeted compounds. Direct injection of water samples MS/MS fragmentation required to identify compounds MRM transitions to quantify targeted contaminants MRM ratios or MRM to trigger MS/MS spectra (EPI) for compound identification with high confidence GUS coupled with library searching and PCA is a powerful research tool for any laboratory 32 2010 AB SCIEX
Trademarks/Licensing For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. 2010 AB SCIEX. 33 2010 AB SCIEX
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