# Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures
## 2 Biological Fluids for Forensic Analyses
### Blood  
Traditionally, blood was considered the main biological matrix to extract and determine drugs or other substances in the clinical and forensic fields. Usually, the literature calls this \"whole blood\" to denote that it refers to blood and its cellular components and to differentiate it from serum or plasma in which solid components have been eliminated. In forensic toxicology, the determination of substances in the blood is strongly affected by their half-life, time, and mode of administration, condition of the corpse, and redistribution phenomena. Analyses are often carried out on blood obtained from peripheral sites, such as the femoral vein, but in some critical situations, as exsanguination caused by severe injury, specimens are collected from the chest or the abdominal cavity considering the substantial differences between the \"two types of blood\". Numerous studies have been conducted to assess the differences between peripheral blood and cardiac blood, which is more abundant and allows the identification of those substances that are extensively metabolized [3,20]. After sampling, the critical stage is the treatment of the sample to extract the analytes of interest, reducing the matrix effect and improving the accuracy of the analysis. The techniques most used in this subject area are protein precipitation, solid or liquid extractant phase extraction, and microextraction. However, the main goal in our day is to simplify these procedures as much as possible to reduce time and solvent consumption [21]. These classic techniques have been revised, modified, and automated to reduce the number of operations performed and optimize the extraction process [22]. [PERSON] et al. have modified QuECHERS extraction to detect some critical drugs and poisons in the forensic field. Their method consists of two main steps: firstly, a fraction of whole blood collected was diluted with distilled water and put into a plastic tube (pre-packed Agilent SimplyQ) containing solid sorbent phase shacked and centrifugated to promote separation of two phases (aqueous layer and acetonitrile layer), with the supernatant directly injected to analyze acidic drugs. The second step, required for basic drugs, consisted in dispersive solid-phase extraction (d-SPE) using a pre-packed tube containing primary-secondary amine, end-capped octadecylsilane, and magnesium sulfate. An aliquot obtained of supernatant by first extraction was added into the tube, mixed, and successfully centrifugated to clean up the blood. The supernatant was utilized for quantification in LC-MS/MS. This method foresees very short times compared to the execution of a SPE cation exchange (necessary for the separation of acidic and basic analytes). It is also important to underline the versatility of the method, which can be applied in the same way even by inexperienced operators without major variations in the results, as also discussed by the authors [16]. [PERSON] et al. have also tried applying d-SPE to extract drugs and pesticides from blood in postmortem cases. The authors evaluated two extraction solvents (acetonitrile and ethyl acetate) and compared the method with solid-liquid extraction low-temperature partitioning (SLE-LTP) regarding selectivity, matrix effect, cleaning, stability of the sample, and operator precision. The two methods were carried out using the same sample and first acetonitrile and then ethyl acetate, and it has been shown that the use of acetonitrile caused less interference from lipids and fatty acids and that the recovery of analytes was maximum for d-SPE compared to SLE-LTP [23]. [PERSON] et al. used solid phase extraction to clean up blood specimens obtained from the femoral vein and heart during their study aimed at identifying synthetic cathinones using LC-MS/MS. For the extraction that was used, mixed-mode cation exchanges cartridges were to retain basic ionized substances. All samples were firstly vortexed, sonicated after adding a phosphate buffer, and then centrifugated.