Development of an UPLC–MS/MS method for the analysis of 16 synthetic opioids in segmented hair, and evaluation of the polydrug history in fentanyl analogue users

Abstract Seizures of synthetic opioids have increased since 2012, with a 45 % increase in synthetic opioid related deaths between 2016 and 2017 in US. Recently, concerns have arisen around these substances and their illicit use also in several European countries. Our aim was to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of 16 synthetic opioids in segmented hair, including fentanyl, norfentanyl, acetylfentanyl, U-47700, AH-7921, acrylfentanyl, crotonylfentanyl, butyrylfentanyl, methoxacetylfentanyl, U-49900, valeryfentanyl, 4-fluoro-iso-butyrylfentanyl, ocfentanyl, furanylfentanyl, tetrahydrofuranylfentanyl, and alfetanyl. Sample preparation involved washing the hair in dichloromethane, water and methanol, and extraction in methanol, followed by solid phase extraction clean-up. This method was validated for linearity, limit of quantification (LLOQ), precision and bias, selectivity, stability, matrix effects, extraction efficiency of the clean up procedure, and carryover. LLOQs ranged from 0.15−1 pg/mg, and the calibration ranged from the LLOQ up to 500 pg/mg. Intra and inter-day precision were evaluated at low and high concentrations, with spiked QCs, during 8 days and the results were satisfactory with RSD  Matrix effects compensated by internal standard fentanyl-d5 (MEIS), were between 77–115 % (RSD  The method was applied to 17 authentic hair samples (body or head hair) from US fentanyl analogue users. When head hair was available, the hair strands were analysed in 1 cm/segment. Concentrations ranges were as follows: fentanyl (n = 16) 2->ULOQ (500) pg/mg, norfentanyl (n = 14) 1−38 pg/mg, acetylfentanyl (n = 7) 0.6->ULOQ (250) pg/mg, furanylfentanyl (n = 5) 2−123 pg/mg, tetrahydrofuranylfentanyl (n = 1) 0.5−63 pg/mg and valerylfentanyl (n = 1) 2.1->ULOQ (50) pg/mg, along the hair strands. To our knowledge, this is the first time where concentrations of tetrahydrofuranylfentanyl, and valerylfentanyl in hair are reported. The same samples were also analysed for the determination of other drugs of abuse using our routine method (also in 1 cm/segment for head hair when available). The results demonstrated poly-drug use in these fentanyl-analogue users population (mean drugs: n = 5): amphetamine and/or methamphetamine (n = 10), buprenorphine (n = 5), cocaine (n = 8), methadone (n = 8), 6-MAM (n = 17), meperidine (n = 1), oxycodone (n = 11), tramadol (n = 3). Evaluation of the concentrations of these drugs, together with the fentanyl analogues is discussed in the present paper. Two authentic samples from two Belgian post-mortem cases, were also analysed showing fentanyl use and in one case polydrug use. The results demonstrated multi-analyte quantitative methods, including fentanyl analogues, are becoming useful in forensic laboratories involved in hair analysis, and in particular when polydrug use is suspected.