Development and validation of RP HPLC method for the estimation of Sofosbuvir and related impurity in bulk and pharmaceutical dosage form

The present work is aimed at development and validation of RP HPLC method which is simple, specific, precise, and accurate for estimation of Sofosbuvir and its process-related impurity in bulk and pharmaceutical dosage forms. Extensive literature survey revealed no method for estimation of the above said. The chromatographic separation was achieved on Agilent Eclipse XDB-C18, 4.6 × 250 mm, 5 μm with mobile phase composed of 0.1% trifluoroacetic acid in 1000 ml of water:acetonitrile (50:50) using an isocratic mode of elution. Detection was made using UV detector at 260.0 nm and LC solution software for analysis of data. The developed method was validated according to ICH guidelines. The linearity of calibration curve for Sofosbuvir in concentration range of 160-480 μg/ml was good. The curve was linear for its process related impurity (Phosphoryl) in concentration range of 10-30 μg/ml. There exists a good correlation between peak area and analyte concentration. Retention time for Sofosbuvir was found to be 3.674 min and its impurity was 5.704 min. Relative standard deviation values for Sofosbuvir is 1.741 and its process related impurity is 0.043. LOD for Sofosbuvir and its impurity was found to be 0.01% (0.04 μg) and 0.03% (0.12 μg) respectively. LOQ for Sofosbuvir and its impurity was found to be 0.50% (0.125 μg) and 1.50% (0.375 μg) respectively. All the results reveal that the proposed method was found to be highly sensitive, simple, precise, accurate, robust, and fast. Large number of samples can be analyzed in shorter time due to shorter retention times, so it can be successfully applied for routine analysis of Sofosbuvir and related phosphoryl impurity in bulk and pharmaceutical dosage forms.


Background
Many drugs are available as the marketed formulations for the treatment of different diseases. So, there is need for control of concentrations of these entities in dosage forms and also in body fluids. Quality assurance and quality control of these marketed formulations are essential for ensuring safety in population. During the course of assay and development of drugs in formulations, there may be interferences caused by a number of sources such as degradation products of the drugs when they are stored for a long time, the presence of other drugs in combination products and the various additives incorporated in formulations have to be kept in view.
HPLC is the most widely used analytical technique. The method is non-destructive and may be applied to thermally labile compounds (unlike GC), so it was most widely used for the analysis of most of the chemicals.
Due to availability of wide range of detectors, it is also said to be sensitive technique.
It is a prodrug of 2′deoxy methyluridine monophosphate that is phosphorylated intracellularly to active triposphate [3].
Very few methods were available for determination of sofosbuvir in tablets [4,5], analysis of combination dosage forms like sofosbuvir, ledipasvir [6], and sofosbuvir, velpatasir in tablet dosage forms [7,8]. But no method was available in literature for estimation of sofosbuvir and its related impurity in bulk and pharmaceutical dosage forms. So the present work was aimed at development and validation of RP HPLC method for the estimation of sofosbuvir and its related impurity in bulk and pharmaceutical dosage forms.

Methods
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
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
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

Accuracy
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
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
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.

Different column
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.

Method development and optimization
The optimized parameters were listed in Table 1. Chromatogram for standard solutions of sofosbuvir and phosphoryl impurity was presented in Figs. 2 and 3 respectively.

System suitability studies
System suitability parameters were shown in Table 2.
Chromatogram for system suitability studies is presented in Fig. 4.

Linearity
Results are shown in Tables 3 and 4, and the linearity curves are shown in Figs. 5 and 6.

Accuracy
The recovery data for accuracy studies was shown in Tables 5 and 6. The accuracy chromatograms for the respective concentrations were shown in Figs. 7, 8, and 9.

Precision
Results were reported in Tables 7 and 8 and chromatograms were presented in Figs. 10 and 11.

Limit of detection (LOD) and limit of quantitation (LOQ)
LOD and LOQ for sofosbuvir were found to be 0.04 μg and 0.12 μg. LOD and LOQ for phosphoryl impurity were found to be 0.125 μg and 0.375 μg respectively     Results were reported in Tables 9, 10, 11, and 12.
Chromatograms for robustness studies for standard and test samples of sofosbuvir and impurity were shown in Figs. 12, 13, and 14.

Discussion
Various solvent system combinations for the determination of sofosbuvir and its related impurity (phosphoryl impurity) in bulk and pharmaceutical dosage forms were studied and finally a mixture of 0.1% trifluro acetic acid in 1000 ml of water and acetonitrile (50:50) was selected as mobile phase as it gave better resolution. The effect of flow rate was studied in the range of 0.9 to 1.2 ml/ min and 1.0 ml/min was preferred to be effective because the analyte peak obtained was well defined and free from tailing. The retention time (RT) was found to be 3.674 min for sofosbuvir and 5.704 min for phosphoryl impurity.

System suitability
The system suitability method acceptance criteria set in each validation run were tailing factor ≤ 2.0 and theoretical plates > 2000. In all cases, the relative standard deviation for analyte peak area < 2.0% as per ICH guidelines [9]. All the parameters like retention time (RT), number of theoretical plates (N), tailing factor (T), and resolution were within the acceptable limits, so the optimized method is suitable for analysis of both compounds.

Linearity
A calibration curve was obtained by plotting a graph between peak area and concentration. Excellent correlation was obtained between peak area and concentration with R 2 = 0.999 for sofosbuvir and 0.999 for phosphoryl impurity as per the limit R 2 ˃ 0.999 [11].

Precision
The % RSD values for the peaks were found within the limits, RSD ≤ 2 [11] as shown in results, so the method was found to be precise.

Robustness
No prominent changes were observed by altering column and flow rate. Hence, the method was found to be robust.

Conclusion
The method proposed for the analysis of sofosbuvir and related impurity in bulk and pharmaceutical dosage forms was found to be specific, precise, accurate, fast, and economical. The developed method was validated in terms of accuracy, linearity, robustness, and precision in accordance with ICH guidelines. Short retention time enabled analysis of sofosbuvir and phosphoryl impurity with minimal amount of mobile phase. The method was found to be precise and accurate. Due to low detection and quantitation limits, the method was said to be sensitive. Robustness data indicate that the method is unaltered due to small changes in chromatographic conditions. This method can be applied successfully for the determination of sofosbuvir and its related phosphoryl impurity in bulk and pharmaceutical dosage forms.