DEVELOPMENT AND VALIDATION OF RP-CHIRAL HPLC METHOD FOR QUANTIFICATION OF (S)-ISOMER IN TENOFOVIR DISOPROXIL FUMARATE
Keywords:Tenofovir disoproxil fumarate, (S)-Isomer, RP-Chiral HPLC, Identification, Quantification and Validation
Objective: The main objective of present study was to develop and validate a reverse phase enantioselective chiral high performance liquid chromatographic method was developed for enantiomeric resolution of Tenofovir disoproxil fumarate; it decreases the HIV infection in human body. The method is specific, rapid, precise and accurate for the separation and determination of (S)-isomer in tenofovir disoproxil fumarate drug substance form.
Methods: The S-Isomer of Tenofovir disoproxil fumarate was resolved on a Chiral AGP (150 Ã— 4.0 mm, 5 Âµm) column (L-41) using a mobile phase system containing 0.1 M ammonium acetate in water pH 6.8 with ammonia solution and methanol in the ratio of (85:15 v/v). The mobile phase was set at a flow rate of 0.8 ml/min and the volume injected was 10Î¼l for every injection. The detection wavelength was set at 260 nm and the column temperature was set at 15 Â°C.
Results: The proposed method was productively applied for the quantitative determination of (S)-isomer in Tenofovir disoproxil fumarate drug substance form. The linear regression analysis data for calibration plots showed a good linear relationship over a concentration range of 0.125 to 3.75 Âµg/ml for (S)-isomer, 0.125-3.75 Âµg/ml for Tenofovir disoproxil fumarate. The mean values of the correlation coefficient were 0.999 and 0.999 for (S)-isomer and Tenofovir disoproxil fumarate. The method was validated as per the ICH guidelines. The detection limit (LOD) was about 0.05 Âµg/ml and quantitation limit (LOQ) was about 0.125 Âµg/ml for (S)-isomer and Tenofovir disoproxil fumarate. The relative standard deviation was found to be 0.78 % for (S)-isomer in Tenofovir disoproxil fumarate.Conclusion: The developed and validated HPLC method and the statistical analysis showed that the method is repeatable and selective for the estimation of the (S)-isomer of the Tenofovir disoproxil fumarate drug substance.
Neil MJO. Ed. by the Merck Index-An Encyclopedia of Chemicals, Drugs and Biological; Merck and Co. Inc; 2006.
Sweetman SC. Ed. by Martindale, the Complete Drug Reference; Pharmaceutical Press; 2007.
Keamey BP, Flherty JF, Shah J. Tenofovir disoproxil fumarate: clinical pharmacology and pharmacokinetics. Clin Pharmacokinet 2004;43:595-612.
Seshachalam U, Rajababu B, Haribabu B, Chandrasekhar KB. Enantiomeric separation of tenofovir on an achiral C18 column by HPLC using L-phenylalanine as a chiral mobile phase additive. J Liq Chromatogr Relat Technol 2008;31:410-20.
Kandagal PB, Manjunatha DH, Seetharamappa J, Kalanur SS. RP-HPLC method for the determination of tenofovir in pharmaceutical formulations and spiked human plasma. Anal Lett 2008;41:561-70.
Sentenac S, Fernandez C, Thuillier A, Lechat P, Aymard G. Sensitive determination of TNF in human plasma samples using reversed-phase liquid chromatography. J Chromatography B 2003;793:317-24.
Jullien V, Treluyer JM, Pons G, Rey E. Determination of TNF in human plasma by high-performance liquid chromatography with spectrofluorimetric detection. J Chromatography B 2003;785:377-81.
Sparidens RW, Crommentuyon KM, Scheelens JH, Beijnen JH. Liquid chromatographic assay for the antiviral nucleotide analogue TNF in plasma using derivatization with chloroacetaldehyde. J Chromatography 2003;791:227-33.
Delahunty T, Bushman L, Fletcher CV. Sensitive assay for determining plasma TNF concentrations by LC/MS/MS. J Chromatogr B: Anal Technol Biomed Life Sci 2006;830:6-12.
Massaki T, Yuichi K, Naoya O, Atsushi H, Kazuhide B, Tsuguhiru K. Determination of plasma tenofovir concentrations using a conventional LC-MS method. Biol Pharm Bull 2007;30:1784-6.
Bezy V, Morin P, Couerbe P, Lepen G, Agrofoglio L. Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer Test of this new volatile medium-pH for HPLCâ€“ESI-MS/MS. J Chromatogr B: Anal Technol Biomed Life Sci 2005;821:132-43.
Shirkhedkar AA, Bhirud CH, Surana SJ. Application of UV-spectrophotometric methods for estimation of tenofovir disoproxil fumarate in tablets. Pakistan J Pharm Sci 2009;22:27-9.
ICH. Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH harmonized Tripartite Guideline; 2005.
Kanarek AD. Analytical methods: a statistical perspective on ICH Q2A and Q2B guidelines for validation of analytical methods. BioPharm Int 2005;19:28-33.
ICH guidance on method validation; validation of analytical procedures Text and Methodology Q2 (R1); 2005
ICH harmonized tripartite guideline impurities in new drug substances Q3 A (R2); 2006.