Application of High-Performance Thin Layer Chromatography in the Quantitative Estimation of Serum Clobazam Levels

Abstract

This study was aimed to develop and validate a High-Performance Thin Layer Chromatography (HPTLC) method for the quantitative determination of serum clobazam levels. This simple and sensitive method will help in therapeutic drug monitoring that aids in the clinical management of patient therapy. Chromatographic separation was carried out on a silica gel 60F254 HPTLC plate using a mixture of Toluene:Methanol:Glacial acetic acid (15:1:0.16, v/v/v) as the mobile phase. Densitometric detection was carried out at 231 nanometers. The method was validated for specificity, precision, accuracy, robustness, linearity, limit of detection, and limit of quantification. Linear calibration curves in the range of 5-80 microgram per band gave a correlation coefficient of 0.99077. The intra-day (n=6) and inter-day (n=18) precision, expressed as the relative standard deviation were in the range of 0.36 to 4.44% and from 0.76 to 2.13%. Clobazam gave a well separated peak at Retardation factor (Rf) 0.30. Caffeine was used as an internal standard, which gave a well separated peak at Retardation factor (Rf) 0.20 without interfering with clobazam. The method was found to be specific with no matrix interference. Thus, the method developed for the estimation of serum clobazam level is simple, cost-effective and reliable for therapeutic drug monitoring.