Method development and validation for the determination of 49 new psychoactive substances (NPS) in serum, urine and hair by ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS): application to real samples

Detection of new psychoactive substances (NPS), both in conventional and nonconventional biological samples, represents a hard challenge for forensic toxicologists. The number of newly NPS, increases each year. As soon as NPS are scheduled, new derivatives appear in the market. Despite the increasing number of NPS and the fact that fatal and acute intoxication cases have been already attributed to this novel class of compounds this phenomenon appears to be considerably underestimated, mainly due to the substantial lack of comprehensive screening methods for their detection in biological specimens. Development of analytical methods for the determination of NPS in biological specimens is of great importance to keep in pace with this phenomenon. Thus, we sought to develop and validate a simple and rapid UHPLC–MS/MS screening method for the determination of 49 NPS belonging to different chemical classes (synthetic cannabinoids, cathinones, benzofurans, aminoindanes, phenethylamines, piperazines and piperidines) in serum, urine and hair extracts in a single run, following rapid and easy sample pre-injection treatment. The method was very fast, easy to perform, cheap and minimum amount of sample (0.1 ml serum or urine and 50 mg hair) was required. Chromatography was carried out using an Acquity UPLC BEH reversed phase C18 column (2.1 x 75 mm, 1.7 µm) and a gradient elution with two solvents: 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The method was sensitive, linear from 1 to 100 ng/ml for serum and urine and from 1 to 100 pg/mg for hair, precise and accurate for most of the analytes. Matrix effects did not negatively affect the analytical sensitivity. The validated method was successfully applied to authentic samples (serum, urine and hair) collected from an intoxication case after the consumption of NPS; and hair samples obtained by illicit drugs users who attended Drug Addiction Services or Care Emergency Departments (ED).