Quantitation of benzodiazepines in whole blood by electron impact-gas chromatography-mass spectrometry

Benzodiazepines are frequently encountered in forensic toxicology. A literature search was conducted to find a simple method using electron impact-gas chromatography-mass spectrometry (EI-GC-MS) to examine whole blood specimens for the most commonly encountered benzodiazepines in the United States. A recently published method was identified in the literature search and used as a starting point for development of a new procedure to be used for routine analysis of forensic toxicology case samples. The procedure was then developed and validated as a rapid and efficient method for the screening and quantitation of benzodiazepines in blood using liquid-liquid extraction and EI-GC-MS in selective ion monitoring mode. Materials and instrumentation common to most forensic toxicology laboratories were utilized while obtaining LODs from 5 to 50 ng/mL and LOQs of 50 ng/mL or less using 1 mL of sample. Target compounds were chosen based on availability and common use in the United States and include diazepam, desalkylflurazepam, nordiazepam, midazolam, oxazepam, temazepam, lorazepam, clonazepam, and alprazolam (relative elution order). The linear range (r2 > 0.990) was validated from 50 to 1000 ng/mL for all analytes. The CV of replicate analyses at both 50 and 200 ng/mL was less than 4%. Quantitative accuracy was within +/- 16% at 50 ng/mL and within +/- 7% at 200 ng/mL. The validated method provides an efficient procedure for the quantitation of a broad range of the most common benzodiazepines in blood at meaningful limits of detection and quantitation using standard laboratory equipment and a small amount of sample.