In Silico Methods in Prediction of Drug Metabolism, Mass Fragmentation, and Chromatographic Behavior
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Hjelt Institute Department of Forensic Medicine University of Helsinki Finland IN SILICO METHODS IN PREDICTION OF DRUG METABOLISM, MASS FRAGMENTATION, AND CHROMATOGRAPHIC BEHAVIOR APPLICATION TO TOXICOLOGICAL DRUG SCREENING BY LIQUID CHROMATOGRAPHY/TIME-OF-FLIGHT MASS SPECTROMETRY Elli Tyrkkö ACADEMIC DISSERTATION To be publicly discussed, with the permission of the Medical Faculty of the University of Helsinki, in the auditorium of Department of Forensic Medicine on April 25th 2014, at 12 noon. Helsinki 2014 SUPERVISORS Professor Ilkka Ojanperä Hjelt Institute Department of Forensic Medicine University of Helsinki, Finland Dr. Anna Pelander Hjelt Institute Department of Forensic Medicine University of Helsinki, Finland REVIEWERS Docent Tuulia Hyötyläinen VTT, Technical Research Centre of Finland Helsinki, Finland Docent Ari Tolonen Admescope Oulu, Finland OPPONENT Dr. Frank T. Peters Forensic and Clinical Toxicology Institute of Forensic Medicine University of Jena, Germany ISBN 978-952-10-9574-0 (paperback) ISBN 978-952-10-9575-7 (PDF) http://ethesis.helsinki.fi Unigrafia Helsinki 2014 Any fool can know. The point is to understand. Albert Einstein CONTENTS CONTENTS ............................................................................................................................4 ABBREVIATIONS ..................................................................................................................6 LIST OF ORIGINAL PUBLICATIONS ................................................................................. 7 ABSTRACT ............................................................................................................................ 8 1 INTRODUCTION ....................................................................................................... 10 2 REVIEW OF THE LITERATURE ............................................................................. 12 2.1 Drug metabolism .............................................................................................. 12 2.1.1 Conventional drug metabolism studies ....................................................... 12 2.1.2 Drug metabolism in silico............................................................................. 13 2.2 Software tools in compound identification .................................................... 16 2.2.1 Mass spectral data processing ...................................................................... 16 2.2.2 Liquid chromatographic retention prediction ............................................ 19 2.3 Accurate mass-based mass spectrometry ....................................................... 19 2.3.1 Accurate mass-based toxicological drug screening .................................... 21 2.3.2 Compound identification without primary reference standards .............. 22 3 AIMS OF THE STUDY .............................................................................................. 24 4 MATERIALS AND METHODS .................................................................................25 4.1 Materials ...........................................................................................................25 4.1.1 Chemicals and reagents ................................................................................25 4.1.2 Urine samples ................................................................................................25 4.2 Sample preparation ..........................................................................................25 4.2.1 Urine samples ................................................................................................25 4.2.2 In vitro incubations ......................................................................................25 4.3 Liquid chromatography/mass spectrometry ..................................................25 4.4 Software ........................................................................................................... 26 4.4.1 Data analysis................................................................................................. 26 4.4.2 Metabolism ................................................................................................... 26 4.4.3 Mass fragmentation ...................................................................................... 27 4.4.4 Chromatographic retention .......................................................................... 27 5 RESULTS AND DISCUSSION ................................................................................. 28 5.1 Metabolite prediction ...................................................................................... 28 5.1.1 Quetiapine metabolism................................................................................ 28 5.1.2 Designer drug metabolism .......................................................................... 29 5.2 Mass fragmentation in silico .......................................................................... 32 5.2.1 Differentiation of structural isomers .......................................................... 32 5.2.2 Metabolite structure identification ............................................................. 34 5.3 Liquid chromatographic retention prediction................................................35 5.4 Software tools applied to accurate mass data................................................ 36 5.5 Preliminary compound identification ............................................................. 37 6 GENERAL DISCUSSION .......................................................................................... 40 7 CONCLUSIONS ......................................................................................................... 46 ACKNOWLEDGEMENTS ...................................................................................................47 REFERENCES ..................................................................................................................... 49 ABBREVIATIONS 2C-H 2,5-dimethoxyphenethylamine 2-DPMP 2-desoxypipradrol 3,4-DMMC 3,4-dimethylmethcathinone bbCID broadband collision-induced dissociation CNS central nervous system CYP cytochrome P450 DMPEA 3,4-dimethoxyphenethylamine EI electron impact FWHM full width half maximum FT-ICR/MS Fourier-transform ion-cyclotron resonance mass spectrometry GC gas chromatography HHMA 3,4-dihydroxymethamphetamine HLM(s) human liver microsome(s) HMA 4-hydroxy-3-methoxyamphetamine HRMS high-resolution mass spectrometry ISCID in-source collision-induced dissociation LC liquid chromatography M(1-12) metabolite (numbering 1-12) [M+H]+ protonated molecule mDa millidalton MPA methiopropamine MS mass spectrometry MS/MS tandem mass spectrometry m/z mass to charge ratio NMR nuclear magnetic resonance NPS(s) new psychoactive substance(s) ppm parts per million PRS(s) primary reference standard(s) -PVP -pyrrolidinovalerophenone Q quadrupole QSAR quantitative structure-activity relationship QSRR quantitative structure-retention relationship QTOFMS quadrupole-time-of-flight mass spectrometry QTP quetiapine RP resolving power RS resolution tR retention time TOFMS time-of-flight mass spectrometry UHPLC ultra-high performance liquid chromatography 6 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following articles, which are referred to in the text by Roman numerals I-IV: I Pelander A, Tyrkkö E, Ojanperä I. In silico methods for predicting metabolism and mass fragmentation applied to quetiapine in liquid chromatography/time-of-flight mass spectrometry urine drug screening. Rapid Commun Mass Spectrom 2009; 23: 506-514. II Tyrkkö E, Pelander A, Ojanperä I. Differentiation of structural isomers in a target drug database by LC/Q-TOFMS using fragmentation prediction. Drug Test Analysis 2010; 2: 259-270. III Tyrkkö E, Pelander A, Ojanperä I. Prediction of liquid chromatographic retention for differentiation of structural isomers. Anal Chim Acta 2012; 720: 142-148. IV Tyrkkö E, Pelander A, Ketola RA, Ojanperä I. In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time-of-flight mass spectrometry. Anal Bioanal Chem 2013; 405: 6697-6709. The original publications have been reproduced with the permission of the copyright holders. 7 ABSTRACT Analysis of drugs in forensic and clinical toxicology has conventionally relied on the use of primary reference standards (PRSs). However, the availability of PRSs for novel pharmaceuticals, new psychoactive substances (NPSs), and their metabolites is often limited. Full metabolite data on new pharmaceutical drugs might be unpublished, and in the case of emerging NPSs, the metabolism is often unknown. Knowledge of the metabolism of these substances is important not only for toxicological risk assessment, but also in terms of analytical method development and forensic or clinical interpretation. Mass spectrometry (MS) techniques with accurate mass measurement capability allow determination of the compound’s elemental composition, which facilitates structural elucidation. Computer systems, i.e. simulation in silico, are available to speed up and assist with the interpretation of analytical data. In the present thesis, current in silico systems were evaluated for their usefulness within accurate mass-based toxicological drug screening. Different software tools were employed to predict drug metabolism, mass fragmentation and chromatographic retention. The aim was to produce supportive information for tentative compound identification