Detection of Acute Diazepam Exposure in Bone and Marrow: Influence of Tissue Type and the Dose‐Death Interval on Sensitivity of Detection by ELISA with Liquid Chromatography Tandem Mass Spectrometry Confirmation*

Abstract: Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC/MS/MS) were used to detect diazepam exposure in skeletal tissues of rats (n = 15) given diazepam acutely (20 mg/kg, i.p.), and killed at various times postdose. Marrow, epiphyseal, and diaphyseal bone were isolated from extracted femora. Bone was cleaned, ground, and incubated in methanol. Marrow underwent ultrasonic homogenization. Extracts and homogenates were diluted in phosphate buffer, and then underwent solid‐phase extraction and ELISA. Relative sensitivity of detection was examined in terms of relative decrease in absorbance (ELISA) and binary classification sensitivity (ELISA and LC/MS/MS). Overall, the data showed differences in relative sensitivity of detection of diazepam exposure in different tissue types (marrow > epiphyseal bone > diaphyseal bone), which is suggestive of heterogenous distribution in these tissues, and a decreasing sensitivity with increasing dose‐death interval. Thus, the tissue type sampled and dose‐death interval may contribute to the probability of detection of diazepam exposure in skeletal tissues.