# Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures
## 2 Biological Fluids for Forensic Analyses
### Vitreous Humor  
Vitreous humor is one of the most widely used matrices under conditions where blood or urine do not provide the necessary information or are unavailable. This fluid is composed primarily of water and other components such as lipids, proteins, and electrolytes. It is taken with a thin needle between the crystalline lens and retina during the autopsy. Compared with other matrices (blood or solid matrices), vitreous humor is a clear matrix; thus, it contains fewer interferences and is sheltered from bacterial contaminations [46]. This is a significant advantage, especially in some situations, as discussed by [PERSON] et al. in their work about the determination of ethanol in postmortem specimens, where they have highlighted that in some cases, quantification of alcohol can be altered by microbial activity during putrefraction time capable of producing ethanol with the fermentation of glucose. In this context, the authors have shown the correlation between ethanol concentration in blood and vitreous humor, confirming the possibility of using vitreous humor as an alternative to other matrices [31]. Many classes of drugs can be detected in this matrix, such as benzodiazepines, opioids, cocaine, and their metabolites. However, the determination of compounds that are highly protein-bound is more difficult [20]. Techniques used to treat and clean up vitreous humor samples are the same as other fluid matrices, but the operations and results can be more accurate by being a cleaner matrix. [PERSON] et al. used a simple protein precipitation protocol to treat postmortem samples while developing their method for simultaneous quantifying benzodiazepines. 150 uL of the sample was mixed with 200 uL of acetonitrile:acetone:methanol (1:1:1, _v:v:v_) and internal standard. The mixture was vortexed and centrifugated to obtain the extract for analysis [47]. [PERSON] et al. treated vitreous humor with dispersive liquid-liquid microextraction before the analysis. They compared the results with the same protocols using a urine sample to quantify methadone and tramadol in forensic cases. Briefly, the sample was added into a vial containing chloroform as extraction solvent and methanol as dispersion solvent. The mixture was firmly vortexed to facilitate the dispersion of the two phases. The precipitate phase (extraction solvent) obtained after centrifugation was taken and evaporated, and the dry product was reconstituted with methanol for analysis in UHPLC and GC-MS [48]. Interesting research was conducted by [PERSON] et al., who have validated an automated process based on immunoaffinity to detect insulin analogous to postmortem vitreous humor. Finding insulin analogs is a very ambitious task due to the size and complexity of these molecules and their close similarity with endogenous insulin, which often requires lengthy and laborious work. The authors proposed a method using robotic immunoaffinity-micro chromatography followed by the separation of alpha and beta chains, followed by only beta chains mass analysis. The insulin extraction from the sample was conducted using Agilent AssayMap, and the micro chromatography was obtained using two monoclonal mouse anti-insulin antibodies immobilized on cartridges. The insulin was purified in the column and eluted with acid conditions [49]. [PERSON] et al. validated a method to study the distribution of antidepressants in postmortem humor vitreous, using for the extraction of analytes a solid-phase extraction containing a mixed mode sorbent (C8 nonpolar sorbent and strong cation exchange sorbent). 1 mL of the unknown sample was added to the internal standard, and 5 mL of phosphate buffer (pH 6). All were vortexed and centrifugated to obtain the supernatant loaded onto the column. For the wash, deionized water was used, and then 1 mL of acetic acid was added and rewashed with methanol before being dried under a vacuum.