Abstract
A stability indicating high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of isoniazid and its related substances of isonicotinic acid and isonicotinamide in fixed dose combination of isoniazid and ethambutol HCl tablet. Inertsil, C-18, 250 × 4.6 mm, 5 m HPLC column with a mobile phase of potassium dihydrogen phosphate buffer of pH 6.9 with a flow rate of 1.5 ml/min has been used for the estimation. The detection wavelength was set at 254 nm in the photodiode array detector. The response of the detector was linear in the range of 0.25 to 1500 µg/ml for isoniazid, 0.25 to 3.00 µg/ml for isonicotinic acid and 0.50 to 3.00 µg/ml for isonicotinamide. The limit of detection (LOD) values for isoniazid, isonicotinic acid and isonicotinamide were 0.083, 0.083 and 0.165 µg/ml, respectively. This validated stability indicating method may be used for the estimation of related substances of isoniazid in tablets. Key words: Isoniazid, high-performance liquid chromatography (HPLC) method, fixed dose combination.
Highlights
Isoniazid (INH) and ethambutol HCl fixed dose combination is used for the treatment of tuberculosis
Many high-performance liquid chromatography (HPLC) columns were evaluated for the peak shape, retention time and resolution of isoniazid, isonicotinic acid and isonicotinamide peaks
The area of isoniazid peak increased with increase of mobile phase pH whereas the area of isonicotinic acid peak decreased with increase in mobile phase pH
Summary
Isoniazid (INH) and ethambutol HCl fixed dose combination is used for the treatment of tuberculosis. The WHO monograph for this fixed dose combination does not include the related substances test (International Pharmacopoeia, 2008). This work reports the development and validation of a high-performance liquid chromatography (HPLC) method for the estimation of isoniazid and its related substances of isonicotinic acid and isonicotinamide in the fixed dose combinations containing isoniazid with ethambutol HCl. EXPERIMENTAL. Isoniazid USPRS (purity 100%) and ethambutol HCl USPRS (purity 100%) were obtained from United States Pharmacopoeia. Isonicotinic acid (purity 99%) and isonicotinamide (purity 99%) reference standards were obtained from Lancaster. Water used for preparation of mobile phase and solutions was purified using Millipore Water Purification System (Elix 10 C model).
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