Isocratic Reversed-phase High Performance Research Articles

Reverse Phase HPLC Methodology for the Determination of Bay K8644.

Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2mL/min. Mean retention time for Bay K8644 was 3.030minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40μg/mL at 274nm wavelength. Analytical limits were 16.56 ± 1.04ng/mL for detection and 55.21 ± 3.48ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products.

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

Comparative evaluation of two intranasal forms of Curcumin: Quantitation and impact on mouse model of asthma

Aim of the studyPresent study aimed to investigate impact of two different intranasal forms of curcumin on mouse model of asthma and detection of its absorption after different time points of administration. Since the systemic bioavailability of curcumin is very low, therefore, intranasal route of administration has been explored here where systemic absorption of two intranasal forms of curcumin was evaluated. Materials and methodsTo explore the possibilities of its absorption and role in amelioration of asthma, two intranasal forms of curcumin, nasal drop and aerosol were administered to control and asthmatic mice. Blood plasma and lungs were collected at different time points of curcumin administration. Separation was done by using isocratic reversed phase high performance liquid chromatography (HPLC). Airway inflammation and oxidative stress parameters like SOD, Catalase, ROS, lipid peroxidation, and total inflammatory cell recruitments were analyzed to study efficacy of two intranasal forms of curcumin. ResultsMaximum absorption was noted at 3 hrs of different intranasal forms of curcumin, administration, i.e., nasal drop (1.93 ± 0.050 plasma, 2.87 ± 0.26 in lungs) and aerosol form of curcumin where better absorption was noted in aerosol form as compared to nasal drop (Lungs 3.08 ± 0.12 aerosol, 2.05 ± 0.020 nasal drop) at 1 h. Both, curcumin aerosol and nasal drops were effective in ameliorating oxidative stress (p < 0.05) associated with asthma in mice model. The present study may pave the way towards the development of intranasal curcumin as complementary medication because of its better absorption in plasma and lungs.

Estimation of Pramipexole Dihydrochloride in Tablet Formulation by the Developed Reverse Phase High Performance Liquid Chromatography Method and its Validation

A simple, rapid isocratic reverse phase high performance liquid chromatography method was developed and validated for the determination of pramipexole dihydrochloride in tablet dosage form as per International Council on Harmonisation guidelines. In this method the chromatographic separation was carried out using C18 column with ultraviolet detector set at 264 nm. The eluent consisted of acetonitrile:phosphate buffer (pH 4.0) (70:30 by volume) with a flow rate of 1 ml/min. The method showed linear response with the retention time of 2.28 min. The main advantage of the developed method is its cost effectiveness and shortened run time. Hence this method can be applied for routine analysis of pramipexole in pharmaceutical dosage form.

Novel Rapid Isocratic RP-HPLC Method for Simultaneous Estimation of Phenylephrine Hydrochloride, Paracetamol, Caffeine, Diphenhydramine Hydrochloride

Background: A tablet dosage form widely used in the treatment of cough and cold, containing phenylephrine hydrochloride, paracetamol, caffeine, and diphenhydramine hydrochloride as active pharmaceutical ingredient was selected for the development of a novel, rapid, simultaneous isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method. Objective: The objective of this paper was to develop and validate a novel, rapid, simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride in bulk and pharmaceutical dosage form. Method: Optimized chromatographic conditions were an isocratic elution with prontosil C18-column (250×4.6 mm, 5 μ), methanol and 20mM phosphate buffer (55:45 v/v, pH 3 ) as mobile phase, flow rate 1.0 mL/min and UV detector set at λ max 220 nm. The method was validated for specificity, precision, linearity, accuracy, sensitivity, and robustness as per the International Council for Harmonization guidelines. Result: The retention times of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride were found to be 2.8 min, 3.3 min, and 4.0 min and 7.3 min, respectively. This novel method was found to be rapid, simple, linear (R2&gt; 0.99), preciserelative standard deviation &lt; 2.0 %), accurate (recovery 98-102%), sensitive and robust. Conclusion: The proposed novel isocratic RP-HPLC method is rapid (short run time below 10min), highly selective, precise, accurate, sensitive and robust. The method was successfully applied for the simultaneous analysis of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride in a pharmaceutical dosage form.

DEVELOPMENT AND VALIDATION OF REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ASSAY METHOD FOR ESTIMATION OF FENOFIBRATE IN TABLET DOSAGE FORM PREPARED USING CRYSTALLO-CO-AGGLOMERATES OF THE DRUG

The aim of the present study was to develop a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method and validate for the determination of fenofibrate in tablet dosage forms. RP-HPLC method was developed using Hi Q Sil C18 (250 cm × 4.6 mm, 5 μm) and mobile phase comprising 1 mM ammonium acetate buffer: Acetonitrile (10:90 v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 290 nm. The retention time was found to be 6.15 ± 0.03 min. Validation of the method was performed for precision, accuracy, linearity, robustness, specificity and sensitivity to conform to the International Conference on Harmonization (ICH) guidelines. The data of linear regression analysis indicated a good linear response in the concentration range of 5 μg/mL–30 μg/mL with correlation co-efficient (R2) of 0.997. The developed method was found to be simple, sensitive, accurate and repeatable for assay of tablets of fenofibrate prepared using crystallo-co-agglomerates of the drug.

Determination of Nateglinide in Tablet Formulation by HPLC Using a Pentafluorophenyl Core-Shell Column

Nateglinide is an insulin secretagogue which has been used for the treatment of Type 2 Diabetes mellitus. A simple analytical methodology for determination of nateglinide in tablet formulation is described. An isocratic reversed phase high performance liquid chromatographic (HPLC) method was developed using Kinetex pentafluorophenyl (PFP) (5μm particle size and 4.6 x 150 mm id) core-shell column as stationary phase and 0.05 M Na2HPO4 (pH=2.0) / methanol (30:70 v/v) mixture as mobile phase with the flow rate of 1.0 mL min-1 and diode-array detector at 215 nm. Column was termostated at 22°C. Under these conditions nateglinide was eluted with a symmetrical peak shape and the retention time was 4.779 minutes. The system suitability parameters such as tailing factor, capacity factor and theoretical plate were evaluted. The method was validated as per International Council on Harmonisation (ICH) guidelines. Linearity was obtained in the 2.54 - 40.70 μg mL-1 concentration range with equation y= 10.818x + 4.7048 (R2= 0.998). Limit of detection and limit of quantification were 1.07 and 2.54 μg mL-1. Accuracy of the method was tested by recovery studies which were in the range of 98.78 - 100.76 %. The intra-day and inter-day precision studies were carried out and the relative standard deviation values of peak areas were within ICH limits. The proposed method was successfully applied to the determination of nateglinide in tablet dosage form.

Determination and validation of pregabalin in bulk and pharmaceutical formulations by reversed phase-high performance liquid chromatography

A simple, specific, quick, isocratic Reversed Phase High Performance Liquid Chromatographic method was developed and validated for the analysis of Pregabalin in bulk and 5-different pharmaceutical formulations, the separation was accomplished on a C18, 5μm Reverse Phase column (250 mm × 4.6 mm) using a methanol : water (95:5, v/v) mobile phase. The compound was eluted isocratically at a flow rate of 0.8 ml /min. The UV detector was set at 288 nm for the detection of Pregabalin (PRG). The method was linear over the range of 5-45 μg/ml and validated with respect to accuracy, precision, linearity, and specificity, limit of detection and limit of quantization. Robustness testing was also conducted to evaluate the effect of minor changes to the chromatographic system and to establish appropriate system suitability parameters. This method was used successfully for the quality assessment of 5-different pharmaceutical formulations with good precision and accuracy.   

Stress testing of crisaborole by a novel stability indicating RP-HPLC method

An isocratic reverse phase high performance liquid chromatographic method has been developed for the determination of crisaborole (CRB), a novel non-steroidal topical phosphodiesterase-4-inhibitor. The analyte was separated on an Agilent eclipse XDB-C18 (3.0 × 150 mm, 3.5 µm) analytical column using water: acetonitrile (30: 70 v/v) as the mobile phase at a flow rate of 0.75 ml/min under ambient temperature and PDA detection at 254 nm. Added to this, several validation parameters were exercised on the developed HPLC method as per ICH guidelines. Method variables, viz., mobile phase flow rate (0.75 ± 0.1 ml/min) and percentage of organic modifier in mobile phase (70 ± 5%) were studied by a central composite design for testing the robustness of the proposed method. The method was found to be linear in the concentration range of 2–100 µg/ml (r2 = 0.999) and precise (RSD ˂ 2%) satisfying regulatory criteria. The method was found to be robust and ambiguity of worst result for certain responses still are acceptable and likely to occur due to the effect of extreme experimental conditions. Stress testing of CRB was carried out under acid, base, thermal, photolysis, peroxide and neutral conditions. Separation of the forced degradation products from the main analyte was accomplished using a gradient elution of the mobile phase consisting acetonitrile from 0 to 100% in water at a flow rate of 1.2 ml/min.

Central composite design based development and validation of an rp-hplc method for paclitaxel in bulk and pharmaceutical dosage form

ObjectiveDevelopment of an accurate, precise, robust, sensitive, economical and rapid isocratic reverse phase high performance liquid chromatography (RP-HPLC) technique complying quality by design (QbD) and validate according to ICH guidelines for the quantitative estimation of Paclitaxel in bulk and pharmaceutical dosage form.MethodThe simultaneous assessment of the Paclitaxel with Nilotanib as internal standard in bulk and pharmaceutical dosage forms with the help of chemometrics, multicriteria decision-making approach. The separation was achieved by utilizing Phenomenex Enable C₁₈ column (Gemini, 15x4.6mm, 5μm particle size) and PDA-UV detection set at 230nm was developed and validation of Paclitaxel in pure form and pharmaceutical formulation, optimized by Derringer’s desirability functions. Chromatographic separation was consisted of a mixture of acetonitrile and KH₂PO₄ (70:30 %v/v, pH 4) adjusted with orthophosphoric acid and the flow rate of 0.8ml/min.Newly developed method resulted in eluting the drug at 3.928min, respectively. The regression coefficients (R²) were observed to be 0.999 for all models. The detection of limits (LOD) was about 9.498ng/ml and quantitation limits (LOQ) were about 31.66ng/ml. The relative standard deviation was observed to be 1.842%. Peak area ratio of the analyte and internal standard was used for the estimation of pharmaceutical formulations.The method was validated by determining its precision, accuracy, and system stability. The results of the investigation demonstrated that the suggested RP-HPLC method is simple, rapid, precise and accurate, which is convenient for the routine determination of Paclitaxel in bulk and pharmaceutical dosage forms.

SIMPLE AND RAPID HPLC METHOD FOR SIMULTANEOUS QUANTIFICATION OF LEVODOPA AND CARBIDOPA IN A FAST DISINTEGRATING TABLET FORMULATION

Objective: Fast disintegrating tablets (FDTs) are found helpful in dysphagia (difficulty in swallowing) especially in Parkinson patients. Levodopa is still the first choice in Parkinson disease treatment and is co-administered by carbidopa for better efficacy.&#x0D; Methods: In the present study, a rapid and simple isocratic Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for simultaneous quantification of levodopa and carbidopa in optimized Fast Disintegrating Tablets (FDTs). The linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) of the method were determined. FDTs were prepared using direct compression, dry and wet granulation and were optimized for faster disintegration time. Tablets thickness, weight, hardness, friability, drug content and dissolution profile were also evaluated.&#x0D; Results: A RP-HPLC system with C18 column and mobile phase 90:10 (v/v) phosphate buffer: methanol was used. The method linearity was found to be within the concentration range of 3.125-50 μg/ml for levodopa, and 3.125-25 μg/ml for carbidopa. The intra and inter-day precision and accuracy were acceptable. LOD and LOQ of levodopa-carbidopa were 0.2-0.8 μg/ml and 0.5-2.4 μg/ml, respectively. The total chromatographic run time was 5 min. The optimized FDTs hardness was 3.81±0.4 and tablets were disintegrated within 30 sec. Levodopa and carbidopa were dissolved in dissolution media within 5 min.&#x0D; Conclusion: Results indicated that this method was suitable for simultaneous quantification of levodopa and carbidopa in the presence of different ingredients of a pharmaceutical solid dosage form. Therefore, this method could be applied in pharmaceutical quality control for rapid quantification of structurally similar substances with different physicochemical properties.

A new stability indicating reverse phase high performance liquid chromatography method for the determination of enantiomeric purity of a DPP-4 inhibitor drug linagliptin.

A simple, sensitive, and stability indicating isocratic reverse phase high performance liquid chromatography method has been developed, optimized and validated for the separation and quantification of S-enantiomer in linagliptin (R-enantiomer) drug substance. Enantiomeric separation was achieved on a Cellulose tris(4-chloro-3-methylphenylcarbamate) stationary phase. Mobile phase consists of aqueous diammonium hydrogen phosphate buffer and acetonitrile in the ratio of 35:65 v/v. Isocratic elution was performed at a flow rate of 1.0 mL/min, the column oven temperature was set at 40°C and detection was at 226 nm. The resolution between R and S enantiomers is found to be more than 4.0. The impact of mobile phase composition, pH of buffer and temperature on the resolution has been studied. The detector response is found to be linear over the concentration range of 0.17-1.7μg/mL. LOD and LOQ levels of S-enantiomer are found to be 0.057 and 0.172μg/mL respectively. The recovery of S-enantiomer is 99.8% w/w. The proposed method is validated for specificity, precision, linearity, accuracy and robustness.

Method Development and Validation of Alectinib Drug by RP-HPLC in Bulk and Pharmaceutical Dosage Form

In this study we developed and validated an isocratic Reversed Phase High Performance Liquid Chromatography method with PDA detection for the estimation of Alectinib drug. The method was selective and is capable of detecting at traces levels in the drug substance. The method was validated on an Inertia ODS C18 analytical column (4.6 X 150 mm, 5 μm), mobile phase consisting of Methanol: phosphate buffer at pH 3 in the ratio of 70: 30% v/v. The flow rate was at 1 ml/min and the column was maintained at ambient temperature condition. The detector using photo diode array was set at a wavelength of 265 nm. Calibration plot was linear in concentration range of 10–50 ppm levels with the correlation coefficient (r2) of 0.999. The intra and inter-day precision% RSD values were 0.48 and 0.51% respectively. The Limit of Detection and Limit of Quantification values were found to be 0.77 μg/mland 2.35 μg/ml respectively. The method was validated in terms of system precision, method precision, linearity, accuracy, limit of detection, quantification and robustness. Due to its speed and accuracy, this method can be used for estimation and analysis of Alectinib drug in Active Pharmaceutical Ingredients and Pharmaceuticals.

RP-HPLC Method Development and Validation for Determination of Eptifibatide Acetate in Bulk Drug Substance and Pharmaceutical Dosage Forms.

A new, rapid, economical and isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms. The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column (150 x 4.60 mm i.d., 5 µM particle size) at ambient temperature using acetonitrile (ACN), water and trifluoroacetic acid (TFA) as mobile phase at flow rate of 1 mL/min and UV detection at 275 nm. Eptifibatide acetate exhibited linearity over the concentration range of 0.15-2 mg/mL (r2=0.997) with limit of detection of 0.15 mg/mL The accuracy of the method was 96.4-103.8%. The intra-day and inter-day precision were between 0.052% and 0.598%, respectively. The present successfully validated method with excellent selectivity, linearity, sensitivity, precision and accuracy was applicable for the assay of eptifibatide acetate in bulk drug substance and pharmaceutical dosage forms.

Development and validation of a reversed phase High Performance Liquid Chromatography method for content evaluation of some brands of paracetamol tablets sold in Abuja, Nigeria

A simple, rapid, accurate and economical isocratic Reversed Phase High Performance Liquid Chromatography (RPHPLC) method was developed, validated and used for the evaluation of content of different brands of paracetamol tablets. The method was validated according to ICH guidelines and may be adopted for the routine analysis of paracetamol in dosage forms both as single or combined formulation. Twelve brands of paracetamol tablets were randomly purchased from pharmacy stores around Abuja, the Federal Capital Territory (FCT) of Nigeria and coded accordingly. Samples were analyzed using a validated isocratic RP-HPLC method with UV detection at 254 nm at ambient temperature. Analysis time was 6 minutes. The inter and intraday precision coefficient of variance for 2 and 8 μg/ml were less than 5% and the percentage recovery was more than 90%. Percentage contents ranged between 93.03- 126.43% (±0.81-11.12). 41.7% of the assessed brands passed while 58.3% failed with the values being either above or below the BP specification of 95-105% for percentage content of paracetamol in tablets. Keywords: Paracetamol; Reversed Phase HPLC; Routine Analysis; Pharmacy Stores

A VALIDATED ANALYTICAL HPLC METHOD FOR THE QUANTIFICATION OF LINCOMYCIN HYDROCHLORIDE IN BULK AND SOLID DOSAGE FORM

Objective: Develop a simple isocratic reverse phase high performance liquid chromatographic method (RP-HPLC) and validate for the determination of lincomycin hydrochloride (LMH) in bulk and pharmaceutical preparations.Methods: RP-HPLC quantification was carried out by using fine pack SIL RPC18 column. The mobile phase (methanol: water) was pumped at a flow rate of 1 ml/min in the ratio of 90:10 v/v and the eluents were monitored at 254 nm.Results: The retention time of the drug was 3.73 min and produced at a linear response in the concentration range of 5-25µg/ml. The percentage RSD was found to be below 2%. The LOD and LOQ were found to be 0.854µg/ml and 0.258µg/ml respectively.Conclusion: Validation of the method was performed for precision, accuracy, linearity, ruggedness, specificity and sensitivity to conform to ICH guidelines for valuation for analytical methods.

Method Development and Validation of Fingolimod in Bulk and Tablet Dosage Form by RP-HPLC

A facile and rapid isocratic reverse phase high performance liquid chromatography assay method has been developed and validated for the determination of fingolimod in bulk and tablet dosage form. The column was equilibrated for at least 30 min and separation was achieved by using kromofil-ODS C18 (150 x 4.6 mm, particle size 5 μm). The column was maintained at ambient temperature (25°C). The mobile phase employed was methanol: water (60: 40 v/v). The eluent was monitored using UV detector at 220 nm. A volume of 10μL of standard and sample solutions was injected in to the HPLC. The flow rate was 1.0 mL/min. The retention times were 4.525 min for fingolimod. The method was found to be linear in the range of 25–150 μg/mL. The developed method was validated as per ICH guidelines. The developed method was found to be simple, precise, accurate, selective and sensitive.

VALIDATED RP-HPLC METHOD FOR DETERMINATION OF ENZALUTAMIDE IN BULK DRUG AND PHARMACEUTICAL DOSAGE FORM

Novel, isocratic reversed phase high performance liquid chromatography method was developed and validated for the determination of enzalutamide (EZA) in bulk drug and pharmaceutical formulation. Efficient separation was achieved on PrincetonSPHER C18 100A, 5μ (250×4.6 mm) under the isocratic mode of elution using acetonitrile: water (80:20) % V/V as a mobile phase pumped in to the column at flow rate 1.0 mL/min. The effluent was monitored at 237.0 nm using UV detector. EZA was eluted in the given mobile phase at retention time (tR) of 3.2 minutes. The standard calibration curve was linear over the concentration range 10 - 60 μg/mL with correlation coefficient 0.997. The method was validated for accuracy, precision, sensitivity, robustness, ruggedness and all the resulting data treated statistically. The system suitability parameters like retention time, theoretical plates, tailing factor, capacity factor were found within the limit.

Establishment and reliability evaluation of the design space for HPLC analysis of six alkaloids in Coptis chinensis (Huanglian) using Bayesian approach

Coptis chinensis (Huanglian) is a commonly used traditional Chinese medicine (TCM) herb and alkaloids are the most important chemical constituents in it. In the present study, an isocratic reverse phase high performance liquid chromatography (RP-HPLC) method allowing the separation of six alkaloids in Huanglian was for the first time developed under the quality by design (QbD) principles. First, five chromatographic parameters were identified to construct a Plackett-Burman experimental design. The critical resolution, analysis time, and peak width were responses modeled by multivariate linear regression. The results showed that the percentage of acetonitrile, concentration of sodium dodecyl sulfate, and concentration of potassium phosphate monobasic were statistically significant parameters (P < 0.05). Then, the Box-Behnken experimental design was applied to further evaluate the interactions between the three parameters on selected responses. Full quadratic models were built and used to establish the analytical design space. Moreover, the reliability of design space was estimated by the Bayesian posterior predictive distribution. The optimal separation was predicted at 40% acetonitrile, 1.7 g·mL−1 of sodium dodecyl sulfate and 0.03 mol·mL−1 of potassium phosphate monobasic. Finally, the accuracy profile methodology was used to validate the established HPLC method. The results demonstrated that the QbD concept could be efficiently used to develop a robust RP-HPLC analytical method for Huanglian.

Stability-Indicating Method and LC-MS-MS Characterization of Forced Degradation Products of Sofosbuvir.

Sofosbuvir is a novel direct acting antiviral agent against hepatitis C virus. In the present work, a rapid, specific and reproducible isocratic reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the determination of sofosbuvir in the presence of its stressed degradation products. Sobosbuvir was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per international conference on harmonization (ICH) conditions. The drug showed degradation under oxidative, photolysis, acid and base hydrolysis stress conditions. However, it was stable under thermal and neutral hydrolysis stress conditions. Chromatographic separation of the drug from its degradation products was performed on Inertsil ODS-3 C18 (250 mm×4.6 mm i.d., 5 µm) column using a green mobile phase of methanol:water 70:30 (v/v). The degradation products were characterized by LC-MS-MS and the fragmentation pathways were proposed. The developed method was validated as per ICH guidelines. No previous method was reported regarding the degradation behavior of sofosbuvir.