Development and Validation of Ase Extraction Compared to Standard Spe Technique in Forensic Toxicology: A Study on Whole Blood Samples for Psychoactive Drugs, Antagonists, Medications, and Anesthetic

Sample extraction procedures are often considered as a bottle-neck step in analytical methods. Many techniques have been used with the purpose of achieving greater efficiency, a reduction of the time spent for the extraction and of the amount of solvent used. The Solid-Phase Extraction (SPE) is still considered as a reliable extractive technique due to its many advantages compared to the other traditional methods, such as liquid/liquid extraction [1], reducing the use of great amounts of solvent, the operation time/procedure steps, and others [2]. Among the various fields in which SPE is applied, the forensic-toxicological field still takes great advantages and is therefore widely used in forensic laboratories [3]. Solid-phase extraction is a well-known and tested method of preparation for samples involved in several scientific fields and represents a fundamental column in toxicological analyses. This type of extraction is based on a principle similar to the partition chromatography, in which several stationary phases were developed for more targeted extractions [4]. Different matrices can be collected for toxicological analysis (blood, urine, bile, hair, etc.) and required a thoughtful preparation of the specimens influencing the outcome and the reproducibility of the analysis. Over the years, SPE has become a performant method in the extraction of molecules and nowadays is used as a standard procedure in forensic field in many laboratories around the world. It allows to efficiently purify and concentrate the samples before performing a liquid or gas chromatographic analysis. Indeed, its impact on the quality of the analysis is greater and it allows concentrating the substances enabling the detection and dosing in a biological matrix excluding endogenous substances. In some cases, it allows also to target the extraction of several compounds permitting the detection of substances that can be considered difficult to extract and would not be detectable with standard procedures. However, this technique requires a fair amount of time, is operator-dependent, has high costs and is subjected to possible human mistakes that could invalidate the extraction [5]. The Accelerated-Solvent Extraction (ASE) is an alternative automatized procedure for the extraction and purification of substances from biological matrices, commonly used in non-forensic laboratories treating, mostly, animal and vegetable samples [3,5,6]. ASE uses operator-independent protocol, decreasing the bias due to operator-dependent procedures, process a highest number of samples in sequence, it improves the extractive processes, reduces the time required for multiple extractions and increase the samples throughputs. This technique uses organic solvents at variable pressures and temperatures above the boiling point, creating a time-saving procedure with low consumption of solvents [7-11]. With this procedure, a sample is enclosed in a sample cartridge that is filled with an extraction fluid and used to statically extract the sample under elevated temperature (50°C-200°C) and pressure (500 psi-3000 psi) conditions in a short time period (15-25 minutes). The compressed gas is used to purge the sample extract from the cell into a collection vessel. Many studies have validated the ASE extraction method in animal [12-19] and botanical [7,8,20-28] field. However, there is a paucity of studies regarding the ASE extraction validation on human biological samples, limited to the meconium [29,30], bone matrix [31,32] and blood samples [33,34], searching for cocaine, dioxins, fatty acids and nicotine and cotinine. For this reason, the purpose of this study was to investigate, under a validation method from qualitative and quantitative point-of-view, whether the accelerated-solvent extraction technique can be used in substitution of the operator-dependent solid-phase extraction in forensic toxicological field. Its reliability is tested on a large number of substances and on whole blood matrix commonly used in forensic toxicological analysis.