Screening and confirmation of suspected benzodiazepine abuse: TLC and HPLC analysis in urine

Benzodiazepines are currently among the most frequently prescribed CNS drugs. They effect their anxiolytic-hypnotic activity by allosterically increasing the inhibitory activity of GABAergic transmission in the brain. Due to this indirect mechanism of action, benzodiazepines are generally considered safe with respect to suicidal attempts. So, they are routinely administered in polypharmacy to many psychiatric patients, in order to alleviate anxiety and sleep disturbances related to their main condition. Moreover, they are widely used as a treatment for generalized anxiety and panic disorder, insomnia, some forms of epilepsy and alcohol withdrawal [1]. Unfortunately, benzodiazepines also have a high potential for abuse: abrupt therapy interruption can cause a withdrawal syndrome and benzodiazepine addiction is relatively common among "poly-drug users" [2]. Two assays are presented for benzodiazepine abuse testing; the screening method is based on thin-layer chromatography, while confirmation uses HPLC. After hydrolysis of the glucuronides, most benzodiazepines can be easily detected in urine by TLC on silica plates. Semi-quantitative results can be obtained, with some discrimination among different kinds of benzodiazepines. The HPLC method is based on the use of a C8 column and an acetonitrile-phosphate buffer mixture as the mobile phase. Most commonly prescribed (and abused) benzodiazepines can be quantitatively analyzed with this method after urine sample pre-treatment by hydrolysis and solid-phase extraction (SPE). Good extraction yields (> 90%) and precision values (RSD < 6.7%) were obtained for all drugs considered. Both tests have been applied to cases of suspect illicit benzodiazepine intake and allowed to confirm the abuse. Assays are in progress in order to extend the method applicability to other benzodiazepines.