Redox and Complex Formation Reaction–Based Spectrophotometric Assay of Rifampicin in Pharmaceuticals and Urine

Abstract

Introduction: Rifampicin (RIF), 3-[(4-methyl-1-piperazinyl) imino methyl] rifamycin SV [Figure 1], is a semi synthetic bactericidal antibiotic, active in vitro against gram-positive microorganisms and mycobacteria [1]. It is used in the treatment of tuberculosis and other infections and widely applied in human and veterinary medicine. There is drug toxicity during the treatment with RIF, especially in patients with human immunodeficiency virus (HIV) infection, and there has been a global increase in the prevalence of drug-resistant tuberculosis. Therefore, drug monitoring in patients during anti-TB therapy is important in the case of drug resistance and toxicity [2,3].

Objectives: Two rapid, simple, sensitive and selective spectrophotometric methods were developed and validated for the assay of rifampicin (RIF) in pure form, capsules and spiked human urine.

Methods: The methods were based on the reduction of iron(III) chloride by RIF and subsequent chelation of iron(II) with 1,10-phenanthroline (Phen method) and 2,2’-bipyridyl (Bipy method). The absorbances of resulting colored products were measured at 520 and 540 nm, respectively. Experimental conditions for the assay were optimized.

Results: Regression analysis of Beer’s law plots showed good correlation in the concentration ranges, 2.5-45 and 2.5-50 µg mL^-1 with apparent molar absorptivities of 1.91×10⁴ and 1.68×10⁴ L mol^-1 cm^-1 for Phen method and Bipy method, respectively. The Sandell sensitivity values, limits of detection (LOD) and quantification (LOQ) values have also been reported for both methods. The accuracy and precision of the methods were evaluated on between-day and day-day basis; the relative error (%RE) was ≤ 2.36% and the relative standard deviation (RSD) was ≤ 1.94%. The developed methods were successfully applied to the determination of drug in RIF capsules and spiked human urine with good recovery. Statistical comparison of the results with the reference method showed good agreement and indicated no significant difference in accuracy and precision.

Conclusions: Two visible spectrophotometric methods for the assay of RIF in drug substance, drug product and in spiked human urine were developed and validated for accuracy, precision, linearity, robustness and ruggedness. The methods employ normal conditions compared to those previously reported, and rely on well-characterized redox-complexation reactions (chelation). Besides, these methods have the advantages of simplicity without involving heating or extraction step; use aqueous solutions of eco-friendly reagents. When extraction difficulties arise with other published methods (Table 7), with these methods, one can do the analysis at low cost without losing accuracy. The methods can be used as an alternative method to reported ones for the routine determination of RIF in drug substance, drug product and in spiked human urine.