Utility of Immunoassay in Drug Screening in Skeletal Tissues: Sampling Considerations in Detection of Ketamine Exposure in Femoral Bone and Bone Marrow Following Acute Administration Using ELISA*

Abstract

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.

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

“…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].…”

Microwave-assisted extraction in toxicological screening of skeletal tissues

“…In this study, we examined the utility of MAE for the extraction of model drugs from ground bone, as part of an effort to streamline methodologies to improve the efficiency of studies of drug disposition in skeletal tissues. The objective of this work was to examine the effects of target drug, solvent, irradiation time and sample mass on the time required for drug recovery, and to compare this methodology against a passive methanolic extraction strategy which has been used in our laboratory previously [1,2,5]. This work involved the extraction of three model drugs (ketamine, diazepam and pentobarbital) from pooled, ground bone tissues obtained from acutely exposed animals following a period of significant decomposition.…”

“…For example, drug and metabolite uptake into bone is poorly understood, as is the time course of drugs within those tissues. Only a handful of studies have considered the distribution of drug within a bone (i.e., trabecular vs. cortical bone) [1,2,5]; drug distribution between different bones (e.g., femur vs. vertebrae) has not been studied under controlled conditions. Drug recovery from mineralized bone may be challenging, and remains poorly characterized.…”

“…Drug recovery from mineralized bone may be challenging, and remains poorly characterized. Review of the literature shows that drug isolation from bone has been attempted using a number of different methods, including passive methanolic extraction of transverse slices from the mid-femoral region [4], bone slivers [8], or ground bone [1,2,5], Soxhlet extraction [9], and acid digestion [7,10]. Typically, the methods reported thus far have been timeconsuming, typically requiring 12-72 h of incubation time (Table 1).…”

“…Therefore, bone tissue may be the only source of toxicological information. Although a growing body of research has been performed in this area [1][2][3][4][5][6][7][8][9][10], with drugs such as ketamine [1,2], tricyclic antidepressants [3,4], benzodiazepines [4][5][6], antipsychotics [4] and morphine [7] being detected in bone or bone marrow, understanding of the implications and limitations of drug measurements in skeletal tissues remains poor. For example, drug and metabolite uptake into bone is poorly understood, as is the time course of drugs within those tissues.…”