Simplified High Performance Liquid Chromatographic Method for the Determination of Clonazepam and Other Benzodiazepines in Serum

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Abstract
A convenient and sensitive high performance liquid chromatographic method was developed for determination of clonazepam in serum using a C-18 reverse-phase column, and mobile phase consisting of a 50:35:15 mixture by volume of pH 6.0 phosphate buffer:methanol:acetonitrile. Quantitation was performed at 313 nm with flunitrazepam as the internal standard. Using 1 ml of serum for extraction, the assay is linear for clonazepam concentrations between 10 and 250 ng/ml. The relative recovery averaged 100.3%, and the coefficient of variation for between-day and within-day assays was < 7%. A simple modification permits analysis of 200 .mu.l of serum, with little loss of precision and with a detection limit of 20 ng/ml. Only three drugs tested (nitrazepam, methaqualone, and norchlordiazepoxide) interfered with the assay, and none are likely to be used therapeutically with clonazepam. Importantly, carbamazepine and carbamazepine-10,11-epoxide do not interfere under the conditions of the assay. The method is equally suitable for the determination of nitrazepam. By adjusting the mobile phase so that volume ratios of phosphate buffer, methanol, and acetonitrile are 45:35:20, and using nitrazepam as the internal standard, seven other benzodiazepines (demoxepam, oxazepam, chlordiazepoxide, norchlordiazepoxide, temazepam, diazepam, and noradiazepam) can be resolved at 254 nm.

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