A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF SOFOSBUVIR FROM HUMAN PLASMA
Objective: The present work aimed to develop a simple, rapid, specific and precise ultra-performance liquid chromatography-tandem mass spectrophotometric (LCâ€“MS/MS) validated method for quantification of sofosbuvir and internal standard (ISTD) Sofosbuvir-d3 in human plasma.
Methods: Samples prepared by employing liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Gemini 5Âµ C18, 50 x 4.6 mm column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 30:70 v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 Â°C. Quantitation was performed by transitions of 428.35/279.26 (m/z) for sofosbuvir and 431.38/282.37 (m/z) for sofosbuvir-d3.
Results: The concentrations of ten working standards showed linearity between 4.063 to 8000.010ng/ml (r2Â â‰¥Â 0.9985). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 75.36% for sofosbuvir and were within the acceptance limits. The coefficient of variation was â‰¤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 0.35% and 3.09%. The % stability of short term and long term stock solution stability studies was found to be 97.25% and 98.81% respectively.
Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within the acceptance limits. Thus the validated economical method was applied for pharmacokinetic studies of sofosbuvir.
2. Hoofnagle J, Di Bisceglie A. The treatment of chronic viral hepatitis. N Eng J Med 1997;336:347-56.
3. Ravikumar V, Subramanyam CVS, Veerabhadram G. Estimation and validation of sofosbuvir in bulk and tablet dosage form by RP-HPLC. Int J Pharm 2016;6:121-7.
4. Mohan Vikas P, Satyanarayana T, Vinod Kumar E, Mounika E, Sri Latha M, Anusha R, et al. Development and validation of new RP-HPLC method for the determination of sofosbuvir in pure form. World J Pharm Pharm Sci 2016;5:775-81.
5. Rezk MR, Basalious EB, Karim IA. Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: application to a bioequivalence study. J Pharm Biomed Anal 2015;114:97-104.
6. Rezk MR, Basalious EB, Amin ME. Novel and sensitive UPLC-MS/MS method for quantification of sofobuvir in human plasma: application to a bioequivalence study. Biomed Chromatogr 2016;30:1354-62.
7. Shi X, Zhu D, Lou J, Gan D. Evaluation of a rapid method for the simultaneous quantification of ribavirin,sofobuvir and its metabolites in rat plasma by UPLC-MS/MS method. J Chromatogr B: Anal Technol Biomed Life Sci 2015;1002:353-7.
8. Analytical procedures and methods validation for drugs and biologics guidance for industry. U. S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER); 2015.
9. Sindhusri M, Swetha T, Ramadevi A, Ashok Kumar A. A novel rapid rp-hplc method development and validation for the quantitative estimation of balofloxacin in tablets. Int J Pharm Pharm Sci 2014;7:319-22.
10. Raveendra Babu G, Lakshmana Rao A, Venkateswara Rao J. A rapid RP-HPLC method development and validation for the quantitative estimation ribavirin in tablets. Int J Pharm Pharm Sci 2014;7:60-3.
11. Srinidhi M, Mushabbar Basha MD, V Raj Kumar, Rajendra Kumar J. Stability indicating RP-HPLC method development and validation for the estimation of sumatriptan in bulk and pharmaceutical dosage form. J Appl Pharm Sci 2016;6:20-5.