Quantification of U-47700 and its metabolites: N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 in 12 autopsy blood samples employing SPE/LC–ESI-MS-MS

Abstract

Purpose: The paper presents a report on the comprehensive assessment of a novel synthetic opioid (NSO) termed U-47700, and its two metabolites: N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 in autopsy blood samples taken from 12 cases of fatal poisonings. Methods: The analysis of the examined samples was based on the solid-phase extraction/liquid chromatography–electrospray ionization-tandem mass spectrometry method, which was developed in the validation process. In the quantitative analytical studies, deuterium analytes analogs were used, namely U-47700-d6 and N-desmethyl-U-47700-d3. Results: The validation parameters of the method were determined, including the limits of quantification on the 1 ng/mL level, calibration curves range of 1–1000 ng/ml, intra-assay precisions and accuracies of 1.1–20.2% and −18.9–9%, respectively, and inter-assay precisions and accuracies of 2.9–13.0% and -11.4–3.3%, respectively. The matrix effects and the extraction efficiencies were formed at the levels of 54.0–119% and 53.0–118%, respectively. The parent substance and its metabolites in blood samples have been shown to be relatively stable under various conditions within the 21-day study. The concentration levels demonstrated in the analyzed blood samples were: U-47700 in the range of 83–24,000 ng/mL, N-desmethyl-U-47700 in the range of 2.0–7520 ng/mL and N,N-didesmethyl-U-47700 in the range of 18–1947 ng/mL. Conclusions: The simultaneous quantification of U-47700, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 seems very useful to confirm the cause of death and to estimate antemortem interval. To our knowledge this is the first trial to measure phase I metabolites of U-47700 in authentic human blood samples.