Objective: The current research work focus to simple and rugged bioanalytical method development and validation of brivudine in human plasma using high-performance liquid chromatography. Methods: The analyte (Brivudine) and internal standard (Sofosbuvir) were extracted using the Solid Phase Extraction (SPE) technique. The chromatographic separation was accomplished by using Zorbax eclipse XDB-C18 Column (150×4.6 mm, 5 μm) with a mobile phase consisted of Methanol: 0.5% Ortho-phosphoric acid (65:35%, v/v) respectively, at a flow rate of 0.7 mL/min. The developed method was validated by performing system suitability, carryover effect, linearity, selectivity, sensitivity, precision, accuracy, recovery, ruggedness, and stability studies. The method was validated as per USFDA guidelines. Results: The selected chromatographic condition was found to efficiently separated brivudine (RT-3.55 min) and ISTD (RT-7.87 min). The assay demonstrated a linear dynamic range of 85.205 to 4500.246 ng/ml for brivudine in human plasma with r2>0.99. Demonstrated the lowest limit of detection at 85.205 ng/ml. This method established an intra-run and inter-run precision within the range of 2.99-6.31%CV and 3.67-5.80%CV, respectively. Additional intra-run and inter-run accuracy were within the range of 97.55-105.37% and 99.27-102.15%, respectively. The mean percentage recovery of brivudine and ISTD studies proved good extraction efficiency and the robustness was also evaluated. Conclusion: A simple, accurate, precise, linear and rugged RP-HPLC method was developed and validated for the estimation of brivudine in human plasma with K2EDTA anticoagulant and suitable for conducting BA/BE and TDM.

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The focus of bioanalysis employed for the quantitative determination of an active analyte(s) and their metabolite(s) in the biological matrix such as plasma, serum, blood, cerebrospinal fluid, and tissues. The extraction of analyte and metabolite in the biological fluids is carried out using different separation methods such as protein precipitation, liquid-liquid extraction, and solid phase extraction. Bioanalytical method development and validation in the pharmaceutical industry are to provide an assessment and interpretation of pharmacokinetics, pharmacodynamics, toxicokinetics, bioavailability/bioequivalence, and therapeutic drug monitoring relationships. This review paper aims to provide a simple and accurate scientific background to improve the quality for development and validation of a bioanalytical method for small molecules with industrial technique as per regulatory agency requirements (United States Food and Drug Administration, EMEA, International Council for Harmonisation and ANVISA).

A simple, specific, sensitive and rapid stability-indicating high performance thin layer chromatographic method has been developed for the simultaneous estimation of ledipasvir and sofosbuvir in combined dosage form. In this method, the separation was achieved on silica gel 60F254 thin layer chromatography plates using mobile phase comprising of hexane: ethyl acetate: methanol (5:3:2,v/v/v) with 3 drops of ammonia at 288 nm as selected wavelength on a densitometer. The Rf value of sofosbuvir and ledipasvir were observed to be 0.21±0.02 and 0.43±0.02, respectively. The linearity, precision, accuracy, robustness, specificity, limit of detection and limit of quantitation of the method were validated according to the ICH guidelines. The linear regression analysis for calibration plots produced r2=0.9948±0.0005 and r2 = 0.9927±0.0003 for ledipasvir and sofosbuvir, respectively. Percent recovery of the drugs from tablet formulation was carried out by standard addition method and was found to be close to 100 and relative standard deviation was less than 2%, which indicated good accuracy and precision of the method. The factors evaluated in the robustness test were determined to have an insignificant effect on the selected responses. To make the method more specific stress studies were conducted for both drugs individually as well as in the combined form by subjected to acid, alkali and neutral hydrolysis, oxidation, thermal and photolytic degradation. Degradation studies indicated ledipasvir to be susceptible to acid and alkaline hydrolysis, thermal, photolytic and oxidative degradation whereas sofosbuvir was susceptible only to alkaline hydrolysis. The degradation product peaks were well resolved from the pure drug with significant differences in their Rf values. The results indicate that the method is suitable for the routine quality control testing of marketed tablet formulation.