HPTLC method for the assay of thiopental in post-mortem blood in a fatal case of suicide

Abstract

Thiopental is an ultra-short-acting barbiturate, used as an induction agent during general anesthesia and to manage intra cranial pressure in traumatic brain injuries. Because of its rapid onset of action, the potential for accidental or intentional abuse of thiopental is high. In this paper, a case is presented in which a 25-year-old female deliberately injected a fatal dose of thiopental.A method is developed for the evaluation of thiopental levels in the post-mortem blood (PMB) by simple and rapid HPTLC. Three different extraction procedures were compared for optimum recovery of thiopental from spiked blood samples. The effect of pH on the extraction yield of thiopental over a pH range of 5–6.5 was examined. An average analytical recovery of 90.5% was achieved from an ethyl acetate extract at pH 5.5. Chromatographic separation was achieved on silica gel 60F254 plates with an optimized mobile phase consisted of hexane–dichloromethane–ethyl acetate in the ratio 7.5:2:0.5 (v/v). Densitometric detection was carried out at 290nm in absorbance mode. No significant chromatographic interference was observed from other drugs used to diagnose the brain death. Calibration curve for thiopental in blood were linear from 1 to 100μgml−1 with r2=0.994. The detection limit was 0.5μgml−1 and its lower limit of quantification was 1.5μgml−1. The method showed excellent intra-assay precision (R.S.D. 1.07–6.28%) and inter-assay precision (R.S.D. 0.39–1.43%) for spiked blood samples at concentration of 1, 10, and 50μgml−1.The toxicological analysis revealed high concentrations of thiopental in PMB (204.75μgml−1 ±0.34), which is of immense help to conclude that the death occurred due to fatal doses of thiopental.