Analytical Method Validation for Determination of Tuberculostatics in Fixed Dose Combination by Capillary Electrophoresis

Abstract

The World Health Organization (WHO) has proposed a standard therapeutic protocol for tuberculosis using associations of drugs formulated in fixed and appropriated doses according to the disease’s phase and weight range of patients. This guidance aimed to reduce the occurence of new cases and to minimize the developlment of resistance to the drugs involved in this therapy. In this associations, isoniazide and rifampicin are always present. Due to this demand, pharmaceutical industry searchs continually keeping the quality of antituberculosis products in order to fulfill the requirements of sanitary registration and the legal responsibility with safety, quality and efficacy of medications. Therefore, it is necessary the practice of a system that joins the good manufacturing and laboratory practices in which the validation studies compose a primary role of this process. At the same time, to fight such a disease like tuberculosis, which is funded by the public health system, the availability of analytic methodologies that are reliable, short time, low cost and less pollutant show to be extremely important to the public budget, particularly in Brazil, that has achieved the sixth position of world drugs market. As Capillary Electrophoresis (CE) is an analytic tool that has been rapidly growing in the last decades for being a technique of high performance, low cost, low volume samples, low organic solvents consumption and low environmental impact, this study validated a methodology for the analysis of isoniazide and rifampicin in tablets of Fixed Dose Combination (FDC) by CE. The conditions of CE were: capillary of fused silica covered with pollyimide of 64.5 cm of total length (56 cm of effective length) and 50 µm of internal diameter; 20 kV voltage; 25°C cassette temperature; Diode Array Detector (DAD) scan monitoring the wavelength of 200 nm; inserting samples by hydrodynamic of pressure of 25 mbar/3s of sample, followed of 25 mbar/2s of electrolyte. Electrolyc solutions containing 60 mM of sodium borate pH 9.85. The analytic methodology proved to be useful for quantitative determination of isoniazide and rifampicin in FDC tablets. It was proved that the working range is linear for the concentration interval of 200 to 700 µg/mL and 500 to 1000 µg/mL for isoniazide and rifampicine, respectively. The matrix of 2 in 1 tablet does not interfere the results for the analysis of isoniazide and rifampicine. Repeatability of rifampicine and isoniazide of 3.17 % and 2.64 %, respectively were acceptable when compared by the acceptance criterion of 3.30 % for variation coefficient of repeatability calculated by Horwitz equation. Intermediate precision of isoniazide and rifampicine of 1.41 % and 1.67 %, respectively were acceptable when compared to the variation coefficient of reprodutibility of 1.97 % and 1.67 % for isoniazide and rifampicine, respectively, calculated by Horwitz equation. Recuperation range of 92.16 – 95.00 % for isoniazide and 97.59 – 98.41 % for rifampicine were acceptable for the criterion of acceptance of the International Conference Harmonization.