HPLC Method for Simultaneous Determination of 1,4-Benzodiazepines and Tricyclic Antidepressants in Pharmaceutical Formulations and Saliva—A Useful Tool in Medicinal Chemistry

Abstract

Herein, a sensitive and rapid reversed phase HPLC assay was developed for the simultaneous determination of 1,4-benzodiazepines and tricyclic antidepressants in human saliva and pharmaceutical formulations using colchicine as internal standard. Diode array detection (DAD) was carried out at a monitoring wavelength of 240 nm. The analytes and the internal standard were extracted by Nexus (Varian) SPE cartridge and separated by 15 min, utilizing a Kromasil C₈ (150 × 4.6 mm; 5 µm) column with a gradient mobile phase containing methanol, acetonitrile, and 0.05 M ammonium acetate, delivered at a flow rate of 1.0 mL/min. Calibration curves were linear up to the examined range of 20 ng/µL with coefficients of determination (r ²) 0.999. The limits of detection (LOD) and quantification (LOQ) were 0.08–0.34 and 0.28–1.13 ng/mL, respectively, using 20 µL injected volumes. The mean relative recoveries of all analytes in saliva were 85–105% (n = 6) and 86–84 % (n = 6) for intra- and inter day, respectively. Analysis for benzodiazepines and tricyclic antidepressants gave good precision (RSD <10%). The sample preparation was very simple and the method was sensitive enough and reproducible. The method described here is a useful tool in medicinal chemistry, suitable for use both in clinical analysis, as well as for quality control of pharmaceutical formulations.

Keywords:

• Benzodiazepines
• HPLC
• Pharmaceuticals
• Saliva
• SPE
• Tricyclic antidepressants

Notes

∗Program with flow rate 1.00 mL/min has been followed throughout the experiment.