AbstractSynthetic cathinones (SCt), have become one of the most prevalent drug classes of new psychoactive substances (NPS) for their psychostimulant and euphoric effects. The speed and variety of these substances entering the drug market pose an extra challenge to the forensic toxicology communities, making the detection of these substances in biological samples rather difficult. In addition, these stimulant drugs can be unexpectedly toxic since they are originated from clandestine laboratories and their ingredients are mostly unknown. Therefore, toxicologists must be able to unambiguously detect and interpret results in order to properly correlate pharmacological or toxicological effects with SCt use.
Several analytical methods have been published for the detection and quantification of a wide range of SCt in various biological samples, but so far rarely incorporate analysis of their metabolites, which are of utmost importance as potential biomarkers in view of the low stability of parent drugs. In response to this, a sensitive analytical method was developed for the quantitative analysis of 16 SCt and 10 dihydro-metabolites by using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry. Dihydro-metabolites were synthesised inhouse using a simple method involving sodium borohydrate reduction of parent drugs. The final method was validated in accordance with ANSI/ASB Standard 036 guidelines over a wide concentration range for both human urine and whole blood samples. The method was then applied to 52 suspicious urine sample collected from toxicology department in Prince Sultan Military Medical City, Riyadh, Saudi Arabia. Qualitative analysis of conventional stimulants such as methamphetamine and methylenedioxymethamphetamine was also investigated in these samples. SCt, alongside their corresponding metabolite, were detected in several samples, whereas one sample was found to contain dihydro-metabolite in the absence of their parent drug. At least one traditional stimulant was detected in those positive samples, alongside SCt, highlighting polydrug use.
The concentration of analytes present in biological samples at the time of analysis may significantly differ from the time of sample collection. This is the case for SCt as their stability is highly susceptible to degradation and thus, the stability of data of these drugs is crucial. The validated methods (in both whole blood and urine) were used to assess the stability of SCt and selected dihydro-metabolites, examining the effects that prolonged storage (six months in whole blood and 12 months in urine) and preservatives had on analyte stability. In whole blood and urine samples, instability was observed under all storage temperatures, but at a lower rate when stored frozen. The effect of preservatives (2% sodium fluoride) was considered insignificant since minor/no changes were observed between preserved and unpreserved blood samples, except for room temperature. The stability differences within synthetic cathinone classes were diverse, in which analytes containing halogens were characterised by poor stability. Moreover, degradation of dihydro-metabolites was significantly slower than their parent drugs under all of the storage conditions, making them more suitable biomarkers of SCt use.
Prior knowledge of enantiomers stability is also crucial to understand their role in toxicity effects. A chiral stability study was performed by applying a validated liquid chromatographytandem mass spectrometry method in reverse-phase mode, revealing that degradation of selected synthetic cathinones namely, R,S-(±)-4-F-PHP, R,S-(±)-α-PVP and R,S-(±)-4-Cl-α-PVP in whole blood were enantioselective, with E2-enantiomers being more rapidly degraded when stored at higher temperatures. Moreover, the obtained results for most of the analytes under frozen conditions did not show any significant changes in enantiomeric fraction. Hence, the potential impact of storage temperatures was not only on the degradation of the analytes but also on their enantiomeric profile. In addition to storage temperature, potential inversion between R-(–) and S-(+)-enantiomers was notably dependent on the solvent system used, in which enantiomeric inversion was observed when using methanolic solution while none with acetonitrile.
|Date of Award||1 Sept 2023|
|Supervisor||Vincenzo Abbate (Supervisor)|