# Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures
## 2 Biological Fluids for Forensic Analyses
### Blood
After these preliminary steps, the sample was inserted in a column, and after washing with acetic acid and methanol, the analytes were eluted with dichloromethane:isopropanol:ammonium hydroxide (93:5:2, _v:v_). Theuate was dried and, after reconstitution, was injected for analysis. Based on our knowledge, this validation method appeared to be the most sensitive among those found in the literature. Thanks to the careful choice of mixed-mode cation exchange cartridges, based on the chemical properties of the analytes sought, the authors obtained a limit of quantification (LOQ) around 1 ng/mL using only 0.25 mL of blood [24]. [PERSON] et al. have validated a modified mini-QuECHERS to extract 15 psychotropic drugs from blood samples obtained after autopsy. The extraction method was optimized using different pH conditions. First, potassium carbonate (for basic condition), sodium chloride (for acid condition), and magnesium sulfate were mixed with 100 \(\mu\)L of sample and 600 \(\mu\)L of acetonitrile. Then, the mixture was vortexed and centrifugated, and the supernatant was dried. The use of basic conditions showed the best results, using acetonitrile and excluding other solvents such as cyclohexane, butyl acetate, and dichloromethane. This single-step method was also compared with two steps of extraction that consisted of the pretreatment of the sample as described above and a second clean-up step with magnesium sulfate and primary secondary amine, which, however, caused a lower recovery of the analytes [25]. [PERSON] et al. have applicated the dried blood spot method to detect a wide range of drugs of abuse. This method is already widely used in clinical analysis due to its minimal invasiveness. The authors have promoted its use in forensic analysis, allowing a further reduction of solvents and time. This study used Bond Elut Dried Matrix Spotting cards from Agilent, where three drops of the mixture of blood and internal standard were proposed and allowed to dry at room temperature. Three spots formed were cut and put into a tube with methanol (0.1% of formic acid), and after the appropriate time, were centrifugated, and the supernatant was dried [22]. [PERSON] et al. have used liquid-liquid extraction (LLE) to treat postmortem whole blood to detect cocaine and its metabolites and cocaine crack biomarkers, exceeding the limits of other techniques such as SPE and protein precipitation that led, firstly, to too high costs, the secondly, to a sample cleaning process which was sometimes not sufficient. For the extraction, 100 \(\mu\)L of blood, 25 \(\mu\)L of methanol:water (50:50, _v:v_), and 25 \(\mu\)L of internal standard was mixed, after which was added 100 \(\mu\)L of carbonate buffer (pH 9.3) and 1 mL of a mix methyl tert-butyl ether:2-propanol (70:30, _v:v_). After this, the mixture was vortexed, centrifugated, and the upper phase was dried. There are numerous applications of this technique for the extraction of cocaine and metabolites from biological matrices, but they often involve the use of very toxic chlorinated solvents. It was interesting to note how in this case the use of these solvents was bypassed [26]. [PERSON] developed a method to evaluate the presence of tricyclic antidepressants and benzodiazep in postmortem blood using a microextraction by packed sorbent coupled to electrospray-time of flight mass spectrometry ((MEPS)-ESI-TOF-MS). Before putting the sample in MEPS, the blood was deproteinized by adding acetonitrile and the mixture was vortexed and centrifugated to obtain two phases. The supernatant (acetonitrile layer) was dried to eliminate the acetonitrile, which is incompatible with MEPS. The dried residue was reconstituted with acid water (formic acid, pH 3), loaded in MEPS for clean-up, and directly analyzed [27]. [PERSON] et al. have proposed a new fast method for qualitatively detecting illicit drugs and drugs in postmortem blood samples and other matrices for rapid and reliable screening.