Simultaneous Bioanalysis of Naproxen and Diphenhydramine in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

A simple, sensitive, and rapid bioanalytical method was developed for the first time for simultaneous es-timation of naproxen sodium (NPX) and diphenhydramine hydrochloride (DPH) in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The isotope-labelled analogs, naproxen 13CD₃ and diphenhydramine D₅ hydrochloric acid, were used as the internal standards. The analytes were extracted from 50 µL of human plasma employing a simple protein precipitation technique. The separation of analytes was carried out on a Zodiac C₁₈ column (50x4.6 mm, 3 µm) using a mixture of HPLC grade acetonitrile and 5 mM ammonium acetate buffer in 0.025% formic acid (60:40, v/v) at a flow rate of 1.0 mL/min. The method showed linearity within the concentration ranges 400 to 120,000 ng/mL for NPX and from 0.80 to 240 ng/mL for DPH with r² > 0.99. The method was validated as per the US FDA guidelines and the results were found to be within the acceptance limits. The method was successfully applied for the pharmacokinetic study of both drugs simultaneously after an oral dose of two caplets, each containing NPX 220 mg and DPH 25 mg under fed conditions in human volunteers. Incurred sample reanalysis was also performed to authenticate the reproducibility of the method.