# Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures
## 3 Main Solid Matrices for Forensic Purposes
### Skeletal Tissue  
In cases of severe decomposition, fragmentation, or skeletonization, the analysis of bones is the only source to determine past exposure to drugs and other substances. However, skeletal tissue is considered a complex matrix, so the extraction of drugs is much more complicated than conventional matrices. [PERSON] et al. have developed a method to determine the presence of most drugs of abuse in bone obtained from autopsies. For extraction of the analytes from the matrix was applied a simple method already validated by [PERSON] et al.; briefly, the bone was cleaned, and any remains of another kind of tissue were removed, then to 1 g of bone was added 3 mL of methanol, after which the mixture was adjusted to pH 10 with ammonium hydroxide. The mixture was stirred for 5 h and was put into an ultrasonic bath for 1 h. When the extraction was completed, the sample was centrifugated, and the supernatant was dried under a stream of nitrogen and reconstituted before analysis [68; 69].  
[PERSON] et al. have also used methanol to extract opioids and their metabolites from skeletal tissue. First, they validated the method using animal specimens, evaluating the distribution of methadone and its metabolite in bones and other work evaluating the distribution of clomipramine, citalopram, and midazolam after chronic dosing in rats [70; 71; 72]. Particularly interesting is the study of [PERSON] et al., in which a method for quantifying psychotropic drugs in bone marrow was developed. They used Direct Immersion Solid-Phase Microextraction (DI-SPME) with liquid chromatography coupled with a mass spectrometer (LC-TOF-MS). The work aimed to optimize all the parameters to obtain the maximum extraction with DI-SPME, such as sample volume, absorption time, post-absorption washing, and desorption time [73].  
[PERSON] et al. have developed a method for the quantification of antidepressants in human bone marrow aspirated. This method has provided microwave-assisted extraction that includes several steps, illustrated in Figure 2, after which the analysis was conducted in UHPLC-TOF-MS [74].  
Figure 2: The sample preparation procedure of human bone marrow aspirate.  
[PERSON] et al. have used human bone as a matrix to detect carbamazepine in one case of overdose after psychiatric conditions. After cleaning and pulverizing bone, the powder was vortexed with 2 mL of methanol and then incubated for 1 h under ultrasounds. The mixture was centrifugated; the supernatant was dried and resuspended in phosphate-buffered saline pH 6 for solid-phase extraction [75].  
Additionally, dry bone samples of people who have died over 23 years were used by [PERSON] et al. to detect some drugs. For this study, they used samples from the cranium, vertebrae, and ribs. After pulverization, all specimens were subjected to accelerated solvent extraction (ASE) using ASE 350 instrument by Thermo-Fischer Scientific. The authors used a mixture of \(n\)-hexane and acetone (4:1, _v:v_) and led extraction at 60 \({}^{\circ}\)C and 1500 psi, following the times foreseen by the protocol. The extraction results were dried and restored before the analysis [76]. [PERSON] et al. have examined the presence of Diclofenac and its distribution in different biological matrices obtained from patients who have died for various reasons. In a case reported by them, dry bones of a fetus were used to conduct the forensic analysis. After homogenization, a small amount of water was added, and the sample was inserted in an ultrasonic bath. The operators successfully performed a liquid-liquid extraction using ethyl acetate followed by a classical operation such as centrifugation, evaporation, and reconstitution prior to the analysis [77].