A Simple and Reliable Liquid Chromatographic Method for Simultaneous Determination of Five Benzodiazepine Drugs in Human Plasma

Abstract

Benzodiazepines (BZDs) are one of the most important drugs that have been used in the treatment of neuropsychological disorders. Indeed, BZDs are abused by drug addicts regardless of their therapeutic uses. Therefore, it was important in forensic and clinical toxicology to reach an easy and reliable method for the screening and quantification of BZDs in the human plasma matrix. In the current work, five BZDs, namely bromazepam, clonazepam, lorazepam, nordiazepam and diazepam were simultaneously separated and detected by a simple and reliable RPLC method in a human plasma matrix. Isocratic mobile elution consisting of 20 mmol L−1 phosphate buffer (pH 7.0) and methanol (50:50, v/v) on a Symmetry C18 column was employed. The flow rate, wavelength and column temperature were fixed at 1.0 mL min−1, 214 nm and 40 °C, respectively. The proposed method was validated, giving a linearity within the concentration ranges 5–500 ng mL−1 for bromazepam and diazepam, 3–500 ng mL−1 for clonazepam and lorazepam and 1–500 ng mL−1 for nordiazepam with a determination coefficient (R2) more than 0.9992. The LOD values for the selected BZDs ranged from 0.54 to 2.32 and from 1.78 to 7.65 ng mL−1 for standard methanolic and plasma matrices, respectively. Precision, accuracy, selectivity, stability, and robustness were some of the terms considered in validating the current RPLC method. Based on these results, a simple and reliable RPLC method was successfully applied to quantify BZDs in human plasma matrix appearing with recoveries ranging from 96.5 to 107.5% and interday RSD less than 4%. The current developed method was useful for rapidly screening the most commonly used BZDs in the market within their therapeutic concentration ranges.

Keywords

• benzodiazepines
• reversed-phase liquid chromatography
• plasma