Development and validation of a method for quantification of new psychoactive substances in post-mortem samples by micro-QuEChERS and LC-MS/MS

Abstract

The group of drugs of abuse known as new psychoactive substances (NPS) is composed of substances derived from or analogous to classic drugs of abuse, produced to mimic their effects while circumventing law enforcement agencies. NPS represent a major public health problem, with a large number of substances being made available on the illicit market in a short period of time, an increase in the chemical diversity of these compounds, and a lack of knowledge about their acute and chronic toxic effects on humans, leading to hospitalizations and deaths of users following severe intoxications by these agents. The increasing emergence of new compounds and unknown risks of harm to users lead to the challenge of developing new analytical methodologies to detect and identify these substances, and to establish toxic and lethal concentrations. The complex analytical framework of forensic laboratories involves the analysis of a large number of possible toxicants present in extremely varied concentrations in complex biological samples. Instrumental tools must provide flexibility, sensitivity, selectivity, and reliability for detection and determination of multiple targets in these complex matrices where the analyte is usually found in low concentrations. To extract compounds from biological matrices, forensic laboratories generally use conventional sample preparation methods, such as liquid-liquid extraction or solid phase extraction, where there is a high consumption of biological material and organic solvents. Thus, there is interest in the continuous search for new sample preparation techniques that are fast, efficient, automatable and environmentally friendly. Techniques based on microextraction processes are being developed as an alternative to classical methods. These offer major advantages over conventional techniques, such as lower reagent consumption and consequently lower operating costs for the laboratory with the acquisition and disposal of these chemicals; short extraction time; minimum necessary sample volume, important in cases of biological samples with limited availability and; potential automation, which becomes highly relevant in laboratories that need to analyze hundreds of samples per day. In addition, these techniques are allied to the principle of Green Analytical Toxicology, which, analogous to Green Analytical Chemistry (GAC), promotes the development of alternative analytical methods that seek to reduce the use of organic solvents, which can be harmful to both analysts and the environment, as the use of increasingly smaller volumes of biological samples, minimizing the amount of biological material collected (which is often scarce) and the disposal of infectious materials. Among the microextraction techniques, the micro-QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) technique for analyte extraction is simple and fast, requires a low sample volume, is suitable for compounds with different physicochemical properties, provides high yield extractions and is environmentally friendly due to low solvent consumption. The objective of this project is to develop methods for forensic toxicological analysis using the micro-QuEChERS technique for preparing post-mortem blood, urine, and vitreous humor samples. The method for identifying and quantifying NPS will be applied to real biological samples, where the levels found will be correlated with the effects caused by these drugs. Furthermore, the results will be evaluated against traditional extraction techniques as well as the practical applicability of the new techniques in routine forensic toxicology laboratories. (AU)