Nano formulation analysis: Analytical method development of isoniazid and simultaneous estimation of antitubercular drugs isoniazid and rifampicin by reverse phase high pressure liquid chromatography

Objective: The objective of the study was to develop and validate a simple and reproducible reverse phase high pressure liquid chromatography (RP-HPLC) method for hydrophilic drug isoniazid (INH) to apply for the analysis of the INH in nanoparticle drug formulations. Furthermore, to estimate simultaneously rifampicin (RIF) and INH in combined form. Methods: Isocratic elution with 10 minutes runtime on a C-18 Luna, 5 µ, 100Å, 150 mm column, methanol, and water as mobile phase with detection wavelength at 268 nm was used. INH nanoformulations were prepared by double emulsion solvent evaporation technique. Quantitative analysis of encapsulated drug was estimated via developed RP-HPLC method. Simultaneous estimation for the two drugs was carried out by gradient elution. All chromatographic separation and estimations were obtained on Shimadzu HPLC system. Results: INH eluted with a short retention time (RT) of 4.06 minutes. Method showed good linearity in the range of concentrations 0.01-100 µg/ml. The limit of detection (LOD) and quantification (LOQ) for INH was 0.03 and 0.12 µg/mL, respectively, and developed method has been successfully applied for the analysis of drugs in nanoparticle formulations. Simultaneous estimation of antitubercular drugs INH and RIF showed two separate peaks within specified runtime. Conclusion: Developed method showed good resolved peaks. Since the RT is short, in a shorter duration more samples could be completed and developed method will be easy for analyzing greater number of samples. Analysis of nanoformulation results have shown that this method is simple, reliable, reproducible, hence can be applied for drug delivery analysis.