HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY DETERMINATION OF CLONAZEPAM INPLASMA USING SOLID-PHASE EXTRACTION

The use of high-performance liquid chromatography for therapeutic drug monitoring of clonazepam has previously been limited by low sensitivi ty and labor-intensive liquid-liquid extractions. The present method w as developed employing a rapid solid-phase extraction, thus minimising sample workup and providing analytical sensitivity down to 2 mug/L us ing 1 ml of plasma. Plasma samples were loaded onto C-18 solid-phase e xtraction columns, and clonazepam and its internal standard (methyl-cl onazepam) were eluted with methanol, dried, and reconstituted in 130 m ul of mobile phase. Chromatographic separation was achieved using a 3- mum RP18 column at 40-degrees-C and a mobile phase of 32% acetonitrile and 0.5% glacial acetic acid in distilled water at 0.5 ml/min. Detect ion was carried out using ultraviolet absorbance at 306 nm. Retention times for clonazepam and methyl-clonazepam were approximately 7 and 12 min, respectively. Standard curves were linear over a range of 5-200 mug/L with intraassay coefficients of variation of 1.2 and 4.8% at 200 and 5 mug/L, respectively. Plasma concentrations measured in patient samples were not statistically different from those obtained using an established gas chromatographic method, and quality control specimens from the Heathcontrol EQA Scheme were consistently within +/- 1.2 SD o f the group means. There was no chromatographic interference from othe r benzodiazepines or other drugs used for the treatment of epilepsy.