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

Abstract

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 (250 mm × 4.6 mm I.D., 5 µm) with a mobile phase consisting of acetonitrile-sodium formate buffer (10 mM, pH = 5.5) (90:10, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was 254 nm. The chromatographic run time was less than 5 min 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 25–5000 ng/mL (r² > 0.99) for diazepam and estazolam using 0.2 mL of human plasma. The limit of quantification (LOQ) and limit of detection (LOD) for both compounds were 25 and 10 ng/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.

Keywords:

• diazepam, estazolam
• human plasma
• hydrophilic interaction chromatography (HILIC)
• method validation
• solid phase extraction

ACKNOWLEDGMENT

The authors gratefully acknowledged projects supported by the National Natural Science Foundation of China (No. 20977058), the State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environment Sciences, Chinese Academy sciences (No. 2011-06), and Natural Science Foundation for innovation group of Hubei Province, China (No: 2009CDA020).

Notes

^a Human plasma 0.2 mL, spiked to yield final concentrations for each analyte to 50, 200, and 1000 ng/mL.

^b Average of three results of the spiked recoveries.

^c The RSDs (%) are given in parentheses.