Chiral and stable isotope analysis in forensic chemistry: novel psychoactive substance analysis

Synthetic cathinones, a group of novel psychoactive substances, have appeared in drug markets worldwide, causing a range of serious health risks and social problems and hence are controlled by legislation in many countries. Intelligence of synthetic cathinones has become important for law enforcement agencies to prevent the spread of these products. However, literature findings about synthetic cathinones focused on identification and quantification of these compounds in their illicit products and biological specimens. Limited studies have been published concerning the intelligence of this group of novel psychoactive substances. Two analytical techniques, chiral and stable isotope analysis, were hypothesised to have the potential of providing intelligence of illicit synthetic cathinone products due to possible variations of relative enantiomeric abundances of synthetic cathinones and relative stable isotopic abundances of any chemicals among the products. Therefore, analytical methods were aimed to be developed in this thesis for performing the two types of analysis to study their potential of providing intelligence of illicit synthetic cathinone products. To study the potential of utilising chiral analysis for providing intelligence, two chiral analytical methods involving chromatography and mass spectrometry were investigated for their ability of determining relative enantiomeric abundances of some synthetic cathinones. In both methods, chiral derivatisation was performed before instrumental analysis. In one of the methods, gas chromatography–mass spectrometry was involved, and thirteen out of fourteen tested pairs of synthetic cathinone enantiomers could be baseline separated using gas chromatography after being derivatised by the reagent, (R)-(−)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride. Eight out of the thirteen were further subjected to optimise the derivatisation and instrumental conditions for the method to perform quantitative chiral profiling of them. Method validation results showed that this method was able to quantify five out of the eight pairs of the individual enantiomers accurately and precisely across an approximately 10-fold dynamic range when a quadratic calibration model was used with appropriate deuterated synthetic cathinones as the internal standards. The limits of detection and quantification were ranged from 0.0075 to 0.025 μg and from 0.0225 to 0.05 μg respectively, which are low enough for illicit product analysis. The mass spectrometric method was also found to give the required specificity for illicit synthetic cathinone product analysis. In the other method, high performance liquid chromatography–tandem mass spectrometry was involved, and eleven out of fourteen tested pairs of synthetic cathinone enantiomers could be baseline separated using high performance liquid chromatography after being derivatised by the reagent, (1R)-(−)-menthyl chloroformate. Three out of the eleven, including two which were not enantio-separated well by the method involving gas chromatography–mass spectrometry, were further subjected to optimise the derivatisation and instrumental conditions for the method to perform quantitative chiral profiling of them. Method validation results showed that this method was able to quantify one of the three pairs of individual enantiomers accurately and precisely across an approximately 67-fold dynamic range when a quadratic calibration model was used with the exact deuterated analogues as the internal standard. The limits of detection and quantification were 0.001 μg and 0.015 μg respectively, which are low enough for illicit product analysis. The tandem mass spectrometric method was also found to be highly specific for illicit synthetic cathinone product analysis. Despite not a large set of illicit products were obtained as samples to study the potential of utilising chiral analysis for providing intelligence, eight illicit products were analysed to see if any synthetic cathinone with varying relative enantiomeric ratios could be found. All samples were analysed by the method involving GC–MS and were found to be synthetic cathinones. The identities of synthetic cathinones in the samples were further confirmed by 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopy. Four different synthetic cathinones were identified, with two newly identified (not one of the fourteen which were studied in method development) members also being baseline enantio-separated by the method. Three samples which were identified as 4-methylmethcathinone, were found to be racemic and hence one could not exclude the possibility that they were produced from the same source. Due to limited number of samples obtained for the study, it is unclear if a wide range of variation of relative enantiomeric abundance of a synthetic cathinone can be observed among illicit products and hence whether chiral analysis could be useful for providing intelligence. Therefore, more samples collected from various known sources should be analysed in future to study the variation of relative enantiomeric abundances of synthetic cathinones. However, the applicability of one of the developed methods has been demonstrated and both gas chromatography and high performance liquid chromatography mass spectrometry are capable for quantitative chiral analysis of various synthetic cathinones. On the other hand, to study the potential of utilising stable isotope analysis for providing intelligence, analytical methods involving gas chromatography–isotope ratio mass spectrometry were developed, and caffeine, an adulterant commonly found in illicit synthetic cathinone products, was targeted for analysis to determine if stable isotope signature of it could be utilised to distinguish the products which contains it as an ingredient. Commercial sport supplements, which are readily available and may contain caffeine as an ingredient, were used to study the potential of this approach for intelligence of illicit synthetic cathinone products. Methods of carbon and nitrogen stable isotope analysis of caffeine in sport supplements were developed and validated. Both carbon and nitrogen stable isotope ratio measurements for caffeine were reproducible and independent of the sample amount used for analysis within the dynamic range of about 10s – 100 mg. The extraction method was found to be efficient without causing stable isotope fractionation. In addition, method of hydrogen stable isotope analysis of caffeine was also investigated and found to be reproducible, although the method validation has not yet completed. Six brands of sport supplements were analysed by the methods. Relative carbon and nitrogen stable isotopic abundances of the caffeine in these products were found to agree with other findings of pure caffeine and various products containing caffeine. The six brands of sport supplements were all distinguishable from each other by comparing the caffeine carbon and nitrogen stable isotope signature, but supplements of a single brand were not distinguished. To conclude, the potential of utilising caffeine stable isotope signature for providing intelligence of illicit products which contain caffeine as an ingredient, was observed. Real illicit synthetic cathinone products, which contain caffeine, with known sources of production should be analysed in future to determine the usefulness of the approach

Antivirulence Agent as an Adjuvant of β-Lactam Antibiotics in Treating Staphylococcal Infections

Staphylococcus aureus can cause a plethora of life-threatening infections. Antibiotics have been extensively used to treat S. aureus infections. However, when antibiotics are used at sub-inhibitory concentrations, especially for β-lactam antibiotics, they may enhance staphylococcal pathogenicity and exacerbate the infection. The combination of antivirulence agents and antibiotics may be a novel approach to controlling antibiotic-induced S. aureus pathogenicity. We have illustrated that under in vitro conditions, antivirulence agent M21, when administered concurrently with ampicillin, suppressed the expression and production of virulence factors induced by ampicillin. In a mouse peritonitis model, M21 reduced bacterial load irrespective of administration of ampicillin. In a bacteremia model, combinatorial treatment consisting of ampicillin or ceftazidime and M21 increased the survival rate of mice and reduced cytokine abundance, suggesting the suppression of antibiotic-induced virulence by M21. Different from traditional antibiotic adjuvants, an antivirulence agent may not synergistically inhibit bacterial growth in vitro, but effectively benefit the host in vivo. Collectively, our findings from this study demonstrated the benefits of antivirulence–antibiotic combinatorial treatment against S. aureus infections and provide a new perspective on the development of antibiotic adjuvants.Science, Faculty ofNon UBCMicrobiology and Immunology, Department ofReviewedFacultyResearche

SaeR as a Novel Target for Antivirulence Therapy against Staphylococcus aureus

ABSTRACT:Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilising a GFP-Lux dual reporter system driven by saeP1 promoter. ,. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and hemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR's DNA-binding domain When saeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target's specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure-activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four-times more potent than the HR3744 and demonstrated identical antivirulence property and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence property, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections