Liquid chromatographic analysis of clonazepam in human serum with solid-phase (Bond-Elut) extraction

A simple, sensitive, selective and precise liquid-column chromatographic assay for clonazepam is described, in which 1 ml of serum containing 50 micrograms/l methylclonazepam as an internal standard is extracted by elution from a Bond-Elut column with 400 microliter of methanol. An aliquot of the eluate is injected on to a reversed-phase column and eluted with a mobile phase of acetonitrile--phosphate buffer (30:70) at a flow-rate of 2 ml/min at a column temperature of 50 degrees C. Detection is at 254 nm. Chromatography is complete in 12 min. A sensitivity of 2 ng/ml is attained when 1 ml of serum is extracted. Analytical recovery of the clonazepam added to serum ranged from 91% to 99% with a coefficient of variation of 6.0%. This assay for clonazepam has good precision, with coefficients of variation of 11% at 15 ng/ml and 2.6% at 50 ng/ml. There was no interference from any of the commonly used antiepileptics.