Sofosbuvir and its impurity were obtained as a gift sample from Mylan Labs, Hyderabad. Sovaldi tablets were purchased from local pharmacy, Hyderabad. Trifluro acetic acid, acetonitrile (HPLC grade) were procured from Sigma Aldrich, Hyderabad. Water (HPLC grade) was obtained from Merck.
Chromatographic equipment and conditions
The development and validation was performed using liquid chromatographic system which is equipped with UV detector and LC solution software. The chromatographic column used for separation was Agilent Eclipse XDB-C18, 4.6 × 250 mm, 5 μm. The mobile phase used for the separation of both API and impurity was 0.1% trifluoroacetic acid in 1000 ml of water: Acetonitrile taken in the ratio of 50:50. Ambient temperature was maintained. Detection was made at a wavelength of 260.0 nm. Validation study was carried out using same optimized condition with suitable preparation of standard and sample solutions.
Preparation of standard solution
Standard solutions of drug and impurity were prepared by dissolving 400 mg of Sofosbuvir and 25 mg of phosphoryl impurity in 100 ml of diluent (water:acetonitrile 50:50).5 ml of the above solution was taken in 50 ml volumetric flask and diluted with diluent up to the mark.
Preparation of test solution
650 mg of Sovaldi formulation was taken in 100 ml volumetric flask, dissolved and diluted to 100 ml with diluent. 5 ml of above solution was taken in 50 ml volumetric flask and diluted with diluent up to the mark.
Validation was carried out by studying the parameters like specificity, system suitability, accuracy, linearity, precision, limit of detection, limit of quantitation, and robustness as per ICH guidelines Q2 (R1) [9, 10].
Linearity and range
Linearity was checked for standard solutions of drug and also impurity at concentrations of 40%, 60%, 80%, 100%, and 120%. Aliquot solutions of sofosbuvir and phosphoryl impurity were prepared in the range of 160-480 μg/ml and 10-30 μg/ml respectively. The chromatographic system was set to equilibrate and samples of study were injected, keeping the injection volume constant, i.e., 20 μl.
System suitability studies form an integral part of method development and ensures adequate performance of chromatographic system. The standard solutions of sofosbuvir (0.4 mg/ml) and phosphoryl impurity (0.025 mg/ml) of about 20 μl were injected under optimized chromatographic conditions to evaluate the suitability of the system.
Accuracy is defined as the closeness of obtained value to true value. Accuracy studies were done by standard addition method. Accuracy is expressed as % recovery of the standard spiked to previously analyzed test sample of tablet. It was measured in drug products by spiking known amounts (80%, 100%, and 120%) of the analyte into the analyzed tablet powder and each concentration was injected into the column for three times and percent recovered was calculated.
Precision is the agreement between replicate measurements of the same sample. It is expressed as relative standard deviation of replicate measurements. A total of 0.4 mg/ml of sofosbuvir and 0.025 mg/ml of phosphoryl impurity standard solutions were injected six times and the responses were recorded. Sample solution was also injected six times to record the response. The chromatograms were recorded. The peak area and retention time of both solutions under study was determined and relative standard deviation was calculated by the formula % RSD = (S.D/Mean) × 100%.
Limit of detection and limit of quantitation
Limit of detection (LOD) is the lowest concentration of analyte which can be detected in a sample under the optimized experimental conditions. The limit of quantification (LOQ) was identified as the lowest concentration of the standard curve that could be quantified with acceptable accuracy, precision, and variability. LOD and LOQ were determined based on the signal to noise ratio as per ICH guide lines. Serial dilutions of the standard solutions of drug and impurity were prepared in the dilution levels of 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005% and injected into the chromatographic system. Responses were recorded and LOD and LOQ were determined.
Robustness was studied to check whether the drug solution was subjected to small, deliberate changes like flow rate, wavelength, and change in mobile phase ratio. Robustness studies were performed by altering column and variation of flow.
A total of 0.4 mg/ml of sofosbuvir and 0.025 mg/ml of phosphoryl impurity standard solutions were injected in different column 3 times and responses were recorded. Sovaldi® sample was also injected 3 times and response was recorded.
Variation of flow
A total of 0.4 mg/ml of sofosbuvir and 0.025 mg/ml of phosphoryl impurity were injected 3 times by making changes in the flow rate, and responses were recorded and same procedure was carried with Sovaldi® sample also.