Development and validation of a stability indicating high-performance liquid chromatography ( HPLC ) method for the estimation of isoniazid and its related substances in fixed dose combination of isoniazid and ethambutol hydrochloride tablets

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 μ 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.


INTRODUCTION
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).Although some methods have been reported for the estimation of isoniazid and its related compounds (Bhutani et al., 2007;Butterfield et al., 1980;Chen et al., 2005;Khuhawar et al., 2005Khuhawar et al., , 2006;;Kompantseva et al., 2005;Kulkarni et al., 2004;Musch et al., 1990;Wang et al., 2007) no method has been published thus far for the estimation of related compounds of isoniazid in the fixed dose combination of isoniazid and ethambutol HCl tablets.This work reports *Corresponding author.E-mail: ayyaps15@gmail.com.Tel: + 91-80-28379197.
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.

Reagents and materials
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.Potassium dihydrogen phosphate AR was from Qualigens, India and triethanolamine AR and potassium hydroxide pellets AR were from Merck, India.Water used for preparation of mobile phase and solutions was purified using Millipore Water Purification System (Elix 10 C model).Mobile phase was filtered using cellulose acetate filter (0.45 micron -Sartorium stedim) and standard and sample solutions were filtered using 0.45 micron PTFE membrane filters (Syringe filter, 25 mm, GD/X, Whatman).

Instrumentation
A Waters HPLC system equipped with a 2695 solvent delivery system, Waters auto injector, thermostatted column compartment and 2996 PDA detector and Empower software was used.Inertsil ODS 3 V HPLC column (250 × 4.6 mm i.d., 5 µ particle size) was used for the analysis.

Mobile phase
A potassium dihydrogen orthophosphate buffer of pH 6.9 was used as the mobile phase.The buffer was prepared by dissolving 27.2 g of potassium dihydrogen phosphate in 1500 ml of water containing 10 ml of triethanolamine solution (prepared by dissolving 1.49 g of triethanolamine in 250 ml of water) and adjusting the pH to 6.9 ± 0.05 using 10 M potassium hydroxide and making up the volume to 2000 ml with water.The buffer was degassed by sonication and filtered through 0.45 micron cellulose acetate membrane filter.The filtered buffer with a flow rate of 1.5 ml/min was used as mobile phase.

Standard preparation
About 5 mg of isonicotinic acid and 5 mg of isonicotinamide were accurately weighed and dissolved in 25 ml of water in a 25 ml volumetric flask.About 100 mg of isoniazid was accurately weighed into a 100 ml volumetric flask, dissolved in 50 ml of water and 1 ml of the related substances solution was added and the volume was made up to 100 ml with water to obtain a solution containing 1000 µg/ml of isoniazid and 2 µg/ml of each of isonicotinic acid and isonicotinamide.

Sample preparation
Twenty tablets of isoniazid and ethambutol hydrochloride were powdered and the tablet powder equivalent to about 100 mg of isoniazid was accurately weighed into a 100 ml volumetric flask.About 70 ml of water was added and sonicated for 5 min and the volume was made up to 100 ml with water and the solution was mixed well to achieve a concentration of 1000 µg/ml of isoniazid.The solution was filtered through 0.45 micron polytetrafluoroethylene (PTFE) membrane filter.

Method validation
The HPLC method was validated for specificity, system suitability, linearity, limit of detection, limit of quantitation, precision, accuracy, solution stability and robustness according to USP and ICH guidelines.

Statistical analysis
The %RSD value and the linear regression analysis by the method of least squares were calculated using Excel program.

Selection of mobile phase buffer and pH
Different buffers were evaluated for system suitability compliance.The potassium dihydrogen phosphate buffer of pH 6.9 containing triethanolamine was found to be the      most satisfactory in terms of compliance with system suitability requirements and sensitivity of the method.The mobile phase pH was found to have a significant impact on the detector response and resolution between the peaks (Table 1).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.The area of isonicotinamide peak decreased to a lesser extent with increase in mobile phase pH.The resolution between isoniazid and the related substances was maximum at the pH of 6.9.Based on these results, the dihydrogen phosphate buffer of pH 6.9 containing triethanolamine was selected as the mobile phase for the method.

Chromatographic parameters of the method
The standard and spiked sample chromatograms obtained by using the selected column and mobile phase have been presented in Figures 5 and 6, respectively.The retention times of isonicotinic acid, isoniazid and  Isonicotinamide are about 5, 15 and 20 min, respectively.The relative retention time of isonicotinic acid was 0.35 and isonicotinamide was 1.38 with respect to isoniazid peak.The minimum resolution between the isoniazid peak and the related substances peak was 7.0, the tailing factor for the peaks was not more than 1.5 and the number of theoretical plates of the each of peak was more than 5000.The isoniazid, isonicotinic acid and isonicotinamide peaks were spectrally pure as calculated by the software on the basis of the purity angle and purity threshold (Figures 7 to 9).The optimized wavelength of the method was 254 nm and the ethambutol hydrochloride which is present in the sample solution did not give any response at this wavelength in the proposed method.

Specificity
The forced degradation of INH, placebo and formulation was carried out as per ICH guideline (Q 2 B) using acid, alkali, oxidation, heat and photolysis as stress agents.The acid stress was carried out by refluxing INH, placebo and formulation at about 80°C for 2 h with 1 N hydrochloric acid.The alkali stress was carried out by refluxing INH, placebo and formulation at about 70°C for 30 min with 0.3 N sodium hydroxide.The oxidation stress was carried out by refluxing INH, placebo and formulation at about 70°C for 30 min with 3% hydrogen peroxide.The thermal degradation was carried out by heating the INH, placebo and formulation at 105°C for 12 h and the photo degradation was performed exposing the drug material as pure drug substance and tablet powder to 1.2 million lux hours.The percentage of degradation observed and the purity of the peaks have been tabulated in Table 2.

System suitability
The resolution between isonicotinic acid and INH peaks is about 22.5 and between INH and Isonicotinamide peaks is about 7.9.The tailing factor for isonicotinic acid, INH and Isonicotinamide peaks are about 1.5, 1.1 and 1.1, respectively.The number of theoretical plates for isonicotinic acid, INH and Isonicotinamide peaks are about 6263, 9547 and 10646, respectively.

Linearity, limit of detection (LOD) and limit of quantitation (LOQ)
The linearity range of INH, isonicotinic acid and isonicotinamide and the LOD and LOQ values have been presented in Table 3.The correlation coefficient for the linearity was more than 0.9990 indicating a good level of correlation between the area of the peak and concentration.

Precision
The precision of the method was checked by analysing six sample preparations which were spiked with known concentrations of the related substances.The % recovery of INH and related substances and the %RSD have been

Accuracy
The accuracy of the method was determined by calculating the % recovery of INH and the related substances at various concentration levels.The results presented in Table 5 show that the % recovery ranges between 98.8 and 100.7% indicating the acceptable accuracy of the method.

Solution stability
The stability of standard and sample solutions in water was checked for 24 h and was found to be satisfactory since the % difference in the area of the peaks when compared to the initial peak area was within ± 2% for INH and ± 10% for isonicotinic acid and isonicotinamide up to 24 h.

Robustness
The robustness of the method was tested by analyzing the standard solution with changes in the flow rate of the mobile phase, pH of the mobile phase and column temperature as presented in Table 5.The system suitability requirements of tailing factor, number of theoretical plates and resolution were within the specified limits even with the altered method parameters indicating that the method is robust and that small changes in the flow rate of mobile phase, pH of mobile phase and column temperature do not affect the performance of the method.

DISCUSSION
Various HPLC columns were tried to achieve better separation of peaks.Out of which the Inertsil ODS 3 V, 250 × 4.6 mm, 5 µ was found to be best choice (Figure 5).Isoniazid and isonicotinamide peaks were not separated with waters symmetry C18, Waters Sunfire C18 and Agilent Zorbaxil C18 columns and the isonicotinamide peak was broad and tailing with Supelcosil LC-18 column.The mobile phase pH was found to have a significant impact on the detector response and resolution between the 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.The area of isonicotinamide peak decreased to a lesser extent with increase in mobile phase pH.The resolution between isoniazid and the related substances was maximum at the pH of 6.9.Based on these results, the dihydrogen phosphate buffer of pH 6.9 containing  triethanolamine was selected as the mobile phase for the method.
The system suitability requirements of resolution of not less than 5.0 between INH and its related substances peak, tailing factor of not more than 2.0, and number of theoretical plates for each peak of not less than 5000 were satisfactorily complied with by the proposed method.The method has been validated in terms of specificity, system suitability, linearity, limit of detection, limit of quantitation, precision, accuracy, solution stability and robustness.The results of validation showed that all the validation parameters are satisfactory.The peak areas of isoniazid, isonicotinic acid and isonicotinamide peaks were linear with respect to concentration.The correlation coefficient values of 0.999 indicate the excellent correlation between the response (peak area) and the concentration.

Conclusion
The analytical method developed for the estimation of INH, isonicotinic acid and isonicotinamide in the tablets is a very useful tool for monitoring of the quality of the fixed dose combinations (FDCs) of INH with ethambutol HCl.The method validation data shows that this is a stability indicating robust method which can be used for checking the quality of the manufactured tablets as well as for stability studies of the tablet.

Table 1 .
Effect of mobile phase pH on sensitivity and resolution of peaks.

Table 5 .
Recovery and robustness experiment data.