Abstract: An analytical method based on micellar liquid chromatography has been developed for simultaneous determination of phenobarbital, estazolam and clonazepam in human plasma. The effects of the concentrations of surfactant, cosurfactant and hydrogen ion in mobile phase (hereafter abbreviated respectively as CM, Cφ and CH) on the retention behavior of the 3 compounds were investigated in terms of three-phase equilibrium theory. The quantitative relationships between the logarithm of retention factor (log k) and the parameters of solutes properties and the composition of mobile phase were set with multiple linear regression method. The experimental results showed that the retention factors (k) of solutes were decreased with the increase of the concentration of surfactant, cosurfactant and hydrogen ion in the mobile phase. The effects of changes of CM, CH and Cφ on retention factors were fully consistent with the theoretical modeling. And there is better multielement linear relationship between the log k of solutes and the logarithm of hydrophobic parameter (log P) and ionization constant (Ka) of solutes and CM, CH and Cφ in the mobile phase. The separation efficiency between phenobarbital, estazolam, clonazepam and other components in human plasma was better under the selected chromatographic conditions. The linearities were good in the ranges of 2.5~50 mg/L of phenobarbital, 0.25~5.0 mg/L of estazolam and 0.05~5.0 mg/L of clonazepam. The method is simple, accurate and reproducible for the determination of phenobarbital, estazolam and clonazepam. The limits of detection (LODs) for phenobarbital, estazolam and clonazepam were found to be 10.27, 1.17 and 0.867 ng (S/N=3), respectively. The recoveries of the method for the determination of phenobarbital, estazolam and clonazepam were 99.80%~102.9%, 94.00%~98.20% and 96.30%~98.70%, respectively.

Key words: clonazepam, estazolam, human plasma, phenobarbital, micellar liquid chromatography