Determination of benzodiazepines in forensic samples by HPLC with photo-diode array detection

A high-performance liquid chromatographic (HPLC) method has been developed for the analysis of several benzodiazepines and some of their metabolites in blood, plasma and urine. The method included a liquid-liquid extraction with n-hexane:ethylacetate, a gradient elution on a C8 reversed phase column with a non-electrolyte eluent and a photo diode array detection. This allowed a rapid detection, a purity check, and identification as well as quantitation of the eluting peaks. The detection limit was 10 to 30 ng and the limit of quantitation was 0.05 microgram/mL, using 1 mL of blood, plasma or urine. The procedure is applied routinely in forensic toxicological analyses involving blood, stomach content, urine and organ samples. About 30 positive cases are reported. The avoidance of the use of an electrolyte buffer in the eluent resulted in a robust procedure, free of technical problems and of long rinsing periods, suitable for routine use in forensic toxicology analysis involving blood, urine, stomach content and tissue samples.