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

Abstract

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.

State-of-the-art of bone marrow analysis in forensic toxicology: a review

“…Chromatographic separation is principally performed by gas chromatography (GC) rather than liquid chromatography (LC). This can be easily explained by the fact that mass spectrometry (MS) is the detection system of choice for toxicological analysis of such complex matrices, and GC/MS systems were in more widespread use than LC/MS systems until a few years ago; recently, however, liquid chromatography tandem mass spectrometry has also been applied to BM [29,30,49]. While mass spectrometry remains the main detection system, as reported in Table 1, other detection systems have been used, such as flame ionization detection for volatile molecules (e.g., ethanol, methanol, isopropanol).…”

“…Lipid removal can be achieved by an additional hexane wash of the final extract [3,22,24,59,60] or by multi-step extraction as proposed by Guillot et al for the quantification of morphine and 6-acetylmorphine (6-AM) [54]. A solid-phase extraction process was successfully applied to BM analysis in other studies [29,30,49,56,61].…”

“…During the following decade, few toxicological data on BM were published, except for some case reports. Recently, there has been renewed interest in BM, in particular with the work on skeletal remains by the Laurentian University (Ontario, Canada) [25][26][27][28][29][30].…”

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues

“…Thus, it seems clear at this point that detection of a drug in skeletal tissues may be best interpreted as being indicative of exposure only. In recent work [12], detection of acute drug exposure in skeletal tissues has been illustrated with the use of ELISA, gas chromatography and liquid chromatography, in combination with other techniques such as liquid-liquid and solid phase extraction. The drugs detected have included ketamine [12] and diazepam [13].…”

“…In recent work [12], detection of acute drug exposure in skeletal tissues has been illustrated with the use of ELISA, gas chromatography and liquid chromatography, in combination with other techniques such as liquid-liquid and solid phase extraction. The drugs detected have included ketamine [12] and diazepam [13]. Here, we simplify the analytical approach through illustration that semi-quantitative detection of fentanyl exposure in extracts of fresh and decomposed skeletal tissues is possible with minimal sample preparation and a sensitive immunoassay based on the ELISA configuration.…”