Comparison of relative distribution of ketamine and norketamine in decomposed skeletal tissues following single and repeated exposures.

Bone was analyzed for ketamine and norketamine to examine whether different patterns of drug exposure could be discriminated. Rats received (intraperitoneally) one 75 mg/kg dose (Acute-1 and Acute-2 groups), three 25-mg/kg doses 1 hour apart (Repeated group), or nine single daily ketamine doses of 75 mg/kg followed by a 24-h washout period (Chronic group). Following euthanasia, all animals decomposed to skeleton outdoors. Ground samples of recovered bone underwent methanolic extraction and analysis by gas chromatography-mass spectrometry after solid-phase extraction. Drug levels (mass normalized response ratios) were compared across bone types and exposure pattern. Bone type significantly influenced drug level for the Acute-1 and Repeated dose groups, and the drug/metabolite level ratio (DMLR) for the Acute-1 group. Mean ketamine and norketamine level and DMLR varied by up to 8-fold, 7-fold and 3-fold, respectively, in the Acute-1 group, and by up to 24-fold, 5-fold and 10-fold, respectively, in the Repeated group. Drug level and DMLR differed significantly between the Acute-1 and Repeated groups for most bone types. In the Chronic group, only 1/16 and 4/16 samples were positive for ketamine and norketamine, respectively. All Acute-2 samples were positive for ketamine and norketamine. The Acute-2 and Chronic groups differed significantly in ketamine and norketamine levels, and DMLR.

Relative distribution of drugs in decomposed skeletal tissue.

Skeletal tissues from a domestic pig exposed to amitriptyline, diazepam, and pentobarbital were analyzed to determine the relative distribution of these drugs in bone. Following drug exposure and euthanasia, remains were allowed to decompose outdoors to complete skeletonization between summer 2007 and fall 2009. Remains were recovered and separated according to bone type. Twelve different bone types were pulverized and sampled in triplicate. Each bone sample underwent methanolic extraction (96 h, 50 °C), followed by solid-phase extraction and gas chromatography-mass spectrometry in the selected ion monitoring mode. Mass-normalized assay responses underwent ANOVA with post-hoc testing, revealing bone type as a main effect for all three drugs, but not for the diazepam metabolite (nordiazepam). The assay response varied with respect to bone type by factors of 27, 39, and 20 for pentobarbital, diazepam, and amitriptyline, respectively. The relative distribution between bone type was qualitatively similar for the three administered drugs analyzed for, with the largest response obtained from rib for all three drugs. This is the first study, to the authors' knowledge, of the distribution of different drugs in various decomposed skeletal tissues in a controlled experiment using an animal model of comparable physiology to humans. These data have implications for the interpretive value of forensic drug measurements in skeletal tissues.

Microwave-assisted extraction in toxicological screening of skeletal tissues.

The use of microwave-assisted extraction (MAE) in screening of decomposed bone tissue for model drugs of abuse is described. Rats received 50 mg/kg (i.p.) pentobarbital (n=2), 75 mg/kg (i.p.) ketamine (n=2) or 16 mg/kg (i.p.) diazepam (n=1), or remained drug-free (control). Drug-positive animals were euthanized within 20 min of drug administration. Animal remains were allowed to decompose in a secure outdoor environment to the point of complete skeletonization. Bones (tibiae, femora, vertebrae, ribs, pelvi, humeri and scapulae) were collected and pooled (according to drug) in order to minimize effects due to inter-bone differences in drug distribution. Bones were crushed and cleaned of marrow and residual soft tissue in alkaline solution or phosphate buffer with ultrasonication. Cleaned bones were then ground and underwent MAE in phosphate buffer (pH 6), methanol or a methanol:water mixture (1:1, v/v) at atmospheric pressure in a domestic microwave oven, or passive extraction in methanol. Bone extracts (control and drug-exposed) containing methanol were evaporated to dryness before reconstitution in phosphate buffer (pH 6) and subsequent analysis by ELISA, while bone extracts containing only phosphate buffer were assayed directly by the same ELISA protocol. Measured absorbance values were expressed as the decrease in absorbance, measured as a percentage, relative to the corresponding drug-free control bone extract. The semi-quantitative nature of the ELISA assay allowed examination of the effects of extraction solvent and bone sample mass on the assay response for each drug examined, and subsequent comparison to assays of extracts obtained through passive methanolic extraction of various bone tissues. Overall, the time required for maximal extraction varied with extraction solvent and bone mass for each drug investigated, with significant extraction occurring with all solvent systems examined. MAE may represent a substantially faster extraction system than passive extraction, with significant extraction recovery observed within 1 min of exposure for all samples examined. The implications of these results in the context of the available literature on drug analysis in skeletal tissues are discussed.