Maurice Wilde 1,2 and Volker Auwärter 1 1 Forensic Toxicology, Institute of Forensic Medicine, Medical Center – University of Freiburg, Freiburg, Germany 2 Hermann-Staudinger-Graduate School, University of Freiburg, Freiburg, Germany Institute of Forensic Medicine Forensic Toxicology IRM Analytical challenges in the forensic toxicological analysis of novel synthetic opioids from the class of ‘U-drugs’ In total, nine novel synthetic opioids from the class of so-called ‘U-drugs’ (developed by Upjohn), another class of new opioids besides fentanyl analogues, have been reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) until the end of 2018. The unambiguous identification of these compounds or their metabolites is often hampered by structural similarities (e.g. isomeric metabolites). The aim of this study was to investigate the phase I in vitro metabolism of eight compounds using pooled human liver microsomes (pHLM) and to identify isomeric and specific metabolites. Structural isomeric compounds (parent compounds) METHODS reaction mix (buffer…) pHLM Incubation 1 h 37°C Protein precipitation ACN Centrifugation 10 min 15.500 x g Extract evaporation & Concentrating step LC solvent 4x Conc. N 2 Substance acquisition (online monitoring) and analyses (structure verification) LC-qToF-MS LC parameters C18 column Mobile phase: A H 2 O (1% ACN, 0.1% HCOOH, 2 mM NH 4 + HCOO - ) B ACN (0.1% HCOOH, 2 mM NH 4 + HCOO - ) Gradient elution MS parameters MS full scan/bbCID scan; scanrate: 1 Hz, CE: 6/30 (±6) eV Calibration in HPC mode (external/internal) Data acquisition: HyStar ® (Bruker) Data processing: DataAnalysis ® (Bruker) 0 1 2 3 4 5 0 20 40 60 80 100 0 5 10 15 20 25 30 Flow rate [ml/min] Mob. Phase B [%] Time [min] Bruker Daltonik Elute UHPLC system impact II qToF instrument AIMS RESULTS CONCLUSION Sufficient chromatographic separation is essential for valid identification of ‘U-drugs’. Furthermore, the extensive metabolism of these compounds may cause interferences by isomers which need to be considered when interpreting analytical findings, especially when analysing biological samples. Maurice Wilde Medical Center – University of Freiburg Institute of Forensic Medicine Albertstraße 9 79104 Freiburg, Germany [email protected] [1] European database on new drugs (EDND) provided by the EMCDDA https://ednd.emcdda.europa.eu/html.cfm/i ndex7246EN.html Literature Contact Cl Cl N H O N Cl Cl N O N Cl N O N Cl Br N O N Cl Cl N O N Br N H O N Cl N O N Cl Cl Cl N O N AH-7921 Isopropyl-U-47700 N-Methyl-U-47931E U-47700 U-47931E (Bromadoline) U-48800 U-49900 U-51754 (Methene-U-47700) Substrates In vitro phase I metabolism studies using pooled human liver microsomes (pHLM) N O O O N Cl Cl N O N 3,4-Methylendioxy-U-47700 U-50488 Six of the investigated ‘U-drugs’ form pairs of constitutional isomers showing the same exact masses and partly forming identical fragment ions in HRMS- bbCID analysis. Evaluation of analytical targets for LC-MS/MS screening method of opioids Max. 2.1e6 cps. 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Time, min 0.0 1.0e6 2.0e6 3.0e6 4.0e6 5.0e6 5.9e6 Intensity, cps Agilent 6890 series gas chromatograph 5973 series mass detector 7683 B series injector Bruker Daltonik Elute UHPLC system impact II qToF instrument Bruker Physik Avance III HD 400 MHz Sampler: BASC 120 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Abundance 100 200 300 360 0 m/z 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Time [min] 0 1 2 3 4 5 6 x10 Intens. GC-MS spectra library Compounds – used for pHLM assay to investigate (in vitro) phase I metabolites Compounds – reported to the EMCDDA until the end of 2018 While U-49900 and Isopropyl- U-47700 only showed few identical fragment ions, AH-7921 and U-47700 exhibited a very similar spectrum of fragment ions, and were distinguishable only by low abundant fragment ions. Furthermore some of the ‘U-drugs’ are regio-isomers, e.g. U-48800 and U-51754, differing in the substitution pattern of the two chlorine substituents (2,4- vs. 3,4-position), which are not distinguishable by the MS- fragment ions (Fig. 2). Cl Cl N O N U-47700 Fig. 1: Fragmentation reactions commonly observed for the class of ‘U-drugs’, examplified for U-47700. 312 284 232 204 Cl Cl N O + Cl Cl N O + Cl Cl NH 2 O + Cl Cl NH 2 O + HN + N + 2 HN + N + 110 154 140 126 173 190 81 Cl Cl O + + Cl Cl NH 3 O + Identical fragment ions Unique fragment ions Unique fragment ions Distinguishable by MS fragmentation Cl Cl N O N Isopropyl-U-47700 Cl Cl N O N U-49900 Molecular formula: C18H26Cl2N2O Molecular formula: C16H22Cl2N2O Cl Cl N H O N AH-7921 Cl Cl N O N U-47700 + + 81 95 Unique fragment ions Unique fragment ions Identical fragment ions 190 Cl Cl O + Cl Cl NH 3 O + 173 204 Cl Cl NH 2 O + Cl Cl NH O + 202/ 204 Cl Cl N O + Cl Cl NH O + 284 284 Distinguishable by MS fragmentation, but loss of sensitivity Molecular formula: C17H24Cl2N2O Cl N O N Cl Cl N O N Cl U-51754 (Methene-U-47700) U-48800 NOT distinguishable by MS fragmentation Identical fragment ions Cl N O + Cl + 81 + 81 Cl N O + Cl Cl NH 2 O + Cl Cl NH 2 O + Cl Cl O + Cl Cl O + Cl 2 HN + 2 HN + 112 112 218 298 158 298 158 218 Fig. 2: Pairs of isomeric compounds. Unique fragment ions for each compound are shown within the green box; the fragment ions in the yellow box are unique by monoisotopic mass, but analytical interference with a heavier chlorine- isotopolgue occurs; fragment ions highlighted by the red boxes are identical by constitution and/or by exact mass. In vitro phase I metabolites – complexity of analytical findings driven by ‘second level‘ isomerism More than a hundred in vitro phase I metabolites and potential degradation products were identified for the eight investigated compounds. Besides N- dealkylation, several hydroxylated metabolites either of the parent compound or the N- dealkylated metabolites were detected. Hydrolysis of the amide function seems to play a minor role for this The compounds showed extensive metabolism in the pHLM assay. The main phase I metabolic reaction observed was N-dealkylation resulting in several isomeric metabolites (Fig. 3). detected for most of the parent compounds or their corresponding N-dealkylated metabolites. Flipbook: HRMS (fullscan/bbCID) spectra of the investigated parent compounds and their 5 most abundant metabolites (and suggestions of the structutal formulae) identified for each of the investigated compounds. Isomers Isomers Isomers Isomers Cl N 2 O N 1 Cl Cl N 2 O N 1 Cl Cl N 2 O 1 NH Cl Cl N 2 O 1 NH Cl Cl N 2 O 1 NH 2 Cl Cl N 2 O 1 NH 2 Cl Br N 2 O N 1 Br 2 NH O N 1 Br N 2 O 1 NH N-Methyl- U-47931E U-47931E (Bromadoline) N-Demethyl-N-Methyl- U-47931E Identical metabolites Br 2 NH O 1 NH N-Demethyl-U-47931E U-48800 U-51754 (Methene- U-47700) N-Demethyl-U-48800 N-Demethyl-U-51754 N,N-Didemethyl-U-48800 N,N-Didemethyl-U-51754 N-dealkylation N-dealkylation N-dealkylation N-dealkylation N-dealkylation Cl Cl N 2 O N 1 Isopropyl- U-47700 N-dealkylation Cl Cl N 2 O 1 NH 2 N,N-Didemethyl-isopropyl- U-47700 Cl Cl N 2 O N 1 U-49900 N-Demethyl-U-49900 N-dealkylation Cl Cl N 2 O HN 1 Isomers Cl Cl N 2 O 1 NH Cl Cl 2 NH O 1 NH N-Demethyl-U-47700 N-Demethyl-AH-7921 Cl Cl N 2 O N 1 U-47700 Cl Cl 2 NH O N 1 AH-7921 N-dealkylation N-dealkylation Fig. 3: Map of isomeric in vitro phase I metabolites for the class of ‘U-drugs’ formed by the most predominant metabolic reaction (N-dealkylation, possibly at N 1 position), and the investigated parent compounds (bold). Yellow boxes frame pairs of isomers, whereas red boxes highlight the formation of identical metabolites from different parent compounds. Marked (*) metabolites may show an additional isomer formed by dealkylation, which is not depicted here (see spectra flipbook). Isomers Cl Cl 2 NH O 1 NH 2 N,N-Didemethyl-AH-7921 N-dealkylation Identical metabolites Cl Cl N 2 O 1 NH 2 N,N-Dideethyl-U-49900 = N,N-Didemethyl-U-47700 N-dealkylation * * * * * * * N-dealkylation class of opioids considering the in vitro data. Dehalo- genation was detected only for the bromine-substituted analoguess U-47931E & N- methyl-U-47931E, though the debrominated meta- bolites showed relatively low abundances. Moreover, further oxidation products such as ketones, diketones, N-oxides or carboxylic acids – possibly formed by ring opening – were also 0 1 2 3 4 5 5 x10 50 100 150 200 250 300 350 m/z Intens. Meas. m/z Ion Formula m/z err [ppm] mSigma 81.0696 C6H9 81.0699 3.7 1.1 126.1275 C8H16N 126.1277 1.6 10.5 172.9551 C7H3Cl2O 172.9555 2.7 8.3 189.9815 C7H6Cl2NO 189.9821 2.9 4.8 232.0284 C10H12Cl2NO 232.0290 2.6 13.3 270.0442 C13H14Cl2NO 270.0447 1.9 5.8 312.0911 C16H20Cl2NO 312.0916 1.9 7.5 357.1492 C18H27Cl2N2O 357.1495 0.8 113.3 * e-poster version: Please find spectra flipchart in print version or download from our homepage via QR-code