RP-DAD-HPLC Method for Quantitative Analysis of Clofazimine and Pyrazinamide for Inclusion in Fixed-Dose Combination Topical Drug Delivery System

Overview

An RP-HPLC method with diode-array detection was developed and validated for simultaneous quantitative analysis of clofazimine and pyrazinamide in a fixed-dose combination topical formulation. The method employs gradient elution on a C18 column with formic acid and acetonitrile mobile phases, detecting the two active pharmaceutical ingredients at distinct wavelengths (254 nm and 284 nm respectively) to ensure selectivity across chemically diverse compounds within a single dosage form.

Methods and approach

Chromatographic separation utilized a C18 column (4.6 mm × 150 mm, 5 µm) with gradient elution at 1 mL/min using 0.1% aqueous formic acid and acetonitrile as mobile phases. Detection was performed at compound-specific wavelengths via diode-array detection. Validation followed ICH Q2 guidelines, encompassing specificity testing against solvents, product matrix components, and degradation products; linearity assessment across 7.8–500.0 µg/mL range; system repeatability evaluation; and intermediate precision determination. Method robustness was assessed using three-level Box–Behnken design with response surface methodology to evaluate effects of detection wavelength, mobile phase flow rate, and column temperature variations.

Results

The method demonstrated linearity with r² = 0.9999 across the specified concentration range. System repeatability yielded %RSD values not exceeding 2.7%, while intermediate precision at 25–500 µg/mL concentrations showed %RSD ≤ 0.85%. Specificity was confirmed with no interference from solvents, matrix components, or degradation products. Box–Behnken design with response surface methodology successfully characterized robustness parameters, establishing method stability across defined operating ranges.

Implications

The developed RP-DAD-HPLC method provides a validated analytical tool suitable for quality control and developmental testing of fixed-dose combination topical formulations containing clofazimine and pyrazinamide. The combination of gradient chromatography with dual-wavelength detection addresses the analytical challenge of simultaneously quantifying pharmaceutically active compounds with divergent chemical properties within a single formulation, supporting both manufacturing quality assurance and regulatory compliance requirements.