Innovative analysis of diazepam and metabolites in rat brain and plasma using an original MEPS – HPLC method

Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is one of the most widely used anxiolytic-hypnotic drugs. It is a long-acting benzodiazepine with anxiolytic, sedative, hypnotic, anticonvulsant, muscle relaxant and amnestic properties. Diazepam is also frequently used for the relief of anxiety during the first period of treatment with selective serotonin reuptake inhibitors (SSRIs) and generally as a coadjuvant during antidepressive therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. In order to clarify the matter, some studies are currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significative set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam, which are the demethylated, hydroxylated and demethylated hydroxylated analogues of diazepam, respectively. All of these compounds are pharmacologically active, have a long half-life and thus significantly contribute to the therapeutic effects of diazepam administration. Their determination can also give important insight into the respective balance of different metabolic pathways. Following our recent studies on benzodiazepine determination, we are developing a feasible and reliable HPLC method for the simultaneous determination of diazepam and its three main metabolites in rat brain and plasma. The method will be applied to “normal” rats and to genetic rat models of depression in order to estimate the drug metabolism in the different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer / acetonitrile mixture as the mobile phase. The detection wavelength is 238 nm. An accurate and innovative sample pre-treatment, based on microextraction by packed sorbent (MEPS) was developed in order to suitably eliminate endogenous interferences, using only 250 µL of matrix (brain homogenate or plasma) for a complete analysis. The analytes are eluted from the cartridge with methanol; the eluate is then dried under vacuum and redissolved in the mobile phase. The results obtained with the MEPS procedure were confirmed by comparison with those obtained with a solid-phase extraction (SPE) procedure. The method has been validated with good results in terms of precision, extraction yield, accuracy, sensitivity and selectivity on both matrices and the determination of diazepam and metabolite levels in rats is currently under way. The results obtained until now are satisfactory form an analytical point of view and will hopefully contribute to the clarification of some metabolic differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.