The Use of LC-MS in Systematic Toxicology Analysis

CAT Meeting Phoenix, AZ June 8, 7 Presentation outline Methods used in Systematic Toxicology Analysis The Use of LC-MS in Systematic Toxicology Analysis LC/MS approach ChromaLynx software Chromatogram deconvolution Library LC/MS vs. GC/MS LC/MS vs. UV detection Jan Bohuslavek,, Ph.D. Waters Corporation Summary 7 Waters Corporation 7 Waters Corporation Forensic laboratory: analytical approaches The known (or suspected toxic analyte) Class identified by screening assay Adverse effects to known medication History of misuse of prescription medication or illicit drugs Evidence at scene Analytical approach: Targeted analysis by LC-MS/MS The unknown Typical situation: ER, forensic, crime labs illicit compounds? Misuse of prescribed drugs? r basically anything else! LC-MS and ChromaLynx software for Systematic Toxicological Analysis Analytical approach: Screen by LC-MS 7 Waters Corporation 3 7 Waters Corporation

Techniques for Toxicology Screening Different Approaches LC-MS & ChromaLynx Approach Traditional screening methods Immunoassays Screening & quantitation Cross reactivity, cost ot available for all drugs LC/UV Identification in complex mixtures UV libraries exist Sensitivity/specificity GC/MS ( gold standard ) Relatively inexpensive Multiple libraries exist Derivatization, laborious ot suitable for ALL compounds Current method combines LC-MS with searchable database based on in-source CID Benefits of using LC large number of samples can be analysed by LC o derivatization Benefits of multiple spectral identification criteria Data matched to a searchable and appendable library 7 Waters Corporation 5 7 Waters Corporation 6 Toxicology Screening & LC/MS Information available from one LC/MS Analysis Mass Spectra Database & Library Building: LC/MS Experimental Conditions HPLC PDA MS Retention (1) UV Absorption Information () Parent Ion (3) Fragment Ions (4-15!) Instrumentation A single or triple quadrupole mass spectrometer, LC, autosampler RIVTRIL Clonazepam 7 Clonazepam 13 34 14.58 15.57 15.57 16.1 Clonazepam 15.56 14.56 15.6 16. 8: Diode Array.75e7 4: Scan ES+ 1.5e9 : Scan ES+ 4.43e8 Clonazepam 1: Scan ES- 15.55 7.7e7 14.44 15. 16.43 16.78 46 15. 16. RIVTRIL Clonazepam 98 (15.57) Cm (95:93-91:914) 13 3.41e5 31 nm 5 3 35 4 RIVTRIL Clonazepam 67 (15.59) 13 6 18 14 7 Clonazepam 67 (15.557) 7 6: Scan ES+ 1.66e8 41 16 7 43 73 57 316 354357 1 155 16 5 9 8 88 Clonazepam 67 (15.549) : Scan ES+ 316 1.33e8 314 318 319 357 371 1: Scan ES- 1.36e7 316 78 139 155 168 19 48 86 317 339377 m/z 15 5 3 35 7 Waters Corporation 7 Liquid Chromatography Generic gradient based on water/acetonitrile mobile phase with ammonium formate and formic acid (~ 3 min run).1 mm id - 3.5µm XTerra C 18 MS column @ 35 C with pre-column LC/MS allows collection of multi-spectral data Pos/ mass spectra recorded at different cone voltages ne analyte can be characterized by up to 1 mass spectra plus retention time 7 Waters Corporation 8

LC/MS and In-Source CID: Generation of Fragments LC/MS and In-Source CID: Generation of Fragments & Library Concept Extractor Ions formed in the atmospheric region are sampled through the sample cone. [MH] + +15 Volts 5 o Fragmentation 179 199 Cl H H 356 [M-H] - -15 Volts +3 Volts 1 14 16 18 4 6 8 3 3 34 36 38 MW: 355 CAS# 86-4- CHCl3 (toxml1884) Amodiaquine [ESI+ @ 15V] -3 Volts Sample Cone These ions can be accelerated by applying a voltage gradient between the sample cone and the extractor cone. +45 Volts +6 Volts Exhaustive Structural Information -45 Volts -6 Volts These ions gaining speed will hit residual neutral molecules in that region and will gain internal energy and eventually fragment into specific ions. +75 Volts +9 Volts 5 18 163 177 5 19 8 4 55 83 Cl H H -75 Volts -9 Volts F + 118 136 68 356 1 14 16 18 4 6 8 3 3 34 36 38 MW: 355 CAS# 86-4- CHCl3 (toxml1884) Amodiaquine [ESI+ @ 9V] Total Fragmentation F - 7 Waters Corporation 9 7 Waters Corporation 1 In-Source CID - LC/MS Library: Some Examples In-Source CID - LC/MS Library: Some Examples TIMLL Free Base C 13 H 4 4 3 S MW 316.4 (ominal 316) CAS# 9154-16- S CH 3 ESI+@9V 144 113 17 158 188 13 44 65 85 317 337 ESI+@75V 144 113 188 17 154 17 44 8 61 317 345 ESI+@6V 188 44 144 113 61 13 17 14 9 87 317 ESI+@45V 61 BUTBARBITAL C 1 H 16 16 MW 1.5 (ominal 1) CAS# 77-8-1 H 3 C H H 155 147 1 156 11 15 167 1 155 1 19 171 11 47 147 13 18 111 141 155 11 168 171 199 15 47 74 89 95 155 168 11 ESI-@9V ESI-@75V ESI-@6V ESI-@45V H Results in getting 6 specific m/z values with different fragmentation patterns for each voltage value: m/z @ 317 [M+H] +, 61,44,188,144 &113 H CH 3 CH 3 188 11613144158 14 13 161 ESI+@3V 317 ESI+@15V 317 117 151 188 333 15 5 3 35 m/z 44 61 44 87 317 H 3 C Barbiturates give a good response to ative ESI. The ion @ m/z 155 is generally observed for most of the barbiturates. 1111 18 139 18 169 11 113 155 1 47 57 m/z 1 14 16 18 4 6 8 3 1 155 168 1 183 11 36 ESI-@3V ESI-@15V 7 Waters Corporation 11 7 Waters Corporation 1

In-Source CID - LC/MS Library: Some Examples In-Source CID - LC/MS Library: Some Examples ITRAZEPAM C 15 H 11 3 3 MW 81.7 (ominal 81) CAS# 146--5 H 158 ESI+@9V 36 18 7 179 18119 8 196 35 37 8 ESI+@7V 8 36 176 8 35 37 54 83 ESI+@5V 8 Acepromazine & aceprometazine UV versus MS Same molecular formula Similar retention times Same UV spectra - + Some molecules may give a good response in both positive & ative ESI thus allowing unambiguous identification. 83 ESI-@9V 5 194 166 195 6 3 53 8 81 ESI-@7V 5 53 8 ESI-@5V 8 5 81 m/z 16 18 4 6 8 3 7 Waters Corporation 13 Identical mass spectra @ 15 & 3 V Differences appear around 45 V Differentiation possible The probability that two compounds have the same mass, same fragmentation patterns over 6 different CV s in both positive and ative ion polarities, and elute at the same retention time is very low! 7 Waters Corporation 14 Toxicology Screening & LC/MS Analysis of the Unknown :: Challenges Chromatogram Examination and Peak Detection Do I really see what I should see? Challenges: Complicated chromatograms - Minor peaks - Hidden, co-eluting peaks Identification of the analytes - Correct mass spectrum for each component - Retention time used in library search in addition to spectra 7 Waters Corporation 15 1911 7: Scan ES+ 1.47 5.39e9 11.65 17.6.8 14.63 1.4.11 1 5. 1. 15.. 1911 6: Scan ES+ 1.46 5.59e9.7 17.61 1.41 1.3 11.64 14.6.16.63 1 5. 1. 15.. 4.47e9 17.66 1.4 1.3 11.63 14.6 15.43.6.15.6 1 5. 1. 15.. 1911 4: Scan ES+ 1.44.7e9 15.43 17.65.5 11.68 1.45 1. 14.61.14.61 6.18 5. 1. 15.. 1911 3: Scan ES+ 13.8 1.43 3.13e9 15.4 1.1 17.64.6 14.6.4 1.8.13 5. 1. 15.. 1911 : Scan ES+ 13.7 3.5e9 1.48 1. 15.41.18.59 17.63.3 1.79 5. 1. 15.. 1911 1: Scan ES-.1 4.51e7 6 1..53 15.46.8 1.84 1.53 18.33 1. 5.33 7.6 1.43 13.1.4 5. 1. 15.. 1911 3: Scan ES+ 13.8 7643378.95e9 Area 4 3 11.67 16638715 1.49 51334 peaks? 13.84 46391 14.7 8415 14.6 11587184 11.5 1. 1.5 13. 13.5 14. 14.5 15. At least 5! 1911 3: Scan ES+ 1. 95 4.31e6 11.15 11.61 11.73 11.96 1.73 11.5 11.5 11.75 1. 1.5 1.5 1.75 13. 13.5 13.5 13.75 14. 14.5 14.5 14.75 13.37 13.6 14.19 14.1 14.31 This area represents only 1 min out of the 5 minute chromatogram of one of the many functions! 14.77 7 Waters Corporation 16

Chromatogram Examination and Peak Detection Mass Spectra Extraction ChromaLynx for Toxicological Screening 1911 3: Scan ES+ 1911 3: Scan ES+ 13.8 14.13 317.961 1.49 7777784.9e9 3.3e7 54138579 Area 4 11.67 19631856 13.78 766456 11.5 1. 1.5 13. 13.5 14. 14.5 Apparent peak RT 14.7 min Mixture 4+5 13 161 5 8 117 33 4 93 77 Apparent peak RT 13.84 min Mixture 1++3 51 65 66 1,,3 318 37 14.66 114495 38 343 367 39 4,5 11 8 4 67 95 361 14 177 371 15 5 3 35 4 m/z 3 11 11.5 1. 1.5 13. 13.5 14. 14.5 Peak #5 RT 14.13 318 13 147 161 79 319 5 33 93 Peak #4 RT 14.1 min 37 51 14 177 19 4 84 95 361 386 393 m/z 15 5 3 7 Waters 35 Corporation 17 357 381 39 65 117 161 8 38 175 5 4 316 367 383 Peak #3 RT 13.84 min 11 13 175 Peak # RT 13.7 min 145 177 65 66 63 8 4 36 8 Peak #1 RT 13.66 min 51 1 3 4 5 339 367 383 7 5 31 39 361 371 398 39 Especially developed for screening using Full scan MS spectral libraries Data processing software based on chromatogram deconvolution techniques - Chromatogram examination - Component detection - Candidate proposal 7 Waters Corporation 18 ChromaLynx for Toxicological Screening Chromatogram Examination : How does it work? Virtually, every scan from each function can be examined and extracted ion chromatograms are generated for the most intense ions. 4.47e9 1.3 11.63 13.8 14.6 15.43 1. 15. Automated Mass Spectrum Extraction @ scan 37 for Function 5 17. 17.66 18.71 1911 37 (13.854) 5: Scan ES+ 8 7.54e7 118 1 161 193 179 63 15 5 65 5 66 m/z 13.85 65.144.75e7 1.81 11.69 1.39 16.84 18.1918.619.1 1. 15. 63.83 6.8e8 1. 15. 13.8 1.1e7 11.63 15.9 17.5 17.83 1. 15. 13.8 8 8.87e7 17.19 1. 15. 13.8 146 1.1e7 11.63 17.48 9.76 14.3 18.19 1. 15. 13.8 118.64e7 1.5 11.86 16.31 16.78 18.77 1. 15. 4.47e9 11.63 14.6 15.43 17.66 1.3 1. 15. 7 Waters Corporation 19 ChromaLynx for Toxicological Screening Component Detection and Mass Spectrum Extraction Peaks in extracted ion chromatograms should be within a limit in terms of scan position (retention time) to be considered as a sole peak. Mass spectrum is then extracted. Scan 37 13.8 8.66 8.87e7 13. 14. 15. 13.85 65.144 1.39.75e7 15.6 13. 14. 15. 63.83 6.8e8 13. 14. 15. 13.8 1.1e7 14.73 15.9 13. 14. 15. 13.8 8 8.87e7 13. 14. 15. 13.8 146 1.16 14.3 1.1e7 1.74 14.73 15.3 15.73 13. 14. 15. 13.8 118 1.4.64e7 1.9 14.79 13.44 15.3 15.6 13. 14. 15. 15.43 4.47e9 13.8 14.9 14.6 13. 14. 15. 1 component: comprising ions m/z 8, 65,, 146, 118 7 Waters Corporation

ChromaLynx Library The library is in IST format Contains hundreds of compounds and metabolites Easily appendable by users Contains multiple entries for every compound (different cone voltage and polarity) Each entry contains: Parent mass Fragment masses Retention time Fragment abundance 7 Waters Corporation 1 7 Waters Corporation ChromaLynx Data Examples Analytical Equipment and Instrumentation Waters ZQ (Single Quadrupole MS) or Quattro Micro (MS/MS) Alliance 695 Liquid chromatography system HPLC Separation Method Waters XTerra TM MS C18, 3.5 µm,.1mm id x 15 mm (1 mm for precolumn) Mobile Phase based on Water/Acetonitrile with 5 mm Ammonium Formate MS perating Conditions Function 1: Full Scan - egative ESI from to 65 amu @ 3 Volts Functions to 7: Full Scan - Positive ESI from to 65 amu @ 15 to 9 Volts 7 Waters Corporation 3 7 Waters Corporation 4

Example: : Strychnine poisoning (From AIT Laboratories) A Example of Strychnine Poisoning Unexplained death Vomit was found at the scene The symptoms were indicative of strychnine poisoning A sample of vomit was submitted for screening Liquid-liquid extraction of the sample was performed, analyzed Fentanyl was used as an internal standard 7 Waters Corporation 5 7 Waters Corporation 6 Example: Polyintoxication - Suicide attempt by massive ingestion of various medications A 65 years old woman was brought to an emergency department. Said she had taken different pills from among her regular medications. Urine samples were analyzed. Toxicologists were looking for molecules coming from the regular treatment and unexpected substances. The known medications taken by the patient were: Thanks for your attention Questions? 7 Waters Corporation 7 7 Waters Corporation 8

A Example of Polyintoxication - Suicide attempt by massive ingestion of various medication Results All expected molecules were found except Tramadol Additional molecules were identified as the main cause of the intoxication: Meprobamate - Mepronizine Aceprometazine - Mepronizine Bromazepam 7 Waters Corporation 9 7 Waters Corporation 3 LC/MS vs. GC/MS Evaluation of an LC/MS Method to Screen for Drugs in Postmortem Whole Blood Specimens 15 post-mortem whole blood specimens were analyzed. In many cases, the GC/MS and LC/MS results were comparable (see examples in Table 1). In the majority of cases, the LC/MS method was able to identify more analytes compared to the GC/MS method (see examples in Table ). Kevin Shanks¹*, Tim Dahn¹,, Andrea R. Terrell, Ph.D.¹,, and Jan Bohuslavek,, Ph.D.² ¹AIT Laboratories, Indianapolis, I, ²Waters Corporation, Milford, MA, USA 7 Waters Corporation 7 Waters Corporation 3

Sample umber 47149 47687 48731 4873 414159 414439 414416 Case Studies: Table 1 and GC/MS Results Amitriptyline ortriptyline lanzapine Paroxetine Bupropion Desmethylsertraline Lamotrigine Chlorpromazine LC/MS Results Amitriptyline ortriptyline lanzapine Paroxetine Bupropion Lamotrigine Chlorpromazine Confirmed Results Amitriptyline ortriptyline lanzapine Paroxetine Bupropion Desmethylsertraline Lamotrigine Chlorpromazine orsertraline 7 Waters Corporation 33 Sample umber 47649 4858 41437 47175 4943 41494 473 GC/MS Results egative egative Acetaminophen Promethazine Chlorpheniramine Dextromethorphan egative LC/MS Results Fentanyl Cocaine ordiazepam Acetaminophen Propoxyphene Promethazine ordiazepam Citalopram Desmethylcitalopram Chlorpheniramine Dextromethorphan Alprazolam Amiodarone Desmethylamiodarone Confirmed Results Fentanyl Cocaine ordiazepam Acetaminophen Propoxyphene Promethazine ordiazepam Chlorpheniramine Dextromethorphan Alprazolam 7 Waters Corporation 34 Conclusion LC/MS vs. LC (Bio Rad s REMEDi) The ChromaLynx software library is thorough. The software is very easy to learn and use. The deconvolution process is very effective. The library is easily appendable by the analyst. The LC/MS method identified more compounds than the GC/MS method. In comparison to the GC/MS method, the LC/MS method is more efficient at identifying polar analytes. A comparison study performed at the Clinical Labs of Hawaii 7 Waters Corporation 35 7 Waters Corporation 36

Sample REMEDi LC/MS W11637 W11639 W11641 F5611 F5615 F61817 Study: F618 H4838 45 samples analyzed using both LC/MS approach and REMEDi W1966 W19658 M6835 X4863 X48637 X48639 Results: W6389 S441 7 samples produced ative results using both methods W6395 F1739 W19961 1 samples produced identical positive results H44533 W58183 8 samples showed more confirmed positive hits using LC/MS F36518 S1714 F36517 W5886 M6833 7 Waters Corporation 37 M6944 7 Waters Corporation 38 Samples with comparable results Samples in which LC/MS approach identified more analytes than REMEDi Sample T4743 T31 F3415 T797 T4718 F5616 T4718 H395 T31 H948 REMEDi Venlafaxine Benzoylecgonine Benzoylecgonine Ranitidine xycodone Propoxyphene Codeine Alprazolam LC/MS Venlafaxine Benzoylecgonine Benzoylecgonine Ranitidine xycodone Propoxyphene Codeine Alprazolam Sample H18493 W18614 X48641 W19675 T4743 W45 H741 H86 REMEDi LC/MS Dextromoramide Dextromoramide Dextromoramide Dextromoramide, Benzoylecgonine 7 Waters Corporation 39 7 Waters Corporation 4

ChromaLynx for Toxicological Screening: Summary Acknowledgements From a technical point of view LC/MS constitutes an excellent additional tool for Toxicological Screening The library contains exhaustive structural information for each compound ChromaLynx utilises an efficient data processing which: - Avoids the risk of missing small or co-eluting peaks - Allows to detect small amount of compounds in Full Scan - Includes examination of multiple functions - Includes retention time control T. Dahn AIT Laboratories, Indianapolis, I Dr. C. Wong and C. issen Clinical Laboratories of Hawaii Potentially usable on any Waters-Micromass Quadrupole-Based Instrument 7 Waters Corporation 41 7 Waters Corporation 4 Thanks for your attention Questions? 7 Waters Corporation 43