Analytical Method Development and Validation for the Simultaneous Estimation of Rifampicin and Isoniazid in its Bulk and Pharmaceutical Dosage Forms by RP-HPLC

Introduction

Rifampicin is 5, 6, 9, 17, 19, 21-hexahydroxy -23- methoxy-2, 4, 12, 16, 18, 20, 22-heptamethyl-8- [N-(4-methoyl-1-piperazinyl)-formamidoyl]-2, 7-(epoxypentadeca-1, 11-13-trienimino)-naphtho-[2, 1-6] furane-1, 11(2H)dion-21 acetate, belonging to the category of anti-bacterial and anti-infective agents. Its acts by inhibiting DNA-dependent RNA polymerase leading to a suppression of RNA synthesis and cell death.¹ Isoniazid is pyridine-4-carbohydrazide, also belonging to the category of anti-bacterial and antiinfective agents. Its acts by inhibiting the formation of myco bacterial cell wall. Isoniazid must be activated by Kat G, a bacterial catalase – peroxidase enzyme in myco bacterium tuberculosis.^2-4

Materials and Methods

Chemicals and Reagents

Water High Performance Liquid Chromatographic (HPLC Grade), Acetonitrile High Performance Liquid Chromatographic (HPLC Grade), Rifampicin (Working standards), Potassium dihydrogen phosphate and Ortho phosphoric acid were procured from standard solutions and the tablets were collected from the local market.^5-7

Instrumentation

HPLC-Auto sampler-UV detector separation module 2695, UV detetctor 2487 empower software versioin-2 waters, UV double beam spectro photo meter UV-3000, pH meter, digital weighing balance, sonicator were used for the study procedures.⁸

Chromatographic Condition

The mobile phase 70:30 methanol:sodium acetate buffer was found to resolve Rifampicin and Isoniazid. Ortho phosphoric acid was pre-owned for pH adjustment of buffer to 3. The mobile phase was strained through 0.45 µm membrane filter and degassed by sonication. The flow rate was set to 1.0 mL/min. The drug showed good absorbance at 240 nm, and this was selected as wavelength for further analysis.^9-10

Preparation of Mobile Phase

To establish a relevant HPLC method, couple of different mobile phases were attempted. Based on the readily available solvents, short run time, results and sensitivity of assay, the final mobile phases concluded were aqueous solvent, i.e., MilliQ water and an organic solvent methanol. Solvents were filtered separately through 0.45 µm membrane filter and degassed by sonication.¹¹

Preparation of Standard Solution

10 mg of the standard drug Rifampicin and Isoniazid were weighed accurately and dissolved in 10 mL methanol separately to produce concentration of 1000 µg/mL. It was sonicated to completely dissolve. Then it was filtered through membrane filter paper. This standard stock solution was used to prepare necessary concentrations to construct calibration curve by proper dilution.

Preparation of Sample Solutions

Twenty tablets of R-CINEX (RIF-100 mg, INZ-100 mg) were taken and powdered. A weighed quantity of Rifampicin and Isoniazid were taken, transferred to 100 mL light resistant flask and made up to the required volume by using mobile phase.^12-13 The solution was filtered through 0.45 µm filter. From the filtrate, 15 mL was taken separately in a 25 mL volumetric flask and a sample solution of concentration of 60 µg/mL of both rifampicin and isoniazid was made.¹⁴

Method Development

Several systematic trials were performed to optimize the mobile phase.¹⁵ Different solvents like methanol, water and acetonitrile in different ratios, different pH values of the mobile phase ratios by using different buffer solutions in order to get sharp peak and base line separation of the components of the excipients were done. Satisfactory peak symmetry, resolved and free from tailing was obtained in mobile phase Methanol: Acetonitrile: Water 60:20:20 (v/v) in isocratic condition for both rifampicin and isoniazid.^16-18

Flow rates of the mobile phase were changed from 0.5–1.2 mL/min for optimum separation. The successful elution of the analytes was done the spectrum showing overlapping spectrum of Rifampicin and Isoniazid was shown in Fig 1.¹⁹

The retention time of Rifampicin and Isoniazid was found to be 2.143mints and 2.862mints respectively was shown in Fig 2. The percentage purity of Rifampicin and Isoniazid in pharmaceutical dosage form was found to be 99.24 and 101.04% respectively was shown Table1.

Results

Retention Time (RT)

The retention time of Rifampicin and Isoniazid was found to be 2.143 mins and 2.862 mins respectively.

Specificity

The system suitability for specificity was carried out to evaluate whether there is any interference of any impurities in retention time of analytical peak.

Linearity

The linearity study was performed for the concentrations 50 ppm to 250 ppm of Rifampicin and 5 ppm to 50 ppm of Isoniazid which is shown in Table 2.

Accuracy

The accuracy study was performed for 50%, 100% and 150% Rifampicin and Isoniazid. The percentage retrieval was found to be 100.56% and 101.47% as shown in Table 3.

Precision (Repeatability)

The precision evaluation was performed for five injections of Rifampicin and Isoniazid. Each standard injection was injected into chromatographic system. The intermediate precision study was performed for five injections which is depicted in Table 4.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD was performed for Rifampicin and Isoniazid which was estimated to be 2.139 and 2.842, respectively. The LOQ was performed for Rifampicin and Isoniazid which was estimated to be 2.138 and 2.843, respectively (Figure3).

Robustness

The robustness was performed for the flow rate variations from 0.4 mL/min to 0.6 mL/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Rifampicin and Isoniazid which indicated that the variation in flow rate affected the method significantly (Table 5).

Figure 3: The LOD for Rifampicin and Isoniazid was estimated to be 2.139 and 2.842, respectively and LOQ was found to be 2.138 and 2.843, respectively.

Discussion The analysis method developed for the separation of Rifampicin and Isoniazid has shown well resolved peaks. Since the RT is short, it indicates that in a shorter duration more samples could be completed and developed method will be easy for analyzing larger samples. The values of LOD and LOQ for these both drugs were significantly low; hence, this method is appropriate for detecting and quantifying the fairly low concentrations of these drugs. Results of statistical analysis, lower %RSD values confirm the ability of the analytical assay. The analytical to ICH guidelines (ICH, Q2 (R1)). Results is simple, reliable, precised, accurate, linear and reproducible, hence can be applied for drug delivery analysis. This method could be even suitable in active pharmaceutical preparations for quality control analysis.

Conclusion

It can be concluded that the proposed RPHPLC method is accurate, precise, sensitive, specific, robust and reproducible for the simultaneous analysis of Rifampicin and Isoniazid with less tailing factor and is also economical. Thermosil RPC18 (4.5×100 nm) 5.0 µm, flow rate was 1 mL/min. Both the samples were in the range of 200 to 400 nm and maximum wavelength was identified at 240 nm.

Consent and ethical approval

It is not applicable

Competing interest

Authors have declared that no completing interests exists

Acknowledgements

We gratefully acknowledge ABIPER and RGUHS University for providing all needed things to prepare this manuscript.