# Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures
## 2 Biological Fluids for Forensic Analyses
### Blood
Their study aimed to develop a green method, minimizing handling, costs, and use of toxic solvents without damaging the accuracy of the analysis. The single expected step was to precipitate proteins using a low concentration of methanol that was added to the sample. The mixture was vortexed and centrifuged to obtain a clear supernatant that was diluted with an aqueous solution (0.1% formic acid) and injected into liquid chromatography tandem mass spectrometry (LC-MS/MS) instrumentation [28]. [PERSON] et al. discussed in their review that ionic liquids are now considered the main alternative to extraction solvents, which are usually toxic for operators. These ionic liquids can be used in a wide range of applications, including the treatment and extraction process of biological samples before forensic analysis [29]. [PERSON] et al. have used ionic liquid-based liquid-liquid microextraction to extract benzodiazepines from the blood for LC-MS/MS analysis. They used BMIm PF6 as an ionic liquid added at 1 mL of blood and dispersed with agitation to promote the extraction by the ionic liquid phase. After the sample was centrifugated, two phases were obtained (upper blood phase and ionic liquid lower phase), and the lower phase was collected and analyzed [30]. [PERSON] et al. used whole blood to determine the concentration of alcohol in 31 forensic cases using GC-FID. Before analysis, blood samples collected from the femoral vein and lyophilized were stored with the addition of sodium fluoride. After reconstitution using water, they were put into hermetic vials where different ethanol concentrations were added. All samples were vortexed and then injected into GC. The investigation and quantification of post-mortem ethanol levels hides various pitfalls, due to the characteristics of the molecule, which is absorbed and rapidly distributed throughout the body, and above all, by some factors such as the production of alcohol by bacteria which can distort the quantified levels. We consider this work important because it highlights the problems of this type of investigation and illustrates the solution [31]. [PERSON] et al. demonstrated the superiority of novel extraction methods using ISO-LUTE PDL+ compared with another extraction method, such as protein precipitation or QuEChERS, in the pretreatment of whole blood for forensic applications. The authors have used this phospholipids and protein removal column to clean up the blood before determining psychoactive drugs in several forensic cases. The sample was diluted with sodium dihydrogen phosphate/disodium hydrogen phosphate, after which internal standard, acetonitrile, and methanol were added. The mixture was loaded in the column and inserted into a centrifuge. After centrifugation, the purified analytes were collected on the bottom of the tube, where 100 uL of 0.1% trifluoracetic acid in acetonitrile was added. After evaporating to dryness, the sample was reconstituted with methanol for analysis [32]. [PERSON] et al. used a new type of extraction based on the use of substrate coated with sol-gel sorbent called fabric-phase sorptive extraction (FPSE) in the analysis of seven antidepressants in whole blood. This innovative extraction technique patented by [PERSON] and [PERSON] has been used over the years in different fields thanks to application versatility and high-performance efficiency, which overcomes the disadvantages of classical techniques [33; 34; 35; 36; 37; 38]. In this work, [PERSON] et al. applied FPSE in the forensic field directly on the collected blood avoiding further pretreatment (e.g., protein precipitation, filtration, etc.). They tested eight different coated membranes and other parameters such as sorbent chemistry, time of extraction, and sample volume extraction solvent. The authors demonstrated that the membrane coated with Carbowax 20 M for 20 min. of extraction time represents the most efficient solution, so they chose this membrane for the real sample application.