DEVELOPMENT AND VALIDATION OF FOUR NEW UVSPECTROMETRIC METHODS FOR THE DETERMINATION OF ISONIAZID (INH) IN PURE AND TABLET DOSAGE FORMS
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ABSTRACT
Tuberculosis (TB) is a serious infectious disease which is on increase since emergence of human immunodeficiency virus (HIV) leading to TB/HIV co infection, causing complications and treatment failure. Isoniazid is a first line drug for the treatment of TB therefore it is important to develop methods that will monitor its quality. In this study, four rapid, simple, relatively accurate, and precise spectrophotometric methods for the quantitative determination of isoniazid in pure and tablet dosage form were developed and validated. Methods 1 and 2 were based on dissolving standard isoniazid powder in distilled water and methanol respectively and scanning using UV-Vis spectrophotometer at a wavelength range of 200 - 400 nm to determine the wavelength of maximum absorption (λmax). Method 3 was based on condensation reaction between isoniazid and 2,4-di-nitrophenyl hydrazine (2,4 DNPH) and heating under acidic condition using 2 mL of 85 % hydrochloric acid (HCl) to form an orange-red hydrazone which was allowed to stand for few minutes for complete color development. Method 4 was based on the diazotization of isonizid with sodium nitrite (NaNO2) and coupling withparaaminotoluidine under acidic condition (2 % HCl) and gently heating to give a colouredchromagen (diazo dye) which was allowed to stand for few minutes for complete color development. The colouredhydrazone and diazo dye formed were scanned at visible range of 400-750 nm to determine their λmaxs. Calibration curves were prepared using these methods. The developed methods were validated using ICH guidelines with respect to linearity, precision, accuracy, percentage recovery, limit of detection (LOD) and limit of quantification (LOQ). Standard isonizid powder and a sample of isoniazid tablet were assayed using each method and compared with BP, 2009 method for the assay of isoniazid. Method 1 and 2 were observed to have a λmax of 264 nm while an orange-red and a yellow chromagencolours were formed for methods 3 and 4 viii with time for complete development and λmax 15 and 20 minutes, 464 and 420 nm respectively. Methods 1 and 2 obeyed Beer’s law within a concentration range of 1-11 μg/mL while the range for methods 3 and 4 was 1-18 μg/mL. The correlation coefficient for the calibration plot was 0.999 in each case. The precision of the methods were ≤ 1.57 (percentage coefficient of variation, % CV) while the accuracy, percentage relative error (% Er) and percentage recoveries were ≤ 4.130 and ≤ 102.01 respectively. The methods have LOD and LOQ of ≤ 0.145 and ≤ 0.559 µg/mL respectively. All the validation parameters were within the normal ranges. The percentage content of isoniazid in the standard powder and brand of the tablet assayed using all the developed methods were within the BP range of 99 - 101 % and 98.0 – 102 % respectively. There was no significant difference (p < 0.05) between the percentages drug content assayed using the developed methods and that of the BP method, thus the developed methods can be interchanged with the BP method for quantitative estimation of isoniazid in pure and tablet dosage form.
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