Validation and application of an UPLC-MS/MS method for the quantification of synthetic cannabinoids in urine samples and analysis of seized materials from the Portuguese market.

An UPLC-MS/MS method using ESI+ionization and MRM was developed and fully validated according to international guidelines for the qualitative and quantitative analysis of nine synthetic cannabinoids and/or their metabolites in urine samples (1mL). Prior to extraction the samples were subjected to an enzymatic hydrolysis using ß-glucuronidase followed by a SPE procedure using Oasis(®) HLB 3cc (60mg) columns. The chromatographic separation was performed with an Acquity UPLC(®) HSS T3 (50mm×2.1mm i.d., 1.8µm) reversed-phase column using a gradient with methanol-ammonium formate 2mM (0.1% formic acid) and with a run time of 9.5min. The method was validated in terms of selectivity, capacity of identification, limits of detection (0.01-0.5ng/mL) and quantification (0.05-0.5ng/mL), recovery (58-105%), carryover, matrix effect, linearity (0.05-50ng/mL), intra-assay precision, inter-assay accuracy and precision (CV<20%). The method was applied to 80 authentic samples, five of them (6.2%) were confirmed or suspected to be positive for the metabolites JWH-018 N-hydroxypentyl and JWH-018 N-pentanoic acid of JWH-018 and for the metabolite JWH-122 N-(5-hydroxypentyl) of JWH-122, and three of them in association with THC and/or THCCOOH (substances included in the method, together with the 11-OH-THC). Additionally, 17 spice products were analyzed, for which were confirmed the presence of the following substances: AM-2201, JWH-018, JWH-022 JWH-073, JWH-122, JWH-203, JWH-210, JWH-250, HU-210 and RCS-4, according to the comparison with authentic reference material and published data. The analytical method developed allowed the analysis of synthetic cannabinoids and the notification of the first cases in Portugal.

Validated method for the determination of misoprostol acid in whole blood by ultra performance liquid chromatography-tandem mass spectrometry.

Misoprostol is a pharmaceutical synthetic compound, analog of prostaglandin E1, frequently used as an abortifacient in not medically supervised or self-induced abortions, particularly in countries with restrictive abortion laws representing a serious public health problem. The aim of this study was to develop and validate a sensitive analytical method for the determination of misoprostol acid in whole blood samples. The samples were prepared by SPE and the chromatographic separation was performed by UPLC-MS/MS using ESI- and MRM mode with an Acquity UPLC(®) BEH C18 (50mm×2.1mm i.d., 1.7µm) column using a methanol-ammonium 0.1% solution gradient in a total run time of 7.0min. The method showed to be selective and linear in range 25-2000ng/L. The LOD and LOQ were 10ng/L and 25ng/L, respectively. The recovery ranged from 89 to 97%. No carryover and significant matrix effect were observed. The intra- and inter-assay precisions and the inter-assay accuracy results were 4.0% and 5.4%, 5.5% and 4.1%, and -1.4% and -2.8%, for the concentrations 50 and 500ng/L, respectively. The method developed allows the analysis of misoprostol acid in whole blood samples with adequate sensitivity to the concentration range obtained from therapeutic doses. The method was successfully used in a controlled misoprostol administration study and has been applied in our laboratory in the forensic toxicology field.

Qualitative and quantitative analysis of THC, 11-hydroxy-THC and 11-nor-9-carboxy-THC in whole blood by ultra-performance liquid chromatography/tandem mass spectrometry.

A qualitative and quantitative analytical method was developed for the simultaneous determination of Δ(9) -tetrahydrocannabinol (THC), 11-hydroxy-Δ(9) -tetrahydrocannabinol (11-OH-THC) and l1-nor-9-carboxy-Δ(9) -tetrahydrocannabinol (THC-COOH) in whole blood. The samples were prepared by solid-phase extraction followed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis using positive ion electrospray ionization and multiple reaction monitoring. The chromatographic separation was performed with an Acquity UPLC® HSS T3 (50 × 2.1 mm i.d., 1.8 µm) reversed-phase column using a methanol/2 mM ammonium formate (formic acid 0.1%) gradient in a total run time of 9.5 min. MS/MS detection was achieved with two precursor-product ion transitions per substance. The method was fully validated, including selectivity and capacity of identification, according to the identification criteria (two transitions per substance, signal-to-noise ratio, relative retention time and ion ratio) without the presence of interferences, limit of detection (0.2 µg/L for THC and 0.5 µg/L for 11-OH-THC and THC-COOH), limit of quantitation (0.5 µg/L for all cannabinoids), recovery (53-115%), carryover, matrix effect (34-43%), linearity (0.5-100 µg/L), intra-assay precision (CV < 10% for the relative peak area ratios and <0.1% for the relative retention time), inter-assay accuracy (mean relative error <10%) and precision (CV <11%). The method has already been successfully used in proficiency tests and subsequently applied to authentic samples in routine forensic analysis.

Liquid chromatography/tandem mass spectrometry for the qualitative and quantitative analysis of illicit drugs and medicines in preserved oral fluid.

A qualitative and quantitative analytical method was developed for the simultaneous determination of 24 illicit drugs and medicines, in preserved oral fluid samples collected with the StatSure Saliva Sampler collection device. The samples were prepared by liquid-liquid extraction followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The chromatographic separation was performed with an Atlantis T3 (100 x 2.1 mm i.d., 3 microm) reversed-phase column using an acetonitrile/2 mM ammonium formate buffer pH 3.4 gradient and the MS/MS detection was achieved with two precursor-product ion transitions per substance. The method was fully validated, including specificity and capacity of identification, limit of detection (0.2-2.1 microg/L), limit of quantitation (0.8-6.4 microg/L), recovery (34-98%), carryover, linearity (the method was linear in the range 1-200 microg/L), intra-assay precision (coefficient of variance (CV) <20% for 20 microg/L and CV <10% for 100 microg/L) and inter-assay accuracy (mean relative error <15%) and precision (CV <20%). The method showed to be specific and sensitive. It has already been successfully used in four proficiency tests and subsequently applied to oral fluid samples collected from road traffic volunteers in the driving population of Portugal (districts of Lisbon, Coimbra and Porto), within the DRUID project.