Down to the bone: Skeletal tissue as alternative matrix in forensic toxicology

In forensic toxicology, the goal is to identify and quantify xenobiotics in order to determine their effect on a certain situation. To reach this goal, a combination of different disciplines such as pharmacology and analytical chemistry are used. These analyses are done on different specimens. In the traditional analyses, there is a wide range of options to pick. The choice of specimen always depends on the context of an investigation. Whole blood, saliva and urine will most of the time be the specimens of choice because many reference data are available. To investigate post mortem cases, the choice of specimens for analysis gets even bigger. During autopsy, one of the common taken samples consists of visceral tissue. However sometimes these traditional matrices are not available anymore due to degradation or decomposition. In those cases, analysis is performed on alternative matrices. In cases of extreme decomposition, most of the time only nails, teeth, hair and bones remain. In the past, research on bone and bone marrow was very limited. One of the possible reasons for the lack of research on bone(marrow) is the difficulty in obtaining bones from cases. Another reason is the inability to reproduce the precise history of drug use in case studies. Previous studies already show the presence of some drugs in bone and bone marrow for acute dosage. However, research on chronic substance abuse is still a gap in the field.This PhD will fill the gaps in the field by exploring the feasibility of skeletal tissue as an alternative matrix in forensic toxicology. The main focus will be on the detection and quantification of psychotropic substances because of their value in forensic cases. In a first phase, analytical methods were developed using an LC-MSMS system. Afterwards these methods were applied to study the effect of chronic dosage on the deposition of drugs in bone tissue. In order to produce reliable data to analyze the disposition, an animal model was used. Like this, dosage parameters can be adjusted in a controlled setting. Blood will be compared to the less known specimens. Aside from the animal model, specimens were also be taken from cases in our forensic research to check if extrapolation of our results to humans is possible. To goal was to develop a standard operating procedure in order to collect uniform data. In this way, a general reference database could be developed. The reference data can be used to have a better understanding and interpretation of drug concentrations found in skeletal tissue. In a last phase, a new way was evaluated to detected and visualise the actual in situ drug distribution of skeletal tissue. Using MALDI-MSI, some fundamental questions concerning drug distribution could be answered.In chapter one, an LC-MS/MS method was optimized and fully validated for the analysis of methadone and its metabolites in skeletal tissue. This method was applied to an animal study to study the chronic disposition in skeletal tissue. Rats were administered a daily methadone dose for 139 days. After dissection, single whole bones or bone parts underwent a methanolic extraction. Methadone and its metabolites were proven to be detectable and quantifiable in skeletal tissue of chronically dosed rats using a fast and easy methanol extraction. Within bone, comparison showed that bone marrow yields the highest concentration. Trabecular bone also showed to be the best type of bone tissue for sampling. Between bone comparison, proved the humerus to be the best bone type for sampling. The concentrations found in tibiae and humeri appeared to be dose dependent for methadone. However, for other bones the variance in methadone concentration was highly variabel. A possible explanation is seen in the lower vascularization of these bones. For the metabolites, no correlation was seen. This could be explained by the highly inter-individual metabolism of methadone. However, skeletal tissue concentration showed no correlation to blood for methadone nor its metabolites. Chapter two is a continuation of the first project, a fully validated method is presented and the distribution of clomipramine, citalopram, midazolam, and metabolites after chronically administration is examined within skeletal tissue. Rats were chronically dosed with respectively clomipramine, citalopram, or midazolam. Clomipramine, citalopram, and metabolites, respectively desmethylclomipramine and desmethylcitalopram are shown to be detectable in all bone types sampled. Midazolam and its metabolite α‐OH‐midazolam could not be detected. The absence of midazolam in extracts gives an indication that drugs with pKa values under physiological pH are badly or not incorporated in bone tissue. Bone and post‐mortem blood concentrations were compared. A range of different bone types was compared and showed that the concentration is strongly dependent on the bone type. In concordance with previous publications, the humerus shows the highest drug levels. Comparison of the same bone type between the different rats showed high variances. However, the drugs–metabolite ratio proved to have lower variances (<20%). Moreover, the drugs–metabolite ratio in the sampled bones is in close concordance to the ratios seen in blood within a rat. From this, we can assume that the drugs–metabolite ratio in skeletal tissue may prove to be more useful than absolute found concentration. In chapter three, the methods from the previous chapter were applied to real forensic cases. First the methods were fully validated for bone marrow. Than blood, bone tissue and bone marrow of different forensic cases were screened for 415 compounds of forensic interest. 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. Afterwards, methadone, clomipramine, citalopram and their respectively metabolites positive samples were quantified using the fully validated methods. 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.In chapter four, a new LC-MS/MS method was developed and fully validated for the analysis of 6 narcotic analgesics and metabolites (tramadol, o-desmethyltramadol, morphine, fentanyl, norfentanyl, codeine) in bone tissue and bone marrow. This method was applied to analyse 22 forensic cases involving narcotics. All 6 narcotics were proven to be detectable and quantifiable in all specimens sampled. When tramadol blood concentrations were correlated with the bone concentrations, a linear trend could be detected. The same was seen between tramadol blood and bone marrow concentration. A similar linear trend was seen when correlating codeine blood concentration with bone and bone marrow concentration. Although some outliers were detected, this shows the potential of skeletal tissue to assist in legal investigations. For morphine no such correlation could be detected. An explanation is found in the highly inter-individual differences in metabolism of morphine. For fentanyl and norfentanyl, the sample size was too small to draw conclusions regarding correlation. As far as the author knows, this is the first-time fentanyl and norfentanyl are quantified in skeletal tissue. In conclusion, due to the absence of reference data for drugs in skeletal tissue, these findings are a big step forward towards a more thorough understanding of drug concentration found in post-mortem skeletal tissue. A side to that they show the big potential of skeletal tissue in forensic toxicology.Chapter five investigates a new way for the detection and visualisation of drugs in skeletal tissue using mass spectrometry imaging. A sample preparation protocol was developed and optimized to make sections of undecalcified bone tissue which are compatible with MALDI-MSI. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle from the previous chapters. The best matrices were CHCA and DHB in positive ion mode for the detection of unknown lipids in mice hind legs based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Publication year:2020