Distribution of clomipramine, citalopram, midazolam, and metabolites in skeletal tissue after chronic dosing in rats.

In recent years, the use of skeletal tissue as an alternative matrix in forensic toxicology has received new interest. In cases where extreme decomposition has taken place, analysis of skeletal tissue is often the only option left. In this article, 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. Extracts were quantitatively analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). 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. Skeletal tissue concentrations found ranged from 1.1 to 587.8 ng/g. 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.

Distribution of Methadone and Metabolites in Skeletal Tissue.

In forensic toxicology, when extended time has elapsed before discovery of the body, the most commonly analyzed specimens are often degraded or not available at all due to decomposition. In this case, skeletal tissue may be the only specimen left. Nevertheless, very limited research is found on the drug disposition in bone, making toxicological interpretation very difficult. Since methadone is linked to almost 50% of drug abuse fatalities in Belgium, an easy extraction and quantification method was developed and validated to investigate the distribution pattern of methadone and its metabolites in skeletal tissue after chronic dosing. In this study, Wistar rats were administered a subcutaneous daily methadone dose of 3 mg/kg for 139 days. After dissection, single whole bones or bone parts underwent a methanolic extraction. The final extract was analyzed using LC-ESI(+)-MS-MS for methadone, EDDP and EMDP. 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 with a variance of <9%. However, for other bones the variance in methadone concentration ranged from 24 to 32%. 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. Using the developed method, quantitative information about methadone after chronic administration was only found in the humeri and tibiae.