Optimized SPE‐HPLC‐FLD Method for the Simultaneous Determination of Olaparib, Propranolol, and Furosemide in Human Urine

ABSTRACT Olaparib belongs to the PARP inhibitors and is widely used as an anticancer drug. Due to frequent side effects, it is usually co‐administered with an antihypertensive, such as propranolol and/or an antidiuretic, such as furosemide. In this study, a highly sensitive HPLC‐FLD method was developed for the simultaneous determination of the three APIs in urine. For their analysis, a Nucleosil C8 column (250 mm × 4.6 mm, 5 μm) at 50°C was used as the stationary phase and an isocratic system of methanol–acetonitrile–20 mM NaH 2 PO 4 at pH 3.6, 12:20:68 v/v/v as the mobile phase. The calibration curves for the analytes were generated both in urine and in the pure diluent. The reliability of the method was evaluated according to ICH guidelines in terms of accuracy and repeatability. For sample purification, protein precipitation techniques involving the addition of organic solvents, acids, or salts, as well as the freezing‐out technique, were studied. Liquid–liquid extraction, using various solvents, and solid‐phase extraction (SPE) were also tested, where the latter was found more suitable. SPE optimization was achieved using experimental design and a D‐optimal mixture design. Reliable analyte recoveries demonstrate the capability of the proposed method, making it a suitable tool for biological studies.