Automated enzymatic hydrolysis of urine samples for improved systematic toxicological analysis of drug‐facilitated sexual assault cases

Abstract

Drug‐facilitated sexual assault (DFSA) is characterized by victim incapacitation due to intoxicating substances. Detection of single drug exposure from DFSA requires a systematic toxicological analysis strategy including sensitive methods covering a broad spectrum of substances. The aim of this study was to develop and validate an UHPLC–MS/MS screening method for analysis of samples from DFSA cases and incorporate an automated enzymatic pre‐treatment of urine samples into a robotic sample preparation for an efficient laboratory workflow. The screening method included 144 drugs of abuse, pharmaceuticals, and metabolites relevant to DFSA. The use of a recombinant enzyme showed an efficient glucuronide hydrolysis with an average parent drug recovery of 97%. Investigation of matrix effect showed no pronounced ion enhancement or suppression for most analytes (96%), and extraction recovery was above 80% for 97% of analytes. Process efficiency ranged from 50% to 138% for most analytes. The LODs ranged from 0.0001 mg/L to 2 mg/L depending on analyte, and most analytes met the SOFT recommended minimum performance limits. The validated method was applied to authentic suspected DFSA cases (n = 38). Results showed that drugs of abuse, benzodiazepines, and antidepressants were most commonly found in suspected DFSA cases. Incorporation of an automated enzymatic hydrolysis step during sample preparation enables a fast and simple workflow for simultaneous analysis of blood and urine samples for an improved systematic toxicological analysis strategy for DFSA cases.