Improved micromethod for determination of underivatized clonazepam in serum by gas chromatography.

We describe a gas-chromatographic micromethod, with use of a solid injection system and an electron capture detector, for determination of underivatized clonazepam in serum. Vigorous mixing of 100 microliter of serum with 20 microliter of borate buffer (pH 9.0) and 100 microliter of a cyclohexane/dichloromethane mixture (4/6 by vol) containing methyl nitrazepam as internal standard suffices to extract 86% of the clonazepam into the organic layer. We obtained a linear response curve for clonazepam in serum in the concentration range 5 to 150 microgram/liter. The lowest detectable concentration by our method is about 1 microgram/liter of serum. Interference by several anticonvulsant drugs, metabolites, and related compounds was investigated. The assay is simple, rapid, and suitable for clinical routine determination of clonazepam in serum in therapeutic concentrations. We illustrate the steady-state concentration of clonazepam in serum during chronic oral administration to 28 patients.