Skeletal tissue, a viable option in forensic toxicology? A view into post mortem cases

Blood analysis is the golden standard in the field of forensic toxicology. However, when extended decomposition of the remains has occurred, alternative matrices are required. Skeletal tissue may provide an appropriate sample of choice since it is very resistant to putrefaction. However, today, the absence of reference data of drug concentrations in skeletal tissue poses a problem to meaningfully and reliably conduct toxicological testing on human skeletal material. The present study investigates the viability of skeletal tissue as an alternative matrix to evaluate xenobiotic consumption in legal cases. Blood, bone tissue and bone marrow of different forensic cases were screened for 415 compounds of forensic interest. Afterwards, methadone, clomipramine, citalopram and their respectively metabolites positive samples were quantified using fully validated methods. Sample preparation was carried out by SPE (whole blood and bone marrow), methanol extraction (bone sections) or protein precipitation (whole blood). All samples were analyzed using liquid chromatography coupled to a triple quad mass spectrometer. Multiple drugs were successfully identified in all sampled matrices. In bone (marrow) not as many substances were detected as in blood but it poses a valid alternative when blood is not available. Especially bone marrow showed big potential with a concordance of 80.5% with blood. Clomipramine, citalopram and their metabolites were proven to be detectable and quantifiable in all specimens sampled. Bone marrow showed the highest concentrations followed by blood and bone tissue. When citalopram blood concentrations were correlated with the bone concentrations, a linear trend could be detected. The same was seen between blood and bone marrow for citalopram concentrations. Methadone was also proven to be detectable in all specimens sampled. However, its metabolites EMDP and EDPP were absent or below the LOD in some samples. Overall, methadone concentrations were higher in bone marrow than in bone. With exception of one case, blood concentrations were higher than bone concentrations. For methadone, a linear trend could be found between blood and bone concentration. Comparing methadone concentrations in blood and bone marrow an exponential trend could be seen. In conclusion, these findings show the potential forensic value of bone and bone marrow as an alternative matrix. Aside to that, a standard protocol for the sample collection and processing is proposed.