Analysis of low-dose benzodiazepines by HPLC with automated solid-phase extraction

We describe a simple, specific, and sensitive reversed-phase HPLC method with automated solid-phase extraction (SPE) for analyzing alprazolam, clonazepam, and nitrazepam concentrations in human serum or plasma. We prepare samples and calibrators with an automated sample preparer, using 100-mg Bond-Elut C18 SPE columns. The HPLC method uses isocratic elution with acetonitrile:methanol:dipotassium hydrogen phosphate, 10 nmol/L, pH 3.7 (30:2:100 by vol), at a flow rate of 1.5 mL/min to separate the drugs. We detect the benzodiazepines with diode-array detector at 240 nm; analyses of peak purity are performed at 210-365 nm. The recoveries were between 94% and 100%. The intraassay and interassay CVs were between 1.0% and 4.1%. The detection limit is 5 nmol/L. The antiepileptic and antidepressant drugs tested did not interfere with the assay. We developed the method for use in a clinical laboratory for therapeutic drug monitoring.