Development of a rapid RP-HPLC method for the determination of clonazepam in human plasma

A rapid and sensitive high-performance liquid chromatography method with UV detection was developed for the determination of clonazepam in human plasma using 3-methylclonazepam, as internal standard. A one-step extraction of both compounds was performed with a mixture of hexane/ethyl acetate (90:10, v/v). The HPLC analysis was carried out on a Nova Pak((R)) C(18) reversed-phase column with a mobile phase of acetonitrile-0.01 M sodium acetate adjusted to pH 7 with dilute acetic acid (40:60, v/v). A linear response was observed over the concentration range 5-100 ng/mL. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The lower limit of quantification was 5 ng/mL without interference of endogenous components. For analytical purpose, the stability of clonazepam in bidistilled water and plasma has been studied. A rapid degradation was noticed when clonazepam was stored in bidistilled water at the daylight following a first-order kinetic rate with a 87 min half life whereas no significant degradation was observed in plasma. This method was applied to measure plasma concentrations of clonazepam either in patients receiving therapeutic doses or in poisoning cases.