A RAPID HILIC METHOD FOR ANALYSIS OF DIAZEPAM AND ESTAZOLAM IN HUMAN PLASMA

A rapid method based on hydrophilic interaction chromatography (HILIC) using UV detection was developed for analysis of diazepam and estazolam in human plasma. The method utilized a Kromasil KR100-5SIL column (250mmx4.6mm I.D., 5 mu m) with a mobile phase consisting of acetonitrile-sodium formate buffer (10mM, pH=5.5) (90:10, v/v) at a flow rate of 1.0mL/min. The detection wavelength was 254nm. The chromatographic run time was less than 5min per injection. All the experiments were performed at room temperature. The isolation of the analytes from plasma samples was achieved by solid phase extraction (SPE) using Oasis HLB cartridges. The standard curve range was 255000ng/mL (r2>0.99) for diazepam and estazolam using 0.2mL of human plasma. The limit of quantification (LOQ) and limit of detection (LOD) for both compounds were 25 and 10ng/mL, respectively. Recoveries of the analytes ranging from 70.7% to 88.5% with intra- and inter-day precision (RSD) less than 11% were obtained at the concentrations above the LOQ. The proposed method is rapid, simple, accurate, and its mobile phase is compatible for mass spectrometry, proving an alternative method for benzodiazepine drug monitoring in forensic and clinical toxicology as well as pharmacokinetic studies.