Development and Validation of RP-HPLC Method for Estimation of Sofosbuvir in Bulk Drug and Dosage Form

Sofosbuvir is an oral nucleoside analogue and potent inhibitor of the hepatitis C virus (HCV) RNA polymerase that is used in combination with other antiviral agents to treat chronic hepatitis C. Elevations in serum enzyme levels during sofosbuvir therapy are uncommon, and it has not been implicated convincingly in cases of clinically apparent liver injury with jaundice. Nevertheless, and for unknown reasons, successful antiviral therapy of hepatitis C with sofosbuvir and other direct acting agents in patients with cirrhosis is occasionally complicated by hepatic decompensation; furthermore, treatment can cause reactivation of hepatitis B in susceptible patients coinfected with the hepatitis B virus (HBV) ^[1].

Sofosbuvir (soe fos' bue vir) is an orally available nucleotide analogue that has potent activity against the RNA-dependent RNA polymerase of the hepatitis C virus (HCV). Sofosbuvir is a monophosphorylated uracil derivative whose single phosphate is protected by an alaninate cap that allows for the absorption and uptake of the molecule by hepatocytes where it is hydrolyzed to sofosbuvir monophosphate. Intracellular host kinases then convert it to the active triphosphate moiety. In multiple clinical trials, sofosbuvir has been shown to cause a rapid and marked decline in serum HCV RNA levels and, in combination with other antiviral agents and with more prolonged therapy, to result in sustained clearance of HCV (sustained virological response: SVR) in a high proportion of patients. Sofosbuvir was approved for use in the United States in 2013 to be used in combination with ribavirin or with both peginterferon and ribavirin in patients with chronic hepatitis C, genotypes 1, 2, 3 or 4. Sofosbuvir is available in tablets of 400 mg under the brand name Solvaldi, the recommended dose being 400 mg once daily in combination with either ribavirin alone (1000 or 1200 mg daily for 12 weeks for genotype 2 and 24 weeks for genotype 3) or in combination with both ribavirin and peginterferon for 12 weeks for patients with genotype 1.Subsequently, a fixed combination of sofosbuvir (400 mg) and the HCV NS5A replication complex inhibitor ledipasvir (le dip' as vir: 90 mg) was approved for use in patients with chronic hepatitis C, genotype 1 in 2014 and for genotype 4 in 2015. This combination is available as a fixed dose, single tablet under the brand name Harvoni and the recommended dose is one tablet daily for 12 weeks, which can be shortened to 8 weeks in selected patients ^[2,3].

In addition, sofosbuvir combined with NS5A inhibitors with broader activity against HCV genotypes, daclatasvir (dak lat' as vir: 2015) and velpatasvir (vel pat' as vir: 2016), has been shown to be effective in treating almost all HCV genotypes with sustained response rates of 95% or greater in response to 12 weeks of treatment in genotypes 1, 2, 4, 5 and 6. In 2016, the fixed combination of sofosbuvir (400 mg) and velpatasvir (100 mg) was approved for use in patients with all 6 genotypes of hepatitis C. This combination is available as a fixed dose, single tablet under the brand name Epclusa. The recommended dose is one tablet daily for 12 weeks. For patients with decompensated cirrhosis (Childs-Pugh Class B or C), ribavirin (1000 to 1200 mg in two divided doses daily) should be added to Epclusa for 12 weeks. Finally, the combination of sofosbuvir with an HCV specific NS3/4 protease inhibitor [2014] was also shown to be highly effective in patients with genotype 1 infection generally in 12 week courses ^[4,5].

For patients who fail to respond to a two or three drug combination of direct acting antiviral agents, combinations of potent agents active against the three major HCV polypeptide products have been developed and have shown excellent activity in these refractory patients. The first such regimen was a single tablet formulation of sofosbuvir (400 mg), velpatasvir (100 mg) and a potent, broad spectrum (pangenomic) HCV protease inhibitor, voxilaprevir (100 mg). This combination was approved for use in the United States in 2017 and is available under the brand name Vosevi. The recommended dosing regimen is 1 tablet daily for 12 weeks. This regimen is not recommended for patients with decompensated cirrhosis (Childs-Pugh Class B or C).As such, sofosbuvir transformed the therapy of chronic hepatitis C and became the most commonly used HCV-specific antiviral agent, replacing peginterferon and combinations of peginterferon, ribavirin and protease inhibitors. Sofosbuvir has few side effects and in placebo controlled trials adverse events occurred at a similar rate with sofosbuvir as placebo. Side effects may include headache, dizziness, nausea and diarrhea. Rare, but potentially severe adverse events include marked bradycardia when sofosbuvir is given with amiodarone ^[6].

Structure Of Sofosbuvir ^[7]

CHROMATOGAPHY

Chromatography is at technique which separates components in a mixture due to the differing time for each component to travel through stationary phase when carried through it by mobile phase. The stationary phase is fixed in place either in column (a hollow tube made up out of a suitable material e.g. Glass) or on a planner surface and the mobile phase moves over or through the stationary phase carrying with it the sample of interest. In practice the stationary phase can be a solid, a liquid adsorbed on a solid or an organic species bonded to a solid surface. In gas chromatography and supercritical fluid chromatography the stationary phase may be fixed in place either in a column or on a planner surface. In HPLC a column is used. The Name given to liquid chromatography on a planner surface is thin layer chromatography (TLC) ^[8,9,].  

Definition of Chromatography

In chromatography depending on how well each component dissolves in a pure solvent, they are transported individually, such as water or gas, as a pure solvent flows slowly over a stationary phase where the mixture has been separated. Greek words meaning "colour" and "to write" are the source of the word “chromatography" ^[10].

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Figure No 1: Schematic diagram of HPLC instrumentation ^[11]

HPLC is one among most useful tools, available for quantitative analysis. Reverse phase chromatography refers to the use of a polar mobile phase with a nonpolar stationary phase in contrast, to normal phase being employed with a nonpolar mobile phase. Liquid chromatography is based upon the phenomenon that, under the same condition, each component in a mixture interacts with its environment differently from other components. Since HPLC is basically a separating technique, it is always used in conjunction with another analytical tool for quantitative and qualitative analysis. Advances in column technology, are high pressure pumping systems and sensitive detectors which have transformed liquid column chromatography into a high speed, high efficiency method of separation. This advanced technology is based upon the of small bore (2.5mm-internal diameter) columns and small particle size (3.5 micro meter) that allow fast equilibrium between stationary and mobile phases. This small particle column technology requires high pressure pumping system capable of delivering the mobile phase at high pressure, as much as 300 atmospheres to achieve flow rates of several ml per minutes. since it is often necessary to use small amounts of analyte with the column packing, sensitive detectors are needed. with this technology, liquid chromatography can give high speed separation when compared with many cases like those achieved by gas chromatography with the advantage that non -volatile or thermally unstable samples can be chromatographed without decomposition or the necessity for making volatile derivatives ^[11,12].

Principle of HPLC

A tiny volume of liquid sample is injected into a tube containing microscopic particles known as the stationary phase during the HPLC separation process. The sample components are subsequently moved down the mobile phase by means of a pump into a densely packed tube or column. Chemical and physical interactions between the packing particles and components occur during column packing. An "HPLC" output is produced at the tube's exit when a detector counts the components that have been separated. Simpler liquid chromatography is still utilized for preliminary work, but HPLC offers faster processing times, more sensitivity, ease of use, and better speed ^[13].

REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Reverse phase chromatography refers to the use of a polar eluants contrast to normal phase chromatography, where a polar stationary phase is employed with a phase chromatography is widely in use due to the following advantages. Many compound such as biologically active substances, have limited solubility in nonpolar solvents that are employed in normal phase chromatography. Ionic or highly polar compounds have high heats of adsorption on straight or alumina column and therefore can elute as a tailing peaks. Column deactivation from polar modifiers is a problem in liquid-solid chromatography, which frequently can lead to irreducibility in chromatography system. Ionic compound can be chromatographed via ion exchange chromatography. This mode of chromatography is tedious because precise control variable such as PH and ionic strength is required for reproducible chromatography ^[14].

MOBILE PHASE CHARACTERISTICS

Following points are considered for the selection of a mobile phase.

• Viscosity.
• Compressibility.
• Refractive index.
• UV cut off
• Polarity.
• Vapours pressure 

The viscosity generally increases with the number of carbons in the solvent. Straight chain alcohol shows a very proposed relationship of this nature. For example, to achieve 1ml/min flow rate in a 4.6 x 250 mm column packed with 5 micro meter Octadecyl silane material, a pressure of 1500 psi is required with methanol. Solvent of low viscosity are needed to be compatible with the limitations of the pump. Also, as viscosity increases, the efficiency of the system, as measured by the number of theoretical plates decreases. The sensitivity of the detection is related to the difference between the refractive index, i.e. the greater the difference, greater is the sensitivity. The UV cut-off is defined as the wavelength below which the solvent will absorb more than1.0 absorbance unit. The polarity of the solvent is a measure of the dielectric constant or the ability to elute particular types of compound. The vapour pressure of a solvent plays an important role in mobile phase selection. solvent reservoir could easily change in composition due to the evaporation of one of the more volatile constituents. The flammability of the mobile phase is a safety consideration. Careful attention should be paid to adequate ventilation and waste solvent disposition ^[15,16].

REVERSE MOBILE PHASE

The mobile phase in RP-HPLC, however, has a great influence on the solutes and the separation of component mixtures. The primary constituent of reverse phase-mobile phase is water. Water miscible solvent such as methanol, ethanol, Acetonitrile, dioxane tetrahydrofuran and DMF are added to adjust the polarity of the mobile phase. The water should be high quality, either distilled or demineralized. The most widely used organic modifier is methanol, Acetonitrile and THF, methanol and Acetonitrile have comparable polarities but the latter is an aprotic solvent. This factor may be important if hydrogen bonding plays a significant role in the separation. When inorganic salt and ionic surfactants are used the mobile phase are generally non inflammable due to high water content degassing is quite or important with reverse phase mobile phases ^[17,18].

NORMAL PHASE

Normal phase chromatography was one of the earliest forms of liquid chromatography. It employs a polar stationary phase (most commonly silica gel) and a non-polar or low-polarity mobile phase (e.g., hexane, chloroform, ethyl acetate). In this setup, analytes with stronger polarity interact more intensely with the stationary phase, resulting in longer retention times and delayed elution ^[19,20].

EXPERIMENTAL WORK

Preparation of standard stock solutions

In order to prepare stock solution, weighed accurately 20 mg Sofosbuvir and transferred into 20 ml volumetric flask, added 15 ml of Methanol and sonicated to dissolve the standard completely and diluted up to the mark with Methanol (1000 PPM). Further diluted 0.5 mL to 25 mL with methanol. (20 PPM)

Preparation of standard stock solution for Chromatographic development:

Sofosbuvir Standard stock solution was prepared by transferring 10 mg Sofosbuvir into a 20 mL clean and dried volumetric flask, added about 15 mL of Methanol to dissolve it completely and made volume up to the mark with methanol. (500 PPM).

Further diluted 2 ml of stock solution to 10 mL with mobile phase (100 PPM). It was prepared in mobile phase of each trial and injected in development trials.

Validation of RP-HPLC Method

FILTRATION STUDY:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample.

This study was conducted with Sofosbuvir Test sample (Tablet solution).

Filtration study carried out with unfiltered and filtered test solution. During filtration activity 0.45 µm PVDF and 0.45 µm Nylon syringe filters used by discarding 5 mL of aliquot sample.

SPECIFICITY:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Following solution shall be prepared and injected to prove the specificity nature of the method.

1. Blank (Mobile phase as Blank)
2. Placebo

Analyzing marketed test sample contains excipients (additives) which are totally unknown. So Placebo prepared at lab level by using formula as follows:

Placebo preparation:

Total 10 gm of placebo prepared.

Weighed 22.10 mg of placebo material (Which is equivalent to 50 mg of Sofosbuvir) and transferred to clean and dried 100 mL of volumetric flask. Added 70 mL of Methanol, sonicate for 10 minutes with intermittent shaking. After 10 minutes allow to cool the solution to room temperature and made volume up to the mark with Methanol. Filtered the solution through suitable 0.45 µ Nylon syringe filter discarding 3-5 mL of initial filtrate. Further dilute 1.0 ml of filtered stock solution to 25 ml with mobile phase, injected the resultant solution and chromatograms were recorded.

LINEARITY AND RANGE

Preparation of linearity solution

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

5 levels of Linearity was performed from 50% to 150% of working concentration

Linearity Sofosbuvir stock solution:

Weighed  25 mg of Sofosbuvir and dissolved in 25 mL with Methanol. Further diluted 5 ml of stock solution to 50 mL with Methanol (100 µg/mL)

Linearity levels prepared as follows:

ACCURACY (% RECOVERY)

The accuracy of the analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value of the value found,

Accuracy will be conducted in the range from 50 % to 150 % of working concentration. Solution of each accuracy level was prepared in triplicate. Calculated % Recovery for each sample, Mean % recovery for each level and overall recovery and also calculated % RSD for each level and % RSD for overall recovery.

Accuracy levels details:

Refer Following table for each sample:

Take clean and dried 9 volumetric flasks of 100 mL. Weighed aprrox 22.10 mg of placebo and transferred in each 100 mL volumetric flask. Weighed Sofosbuvir API as per accuracy level and transferred in same 100 ml volumetric flask. Add 70 ml of Methanol sonicate it for 10 minutes with intermittent shaking. Allowed to cool the solution at room temperature and made the volume up to the mark with Methanol. Filter the solution through suitable 0.45 µ Nylon filter discarding 5 mL of filtrate. Further dilute 1.0 ml of filtrate to 25 ml with mobile phase.

Acceptance criteria

1. % Recovery for each sample and Mean recovery and overall recovery should be in the range of 98-102%.
2. The Relative Standard Deviation should not be more than 2.0%.

PRECISION

Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous test under the prescribed conditions. Precision is of two types, Repeatability and Intermediate precision. It is performed on tablet test sample.

1. Repeatibility:

Preparation of sample solution (6 Samples prepared):

Weighed 20 tablets transferred in mortar pestle and crushed to fine powder. Mixed the contents with butter paper uniformly. Weighed the powder material equivalent to 50 mg of Sofosbuvir (72.10 mg of powder material) and transferred to clean and dried 100 mL of volumetric flask. Added 70 mL of Methanol, sonicated for 10 minutes with intermittent shaking. After 10 minutes allow to cool the solution to room temperature and made volume up to the mark with Methanol. Filtered the solution through suitable 0.45 µ Nylon syringe filter discarding 3-5 mL of initial filtrate. Further diluted 1.0 ml of filtered stock solution to 25 ml with mobile phase. (20 mcg of Sofosbuvir), injected the resultant solution and chromatograms were recorded.

Six samples prepared.

Precision (Repeatability) Sample details are as follows:

% Assay: 90-110% for each sample and mean assay value

% RSD for % assay value of 6 samples: NMT 2%

1. Intermediate precision

It is performed by doing analysis on another day to check reproducibility of results. Samples prepared in same manner as that of Repeatability parameter (6 Samples prepared).

Intermediate Precision Sample details are as follows:

% Assay: 90-110% for each sample and mean assay value

% RSD for % assay of 6 samples of Intermediate precision: NMT 2

% RSD for Total 12 samples: NMT 2% for test results (6 of Repeatability and 6 of Intermediate precision)

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Detection limit:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

Quantitation limit:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

As per ICH Q2R1 guidelines LOD and LOQ was determined by using the approach Based on the Calibration Curve in which residual standard deviation of a regression line was calculated and determined the LOD and LOQ by using following formula:

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line

S = Slope of regression line

ROBUSTNESS

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Determination: Standard solution was injected under different chromatographic conditions as shown below.

1. Changes in flow rate by ±10%. (± 0.1ml/min)
2. Change in column oven temperature. (± 2ºC)
3. Change in wavelength (± 3 nm)

RESULT AND DISCUSSION

FILTRATION STUDY:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on tablet test sample.

Results of Filter study

Chromatograms:

Fig. No. 8 Typical chromatogram of unfiltered sample.

Fig. No. 9 Typical chromatogram of sample filtered through 0.45µ PVDF filter.

Fig. No. 10 Typical chromatogram of sample filtered through 0.45µ Nylon filter.

Acceptance criteria: % Absolute difference of filtered samples NMT 2.0 w.r.t. Unfiltered sample.

Data interpretation: Both filters PVDF and Nylon passes the criteria for filter study, hence both filters can be used. We used Nylon filter because it showed less absolute difference as compare to PVDF filter.

SPECIFICITY:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Blank and Placebo solution prepared and injected to check interference at R.T of Sofosbuvir.

Results of Specificity.

Chromatograms:

Fig. No. 15 Typical chromatogram of Blank solution.

Fig. No. 16 Typical chromatogram of Placebo solution.

Acceptance criteria:

• Blank: There should be no Interference at R.T. of Sofosbuvir
• Placebo: There should be no Interference at R.T. of Sofosbuvir

Data interpretation: Blank and placebo was not having interference at R.T. of Sofosbuvir. Hence developed chromatographic method passed the criteria for specificity.

Linearity and Range

Linearity of an analytical method is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range.

Linearity Data for Sofosbuvir:

Fig.  No. 17 Calibration curve of Sofosbuvir.

Data of linearity of Sofosbuvir:

Y = M X + C

Y = 370585.12 X + 5740

Where, X = concentration of Analyte in µg/mL

Y = is area of peak.

M = Slope

C= Intercept

Chromatograms:

Fig. No. 18 Typical chromatogram of Linearity 50%.

Fig. No. 19 Typical chromatogram of Linearity 75%.

Fig. No. 20 Typical chromatogram of Linearity 100%.

Fig. No. 21 Typical chromatogram of Linearity 125%.

Fig. No. 22 Typical chromatogram of Linearity 150%.

Conclusion:

From the calibration curve it was concluded that the Sofosbuvir shows linear response in the range of 10.0-30.0 μg /ml. The Regression value was found well within the limit.

ACCURACY (RECOVERY):

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method is determined by applying the method to analyzed samples to which known amounts of analyte have been added.

Result and statistical data of Accuracy of Sofosbuvir

% RSD for Overall Recovery: 0.844

Chromatograms:

Fig. No. 23 Typical chromatogram of Accuracy 50%.

Fig. No. 24 Typical chromatogram of Accuracy 100%.

Fig. No. 25 Typical chromatogram of Accuracy 150%.

Acceptance criteria:

% Recovery for each level and overall recovery: 98.0 to 102.0%

% RSD for each level and overall recovery: NMT 2.0

Data interpretation: Recovery of analytical procedure was found well within acceptance criteria at all 3 levels. % Recovery not get hampered by changed in analyte concentration.

PRECISION

Precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method is usually expressed as standard deviation or relative standard deviation. Precision was performed on Test sample.

Result of Intra- day and Inter- Day Precision for Sofosbuvir test sample assay:

Chromatograms:

Fig. No. 26 Typical chromatogram of Repeatability precision (Sample 1).

Fig. No. 27 Typical chromatogram of Inter-day precision (Sample 1).

Acceptance criteria:

% Assay: % Assay value for each sample (Individual sample) and mean assay value for precision (6 sample), mean assay value intermediate precision (6 sample),  and mean assay value for precision plus intermediate precision sample (12 sample): 90-110%

% RSD: : % RSD for precision study samples(6 sample), Intermediate precision study samples (6 sample) and precision plus intermediate precision sample (12 sample): NMT 2.0

Data interpretation: % Assay and % RSD was found well within acceptance limit and hence method is precise (Reproducible).

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

σ = 27110.14508 (Residual standard deviation of a regression line)

s = 370585.12 (Slope)

Detection limit (LOD):

LOD = 3.3 σ / S

LOD = 3.3 x 27110.14508 / 370585.12

LOD = 0.241 µg/mL

Quantitation limit (LOQ):

LOQ = 10 σ / S

LOQ = 10 x 27110.14508 / 370585.12

LOQ = 0.732 µg/mL

ROBUSTNESS

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Following changes made under Robustness:

• Change in Wavelength
• Change in flow rate
• Change in column oven temperature

Tab.No.30 Result of Robustness study:

1. Change in Wavelength by +3 NM:

Fig. No. 29 Typical chromatogram of Standard +3 NM.

2. Change in Wavelength by -3 NM:

Fig. No. 30Typical chromatogram of Standard -3 NM.

3. Change in Flow rate by + 10% (1.1 mL/min)

Fig. No. 31 Typical chromatogram of Standard +10 F.R.%.

4. Change in Flow rate by - 10% (0.9 mL/min)

Fig. No. 32 Typical chromatogram of Standard -10 F.R.%.

5. Change in Column Oven temperature by +2°C:

Fig. No. 33Typical chromatogram of Standard +2°C C.O.T.

6. Change in Column Oven temperature by -2°C:

Fig. No. 34 Typical chromatogram of Standard -2ºC C.O.T.