STABILITY-INDICATING HPLC AND UV SPECTROPHOTOMETRIC DETERMINATION OF SOFOSBUVIR IN PURE FORM AND TABLETS BY

Four simple, sensitive, accurate and precise stability-indicating methods were developed for determination of sofosbuvir (SFB) in pure form as well as in its pharmaceutical preparation and in presence of its alkaline degradate. The first method is an HPLC stability–indicating method, where the intact drug (SFB), the internal standard (atorvastatin) and SFB degradation product were separated on a Athena C18 (250 mm X 4.6 mm ID, 5μm particle size) column using methanol–water (70:30, v/v) as a mobile phase at a flow rate of 1 ml/min and UV detection at 260 nm. The second method is the ratio difference method, where the UV absorption spectra of different concentrations of SFB were divided by the absorption spectrum of a certain concentration (30 μg /ml) of its degradation product (divisor) to get the ratio difference spectra. Afterwards, the difference in peak amplitudes between 270 and 245 nm were measured. The third method is the ratio derivative method, where the amplitudes of first derivative of the obtained ratio difference spectra were measured at 282 nm. The fourth method is the mean centering of ratio difference spectra, where the amplitudes of the mean centered ratio difference spectra were measured at 262.6 nm. The calibration curves were linear over the concentration range of 5-35 μg/ml for all methods. The proposed methods can selectively analyse the drug in presence of up to 86 % of its degradation product with mean recoveries of 100.66±1.310, 101.04±1.662, 101.06±1.026 and 99.92+1.374 for the four methods, respectively. These methods were validated and successfully applied for the determination of SFB in its commercial preparation. Moreover, the obtained results were statistically compared with those of the reported method by applying t-test and Ftest at 95% confidence level. It was found that no significant differences were observed regarding accuracy and precision.

HPLC is an important qualitative and quantitative technique, generally used to separate, identify, and quantify the active compounds in pharmaceutical and biological samples (Martin and Guiochon, 2005).Reversed-phase chromatography is the most commonly used separation mode in HPLC.The reasons for this include the simplicity, versatility and scope of the reversed-phase method as it is able to handle compounds of a diverse polarity and molecular mass (Willard and Dean, 1986;Harvey, 2000;Connors, 2005).
Under computer-controlled instrumentation, first derivative of ratio spectra, ratio difference and mean centering of the ratio spectra methods are playing a very important role in the analysis of binary mixtures without previous separation by UV In this work; HPLC, UV ratio difference, UV ratio derivative and UV ratio mean centering methods were applied to the selective determination of SFB in presence of its alkaline degradate.The proposed procedures were successfully applied for determination of SFB in bulk powder and in its pharmaceutical dosage form.

Pure sample
 SFB was kindly supplied by Al Andalous for Pharmaceutical Industries, 6th of October, Giza, Egypt.The purity was assigned as 99.15%. Atorvastatin was kindly supplied by Amoun Pharmaceutical Company, Cairo, Egypt.
The purity was assigned as 99.25%.

Pharmaceutical preparation
SOVALDI ® tablets, each tablet contains 400 mg of SFB (B.No.100924, manufactured by Gilead Sciences, USA), purchased from local market.

Reagents and solvents
All chemicals and reagents used throughout the work were of analytical grade.

Standard solution of intact SFB
A standard solution of SBF (100 μg/ml) was prepared by dissolving 10 mg of SFB in 50 ml of methanol and complete to 100 ml with the same solvent.

Standard solution of atorvastatin (Internal standard):
A standard solution of atorvastatin (100 μg/ml) was prepared by dissolving 10 mg of atorvastatin in 50 ml of methanol and complete to 100 ml with the same solvent.

Standard solution of degraded sample:
100 mg of pure SFB powder were refluxed with 50 ml 1M NaOH for 38 hours.After cooling, the solution was neutralized by 1M HCl, evaporated to dryness under vacuum.The obtained residue was extracted with methanol (2 × 10 ml), filtered into a 100-mL volumetric flask and diluted with methanol to obtain a stock solution labeled to contain degradate derived from 1 mg/ml of SFB.On the other hand, SFB showed no considerable degradation when acidic, oxidative and UV degradation methods had been used instead of alkaline.

Procedures
Construction of the calibration curve (General procedure)

Chromatographic conditions
At ambient temperature, isocratic separation was carried out on Athena C18 (250 X 4.6 X 5μm particle size) using mobile phase consists of methanol: water (70:30, v/v).The mobile phase was degassed by a degasser before pumped at flow rate 1 ml/min.The injected volume of the standard solution was 20 µl and UV detection at 260 nm.

Linearity
Aliquots of standard SFB solution (100 μg/ml) containing (50-350 μg) of intact SFB were transferred into a series of 10 ml volumetric flasks containing (150 μg ) of atorvastatin (internal standard) and adjusted to volume with mobile phase.Into HPLC column, 20 µl were injected from each concentration under the described chromatographic conditions.Calibration graph was constructed by plotting the peak area ratio against the corresponding drug concentration in µg/ml and the regression equation was derived.

Ratio methods
Different aliquots of SFB standard solution ranging from (50-350) µg were transferred to a 10-ml volumetric flasks and completed to volume with methanol.The absorption spectra (from 200 to 400 nm) of these solutions were recorded using methanol as a blank, and then divided by the spectrum of SFB degradates solution (30 µg/ml).

A. Ratio difference method
The difference in the peak amplitudes (ΔP) at the ratio spectra was measured at 270 and 245 nm (ΔP 270-245 nm ).The measured ΔP values versus the final concentrations in μg/ml were plotted to get the calibration graph and the regression equation was derived.

B. First derivative of ratio spectra method
The first derivative corresponding to each ratio spectrum was recorded, using Δλ = 8 nm.The amplitude values at 282 nm were measured.The measured amplitude values versus the final concentrations in μg/ml were plotted to get the calibration graph and the regression equation was derived.

C. Mean centering of ratio spectra method
The ratio spectra (from 200 to 400 nm) were mean centered and the mean centered values were measured at 262.6 nm.The measured mean centered values versus the final concentrations in μg/ml were plotted to get the calibration graph and the regression equation was derived.

Analysis of pharmaceutical preparation
Five tablets of SOVALDI ® 400 mg were weighed and finely powdered.An accurately weighed amount of the powder equivalents to 10 mg was dissolved in methanol, filtered into 100 ml volumetric flask and the volume was completed to volume with methanol to obtain a solution labeled to contain 100 μg/ml of SFB.Transfer aliquots covering the working concentration range into 10 ml volumetric flasks.Proceed as described under -General Procedure‖ for each method.Determine the content of the tablets either from the calibration curve or using the corresponding regression equation.

Degradation of SFB
Accelerated degradation method of SFB was achieved upon heating under reflux with 1 M sodium hydroxide for 38 hours.Later on, complete degradation of SFB was confirmed by TLC.For this purpose, the solution after reflux with 1M sodium hydroxide for 38 hours was cooled, neutralized with 1 M hydrochloric acid, evaporated under vacuum till dryness, extracted with methanol and filtered.The obtained solution was tested by TLC on silica gel 60 GF 254 plates.Separation of the intact drug and its corresponding degradate was achieved by using mobile phase consists of methanolchloroformglacial acetic acid (50: 50: 0.1 by volume) and UV detection at 254 nm.R f values of the intact SFB and its corresponding degradate were 0.26 and 0.61, respectively.

HPLC method
In the present study, a simple and sensitive reversed phase HPLC procedure was suggested for the selective quantitative determination of SFB in presence of its alkaline degradation product.
Different chromatographic conditions affecting the separation were tested taking in consideration the resolution between the drug, its degradation product and the internal standard.Several mobile phases were tried in order to separate the intact drug from its degradate and the internal standard including methanol: water in different ratios.Good separation was carried out on Athena C18 ( 250 X 4.6 X 5μm particle size) column using a mobile phase consists of methanol: water (70:30, v/v) at flow rate 1 ml min -1 and UV detection at 260 nm.
In HPLC chromatogram, showed in Figure 2, the peak of intact SFB, its degradation product and the internal standard were clearly separated and their corresponding peaks were sharply developed at reasonable retention times of 2.8 ± 0.02, 4.8 ± 0.03 and 6.9 ± 0.03 minutes for intact SFB, atorvastatin (internal standard) and degradation product respectively.

Ratio difference method
The zero-order absorption spectra of SFB and its alkaline degradate (Fig. 3) show severe overlap, which does not permit direct determination of SFB in presence of its degradate.
In this method, the absorption spectra of SFB were divided by a suitable absorption spectrum of its degradation product as a divisor to get the ratio spectra.The difference in peak amplitudes between two selected wavelengths in the ratio spectra was found to be proportional with the concentration of the drug without interference from its degradation product (Fig. 4).The method comprises two critical steps, the first is the choice of the divisor; the selected divisor should compromise between minimal noise and maximum sensitivity.The divisor concentrations of 30 µg /ml gave the best results.The second critical step is the choice of the wavelengths at which measurements are to be recorded.Any two wavelengths can be chosen provided that they exhibit different amplitudes in the ratio spectrum and give good linearity at each wavelength individually.The best results were obtained at 245 and 270 nm (ΔP 270-245 nm ).

First derivative of ratio spectra method
In this method, the absorption spectra of SFB were divided by a suitable absorption spectrum of its degradation product as a divisor to get the ratio spectra.By application of the first-derivative to these ratio spectra, SFB can be quantitatively determined at 282 nm without any interference from its degradation product (Figs.5, 6).
Careful choice of the divisor and the working wavelength were of great importance.The divisor concentration of 30 µg /ml was found to be the best.It produces minimum noise and gives better results in accordance with selectivity.

Mean centering of ratio spectra method
In this method, the absorption spectra of SFB were divided by a suitable absorption spectrum of its degradation product as a divisor to get the ratio spectra.The obtained ratio spectra were mean centered.The mean centered values at 262.6 nm was found to be proportional with the concentrations of the drug without interference from its degradation product (fig.7).Careful choice of the divisor concentration was of great importance.The divisor concentrations of 30 µg /ml gave the best results in accordance with selectivity.5): First derivative of the ratio spectra of SFB (30 µg/ml) and its alkaline degradate (30 µg/ml) using 30 µg/ml of degradate as a divisor.7): Mean centering of the ratio spectra of SFB at various concentrations using 30 µg/ml of degradate as a divisor.

Linearity and range
Under the described experimental conditions, the calibration graphs for the methods were constructed by plotting the response versus drug concentrations in μg/ml.The regression plots were found to be linear over the range of 5-35 μg/ml for the four methods.Linearity ranges, regression equations, intercepts, slopes and correlation coefficients for the calibration were listed in Table 1.

Limits of detection and quantitation
The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated from the following equations: Where σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve.
The small values of LOD and LOQ (Table 1) indicate good sensitivity.

Accuracy and precision
Three replicate determinations of three different concentrations of SFB in pure form within linearity range for each method were performed in the same day (intra-day) and in three successive days (inter-day).Accuracy as recovery percent (R%) and precision as percentage relative standard deviation (RSD%) were calculated (Table 2).The small values of RSD% indicate high precision of the methods.Morever, the good R% confirms excellent accuracy.

Specificity
The specificity of the proposed methods were assured by applying the laboratory prepared mixtures of the intact drug together with its degradation product.The proposed methods were adopted for the specific determination of intact SFB in presence of up to 86% of its degradate with mean recoveries of 100.66+1.310,101.04+1.662,101.06+1.026and 99.92+1.374for HPLC, ratio difference, ratio derivative and mean centering methods respectively (Table 3).

System suitability
System suitability test for HPLC method was applied to a representative chromatogram to check various parameters such as the number of theoretical plates (N), resolution factor (R), capacity factor (K‾), tailing factor (T) and selectivity factor(α).The results obtained revealed that the chromatographic conditions described here allow complete base line separation between drug, its degradate and the internal standard peaks with minimum tailing.

Robustness
The robustness of the HPLC method was evaluated by slight changes in the chromatographic conditions such as flow rate (±0.1 ml/min.)and mobile phase contents ratio (±3%).It was found that; these minor changes did not affect the system suitability parameters, confirming robustness of the procedure.
The robustness of the three ratio methods was evaluated by slight changes in the optimum conditions such as divisor concentration (±0.2 μg/ml) and wavelength at which measurements done (±0.2 nm).It was found that; these minor changes did not affect the results, confirming robustness of the procedures.

Stability of standard solutions
The stability of standard solutions of SFB and the internal standard (atorvastatin) were determined by repeated analysis of solutions stored either at room temperature or in refrigerator at different time intervals and comparing the responses (peak areas) with those of freshly prepared standard solutions.From the results, it was found that, both SFB and atorvastatin standard solutions were stable for at least 3 and 7 days when stored at room temperature and in refrigerator, respectively.

Pharmaceutical Applications
The proposed methods were applied to the determination of SFB in SOVALDI ® tablets.The results were validated by comparison to a previously reported method (Al-Andalous Company).No significant difference was found by applying t-test and F-test at 95% confidence level (Armitage, 1994), indicating good accuracy and precision of the proposed methods for the analysis of the studied drug in its pharmaceutical dosage form (Table 4).

CONCLUSION
Simple, rapid, sensitive, accurate, precise and not expensive methods were developed for the analysis of SFB in pure form, in its tablets form and in presence of its alkaline degradation product.The sensitivity, reproducibility and simplicity of the proposed methods make them valuable in routine analysis of SFB.In addition, the proposed methods were found to be stability indicating methods.

Figure ( 4
Figure (4): Ratio spectra of SFB at various concentrations using 30 µg/ml of alkaline degradate as a divisor.

Figure (
Figure (5): First derivative of the ratio spectra of SFB (30 µg/ml) and

Figure ( 6
Figure (6): First derivative of the ratio spectra of SFB at various concentrations using 30 µg/ml of degradate as a divisor.

Figure (
Figure (7): Mean centering of the ratio spectra of SFB at various concentrations using 30 µg/ml of degradate as a divisor.

Table ( 1
): Spectral data for determination of SFB by the proposed methods: + bx where y is the response and x is the concentration in µgml -1 .

Table ( 4
): Determination of SFB in SOVALDI ® tablets by the proposed and reported methods: * It is an HPLC method using Zorbax phenyl (250 X 4.6 X 5μm particle size) column and phosphate buffer pH3.5: acetonitrile (60:40, v/v)as mobile phase at flow rate 1.5 ml/min and UV detection at 260 nm (Al-AndalousCompany) .
**** The values in the parenthesis are tabulated values of t and F at (p= 0.05).