Novel strategies for the analysis of drugs of abuse
Creators: name: El Shamaileh, Majed
Publisher: name: University of Lincoln
Year: 2023
Subjects: F410 - Forensic science

description: The data presented in this thesis has been organized in three parts: First part included the development and validation of a quantitative HPLC-DAD analytical method of mephedrone after extraction from spiked whole blood and serum samples, alone and with methcathinone. The second part included in vitro metabolism of mephedrone and other NPS, which are methoxetamine and methcathinone, using an in-house prepared in vitro metabolic system, namely liver microsomes, followed by performing analysis for the drugs and their proposed metabolites utilizing LC-MS. Third part included in vitro studies of selected NPS using purchased HepaRG and hepatocytes. In vitro study included in vitro cytotoxicity studies of 4-fluoromethamphetamine, mephedrone, methoxetamine and methcathinone, and analytical studies of these drugs of abuse and their potentially produced metabolites using GC-MS.In the first part of this thesis, a HPLC method for the analysis of mephedrone after LLE from blood matrix was developed and validated and shown to be linear with R2&gt; 0.995, precise with intraday and interday RSD values of 4.36 and 4.77% respectively and LOD and LOQ of 0.025 and 0.082 ?g/mL respectively. Recovery percentages were low and ranged between 28-37%. Emulsion formation was the major problem effaced which negatively affected recovery and precision values. The previously developed method was optimised and fully validated for the simultaneous analysis of mephedrone and methcathinone after liquid-liquid extraction (LLE) from whole blood and serum samples. The LLE method was optimised through selection of extraction solvent and adjustment of pH values achieving the best validation parameters and minimal emulsion formation. The LLE protocol involved extraction with a mixture of dichloromethane: n-butanol (80:20 v: v) after buffering the sample with borate buffer pH=9.2 and using aniline as internal standard. The HPLC-DAD method was optimized, using reverse mode chromatography and buffered mobile phase of (acetate buffer pH 4.1: ACN – 85:15) for qualitative and quantitative analysis of these drugs in less than 10 minutes under isocratic elution and ambient temperature. The method was fully validated for both drugs and showed to be linear over the specified range of 0.1-10 ?g/mL with R2 &gt; 0.99 for both drugs. The accuracy was assessed by calculating percentage recovery at different concentrations forxiiboth drugs, and retained recovery percent between 84-110%. For repeatability and intermediate precision tests, RSD values were ? 6.73%. Specificity was assessed by good resolution between the peaks and by checking peak purities. Limit of detection and limit of quantification, calculated mathematically for both drugs either extracted from whole blood or serum samples, were 0.010- 0.013 ?g/ml and 0.032 - 0.043 ?g/mL, respectively.In the second part, in vitro studies on the metabolism of the selected NPS using pig liver microsomes and liquid chromatography-mass spectrometry (LC-MS) analysis were performed. Microsomes were prepared by a conventional ultracentrifugation method. In brief, pig liver was brought freshly from local abattoir, sliced into small pieces, homogenised and ultra-centrifuged to produce microsomes and S9 fractions. Produced microsomes were incubated with the drugs of interest under optimised conditions and followed by analysis utilizing LC-MS for the detection of the drugs and the potentially produced metabolites. It was possible to detect two metabolites of the drug mephedrone, hydroxytolyl-mephedrone and nor-dihydro mephedrone. For MXE, one metabolite produced by the O-demethylation was detected and it identity confirmed by MS/MS study to be o-desmethyl-MXE. Another metabolite was detected is suggestively produced by the reduction of the ketone moiety to produce dihydro-MXE or by two steps of O-demethylation and hydroxylation to produce O-desmethyl –hydroxy-MXE. However, due to low intensity signal recorded, MS/MS study was not conclusive for the identity of the moleculeIn the third part, two types of hepatocytes were used for the study of the metabolism and cytotoxicity of the selected NPS - Mephedrone, Methoxetamine, Methcathinone and 4-Fluoromethamphetamine. Studying the metabolism of selected NPS followed utilizing HepaRG™ followed by GC-MS analysis, it was possible to detect new peaks in the chromatograms of mephedrone and methcathinone which is suggestively the product of N-demethylation. However, it was not possible to detect any new peaks in the chromatograms of methoxetamine nor 4-flouromethamphetamine. The cytotoxicity study utilizing HepaRG cell line showed that these drugs have cytotoxic effects causing in vitro cell death, within the specified range of 4.0x10-2-1.6x101 mM. These drugs were able to cause 43-83%iicell death, and EC50 values were 0.2323-0.6297 mM. The most potent drug was 4-fluoromethamphetamine, while mephedrone showed the least biological effect to produce.