Methods for the measurement of benzodiazepines in biological samples

A review of methods for the measurement of benzodiazepines in biological specimens published over the last five years is presented. A range of immunoassay procedures using EIA, ELISA, FPIA, agglutination or kinetic interaction of microparticles, or RIA methods are now available. Cross reactivities to benzodiazepines are variable such that no one kit will recognise all benzodiazepines and their relevant metabolites at concentrations likely to be encountered during therapeutic use. Prior hydrolysis of urine to convert glucuronide metabolites to immunoreactive substances improves detection limits for many benzodiazepines. Several radioreceptor assays have now been published and show good sensitivity and specificity to benzodiazepines and offer the advantage (over immunoassay) of being able to detect these drugs with equal sensitivity. Solvent extraction techniques using a variety of solvents were still popular and offer acceptable recoveries and lack of significant interference from other substances. A number of papers describing solid phase extraction procedures were also published. Direct injection of specimens into a HPLC column with back flushing were also successfully described. Seventy two chromatographic methods using HPLC, LC-MS, GC and GC-MS methods were reviewed. HPLC was able to achieve detection limits for many benzodiazepines using UV or DAD detection down to 1-2 ng/ml using 1-2 ml of urine or serum (blood). ECD detectors gave detection limits better than 1 ng/ml from 1 ml of specimen, which was an order of magnitude lower than for NPD. EI-MS offered similar sensitivity, whilst NCI-MS was capable of detection down to 0.1 ng/ml. Methods suitable for the separation of enantiomers of benzodiazepines have been described using HPLC. Electrokinetic micellar chromatography has also been shown to be capable of the analysis of benzodiazepines in urine.