Short- and long-term stability of synthetic cathinones and dihydro-metabolites in human urine samples.

PURPOSE: Synthetic cathinones constitute the second largest group of new psychoactive substances, which are often used for recreational purposes and reported in toxicological analysis. Various factors may influence the stability of synthetic cathinones between sampling and analysis, and therefore, stability studies are required to determine the best storage conditions as well as extend the period of detection. METHODS: This study involved sixteen synthetic cathinones and ten dihydro-metabolites spiked in human urine to evaluate the stability under common storage conditions to imitate real forensic toxicology samples. The samples were stored at either room temperature (22-23 °C) for up to 3 days, refrigerated (4 °C) for up to 14 days or frozen (-40 °C) for up to 12 months, and analyzed in triplicate using a validated liquid chromatography-tandem mass spectrometry method. RESULTS: Analytes' concentrations decreased over time, although slower when stored frozen. All analytes remained stable (> 80%) for 1 month when stored frozen before losses in content were more apparent for some compounds, depending on their chemical structure. Under all storage conditions, the highest instability was observed for analytes containing halogens (i.e., chlorine or fluorine). Thus, halogenated analytes were further investigated by using liquid chromatography coupled to quadruple time-of-flight mass spectrometry to attempt identifying degradation products. CONCLUSIONS: Irrespective of parent analytes, dihydro-metabolites had improved stability at each tested temperature, which highlights their importance as appropriate urine biomarkers when retesting is required after a long period of storage.

Influence of long-term storage temperatures and sodium fluoride preservation on the stability of synthetic cathinones and dihydro-metabolites in human whole blood.

PURPOSE: Synthetic cathinones, one of the largest groups of new psychoactive substances, represent a large analytical and interpretative challenge in forensic laboratories. Of these is the synthetic cathinones' instability in different biological samples, which may lead to drug concentration discrepancies when interpreting toxicological findings. In this study, the stability of a panel of synthetic cathinones and their dihydro-metabolites (n = 26) together with internal standard was monitored in human whole blood stored at various temperatures over 6 months. The influence of sodium fluoride as a preservative in blood collection tubes was also investigated. METHODS: Samples were extracted using a two-step liquid-liquid extraction technique, and analyzed using a validated liquid chromatography-tandem mass spectrometry method following recommendations of published guidelines. RESULTS: The influence of temperature over analytes' stability was an important element in whole blood samples, with - 40 °C being the best storage temperature for all tested analytes. Sodium fluoride did not significantly affect the stability of cathinones except at room temperature. Dihydro-metabolites displayed better stability in whole blood samples and remained detectable for a longer period of time under all tested conditions. CONCLUSIONS: The data suggest that samples containing synthetic cathinones should be analyzed immediately, if possible. Alternatively, whole blood samples should be stored frozen (at - 40 °C or lower); however, (quantitative) results should be interpreted with caution after long-term storage. The data also promote the use of dihydro-metabolites as biomarkers for synthetic cathinones intake, as these reduced metabolites may be detected for longer period of time when compared with parent drugs in whole blood samples.

Development and validation of a chiral LC-MS/MS method for the separation and quantification of four synthetic cathinones in human whole blood and its application in stability analysis.

Synthetic cathinones, a subclass of new psychoactive substances, have gained high popularity on the recreational drugs market over the past years. These drugs typically have a chiral center, so they may exist as two stereoisomers. Therefore the pharmacological, pharmacokinetic or metabolic properties of their enantiomers are expected to differ. However, these drugs are often synthesized and sold as a racemic mixture, and as a consequence, differentiation of their (R)- and (S)- enantiomers is relevant in clinical and forensic toxicology. Information about single enantiomers of synthetic cathinones is relatively scarce due to challenges of their chiral analysis. Hence, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the chiral separation and quantification of four synthetic cathinones in human whole blood samples. The method was fully validated in terms of linearity, limit of detection, limit of quantification, bias, precision, carryover, interferences, matrix effects, recovery and processed sample stability and successfully applied to evaluate the stability as well as enantioselective degradation of synthetic cathinones enantiomers under various storage conditions. For most of the analytes, significant enantioselective degradation was observed when stored at room temperature or refrigerated, with the E2-enantiomers observed to more rapidly degrade under both conditions. This is the first report concerning the stability and enantioselective degradation of synthetic cathinone enantiomers in whole blood. Moreover, the inversion study demonstrated enantiomeric inversion of R-(-)- and S-(+)-methylenedioxypyrovalerone (MDPV) in human whole blood and methanolic solution.

A Quantitative LC-MS/MS Method for the Detection of 16 Synthetic Cathinones and 10 Metabolites and Its Application to Suspicious Clinical and Forensic Urine Samples.

BACKGROUND: Synthetic cathinones currently represent one of the most predominant (sub)-classes of new psychoactive substance (NPS) in illicit drug markets. Despite the increased concerns caused by the constant introduction of new analogues, these drugs are not commonly assayed in routine drug testing procedures and may not be detected in standard screening procedures. This study presents a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of 16 synthetic cathinones and 10 metabolites in human urine. METHODS: The method was validated for all analytes using published guidelines. The evaluated parameters achieved acceptable values according to the set criteria. Potential abuse of synthetic cathinones was investigated in suspicious urine samples from Saudi Arabia originating from workplace drug testing, pre-employment and Accident & Emergency (A&E). Such samples generated a presumptive positive immunoassay for amphetamine; however, they yielded a negative LC-MS/MS confirmation for this analyte, following the recommended cutoff values of Substance of Abuse and Mental Health Services Administration (SAMHSA) guidelines. RESULTS: 5.8% of the analyzed samples were found to contain at least one target analyte, namely mephedrone and N-ethylpentylone, as well as their dihydro-metabolites. The results also revealed polydrug use with the synthetic cathinones being present together with other classical stimulant drugs. CONCLUSIONS: This is the first report of NPS use in Saudi Arabia with respect to designer stimulant drugs. Confirmatory urine analyses for suspicious stimulant use should extend beyond classical stimulants to cover a broad range of NPSs and their metabolites in order to report any otherwise potentially undetected/new analyte.

Stability of synthetic cathinones in clinical and forensic toxicological analysis-Where are we now?

Understanding the stability of analyzed drugs in biological samples is a crucial part for an appropriate interpretation of the analytical findings. Synthetic cathinones, as psychoactive stimulants, belong to a major class of new psychoactive substances. As they are subject to several degradation pathways, they are known to clinical and forensic toxicologists as unstable analytes in biological samples. When interpreting analytical data of synthetic cathinones in biological samples, analysts must be aware that the concentration of analytes may not accurately reflect the levels at the time they were acquired owing to many factors. This review provides (i) an overview of the current scientific knowledge on the stability of synthetic cathinones and/or metabolites in various human biological samples with a focus on factors that may deteriorate their stability-such as storage temperature, length of storage, matrix, pH, type of preservatives, concentration of analytes, and the chemistry of the analytes-and (ii) possible solutions on how to avoid such degradation. The PubMed database as well as Google Scholar was thoroughly searched to find published studies on the stability of synthetic cathinones since 2007 by searching specific keywords. A total of 23 articles met the inclusion criteria and were included in this review. Synthetic cathinones that carry methylenedioxy or N-pyrrolidine ring showed higher degradation resistance over other substituted groups. Acidification of samples pH plays a crucial role at increasing the stability of cathinones even with analytes that were frequently considered as poorly stable. This review also provides several recommendations for best practice in planning the experimental design, preservation, and storage conditions in order to minimize synthetic cathinones' degradation in human biological samples.