Determination of the new psychoactive substances MDMB-4en-PINACA, ADB-BUTINACA and some of their metabolites in blood and urine using DLLE-LC-MS/MS: application to real forensic case samples.

MDMB-4en-PINACA and ADB-BUTINACA are indazole-derived synthetic cannabinoids that have increasingly been encountered in forensic toxicology, partly due to their potent cannabinoid receptor activity and their association with severe intoxication cases involving agitation, hallucinations, paranoia, and psychosis-like symptoms. In this study, a simplified and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method incorporating a simple and rapid sample preparation based on methanol-assisted protein precipitation followed by dispersive liquid-liquid extraction (DLLE) was developed and validated for the simultaneous determination of MDMB-4en-PINACA, ADB-BUTINACA, and their major metabolites, MDMB-4en-PINACA butanoic acid and ADB-BUTINACA N-butanoic acid, in blood and urine. The method provided effective separation and quantification of all analytes over a linear range of 1-100 ng/mL, with limits of detection between 0.51 and 1.20 ng/mL. No significant matrix interferences were observed, confirming adequate selectivity. The validated method was successfully applied to real forensic case samples, in which the inclusion of metabolite monitoring improved the confirmation of exposure particularly in cases where parent compounds were present at low concentrations or were no longer detectable. The results demonstrate that the proposed DLLE-LC-MS/MS method is a simple, robust, and reliable approach for the simultaneous determination of these synthetic cannabinoids and their metabolites in routine forensic toxicology, especially in investigations involving suspected synthetic cannabinoid-related neuropsychiatric manifestations such as acute psychosis.