Validation and application of high-performance liquid chromatographic and spectrophotometric methods for simultaneous estimation of nebivolol and hydrochlorothiazide: A novel approach to multivariate calibrations by R-Software Environment

Abstract

A fast, simple reversed-phase HPLC method and two spectrophotometric methods based on principal component regression and partial least squares calibrations were developed for determination of nebivolol (NEB) and hydrochlorothiazide (HCTZ) in formulations without prior separation or masking. The HPLC assay utilized a Phenomenex-Luna RP-18(2) 250 × 4.6 mm, 5 μm column with acetonitrile-0.03% aqueous formic acid, pH 3.3 (65 + 35, v/v), mobile phase at a flow rate of 1.0 mL/min, and UV detection at 277 nm. The retention times of NEB and HCTZ were 2.133 and 2.877 min, respectively. The total run time was <4 min. Chemometric calibrations were constructed by using an absorption data matrix corresponding to a concentration data matrix, with measurements in the range of 231-310 nm (Δλ = 1 nm) in their zero-order spectra using 16 samples in a training set. The chemometric numerical computations were obtained by using R-Software Environment (Version 2.1.1). The proposed methods were validated for various International Conference on Harmonization regulatory parameters like linearity, range, accuracy, precision, robustness, LOD, LOQ, and HPLC system suitability. Laboratory-prepared mixtures and commercial tablet formulations were successfully analyzed using the developed methods. All results were acceptable and confirmed that the method is suitable for its intended use.