Effects of Tissue Type and the Dose-Death Interval on the Detection of Acute Ketamine Exposure in Bone and Marrow with Solid-Phase Extraction and ELISA with Liquid Chromatography-Tandem Mass Spectrometry Confirmation

Ketamine exposure was detected in skeletal tissues by ELISA and liquid chromatography-tandem mass spectrometry (LC-MS-MS). Rats (n = 9) received ketamine hydrochloride acutely (75 mg/kg, i.p.) and were euthanized within 15, 30, or 90 min. Drug-free control animals (n = 3) were also euthanized. Extracted femora were separated into epiphyseal and diaphyseal fragments, with marrow isolated from the medullary cavity. Bone was ground and incubated in methanol. Extracts were dried and reconstituted in phosphate buffer (0.1 M, pH 7.3), and marrow was homogenized in alkaline solution. Both then underwent solid-phase extraction. Extracts were assayed by ELISA, with data expressed in terms of relative decrease in absorbance (%DA, drug-positive tissues vs. matrix-matched drug-free controls) and binary classification test sensitivity (S). Generally, %DA decreased in the order of marrow > epiphyseal bone > diaphyseal bone, and was negatively correlated with dose-death interval (DDI). Measured S values were 100% in ELISA analysis of extracts of all tissue types. Sensitivity values were computed from LC-MS-MS data using a 5 ng/mL cutoff. Sensitivity values for ketamine detection were 100%, 0-100% and 0%, at the 15, 30, and 90 min DDI, respectively, and sensitivity values for norketamine detection were 0-66%, 0-66%, and 0% at the 15, 30, and 90 min DDI, respectively. These results suggest that the tissue type sampled and DDI may influence the sensitivity of detection of ketamine exposure in skeletal tissues.