• Sushruta S Hakkimane Department of Biotechnology, Manipal Institute of Technology, Manipal University
  • Bharath Raja Guru Department of Biotechnology, Manipal Institute of Technology, Manipal University, India


Objective: The objective of the study was to develop and validate a simple and reproducible reverse phase high pressure liquid chromatography (RPHPLC) 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.


Keywords: Antitubercular drugs, Reverse phase high performance liquid chromatography, Analytical method development.

Author Biographies

Sushruta S Hakkimane, Department of Biotechnology, Manipal Institute of Technology, Manipal University
Sushruta S Hakkimane is a graduate student working for her PhD under the guidance of Dr. Bharath Raja Guru
Bharath Raja Guru, Department of Biotechnology, Manipal Institute of Technology, Manipal University, India
Presently I am working as a Associate Professor and Head, Department of Biotechnology, Manipal Institute of Technology, Manipal University. One of the top five private Engineering colleges of India.


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How to Cite
Hakkimane, S. S., and B. R. Guru. “NANO FORMULATION ANALYSIS: ANALYTICAL METHOD DEVELOPMENT OF ISONIAZID AND SIMULTANEOUS ESTIMATION OF ANTI-TUBERCULAR DRUGS ISONIAZID AND RIFAMPICIN BY RP-HPLC”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 10, no. 5, May 2017, pp. 330-5, doi:10.22159/ajpcr.2017.v10i5.17582.
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