# Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures
## 2 Biological Fluids for Forensic Analyses
### Urine  
Urine, together with blood, is commonly used in forensic toxicology, as urine also represents a deposit of drugs, illicit drugs, and other substances and metabolites that may provide information on the victim's exposure before death. In forensic cases, urine can be obtained with a withdrawal from the bladder and successively analyzed, considering that the urine contains less protein and lipids than blood, resulting in it being more accessible and faster to treat. [PERSON] et al. investigated the presence of new psychoactive substances in both blood and urine collected postmortem, and to compare the two matrices, they applied the same treatment. Using the QuEChenERS method, they tested four extraction protocols with different amounts of acetonitrile and water added to an aliquot of urine. All were vortexed, and then magnesium sulfate was added, as sodium acetate, re-vortexed and centrifuged. The supernatant was separated and put into a vial containing primary secondary ammine and magnesium sulfate and once again vortexed and centrifugated to clean up the sample. Finally, the supernatant obtained was dried and resuspended prior to analysis [40]. [PERSON] et al. compared three different extractions and clean-up methods to determine the concentration of insecticides and fungicides in postmortem urine samples. Simple protein precipitation was carried out by adding 150 uL of methanol: water (50:50, _v:v_) into the sample, and then the mixture was centrifugated, and the supernatant was separated from the precipitate. For solid phase extraction, 2 mL of urine was loaded on a C18 cartridge, and after washing with water, the analytes were eluted using 1.5 mL of methanol. For dispersive solid phase extraction (d-SPE) were tested four different protocols were used. However, a large number of operations to be conducted led the authors to discard this method of extraction, which seemed to induce errors. SPE method did not ensure sufficient recovery of analytes, so protein precipitation was considered the best technique [41]. [PERSON] et al. in their work already discussed in the \"blood section\" have also used urine to develop screening methods for illicit substances. In this case, urine was processed with enzymatic hydrolysis of any metabolites present. To 500 uL of the sample was added 100 uL of enzymatic solution (glucuronidase), which was left in incubation at 60 \({}^{\circ}\)C for 3 h. After dilution with a solution of internal standard, an amount of sample was directly injected into LC-MS/MS. The use of a minimum volume of enzymatic hydrolysis and non-toxic solvents is the real novelty represented by this work, which differs from the others present in the literature [28]. [PERSON] et al. developed a new type of headspace solid-phase microextraction using carbon nanotubes as a sorbent to increase the effectiveness of extraction of amphetamine-type stimulants from postmortem urine. The work involved the preparation of this sorbent and covering appropriate fiber as supporting material and then the headspace solid phase microextraction. Briefly, 5 mL of diluted urine was placed into a vial under stirring with an aluminum cap and then into a thermostatic bath (80 \({}^{\circ}\)C) after the syringe needle of SPME containing the coated fiber was inserted through the aluminum cover for the time of extraction (10-60 min). Then the coated fiber was subjected to thermal desorption at 250 \({}^{\circ}\)C prior to GC analysis. It is interesting to note how the study and development of such an efficient sorbent of this type can increase the performance of an already very high-performing extraction technique like SPME [42]. In recent work, [PERSON] et al. validated a method to discover novel zolpidem metabolites in urine samples collected from victims of fire who consumed Myslee(r) 5 mg (5 mg of zolpidem per tablet). The urine was centrifuged after adding 0.1% acid formic in acetonitrile, and the collected supernatant was dried and resuspended with methanol/water.