Development and Validation of UV Spectrophotometric Methods for Quantitative Estimation of Antihypertensive Drugs in Fixed Dose Combinations: A Green Analytical Approach

The aim of the current research work was to develop simple, precise and accurate UV spectrophotometric methods for quantitative estimation of amlodipine besylate (AMLO) and nebivolol hydrochloride (NEBI) in binary mixtures and their fixed dose combination. The principle employed was based on isobestic point, area under curve (AUC) and dual wavelength approach. In isobestic point method, wavelength for AMLO and NEBI was found to be 268nm. Concentration of NEBI was determined at isobestic wavelength 268nm while AMLO was determined at 360nm and 268nm as there was no interference of NEBI. In AUC, wavelength of maximum absorption for AMLO and NEBI was found to be 360nm and 280nm. The measurements of AUC were carried out at wavelength range of 348-372nm for AMLO and 268-292 nm for NEBI. In case of dual wavelength, two wavelengths were selected for each drug so that the difference in absorbance would be zero for another drug. The wavelengths selected for determination of AMLO were 272.42nm and 292.42nm, whereas, the wavelengths selected for determination of NEBI were 234.37nm and 239.97nm. Validation of methods were carried out as per ICH Q2(R2) guidelines. Linearity was established over concentration range of 14-30µg/mL for AMLO (r² = 0.999 at 268nm and r² = 0.999 at 360nm) and NEBI (r² = 0.999 at 268nm) in isobestic point method, whereas in case of AUC, it was established at concentration range of 5-25µg/mL for AMLO (r² = 0.999) and NEBI (r² = 0.980). However, in dual wavelength method, linearity was determined at concentration range of 10-26µg/mL for AMLO (r² = 0.999 at 272.42 - 292.42nm) and NEBI (r² = 0.993 at 234.37 - 239.97nm). Percentage relative standard deviation (%RSD) value of less than 2% for intra-day and inter-day precision confirmed the precision of the methods. Accuracy of the methods was found to be in range of 98-102%. The assay results pertaining to the methods were within the range of pharmacopoeial limits. The greenness of the method was assessed using AGREE and ComplexGAPI tools. The routine analysis of fixed dose combination in terms of time and cost renders the method suitable and valid as compared to other expensive techniques using sophisticated instruments. The developed analytical methods stand out not only for its accuracy and efficiency but also for its profound commitment to sustainability and environmental safekeeping.