Analysis of new psychoactive substances in oral fluids by means of microextraction by packed sorbent followed by ultra-high-performance liquid chromatography-tandem mass spectrometry.

In recent years, new drugs, commonly known as new psychoactive substances (NPS), appeared on the market, which include, among others, synthetic cannabinoids, cathinones, and tryptamine analogs of psilocin. The aim of this work was to develop and validate a new method for simultaneous screening and quantification of 31 NPS in oral fluid by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The chosen target analytes represented different chemical and toxicological NPS classes, such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites. The procedure involved a rapid sample preparation based on protein precipitation followed by clean-up utilizing microextraction by packed sorbent (MEPS); the quantitative analysis was performed by UHPLC-MS/MS. The MEPS clean-up, regardless of non-quantitative recoveries for some analytes, provided an effective removal of interfering compounds, as demonstrated by reduced matrix effects found at different concentrations for all the analytes. The validation protocol, based on SWGTOX guidelines, demonstrated the suitability of the proposed method for quantitative analysis: linearity range ranged over 3 or 4 orders of magnitude; precision and accuracy tests gave RSD% values below 25%, and accuracy ranged from 85.9% to 107%, accomplishing SWGTOX requirements. Limits of detection (LODs) ranged between 0.005 ng/mL and 0.850 ng/mL and limits of quantification (LOQs) from 0.015 to 2.600 ng/mL.

Application of a rapid µ-SPE clean-up for multiclass quantitative analysis of sixteen new psychoactive substances in whole blood by LC-MS/MS.

Europe is an important market for illegal drugs, and nowadays a lot of new different psychoactive substances (NPS) are widespread. This work reports the development of a method to determine simultaneously different classes of NPS, as synthetic cannabinoids (SC) and their metabolites, cathinones and phenetylamines, directly on whole blood (WB) without anti-coagulants and using miniaturized solid phase extraction (µ-SPE). In order to demonstrate the feasibility of the method 16 different NPS belonging to the mentioned classes were selected for the analysis. Recoveries ranged from 21% to 70% while matrix effect was lower than 15% for all the analytes. LOQ values were 5ngmL-1 for cathinones and phenetylamines, between 0.25 and 1ngmL-1 for SCs and up to 2.5ngmL-1 for SC metabolites. The performance of µ-SPE was compared with different clean-up strategies (i.e. protein precipitation (PPT), liquid liquid extraction, PPT/SPE hybrid) in term of recovery, matrix effect and suitability for multi-class analysis. The developed method was validated according to SWGTOX guidelines. The validation data demonstrated that this approach is potentially very useful as confirmation method for multiclass analysis in WB and post mortem specimens. In fact only 100µL of human WB are used, sample preparation involves few rapid steps and the method is easily implementable for the determination of other NPS.

Identification of MT-45 Metabolites: In Silico Prediction, In Vitro Incubation with Rat Hepatocytes and In Vivo Confirmation.

MT-45 is a synthetic opioid with a pharmacological activity comparable to morphine and it has been involved in intoxications and fatalities reported in Europe and in USA. It was recently subject to control measures, but to date the metabolic pathways of the substance are still unknown. Using rat hepatocytes and LC-HRMS, 14 novel Phase I and II MT-45 metabolites were identified, products of monohydroxylation, dihydroxylation and N-dealkylation; glucuronide conjugation of mono- and dihydroxylated metabolites also occurred. The detected metabolites were firstly predicted in silico, then incubation of the drug with rat hepatocytes was carried out and the obtained metabolites were identified by LC-HRMS, with retention times, mass shift between theoretical mass and observed mass (<5 ppm), peak abundance and fragmentation pattern. Hydroxylated MT-45 was found to be the major metabolite of MT-45 in vitro experiments. The presence of all metabolites was confirmed by in vivo experiments in urine samples of CD-1 male mice; in these samples hydroxy-MT-45-glucuronide and di-hydroxy-MT-45-glucuronide are the most abundant metabolites, while the parent drug is found at concentration <10 ng mL-1 after 300 min. The knowledge of Phase I and II MT-45 metabolite structure is then crucial to develop analytical methods to identify MT-45 consumption in clinical and forensic testing.

Multi-class analysis of new psychoactive substances and metabolites in hair by pressurized liquid extraction coupled to HPLC-HRMS.

In this paper, an analytical method has been developed and validated for the analysis of new psychoactive substances (NPS) and metabolites in hair samples. The method was based on pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) clean-up and high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) analysis. To evaluate extraction efficiency and the applicability of the method, hair samples were fortified by soaking in order to obtain a good surrogate for drug users' hair; the amount of incorporated drugs related to their lipophilicity, similarly to in vivo drug incorporation. To the best of our knowledge, this is the first method that allowed for the analysis of both cathinones (5) and synthetic cannabinoids (7) in hair with a single extraction procedure and chromatographic run. A phenethylamine (2C-T-4), 4- fluorophenylpiperazine and methoxetamine were also included showing that PLE coupled to SPE clean-up was suitable for a multi-class analysis of NPS in hair. In addition, the use of PLE significantly reduced hair analysis time: decontamination, incubation, clean-up, and liquid chromatography-mass spectrometry (LC-MS) analysis were carried out in approximately 45 min. The method was fully validated according to Scientific Working Group for Forensic Toxicology (SWGTOX) and Society of Hair Testing (SoHT) guidelines. Limit of quantification (LOQ) values ranged from 8 to 50 pg mg-1 for cathinones, phenetylamines and piperazines, and from 9 to 40 pg mg-1 for synthetic cannabinoids (10 pg mg-1 for methoxetamine). Matrix effects were below 15% for all the analytes, demonstrating the effectiveness of the clean-up step. Inaccuracy was lower than 9% in terms of bias. Copyright © 2016 John Wiley & Sons, Ltd.

Broad Screening and Identification of Novel Psychoactive Substances in Plasma by High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry and Post-run Library Matching.

Drug abuse is today a growing global problem. Often the consumers are not aware about the type of substances they are using and the correlated risks. In recent years, new psychoactive substances (NPS) appeared in the illicit market. The presence of NPS, such as synthetic cathinones, cannabinoids and phenethylamines, which are known to be pharmacologically and toxicologically hazardous, has been frequently reported. The aim of this study was the development of a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for a broad screening of NPS in plasma. Data acquisition was in MS/MS and full-scan modes and the method was validated for 25 NPS belonging to different chemical classes. Quantitative results have been obtained for these analytes with limits of quantification ranging from 0.03 to 0.4 ng/mL. The method was proven to be suitable for the screening of additional substances; to this aim, a post-run library matching was conducted for every sample with an in-house database containing over 300 NPS and known metabolites. The library may be constantly expanded with new drugs, in order to obtain a broad screening of NPS in biological matrices.

Pressurized liquid extraction for the determination of cannabinoids and metabolites in hair: Detection of cut-off values by high performance liquid chromatography-high resolution tandem mass spectrometry.

Hair analysis has become a routine procedure in most forensic laboratories since this alternative matrix presents clear advantages over classical matrices; particularly wider time window, non-invasive sampling and good stability of the analytes over time. There are, however, some major challenges for the analysis of cannabinoids in hair, mainly related to the low concentrations of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH), that is the major metabolite. In this study a fast, accurate and sensitive method for the determination of cannabinol, cannabidiol, THC and THC-COOH in hair has been developed. The extraction of analytes from hair (50mg) is based on an automated pressurized liquid extraction (PLE) using water modified with the surfactant sodium dodecyl sulphate as eluent phase. PLE extract is then cleaned up by SPE using polymeric reversed phase cartridges Strata XL before the injection in the HPLC-HRMS/MS system. Chromatographic conditions obtained with a fused-core column allowed a good separation of the analytes in less than 4min. The whole procedure has been validated according to SWGTOX guidelines. The LLOQs obtained for THC-COOH and the other analytes were respectively 0.1 and 2pg/mg. To the best of our knowledge, this is the first LC-MS/MS based method that allows the detection of THC-COOH in hair at values lower than the cut-off.