NEW METHODOLOGIES FOR THE DETERMINATION OF AMMONIUM AND SYNTHETIC CANNABINOIDS IN FORENSIC SAMPLES

Forensic science is a discipline that combines the knowledge of chemistry, biology, pharmacology, statistics, mathematics, physics, and medicine, among others. The present doctoral thesis was focused in two areas of this wide discipline. In post-mortem chemistry, the estimation of the post-mortem interval (PMI) is done through the determination of potassium with capillary electrophoresis (CE). However, it is limited up to 100 hours, after which potassium levels reach a plateau. In forensic toxicology, the elucidation of metabolic pathways of emerging Novel Psychoactive Substances (NPS) is highly important to include those substances in screening methods that prove their consumption. Also, the consideration of different matrices for rapid NPS screening is another interesting topic for when the most common matrices, such as blood and urine, are unavailable for testing or are not enough. Therefore, in the present PhD thesis, three new methodologies are presented for the analysis of ammonium and synthetic cannabinoids in a variety of forensic samples. ¨ The objective of the first project was the validation of a capillary electrophoresis (CE) method for the determination of ammonium in vitreous humour and the improvement of the estimation of the post-mortem interval (PMI). Vitreous humour samples were collected from a total of 38 medico-legal autopsies or external examination of corpses of violent or sudden deaths, in which the exact time of death was known. The vitreous humour samples were prepared by diluting 1:20 with the IS solution prior to analysis with capillary electrophoresis instrument. The results were analysed and interpreted on the basis of different statistical analyses: linear and polynomial regression and artificial neural networks (multivariate statistics). The findings show a wider PMI window of up to 168 hours which is useful for a much wider time span than that offered by potassium analysis (limited to about 90–100 hours). Furthermore, the results show the potential usefulness of using ammonium and potassium in combination with ANN for inferring the PMI. ¨ The aim of the second project was the elucidation of the 5F-APINAC metabolic pathway, which was a recently emerged NPS. Firstly, the possible metabolites were theoretically sketched using ChemDraw and scientific articles about substances with similar chemical structures. Secondly, in vitro experiments with HLMs and in vivo ones with rat urine after intravenous administration of the drug were carried out. Thirdly, samples were prepared for analysis with the Toxtyper instrument (LC-IT-MS) and results were confirmed by LC-QTOF. The theoretical metabolites were confronted with the experimental results and the metabolic pathway was elucidated. The metabolic pathway of 5F-APINAC was elucidated finding 15 different metabolites. It was found out that ester hydrolysis was the predominant metabolic reaction found in both in vitro and in vivo experiments. The discovered metabolites may serve for future studies and are likely to be incorporated into routine analytical screening methods as urine markers of 5F-APINAC consumption. ¨ The objective of the last project was the development and validation of a fast and sensitive method for the screening of 13 new synthetic cannabinoids in human hair. The method was developed using the Toxtyper® LC/IT–MS system with different sources (ESI, APCI, and IonBooster) and validated in terms of precision, bias, sensitivity, and matrix effects. The blank hair samples were spiked with 13 synthetic cannabinoids and underwent ultrasonic extraction. In addition, real hair samples from patients that have tested positive for other illegal drugs were tested. The results show the suitability of the developed methodology for the screening of 13 synthetic cannabinoids in human hair. In addition, it is high throughput and can be easily updated with the appearance of new synthetic cannabinoids in the market.