OPTIMIZED METHOD FOR RP-HPLC

Mobile Phase: 0.1% OPA: Methanol (45:55)

Column: Zorbax 250X 4.6mm, 5µm, C8

Flow Rate: 1.0ml/Min

Temperature: 30˚C

Volume: 10µl   

Detector: PDA

Procedure:Inject 10μL of standard, sample into chromatographic system and measure the areas for the Ledipasvir and Sofosbuvirpeaks and calculate the % assay by using the formula

PREPARATION OF MOBILE PHASE 5

Transfer 1000ml of HPLC water into 1000ml of beaker add 0.1%OPA.

Transfer the above solution 450ml of0.1%OPA, 550ml of Methanol is used as mobile phase. They are mixed and sonicated for 20min.

SYSTEM SUITABILITY:

Tailing factor for the peaks due to Ledipasvir and Sofosbuvirin standard solution should not be more than 2.0.Theoretical plates for the Ledipasvir and Sofosbuvirpeaks in standard solution should not be less than 2000.

SPECIFICITY:

Specificity studies were carried for both pure drugs and drug product by comparing with blank and placebo. These blanks and placebo were compared with standard and sample shows that the analyte chromatographic peak is not attributable to more than one component as the impurities are not available.

Forced Degradation Studies 6

In the present investigation, as there was no interference of impurities with the analyte peaks, forced degradation studies were conducted with the same LC conditions developed to separate drug peaks of interest from their degradants which proves the stability indicating power of the method. Intentional degradation was attempted to various stress conditions such as acid hydrolysis (using 1.0N HCl), base hydrolysis (using 1.0 N NaOH), oxidative hydrolysis (using 3.0%v/v H2O2), thermal degradation (heated at 70°C for 14 days) and photolytic degradation (to overall illumination of ≥210Wh/m2 at room temperature with UV light for 14 days), to evaluate the ability of the proposed method to separate Ledipasvir and Sofosbuvir from their degradation products. Acidic hydrolysis: Forced degradation in acidic media was performed by taking accurately weighed samples of 519.7mg of Ledipasvir and Sofosbuvir each in separate 5mL volumetric flasks. Then 2mL of 1N HCl was added, made to dissolve and final volume was made up to the mark with 1N HCl to get mg/mL solutions and these were kept at 70ºC for 2 days and analyzed after suitable dilution.

Basic hydrolysis:

Forced degradation in basic media was performed by dissolving separately an accurately weighed quantities (122.5mg each) of Ledipasvir and Sofosbuvir in 1N NaOH in 5mL volumetric flasks and final volume was made up to 5mL with the same to get mg/mL solutions and these solutions were kept at 70ºC for 2 days and analyzed after suitable dilution.

Oxidative degradation:

Oxidative degradation studies were carried out in 3% (v/v) H2O2. Stock solutions of 122.5mg/mL of Ledipasvir and Sofosbuvir were prepared and kept at 70ºC for 2 days and analyzed after suitable dilution. Photo Degradation: For photolytic stress, samples of drug substances in solid state were irradiated with UV radiation (overall illumination of ≥210Wh/m2 at room temperature with UV radiation), for 14 days. Stock solutions of 1mg/mL were prepared in methanol from the exposed drug substances individually.

Thermal Degradation:

For thermal stress, 122.5mg of samples of drug substances in solid state were packed in glass vials and placed in a controlled temperature oven at 70°C for 14 days. Stock solutions of 1mg/mL were prepared in methanol from the exposed drug substances individually. For HPLC analysis, all the stressed sample solutions were diluted with mobile phase to obtain final concentration of 60μg/mL of Ledipasvir and Sofosbuvir and 100μg/mL of Rabeprazole respectively. Similarly, mixture of both drugs in a concentration of 60μg/mL of Ledipasvir and Sofosbuvir and 100μg/mL of Rabeprazole was prepared prior to analysis by HPLC. Besides, solutions containing 60μg/mL of Ledipasvir and Sofosbuvir and 100μg/mL of Rabeprazole for each drug separately were also prepared without performing the degradation of both the drugs. Then 20μL of above solutions were injected into the HPLC system and analyzed.Solution of standard, sample, blank and placebo were prepared as per test procedure and injected into the HPLC system.

Acceptance criteria:

Chromatogram of standard and sample should be identical with near Retention time.

Blank interference

A study to establish the interference of blank was conducted. Diluent was injected into HPLC system as per the test procedure.

Acceptance criteria

Chromatogram of blank should not show any peak at the retention time of analyte peak. There is no interference due to blank at the retention time of analyte. Hence, the method is specific.

LINEARITY

Prepare a series of standard solutions and inject into HPLC system. Plot the graph of standard versus the actual concentration in µg/ml and determine the coefficient of correlation and basis for 100% response.

Acceptance criteria

Linearity regression coefficient of average peak area response of replicate injections plotted against respective concentration should not be less than 0.999. The % y-intercept as obtained from the linearity data (without extrapolation through origin 0, 0) should be within ±2.0.

Statistical Evaluation

A graph between the concentration and the average area was plotted. Points for linearity were observed. Using the method of least squares, a line of best fit was taken and the correlation Coefficient, slope and, y-intercept were calculated.

PRECISION

Preparation of sample

• Transfer the 122.5mg of sample into a 100ml of volume at flask and add 10ml of water and 10ml of Methanol and sonicate 20min and makeup with water. Transfer the above solution into 2.5ml into 25ml volume metric flask dilute to the volume with water.

• The method precision parameters were evaluated from sample chromatograms obtained, by calculating the % RSD of peek areas from 6 replicate injection.

Acceptance criteria: The injection reproducibility requirements are met if the %RSD for peak areas is not more than 2.0 and for retention times is not more than 2.0.

RECOVERY/ACCURACY

Recovery study can be performed in the concentration range of 80% to 120% of the target concentration of the test. Minimum 3 concentrations are recommended.

Acceptance criteria:

The average percentage recovery was between 98-102% and Relative standard deviation of these recovery concentrations was less than 2%.

SYSTEM SUITABILITY:

SPECIFICITY:

Specificity data for Ledipasvir and Sofosbuvir

RESULT

The forced degradation study showed the method was highly specific, the chromatographic peaks does not interfere with any other impurities. This proves that, excipients have no effect on the analytical method. On the other hand, blank peak did not overlap drug peak. So the method is highly selective.

ACCURACY:

Accuracy data for Ledipasvir

RESULT

Results of accuracy study are presented in the above table. The measured value was obtained by recovery test. Spiked amount of both the drug were compared against the recovery amount.

% Recovery was 100.00% for Ledipasvirand 100.00% forSofosbuvir. All the results indicate that the method is highly accurate.

PRECISION:

RESULT

Results of variability were summarized in the above table. % RSD of peak areas was calculated for various run. Percentage relative standard deviation (%RSD) was found to be less than 2% which proves that method is precise.

LINEARITY:

RESULT

A linear relationship between peak areas versus concentrations was observed for Ledipasvir and Sofosbuvirin the range of 50% to 150% of nominal concentration. Correlation coefficient was 0.999 for both Ledipasvir and Sofosbuvirwhich prove that the method is linear in the range of 50% to 150%.