Determination of diazepam in human plasma by developed and validated a high-performance liquid chromatographic ultraviolet method

Abstract

Background and Aims: Diazepam is accepted as a safer drug to medicate in many serious cases, acting as an anticonvulsant, an anxiolytic and a treatment for many types of poisoning. Monitoring it is important in achieving successful treatment and reducing the risk of toxic effects. In this study, it is aimed to develop and validate a sensitive, repeatable, and reliable method based on high-performance liquid chromatographic analysis for the determination in human plasma.
Methods: Separation was carried out using a reverse-phase C18 column (4.0 mm x 150 mm, 3 μm) at 30 °C. The solution was prepared with a 10 mM phosphate buffer and acetonitrile (1:1, v/v) was employed as a mobile phase at the isocratic flow with 0.5 mL/min rate. Quantification was applied at 230 nm. A solid-phase extraction method was established and optimized, which was then used in the preparation of the plasma (0.5 mL) samples to the analysis.
Results: The method was found to be linear (r2= 0.9805) between 100 and 1200 ng/mL. The analysis run was ≤12 min. Intra- day and inter-day accuracy were found between -5.78 and 5.93 and precision was ≤1.82%. The limit of detection and quan- tification were calculated as 20.42 and 61.86 ng/mL, respectively. Recovery was found between the range of 95.12% and 106.83%. The method was determined to be robust according to changes in UV, mobile phase organic solvent content, mobile phase pH, column temperature, and operator.
Conclusion: This simple, sensitive and reliable method is suggested for accredited-reference laboratories working on the therapeutic drug monitorization and/or overdose-toxicological quantified analysis of diazepam in human plasma.

Keywords

• Diazepam
• Solid-phase extraction
• Method validation
• High-performance liquid chromatography-ultraviolet detection

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