Characterization of iso-LSD metabolism using human liver microsomes in comparison to LSD and its applicability as urinary biomarker for LSD consumption.

Urinalysis of lysergic acid diethylamide (LSD) poses a challenge due to its rapid metabolism, resulting in little to no LSD detectable in urine. Instead, its primary metabolite, 2-oxo-3-hydroxy-LSD, is predominantly detected. In this study, we observed several urine profiles with iso-LSD detected together with 2-oxo-3-hydroxy-LSD. Iso-LSD is derived from illicit preparation of LSD as a major contaminant, and it was detected at higher abundance than LSD and 2-oxo-3-hydroxy-LSD in certain urine samples. Therefore, the metabolism of iso-LSD and its potential as a viable urinary biomarker for confirming LSD consumption is of interest. For metabolism studies, LSD and iso-LSD were incubated in human liver microsomes (HLMs) at 0 min, 60 min and 120 min to characterize their metabolites using LC-QTOF-MS. For urinary analysis, 500 µL of urine samples underwent enzymatic hydrolysis and clean-up using supported-liquid extraction (SLE) prior to analysis by LC-QTOF-MS. From HLM incubation study of LSD, the metabolites detected were dihydroxy-LSD, 2-oxo-LSD, N-desmethyl-LSD (nor-LSD) and 2-oxo-3-hydroxy-LSD with LSD levels decreasing significantly throughout all time points, consistent with the existing literatures. For HLM study of iso-LSD, metabolites eluting at retention times after the corresponding metabolites of LSD were detected, with iso-LSD levels showing only a slight decrease throughout all time points, due to a slower metabolism of iso-LSD compared to LSD. These findings corroborate with the urinalysis of 24 authentic urine samples, where iso-LSD with 2-oxo-3-hydroxy-LSD was detected in the absence of LSD. Based on our findings, iso-LSD is commonly detected in urine (18 out of 24 samples) sometimes with traces of possible 2-oxo-3-hydroxy-iso-LSD. The slower metabolism and high detection rate in urine make iso-LSD a viable urinary biomarker for confirming LSD consumption, especially in the absence of LSD and/or 2-oxo-3-hydroxy-LSD.

Qualitative Confirmation of 94 New Psychoactive Substances and Metabolites in Urine Using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry.

Numerous methods and techniques have been published for the identification of new psychoactive substances (NPS) and their metabolites in urine. However, there lacks a holistic approach to analyze different groups of NPS and their metabolites with decision points for reporting their use. In this study, data-dependent acquisition workflow using liquid chromatography--quadrupole time-of-flight mass spectrometry was developed and validated for the identification of a total of 94 NPS and metabolites in urine using the established decision points. The limit of identification for all analytes was determined at 25% below their respective decision points. The method was demonstrated to be accurate and precise at their respective decision points with extraction recoveries and ion suppression/enhancement ranging from 51.0% to 103.5% and -81.6% to 159.1%, respectively. There was no observed carryover up to 200 ng/mL for all analytes and no interferences from urine matrixes, internal standards and other common drugs of abuse. The extracted drug analytes were stable at 4 and 15°C for up to 3 days. The validated method was successfully evaluated and applied in the testing of urine samples from NPS users. In conclusion, this validated method can analyze a wide range of NPS and their metabolites with the use of decision points for consistency in reporting.