Before the validation runs, a system suitability test was conducted to assess chromatographic parameters such as retention time, number of theoretical plates, capacity factor, and asymmetry factor. The outcomes of these system suitability parameters are presented in Table 2.

The linearity of Elbasvir and Grazoprevir were evaluated at six concentration levels by diluting the standard stock solution to give solutions of Elbasvir and Grazoprevir in the concentration range from 12.5-75 µg/mL µg/ml and 25-150 µg/mL . The regression analysis was carried out for the slope, intercept and correlation coefficient. The results were given in Table 3, Table 4 and Figure 4, Figure 5, Figure 6.

Figure 4

Figure 5

Figure 6

To determine the accuracy of the method, the standard addition method was employed. A known quantity of the standard drug was added to a fixed amount of pre-analyzed standard solution at 50%, 100%, and 150% levels. These solutions were analyzed in triplicate using the proposed method, and the corresponding results are detailed in Table 5.

The assay's precision was examined for both repeatability and intermediate precision. Repeatability was derived from six replicate injections of freshly prepared test solution for Elbasvir and Grazoprevir in the equipment, with the chromatogram results documented in Table 6.

Six replicate injections of the same dilution were analyzed on two different days by different analysts to assess precision variations. The % RSD for Elbasvir and Grazoprevir were found to be 0.9 and 0.8, respectively, well within the acceptable limit of ≤2. This suggests that the method is reproducible on different days, indicating precision. Results are presented in Table 7, Table 8.

For robustness assessment, slight changes in chromatographic conditions, including flow rate of the mobile phase, composition of the mobile phase, and column temperature, were made. The study revealed no significant changes in the chromatograms, indicating the robustness of the developed RP-HPLC method. Robustness study results are outlined in Table 9, Table 10.

Figure 7

Table 9

Condition	Mean area	% assay	% difference
Optimised	796701	99.65	-----
Flow rate at 0.9 mL/min Flow rate at 1.1 mL/min	792534 790125	99.01 99.85	0.64 0.20
Mobile phase: • Buffer-acetonitrile (55:45) • Buffer-acetonitrile (65:35)	785981 745869	100.06 100.12	0.41 0.47
Column Temperature: • at 250C • at 350C	789586 785241	99.05 99.67	0.60 0.02

Table 10

Condition	Mean area	% assay	% difference
Optimized	221969	99.45	-----
Flow rate at 0.9 mL/min Flow rate at 1.1 mL/min	222845 222021	100.35 99.67	0.9 0.22
Mobile phase: • Buffer-acetonitrile (55:45) • Buffer-acetonitrile (45:55)	223139 222685	100.71 100.09	1.43 0.64
Column Temperature: • at 250C • at 350C	223413 222737	101.01 100.23	1.56 0.78

LOD and LOQ were determined following ICH guidelines. The values for Elbasvir were 0.30 and 0.92 µg/mL, while those for Grazoprevir were 0.28 and 0.86 µg/mL, respectively. The low LOD and LOQ values indicate the method's sensitivity.

The validated RP-HPLC method was employed for the assay of a marketed tablet formulation containing 50 mg of Elbasvir and 100 mg of Grazoprevir. Three injections of prepared sample and standard solutions were made. The estimated values for the labeled claim of Elbasvir and Grazoprevir in Zepatier tablets were 99.38±0.5% and 99.19±0.66%, respectively. RSD values for Elbasvir and Grazoprevir are within the limit of ≤2. Results are depicted in Figure 7 and Table 11.

An Zodiac C18 column (4.6x150mm; 5μm) was selected as the stationary phase for separation of both drugs and detection was carried out at 260 nm. Initially, reverse phase liquid chromatography separation was attempted using various ratios of methanol and water and acetonitrile and water as the mobile phases, in which both the drugs were not eluted properly, and the resolution was also poor. Further, trials were also performed to optimize the organic content of mobile phase using 0.1% Orthophosphoric acid. The retention times were found to about 2.324 min and 3.301 min for Elbasvir and Grazoprevir respectively.

The findings and outcomes derived from this investigation, encompassing system suitability, linearity and range, accuracy, precision, and robustness, align comfortably with established criteria. Based on the experimental inquiries, one can infer that the suggested method is viable for the regular analysis of Elbasvir and Grazoprevir in their combined dosage form.