Quantitative analysis of fentanyl, several analogues and metabolites in urine by parallel artificial liquid membrane extraction and liquid chromatography tandem mass spectrometry analysis

The rapid introduction of new psychoactive substances (NPS) has definitively changed the drug market. Among the several NPS that were identified in the last decades, fentanyl and its analogues deserve special attention. These are synthetic opioids with high potency and are associated with increasing number of deaths; for this reason, forensic toxicologists are paying close attention to these analytes and sensitive analytical methods for their detection in biological samples of drug users are needed. The aim of this study was the development of a LC–MS/MS method for the determination of fentanyl, 23 analogues and metabolites in urine by exploiting parallel artificial liquid membrane extraction (PALME). This technique was shown to be particularly suitable for fentanyl extraction and allowed to obtain a high enrichment factor by using a few microliters of organic solvent (1-octanol) immobilized into a polyvinylidene fluoride (PVDF) membrane. The extraction was carried out on a 96 well plate providing high laboratory throughput. The applied strategy allowed to measure concentrations ranging from 0.1 ng mL−1 for fentanyl and most analogues to 5 ng mL−1 for metabolites, by using an entry level mass spectrometer. Because of the different concentration levels generally found in real samples, linearity was studied in different ranges i.e. LOQ to 50 ng mL−1 for parent drugs and LOQ to 200 ng mL−1 for metabolites. All the validation parameters were found within the imposed limits, and notably matrix effect was not significant for all the analytes, showing the selectivity achieved by PALME extraction.