UHPLC-HRMS and GC-MS Screening of a Selection of Synthetic Cannabinoids and Metabolites in Urine of Consumers

Background and Objectives : The use of synthetic cannabinoids has increased around the world. As a result, the implementation of accurate analysis in human biological matrices is relevant and fundamental. Two different analytical technologies, ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) and high-sensitivity gas chromatography-mass spectrometry (GC-MS) were used for the determination of three synthetic cannabinoids JWH-122, JWH 210, UR-144 and their metabolites in urine of consumers. Materials and Methods : Sample preparation included an initial hydrolysis with β-glucuronidase and liquid-liquid extraction. The UHPLC-HRMS method included a Kinetex 2.6 u Biphenyl 100A (100 × 2.1 mm, 2.6 μm) (Phenomenex, Italy) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode was used (mass range 100-1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m × 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40-550 m/z). Results: Both methods have been successfully used for screening of parent synthetic cannabinoids and their metabolites in urine samples of consumers. Conclusions: The screening method applied JWH-122, JWH-210, UR-144 and their metabolites in urine of consumers can be applied to other compounds of the JWH family

High Performance Liquid Chromatography Tandem Mass Spectrometry Measurement of Bimatoprost, Latanoprost and Travoprost in Eyelash Enhancing Cosmetic Serums

Most common prostaglandin analogs, bimatoprost, latanoprost and travoprost, are licensed for the reduction of elevated intraocular pressure in patients with open angle glaucoma and ocular hypertension, but their non approved use as eyelash enhancers is becoming popular, especially in patients with eyelashes hypotrichosis. A fast and sensitive high performance liquid chromatography tandem mass spectrometry method was developed for the measurement of bimatoprost, latanoprost and travoprost in cosmetic serums freely web-sold to increase eyelash length, thickness and darkness. The analytes and the internal standard (reserpine) were separated by reversed phase chromatography with 5 mM ammonium acetate with 0.02% formic acid (mobile phase A) and 5 mM ammonium acetate in acetonitrile/water (95/5; v/v) with 0.02% formic acid (mobile phase B) by gradient elution and detected with tandem mass spectrometry operated in multiple reaction monitoring mode. Linearity between 1 and 500 μg/g shows good correlation coefficients (r2 = 0.99) for all substances. Analytical recovery of analytes under investigation were always higher than 90% and intra-assay and inter-assay precision and accuracy always better than 11%. This method was successfully applied to analyze cosmetic serums freely sold on the Internet websites

Role of Neonatal Biomarkers of Exposure to Psychoactive Substances to Identify Maternal Socio-Demographic Determinants

Simple summary: the rapid identification of newborns exposure to psychoactive drugs allows an appropriate clinical care. this study tried to identify maternal profiles that help to identify newborns exposed to psychoactive drugs during pregnancy. mothers were interviewed using a questionnaire. The biomarkers of fetal exposure were measured in meconium samples. statistical analysis was performed to identify the maternal characteristics that were most likely to be associated with drug use during pregnancy. of a total of 372 mothers, 49 (13.2%) tested positive for psychoactive drugs use: 24 (49.0%) for cannabis, 11 (22.5%) for ethyl glucuronide, six (12.2%) for cocaine, and eight (16.3%) for more than one psychoactive substance. the maternal characteristics that most likely identify substance use during pregnancy are: maternal age < 24 years, lack of pregnancy care, single-mother families, and active tobacco smoking. the profiles of prenatal maternal exposure identified in a clinical setting can be used to request specific detection tests for identifying newborns exposed to these drugs.Background: the accurate assessment of fetal exposure to psychoactive substances provides the basis for appropriate clinical care of neonates. the objective of this study was to identify maternal socio-demographic profiles and risk factors for prenatal exposure to drugs of abuse by measuring biomarkers in neonatal matrices. methods: a prospective, observational cohort study was completed. Biomarkers of fetal exposure were measured in meconium samples. The mothers were interviewed using a questionnaire. univariate and multivariate logistic regression analyses were performed. results: a total of 372 mothers were included, 49 (13.2%) testing positive for psychoactive substances use: 24 (49.0%) for cannabis, 11 (22.5%) for ethyl glucuronide, six (12.2%) for cocaine, and in eight (16.3%) more than one psychoactive substance. mothers who consumed any psychoactive substance (29.7 +/- 6.6 years) or cannabis (27.0 +/- 5.7 years) were younger than non-users (32.8 +/- 6.2 years, p < 0.05). cocaine (50.0% vs. 96.9%, p < 0.05) and polydrug users (37.5% vs. 96.9%, p < 0.05) showed a lower levels of pregnancy care. Previous abortions were associated with the use of two or more psychoactive substances (87.5% vs. 37.8%, p < 0.05). Single-mother families (14.3% vs. 2.5%, p < 0.05) and mothers with primary level education (75.5% vs. 55.1%, p < 0.05) presented a higher consumption of psychoactive substances. Independent risk factors that are associated with prenatal exposure include: maternal age < 24 years (odds ratio: 2.56; 95% CI: 1.12-5.87), lack of pregnancy care (odds ratio: 7.27; 95%CI: 2.51-21.02), single-mother families (odds ratio: 4.98; 95%CI: 1.37-8.13), and active tobacco smoking (odds ratio: 8.13; 95%CI: 4.03-16.43). conclusions: these results will allow us to develop several risk-based drug screening approaches to improve the early detection of exposed neonates

GC-MS/MS Determination of Synthetic Cathinones : 4-chloromethcathinone, N-ethyl Pentedrone, and N-ethyl Hexedrone in Oral Fluid and Sweat of Consumers under Controlled Administration: Pilot Study

This study presents a validated GC-MS/MS method for the detection and quantification of 4-chloromethcathinone or clephedrone (4-CMC), N-ethyl Pentedrone (NEP), and N-ethyl Hexedrone (NEH, also named HEXEN) in oral fluid and sweat and verifies its feasibility in determining human oral fluid concentrations and pharmacokinetics following the administration of 100 mg of 4-CMC orally and 30 mg of NEP and NEH intranasally. A total of 48 oral fluid and 12 sweat samples were collected from six consumers. After the addition of 5 μL of methylone-d and 200 μL of 0.5 M ammonium hydrogen carbonate, an L/L extraction was carried out using ethyl acetate. The samples, dried under a nitrogen flow, were then derivatized with pentafluoropropionic anhydride and dried again. One microliter of the sample reconstituted in 50 μL of ethyl acetate was injected into GC-MS/MS. The method was fully validated according to international guidelines. Our results showed how, in oral fluid, the two cathinones taken intranasally were absorbed very rapidly, within the first hour, when compared with the 4-CMC which reached its maximum concentration peak in the first three hours. We observed that these cathinones were excreted in sweat in an amount equivalent to approximately 0.3% of the administered dose for 4-CMC and NEP. The total NEH excreted in sweat 4 h after administration was approximately 0.2% of the administered dose. Our results provide, for the first time, preliminary information about the disposition of these synthetic cathinones in the consumers' oral fluid and sweat after controlled administration

The recurrent pathogenic Pro890Leu substitution in CLTC causes a generalized defect in synaptic transmission in Caenorhabditis elegans

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders

Determination of the Synthetic Cannabinoids JWH-122, JWH-210, UR-144 in Oral Fluid of Consumers by GC-MS and Quantification of Parent Compounds and Metabolites by UHPLC-MS/MS.

The consumption of synthetic cannabinoids (SCs) has significantly increased in the last decade and the analysis of SCs and their metabolites in human specimens is gaining interest in clinical and forensic toxicology. A pilot study has been carried out using a combination of an initial last generation gas chromatography-mass spectrometry (GC-MS) screening method for the determination of JWH-122, JWH-210, UR-144) in oral fluid (OF) of consumers and an ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) confirmatory method for the quantification of the parent compounds and their metabolites in the same biological matrix. OF samples were simply liquid-liquid extracted before injecting in both chromatographic systems. The developed methods have been successfully validated and were linear from limit of quantification (LOQ) to 50 ng/mL OF. Recovery of analytes was always higher than 70% and matrix effect always lower than 15% whereas intra-assay and inter-assay precision and accuracy were always better than 16%. After smoking 1 mg JWH-122 or UR-144 and 3 mg JWH-210, maximum concentration of 4.00-3.14 ng/mL JWH-122, 8.10-7.30 ng/mL JWH-210 ng/mL and 7.40 and 6.81 ng/mL UR-144 were measured by GC-MS and UHPLC-HRMS respectively at 20 min after inhalation. Metabolites of JWH 122 and 210 were quantified in OF by UHPLC-HRMS, while that of UR144 was only detectable in traces. Our results provide for the first time information about disposition of these SCs and their metabolites in consumers OF. Last generation GC-MS has proven useful tool to identify and quantify parent SCs whereas UHPLC-HRMS also confirmed the presence of SCs metabolites in the OF of SCs consumers

High Performance Liquid Chromatography Tandem Mass Spectrometry Measurement of Bimatoprost, Latanoprost and Travoprost in Eyelash Enhancing Cosmetic Serums

Most common prostaglandin analogs, bimatoprost, latanoprost and travoprost, are licensed for the reduction of elevated intraocular pressure in patients with open angle glaucoma and ocular hypertension, but their non approved use as eyelash enhancers is becoming popular, especially in patients with eyelashes hypotrichosis. A fast and sensitive high performance liquid chromatography tandem mass spectrometry method was developed for the measurement of bimatoprost, latanoprost and travoprost in cosmetic serums freely web-sold to increase eyelash length, thickness and darkness. The analytes and the internal standard (reserpine) were separated by reversed phase chromatography with 5 mM ammonium acetate with 0.02% formic acid (mobile phase A) and 5 mM ammonium acetate in acetonitrile/water (95/5; v/v) with 0.02% formic acid (mobile phase B) by gradient elution and detected with tandem mass spectrometry operated in multiple reaction monitoring mode. Linearity between 1 and 500 μg/g shows good correlation coefficients (r2 = 0.99) for all substances. Analytical recovery of analytes under investigation were always higher than 90% and intra-assay and inter-assay precision and accuracy always better than 11%. This method was successfully applied to analyze cosmetic serums freely sold on the Internet websites

New synthetic opioids in biological and non-biological matrices: A review of current analytical methods

Abuse of new synthetic opioids is a worldwide rising issue posing a serious threat to the health of consumers. Unlike commonly abused drugs, which can be identified through routine drug testing, these new psychoactive substances may escape immunological screening tests and appropriate confirmation methods have not yet been developed and validated. Gas chromatography combined with mass spectrometry (GC–MS) and more frequently liquid chromatography tandem mass spectrometry (LC–MS/MS) as well as ultra high-liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) are the methods required to attack this analytical problem. The technologies are sufficiently sensitive and specific to be used with antemortem and postmortem specimens and also to disclose drug metabolism for substances used in very low doses, resulting in minute concentrations in biological matrices. An updated review and current trends concerning analytical methods for the determination of illicit fentanyl analogues and other new synthetic opioids at risk of abuse is reported here for both ante- and postmortem biological matrices and non-biological specimens

Systematic toxicological analysis of Indian herbal ready-to-chew pouches by gas chromatography mass spectrometry

Objective: Systematic toxicological analysis by gas chromatography-mass spectrometry has been applied for the analysis of Indian herbal ready-to-chew pouches. Material and methods: These small packages containing a powder formed by areca nut slices, tobacco, menthol and lime, are available in traditional markets and web sites. Their consumption has been recently spread in the asiatic countries not only among adults but especially among young people. Acute and chronic side effects, an increase in the incidence of oral and esophagus cancer and the possibility to develop dependence and tolerance phenomena following repeated pouches consumption prompted the systematic toxicological analysis of gutka and panmasala pouches. Gas chromatography mass spectrometry was used to verify the presence of declared substances, and to investigate the possible presence of licit and illicit psychoactive compounds. Results: Arecoline (1.35–1.91 mg/g) and nicotine (0.31–0.71 mg/g) and arecoline alone (1.67–2.74 mg/g) were identified in gutkha and panmasala pouches, respectively,  together with menthol (1.89–2.78 mg/g gutka and 1.91–2.84 mg/g panmasala). No other pharmacological active compounds were identified. Conclusion: The mg amounts of the two alkaloids found in the pouches together with the reported consumption of several pouches a day may be consistent with health risks