Estimation of measurement uncertainty and validation of RP-HPLC for simultaneous determination of five antihistamines in pharmaceutical formulations

Abstract

Nowadays, measurement uncertainty is considered an essential parameter for method evaluation. Measurement uncertainty estimation requires a detailed study of all possible sources of uncertainty. The aim of this work was to develop and validate an isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous separation and determination of five antihistamines in pharmaceutical formulations and to establish a procedure to evaluate the measurement uncertainty of values generated by the validated method. A relatively short chromatographic separation time, less than 9 min, was achieved by means of a Waters XBridge C18 (4.6 mm×250 mm, 5 µm) analytical column under isocratic conditions using a mobile phase consisting of sodium perchlorate buffer (pH 3.5; 0.1 mol L−1)–acetonitrile (55:45, v/v) at a flow rate of 1 mL min−1 and UV detection at 235 nm. The selectivity, method linearity, accuracy, precision, limits of detection (LOD) and limits of quantification were examined as parts of the method validation. The described method shows excellent linearity over a range of 30 μg mL−1 to 70 μg mL−1 for all compounds with correlation coefficients higher than 0.995. The standard deviations of the intraday and inter-day precision were all less than 2 %. The LOD of all studied compounds ranged from 0.029 μg mL−1 to 0.03 µg mL−1. Using EURACHEM/CITAC guide and Valid Analytical Measurement (LGC/VAM) protocol, measurement uncertainty associated with precision and standard preparation were the most important contributing for the five antihistamines, accounting for 87.72 % to 91.03 % and 4.05 % to 10.24 % of total uncertainty, respectively, and overall uncertainties obtained were from 0.05 mg to 0.58 mg. Therefore, the proposed method was suitable for the routine analysis of antihistamines in pharmaceuticals formulations with the required accuracy, precision and measurement uncertainty.