Introduction

Sofosbuvir (SOF)

Sofosbuvir is chemically known to be isopropyl (2S)-2-[[[(2R, 3R, 4R, 5R)-5-(2,4-dioxopyrimidin-1-yl)-4-fluro-3-phosphoryl]amino]propanoate with structural formula of C₂₂H₂₉FN₃O₉P (Vejendla, Subramanyam, & Veerabhadram, 2016; Vikas & Satyanarayana, 2016). The active antiviral agent 2'-deoxy-2'-α-fluro-β-C-methyluridine-5'-triphosphate (Hassouna, 2017; Ramesh, Madhukar, Kannappan, Ravinder, & Sravanthi, 2016). The defective substrate for the NS5B which is served by the triphosphate acts as an inhibitor for the viral RNA synthesis (Naazneen & Sridevi, 2017; Rote & Alhat, 2017).

Ledipasvir (LED)

Ledipasvir is chemically known to be Methyl N-[(2S)-1-[(6S)-6-[5-[9,9-Difluoro-7-[2-[1S,2S,4R)-3-[(2S)-2(methoxycarbonylamino)-3methylbutanoyl] 3azaabicyclo[2.2.1]hetan-2-yl]-3H-benzimidazole-5-yl]fluoren-2-yl]-1H-imidazol-2-yl-5-azapiro[2.4] heptan-5-yl]-3H-methyl-1-oxobutan-2-yl] carbamate with structural formula of C₄₉H₅₄F₂N₈O₆ (Devilal & Durgaprasad, 2016; Nagaraju & Vardhan, 2017). The viral phosphoprotein NS5A is inhibited by Ledipasvir (Rai, Prajapati, & Patni, 2017; Rao, Reddy, & Rao, 2017). It leads to viral replication, assembly and secretion (Hassouna, 2017; Trivedi & Tandel, 2018).

Literature survey reveals that very less methods or studies are done on combined dosage form for Sofosbuvir and Ledipasvir (Madhavi & 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 & Shalini, 2010; Vejendla et al., 2016).

Materials and Methods

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 C₁₈ COSMOSIL column with 4.6 x 250 mm as dimensions and 5 mm particle size packing with flow rate 0.7 mL/min and sample size of 20 ml. The filtration assembly with Nylon 66 membrane filter of 13mm diameter having a pore size of 0.45 micron was used to filter 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 efficient separation of drugs. Acetonitrile and 0.1% orthophosphoric acid mixture in the ratio of 55:50v/v pH adjusted to 3.0 provided an efficient 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.

Results and Discussion

Method Validation

The method validation parameters were determined experimentally according to International Conference on Harmonization (ICH) guidelines.

Figure 1 illustrates structure of Sofosbuvir whereas Figure 2 illustrates structure of Ledipasvir. Figure 3 illustrates Standard chromatogram drug chromatogram of Sofosbuvir and Ledipasvir.

Figure 4 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/Specificity

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 specific 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 flow 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 filtered 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 fluids. Nevertheless, very few methods are reported of these two drugs in fixed 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.