INTRODUCTION:

Sofosbuvir is a nucleotide analog inhibitor of HCV NS5B polymerase. The IUPAC name for sofosbuvir is (S)-Isopropyl 2-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo3,4-dihydro pyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyl tetra hydrofuran-2yl) methoxy)-(phenoxy) phosphoryl amino) propanoate. It has a molecular formula of C₂₂H₂₉FN₃O₉P and a molecular weight of 529.45. It has the following structural formula:

Sofosbuvir is a white to off-white crystalline solid with a solubility of ≥ 2 mg/mL across the pH range of 2-7.7 at 37 °C and is slightly soluble in water.

SOVALDI tablets are for oral administration. Each tablet contains 400 mg of sofosbuvir. The tablets include the following inactive ingredients: colloidal silicon dioxide, croscarmellose sodium, magnesium stearate, mannitol, and microcrystalline cellulose. The tablets are film-coated with a coating material containing the following inactive ingredients: polyethylene glycol, polyvinyl alcohol, talc, titanium dioxide, and yellow iron oxide. Sofosbuvir is used in combination therapy with other antiviral medications to treat chronic hepatitis C virus (HCV) infected patients with HCV genoptypes 1-6, and to treat HCV and HIV co-infected patients. Depending on the level of cirrhosis or decompensation, combination therapy can also include either ribavirin alone or ribavirin and peg-interferon alfa.

When used in combination with Ledipasvir as the combination product Harvoni, sofosbuvir has the following indications: treatment of genotypes 1, 4, 5, or 6 infection without cirrhosis or with compensated cirrhosis; in combination with Ribavirin for genotype 1 infection with decompensated cirrhosis; or in combination with Ribavirin for the treatment of genotype 1 or 4 infection who are liver transplant recipients without cirrhosis or with compensated cirrhosis.

When used in combination with Velpatasvir as the combination product Epclusa, sofosbuvir is indicated for the treatment of adult patients with chronic hepatitis C virus (HCV) genotypes 1, 2, 3, 4, 5, or 6 infection without cirrhosis or with compensated cirrhosis, or in combination with Ribavirin if associated with decompensated cirrhosis.

Few liquid chromatographic methods for determination sofosbuvir either individually or in a combination with other drugs were reported earlier. The methods developed in HPLC ([5-7]) and LC-MS ([8-15]) have been reported. However to my present knowledge, no spectrophotometric method for the determination of sofosbuvir in tablet dosage forms has been published. Herein, a simple, sensitive and direct analysis without complicated sample preparation spectrophotometric method is optimized and validated for Sofosbuvir, determination in bulk and pharmaceuticalformulations^1-5.

EXPERIMENTAL:

Instrument:

Elico Ultraviolet-Visible double beam spectrophotometer SL-164 with 1 cm matched quartz cells was used for all spectral measurements.

REAGENTS:

All the chemicals used were of analytical reagent grade.

1. 1,10-Phenanthroline :0.1 M in distilled water.

2. 2,2’-bipyridyl: 0.1 M in distilled water.

3. Ferric Chloride hexahydrate: 0.3% w/v in distilled water

PROCEDURE:

Standard stock solution was prepared by dissolving 100 mg of Sofosbuvir in 100 mL of distilled water to get a concentration of 1000 μg /mL. This was further diluted to get the working standard solution of 50 μg /mL and 100 μg /mL.

ASSAY PROCEDURE:

Method A:

Aliquots of standard drug solution of Sofosbuvir 1 –6.0 mL (50 μg /mL) were taken and transferred into series of graduated test tubes. To each test tube 2 mL of Ferric chloride and 2 mL of 1,10-Phenanthroline (0.1M) were added. The test tubes were allowed to stand in water bath at 70⁰ c for 15 mins. The test tubes were then cooled to room temperature and the solutions were made upto 10 mL with distilled water. The absorbance of the red colored chromogen was measured at 510 nm against reagent blank and a calibration curve was constructed. The absorbance of the sample solution was measured, and the amount of Sofosbuvir was determined by referring to the calibration curve.

Figurhe-2: Calibration curve of sofosbuvir with 1,10-Phenanthroline and Ferric Chloride

Method B:

Aliquots of standard drug solution of Sofosbuvir 0.5 – 5.0 mL (20 μg /mL) were taken and transferred into series of graduated test tubes. To each test tube 1 mL of Ferric chloride (0.3%) and 0.5 mL of Potassium ferricyanide (0.2%) were added and thoroughly shaken and set aside for 5 mins. The volume in each test tube was made upto 10 mL with distilled water. The absorbances of the solutions were measured at 520 nm against reagent blank, within 30 mins and the calibration curve was plotted. Similarly the absorbance of the sample solution was measured, and the amount of Sofosbuvir was determined by referring to the calibration curve.

Figure-3: Calibration curve of sofosbuvir with 2,2'--Bipyridyl and Ferric Chloride

The methods were extended for the determination of Sofosbuvir in tablets (SOVALDI tablets ® 400 mg ) was choosen. The weight 100 mg of Sofosbuvir was accurately weighed and transferred in to 100 mL volumetric flask, was dissolved in 50 mL of distilled water, sonicated for 15 mins, filtered and washed with distilled water. The filtrate and washings were combined and the final volume was made to 100 mL with distilled water. The solution was suitably diluted and analyzed as given under the assay procedure for bulk samples. The results are represented in Table II. None of the excipients usually employed in the formulation of powder for injection interfered in the analysis of Sofosbuvir, by the proposed methods.

RECOVERY STUDIES:

To ensure the accuracy and reproducibility of the results obtained, adding known amounts of pure drug to the previously analyzed formulated samples and these samples were reanalyzed by the proposed method and also performed recovery experiments. The percentage recoveries thus obtained were given in Table II.

RESULTS AND DISCUSSIONS:

In the present study, the Method A and B are based on the reduction of Ferric chloride to ferrous form by the drug, which forms complex with 1, 10-Phenanthroline and 2, 2’- bipyridyl to yield blood red colored chromogen. The colored chromogens were stable for more than 3 hrs and exhibited maximum absorption at 510 nm and 520 nm respectively. Reaction mechanisms involved in the formation of colored species was shown in fig—3. The conditions required for the formation of colored complexes were optimized. Statistical analysis was carried out and the results of which were satisfactory. The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity and Sandell’s sensitivity are presented in Table I. The regression analysis using the method of least squares was made for slope (m), intercept (b) and correlation obtained from different concentrations and the results are summarized in Table I.

The reproducibility and precision of the methods are very good as shown by the low values of coefficient of variance (CV). Recovery studies were close to 100 % that indicates the accuracy and precision of the proposed methods, and also indicates non-interferences from the formulation excipients. All the validated parameters are summarized in Table II.

In conclusion, the proposed methods are simple, sensitive, accurate and economical for the routine estimation of Sofosbuvir in bulk and in its formulations.

Table: I-OPTICAL CHARACTERISTICS AND PRECISION DATA

Parameters	Method A	Method B
l _max (nm)	510	520
Beer’s law limits μg /mL	10-140	5-50
Molar absorptivity (l mol.cm)	7.8x10⁴	7.04x10⁴
Sandell’s sensitivity (μg s/cm²/0.001absorbance unit)	0.128	0.142
Regression Equation* (Y) Slope (m) Intercept (c)	0.011 0.0188	0.037 0.0007
Correlation Coefficient(r)	0.998	0.996
Precision (%Relative Standard Deviation)	0.62	0.25

*Y=mx+c, where X is the concentration in micrograms/ml and Y is absorbance unit.

Table II: ASSAY OF SOFOSBUVIR IN TABLETS.

Sample No.	Labeled Amount in [mg]	% Obtained* by proposed method (mg)		** % Recovery by the Proposed method
Sample No.	Labeled Amount in [mg]	Method-A.	Method-B	Method -A	Method- B
1	400	99.76	98.99	95.4	97.2
2	400	99.52	98.74	96.8	97.54

*Average of three determinations.** After spiking the sample.

Reaction Schemes involving in the formation of Chromogens:

Proposed Scheme-1 for Mehtod-A: Step- I:

When treated with known excess of Fe(III) drug undergoes oxidation giving oxidation products of drug (CEF) inclusive of reduced form of Fe(III) i.e Fe(II) besides unreacted Fe(III). Fe(II) has a tendency to give colored complex on treatment with 1,10-phenanthroline.

Step II:

The next step concerns with the estimation of Fe (II) with 1, 10-phenanthroline which forms colored tris complex (Ferroin).

Proposed scheme-2 for Method-B: Step-1:

When treated with known excess of Fe(III) drug undergoes oxidation giving oxidation products of sofosbuvir inclusive of reduced form of Fe(III) i.e. Fe(II) besides unreacted Fe(III). Fe(II) has a tendency to give colored complex on treatment with 2,2-Bipyridyl.

SFSB+Fe(III) Oxidation product of SFSB + Fe(II) + Unreacted Fe(III)

Step II:

The next step concerns with the estimation of Fe(II) with 2, 2'--bipyridyl, which forms colored tris complex (ferroin).

Figure 5: Proposed scheme of Sofosbuvir with 2,2-Bipyridyl/ FeCl₃

ACKNOWLEDGEMENT:

The authors are grateful to for the supply of Sofosbuvir as a gift sample and to the Cipla Pharmaceuticals, and to the Management, Vishnu Chemicals, Jeedimetla, Hyderabad, for providing the necessary facilities to carry out the research work.

CONFLICT OF INTEREST:

The author no conflict interest.

REFERENCES:

1. The American Society of Health-System Pharmacists. Archived from the original on 2016-12-01. Retrieved Nov 30, 2016.

2. Divya Rajagopal for the Economic Times. Sept 12, 2015. Can Indian generic makers find gold with a blockbuster Hepatitis C drug?

3. 3."Sofosbuvir (Sovaldi)-Treatment-Hepatitis C Online". www.hepatitisc.uw.edu. Archived from the original on 23 December 2016. Retrieved 8 January 2017.

4. FDA, News Release (June 28, 2016). "FDA approves Epclusa for treatment of chronic Hepatitis C virus infection". Archived from the original on June 3, 2017.

5. "Sovaldi 400 mg film coated tablets - Summary of Product Characteristics (SPC) - (eMC)". www.medicines.org.uk. Archived from the original on 31 January 2017. Retrieved 8 January 2017.

Received on 03.05.2018 Modified on 10.06.2018

Research J. Pharm. and Tech. 2019; 12(4):1517-1520.

DOI: 10.5958/0974-360X.2019.00251.8