Development and Validation of RP-HPLC Method for the estimation of Sofosbuvir and Ledipasvir in combined dosage form

Nowadays scientists aim at developing generic and speci icmethods for drugs in combined pharmaceutical dosage forms. The main aim was to develop and carry out validation using precision and accuracy for Sofosbuvir and Ledipasvir by applying RP-HPLC method. The dosage form in ixed combination of Sofosbuvir-Ledipasvir helps in effective treatment of genotypes of chronic hepatitis C virus. This combination of Sofosbuvir-Ledipasvir was the irst FDA approved direct acting antiviral to treat hepatitis C. Both the drugs were optimized using mobile phase as acetonitrile: 0.1% orthophosphoric acid (55:50v/v) with pH 3.0. The mobile phase was optimized at maximum wavelength of 283nm. The separation was achieved using C18 Cosmosil (4.6 x 250mm) column with a particle size of 5μ. The method was speci ic eluting retention times of 3.7 for Sofosbuvir and 6.0 for Ledipasvir. Intra and interday precision was carried and %RSD was found to be less than 2%. The results were found to be accurate with percentage recovery of 99.76% and 99.10% for Sofosbuvir and Ledipasvir respectively. Linearity was carried out in the concentration ranging from40-200μg/mL and 9-45μg/mL for Sofosbuvir and Ledipasvir respectively. Both the drugs showed the regression coef icient of 0.999. Deliberate changes in low rate, pH and wavelength were made and found that method was robust. The developed method was found to be accurate, simple, speci ic, robust and precise for the simultaneous estimation of Sofosbuvir and Ledipasvir in tablet dosage form.

Literature survey reveals that very less methods or studies are done on combined dosage form for Sofosbuvir and Ledipasvir (Madhavi and Rani, 2017). Thus the objective of the present work was to develop a very simple, economical, precise and accurate method with reproducible assay results for Sofosbuvir and Ledipasvir in simultaneous dosage form to validate as per ICH Guidelines. (Ravichandran et al., 2010;Vejendla et al., 2016).

Chemicals
The pure standard gift samples of Sofosbuvir and Ledipasvir were obtained from Zydus Cadila Pvt. Ltd. (Goa India). Harvoni, the commercial formulation in tablet form was procured from medical stores.

Instrumentation
The HPLC instrument included Agilent HPLC gradient system equipped with G13104A ISO Pump, G1314A Detector and Chemstation software. Separation and quantitation was done using C 18 COS-MOSIL column with 4.6 x 250 mm as dimensions and 5 mm particle size packing with low rate 0.7 mL/min and sample size of 20 ml. The iltration assembly with Nylon 66 membrane ilter of 13mm diameter having a pore size of 0.45 micron was used to ilter both the solutions. Wavelength of detection was obtained from the PDA detector where the two drugs showed the absorption maxima at 283nm. A Digital Weighing Balance (Make: Shimadzu AUX 220) was used for preparations of all standard and sample solutions required.

Chromatographic conditions
The Chromatographic conditions were optimized using isocratic mode. A mixture of Acetonitrile and orthophosphoric acid (0.1%) with a ratio of 55:50 v/v at 3.0 pH expressed a good peak symmetry with ef icient separation of drugs. Acetonitrile and 0.1% orthophosphoric acid mixture in the ratio of 55:50v/v pH adjusted to 3.0 provided an ef icient separation of the drugs with good peak symmetry. The eluents were monitored at 283nm with a runtime of 10mins. 0.7mL/min expressed optimum retention time of 3.7min and 6.0min for SOF and LED respectively. An ambient column oven temperature was maintained.

Preparation of Mobile phase
Acetonitrile and orthophosphoric acid (0.1%) in the ratio of 55:50 v/v was optimized as the mobile phase with pH adjusted to 3.0. The mobile phase was sonicated for 15 minutes and the same was used as diluent.

Preparation of Standard stock solution
An accurate amount of Sofosbuvir (40mg) and Ledipasvir (90mg) was weighed and dissolved in 10 mL of methanol. The stock solution obtained of 4000 µg/mL for Sofosbuvir and 90 µg/mL of Ledipasvir was diluted further to obtain the required concentration.

Optimization of method
Wavelength of detection of the standard solution of the drugs was obtained from the PDA detector. Both the drugs were scanned between 190 nm to 400nm. The isobestic wavelength after scanning overlain spectra was 283nm.

Method Validation
The method validation parameters were determined experimentally according to International Conference on Harmonization (ICH) guidelines.           illustrates Chromatogram of Sofosbuvir and Ledipasvir in assay sample. Figure 5 and Figure 6 illustrates Linearity graph of Sofosbuvir and Ledipasvir respectively. Table 1 illustrates Chromatographic conditions of Sofosbuvir and Ledipasvir. Table 2 illustrates System suitability parameters of Sofosbuvir and Ledipasvir. Table 3 illustrates Intraday precision results of Sofosbuvir and Ledipasvir. Table 4 illustrates Intermediate Precision results of Sofosbuvir and Ledipasvir. Table 5 and Table 6 illustrate Linearity data of Sofosbuvir and Ledipasvir respectively. Table 7 illustrates Linearity data of Sofosbuvir and Ledipasvir. Table 8 illustrates Robustness data of Sofosbuvir and Ledipasvir. Table 9 illustrates Assay results of Sofosbuvir and Ledipasvir.

Selectivity/Speci icity
Selectivity is demonstrated by the resolution of the two compounds, Sofosbuvir and Ledipasvir. The sample was assessed unequivocally in the presence of other matrix components. Retention times were speci ic at 3.7 and 6.0 for Sofosbuvir and Ledipasvir respectively.

System Suitability
System suitability was evaluated for analytical operations and samples as whole. Six replicated were recorded of the standard chromatogram and the parameters determined were tailing factor (T), capacity factor (k'), resolution (R) and theoretical plates (N).

Precision
Precision is measured either as degree of reproducibility or of repeatability. The assessment was achieved by evaluating relative standard deviation.

Linearity and range
Linearity was expressed in terms of relationship between concentration and assay method. This relationship was achieved by studying the concentration levels of Sofosbuvir and Ledipasvir in the range of 40-200 µg/mL and 9-45 µg/mL respectively in HPLC system noting the peak areas.

Accuracy
Accuracy of the method is determined by applying the analytical procedure to an analyte of known purity and evaluating its recovery studies. The standard addition method at concentration levels of 80%, 100%, 120% of both the drugs was performed to evaluate the percentage recovery of spiked samples.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The limit test of detection and quantitation are characteristic of low level of compounds in sample matrices. The method expresses the concentration of analyte based on signal to noise ratio of 3:1 for LOD and 10:1for LOQ.

Robustness
The robustness expresses deliberate variations in the method developed by changing the conditions of the experiment. Such changes were made in the parameter conditions of low rate, pH and wavelength.

Assay
An accurate amount of tablet powder equivalent to 40 mg of Sofosbuvir was weighed. The powder was transferred in methanol and mixed for around 20 mins to dissolve the drugs. The volume was made up to 10 mL. Further dilutions were made as per requirement and iltered using Sample Filtration Assembly.

CONCLUSIONS
Fixed dose combination containing Sofosbuvir (400mg) and Ledipasvir (90mg) is introduced lately as a direct-acting antiviral and also for Hepatitis C treatment. Literature survey reveals about the several methods used to assess the drug individually and in combination along with other drugs in various pharmaceutical drug dosage form and biological luids. Nevertheless, very few methods are reported of these two drugs in ixed dosage form. Thus an effort was made to undertake and develop an accurate, economical and sensitive HPLC method for estimating these drugs in combined dosage form. Both the drugs were resolved using acetonitrile: orthophosphoric acid in a ratio of 55:50 v/v with a pH adjustment of 3.0. The resolution was obtained on C18 Cosmosil (4.6 x 250 mm) column at a maximum wavelength of 283 nm. An outstanding resolution was obtained between both the drugs with an optimum retention time.
The developed analytical method accounted to be simple, precise and accurate for Sofosbuvir and Ledipasvir in combined tablet dosage form.