Development and validation of a quantitative analytical method for urine samples in drug-facilitated sexual assault cases.

Drug-facilitated sexual assault (DFSA) is a type of drug-facilitated crime defined as a form of sexual violence against an individual incapacitated or unconscious due to the effects of psychoactive substances. These substances alter the victim's level of consciousness, judgment, and/or memory. This study aimed to develop and validate a quantitative LC-MS/MS method for psychoactive substances and biotransformation products with detection limits suitable for DFSA investigations. The scope was based on ANSI/ASB Standard 121 guidelines (2021). Urine samples were submitted to enzymatic hydrolysis, followed by liquid-liquid extraction. An aliquot of urine underwent protein precipitation with acetonitrile. After agitation, centrifugation, and evaporation of solvent, samples were resuspended with mobile phase and injected into LC-MS/MS system. The method was validated according to ANSI/ASB Standard 036 recommendations, with quantification limits of 0.5 to 50 ng/mL and linearity of 0.5 to 750 ng/mL. Bias and imprecision were better than 14.3% and 14.8%, respectively, and matrix effect ranging from -72.9% to 21.5%. No evidence of carryover and interference was observed. All substances were stable at 10 °C for 24 hours and recovery results were better than 5.2%. In total, 42 samples from sexual assault victims were analyzed, with at least one substance detected in 26 samples. Ethanol was the most predominant substance, followed by 11-nor-9-carboxy-Δ-9-tetrahydrocannabinol and cocaine. A sensitive method was developed and validated to quantify psychoactive substances in urine, with low limits of detection and quantification, adequate linearity, bias, imprecision, and successfully applied to the analysis of authentic urine samples.