DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF DORZOLAMIDE HYDROCHLORIDE AND TIMOLOL MALEATE IN OPHTHALMIC DOSAGE FORM

RAMAKRISHNA K.; SRUJAN KUMAR M.

doi:10.22159/ijap.2025v17i6.55359

Authors

RAMAKRISHNA K. Institute of Pharmaceutical Sciences and Research, Bhagwant University, Sikar Road, Ajmer-305023, Rajasthan, India https://orcid.org/0009-0002-4158-1764
SRUJAN KUMAR M. Institute of Pharmaceutical Sciences and Research, Bhagwant University, Sikar Road, Ajmer-305023, Rajasthan, India https://orcid.org/0009-0006-1385-1877

DOI:

https://doi.org/10.22159/ijap.2025v17i6.55359

Keywords:

COSOPT® PF (RLD), Dorzolamide hydrochloride (DZH), Timolol maleate (TIM), RP-HPLC, Stability-indicating method, Forced degradation, Ophthalmic solution, Method validation

Abstract

Objective: This new reverse phase high-performance liquid chromatography (RP-HPLC) method demonstrates superiority over the methods found in official monographs, as it is not included in the United States Pharmacopeia, British Pharmacopeia, or European Pharmacopeia (USP, B. P, Ph Eur). It effectively separates all impurities with excellent resolution from the main analyte, and the spectral purity of the main analyte was confirmed through peak purity analysis in specificity studies involving spiked sample solutions and degradation studies. According to the British Pharmacopeia, impurities B and D are associated with dorzolamide hydrochloride (DZH), while timolol maleate (TIM) includes Impurity G. The USP lists maleic acid and DZH-related compounds B and D for DZH, along with G, B, and D for TIM.

Methods: Chromatographic separation was carried out on a Symmetry C8 column using buffer pH 3.0, acetonitrile, and methanol (50:40:10 v/v/v) as mobile phase, at a flow rate of 1.2 ml/min, and a step gradient elution was used. DZH and TIM were detected at 253 nm and 295 nm, respectively, with retention times of 17.6 and 28.4 min.

Results: The method was found to be linear (r²>0.999) in the concentration ranges of 50-400 μg/ml for DZH and 12-100 μg/ml for TIM. It was validated for precision (both analytes %RSD = 0.3%), accuracy (recoveries 98.7-100.5%), specificity, and robustness. Forced degradation studies also showed the validity of the method with good resolution between the degradation products and the main peaks.

Conclusion: The developed RP-HPLC method is novel, simple, sensitive, precise, accurate, and robust for DZH and TIM in ophthalmic formulations as per the International Council for harmonisation (ICH) for stability studies to perform Quality control (QC) of bulk and finished dosage forms.

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