Analytical Strategies For Formulation Assessment: A Review Of RP-HPLC Methods For Sofosbuvir And Velpatasvir

Hepatitis C is a serious global health concern caused by the hepatitis C virus (HCV), a single-stranded RNA virus that primarily affects the liver and can lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. The development of direct-acting antiviral (DAA) agents has significantly improved the treatment outcomes of HCV infection. Among these, the combination of Sofosbuvir and Velpatasvir has emerged as a highly effective, pan-genotypic therapy with high cure rates and improved patient compliance.

Sofosbuvir is a nucleotide analog prodrug that acts as an inhibitor of the HCV NS5B RNA-dependent RNA polymerase, thereby preventing viral replication. It is widely used in combination therapies due to its high efficacy, favourable safety profile, and reduced treatment duration. Velpatasvir is an NS5A inhibitor that interferes with viral replication and assembly by targeting a key non-structural protein essential for the HCV life cycle. The combination provides synergistic antiviral activity and is effective against all major HCV genotypes ^[1,2].

With the increasing clinical use of this combination, there is a growing need for reliable and validated analytical methods for their quantitative estimation in bulk drugs and pharmaceutical dosage forms. Analytical techniques play a crucial role in drug development, quality control, and regulatory compliance. Among the various methods, RP-HPLC is the most widely employed technique due to its high sensitivity, specificity, accuracy, and reproducibility. Several RP-HPLC methods have been reported using different chromatographic conditions, including variations in mobile phase composition, column type, flow rate, and detection wavelength. These methods are developed and validated according to International Council for Harmonisation (ICH) guidelines, ensuring parameters such as linearity, precision, accuracy, robustness, LOD, and LOQ meet acceptable criteria ^[2–5]. This review article compiles and critically analyses these RP-HPLC methods to compare chromatographic conditions, validation parameters, and analytical performance.

Review of Reported Analytical Methods

S. Bhargavi et al. (2023) ^[1]

• Chromatographic Conditions: The method used a Discovery C18 column (150 mm × 4.6 mm, 5 µm). The mobile phase consisted of 0.1% orthophosphoric acid buffer and acetonitrile (40:60 v/v). Flow rate was 1.0 mL/min, column temperature 30°C, and detection wavelength 272 nm.
• Performance: Sofosbuvir and Velpatasvir eluted at ~2.18 minutes and ~3.03 minutes, respectively.
• Validation: Linearity was established in the 25–150% concentration range with correlation coefficients of 0.9992. Accuracy showed 98% to 101% recovery, and precision revealed %RSD values below 2% (0.4% for Sofosbuvir; 0.9% for Velpatasvir).
• Sensitivity: LoD values were reported as 0.91 µg/mL for Sofosbuvir and 2.75 µg/mL for Velpatasvir, with LoQ values of 0.34 µg/mL and 1.03 µg/mL, respectively.

Dr. K. Bhavyasri et al. (2023) ^[2]

• Chromatographic Conditions: Separation was achieved using a Hypersil C18 column (4.6 × 150 mm, 5 µm). The mobile phase was water and acetonitrile (30:70 v/v) at a flow rate of 1.0 mL/min. Detection was carried out at 230 nm.
• Performance: The retention times were approximately 3.88 minutes for Sofosbuvir and 2.81 minutes for Velpatasvir. Total run time was about 7 minutes.
• Validation: Linearity ranged from 20–100 µg/mL for Sofosbuvir and 10–50 µg/mL for Velpatasvir. Precision showed low %RSD values (0.42% for Sofosbuvir, 0.9% for Velpatasvir). Percentage recovery was ~99.4% for Sofosbuvir and ~99.8% for Velpatasvir.
• Sensitivity: LOD values were 3.7 µg/mL (Sofosbuvir) and 0.6 µg/mL (Velpatasvir), while LOQ values were 11.3 µg/mL and 1.9 µg/mL, respectively.

T. Hanuman et al. (2020) ^[3]

• Chromatographic Conditions: The method utilizes a Kromosil C18 column with a mobile phase composed of phosphate buffer and acetonitrile (70:30 v/v) adjusted to pH 3.5. The flow rate was maintained at 1.0 mL/min, with detection at 260 nm.
• Performance: Retention times were approximately 2.40 minutes for Sofosbuvir and 2.98 minutes for Velpatasvir.
• Validation: Linearity was observed over 40–240 µg/mL for Sofosbuvir and 10–60 µg/mL for Velpatasvir (correlation coefficients close to 0.999). Accuracy studies showed 98% to 100% recovery, and precision studies revealed %RSD values less than 2%.
• Sensitivity: LOD values were 0.59 µg/mL (Sofosbuvir) and 0.29 µg/mL (Velpatasvir); LOQ values were 1.80 µg/mL and 0.89 µg/mL, respectively.
• Application: Successfully applied to VELPANAT tablets, showing assay values of ~99.48% (Sofosbuvir) and ~99.25% (Velpatasvir) without excipient interference.

Kalpana Nekkala et al. (2018) ^[4]

• Chromatographic Conditions: Separation was carried out using a Luna C18 column (150 × 4.6 mm, 3 µm) under isocratic conditions. The mobile phase consisted of 0.1% formic acid in water, methanol, and acetonitrile (35:40:25 v/v). The flow rate was 0.8 mL/min, with detection at 269 nm.
• Performance: Retention times were approximately 3.72 minutes for Sofosbuvir and 2.62 minutes for Velpatasvir.
• Validation: The method was linear over 80–240 µg/mL for Sofosbuvir (r² = 0.9992) and 20–60 µg/mL for Velpatasvir (r² = 0.9998). Precision demonstrated %RSD values less than 1%. Recovery ranged from 98.6% to 100.6% for Sofosbuvir and 98.4% to 100.4% for Velpatasvir.
• Sensitivity: LOD was 0.01 µg/mL (Sofosbuvir) and 0.001 µg/mL (Velpatasvir); LOQ was 0.03 µg/mL and 0.004 µg/mL, respectively, indicating high sensitivity.

Comparative Analysis of Chromatographic Conditions and Parameters

The review clearly indicates that RP-HPLC is an effective and reliable technique for quantitative analysis of these drugs. All developed methods used C18 columns and achieved short retention times (typically 2–4 minutes) while complying with ICH validation guidelines. Linearity studies exhibited strong correlation coefficients (r² ≈ 0.999), accuracy ranged between 98% and 102%, and precision (%RSD) remained <2% across the research works. The comparative conditions are detailed below: