Linear correlations were established while plotting the peak area against the concentrations of each of: SOF, LDV, and PAR within their concentration ranges of 5–60.0 µg/ml, 2–22 µg/ml, and 1–22 µg/ml, respectively. The obtained results are presented in Table 1 and Fig. 2. It's worth noting that oral doses of the mentioned drugs was reported to show maximum plasma concentrations (Cmax) of 567 ng/ml and 323 ng/ml for sofosbuvir and ledipasvir, respectively. While after oral administration of acetaminophen, Cmaxis 12.3 μg/ml.

Linearity curves with regression equations in pure bulk. A SOF (5–60.0 µg/mL), B LDV (2–22 µg/mL) and C PAR (1–22 µg/mL)

Regarding drug spiked human plasma, linear relationships were observed by plotting the ratio of the peak area for each analyte (SOF and LDV) to the peak area of the PAR internal standard (10 µg/ml) against the concentrations of SOF (ranging from 5 to 35 µg/ml) and LDV (ranging from 4 to 20 µg/ml). These findings are depicted in Fig. 3.

Linearity curves with regression equations in drug spiked plasma. A SOF (5–35 µg/ml) and B LDV (4–20 µg/ml) in presence of PAR as internal standard (10 µg/ml)

According to ICH recommendations [27], proposed methods' accuracy was assessed by analyzing different concentrations of SOF (15, 25and 40 μg/ml), LDV (8, 15and 18 μg/ml) and PAR (2, 6and18μg/ml). Standard deviation and mean recoveries were determined to be around acceptable parameters, with high accuracy (Table 2). For drug spiked human plasma method, different concentrations of drug spiked human plasma standard SOF (8, 18 and 25 µg/ml) and LDV (7, 14 and 18 µg/ml) were studied (Table 3).

Precision of the suggested method was valid for both bulk and drug spiked human plasma samples, considering intra-day and inter-day variations. In the case of bulk analysis, intra-day precision was assessed by analyzing three different concentrations of SOF (10, 30, and 60 µg/ml), LDV (5, 10, and 14 µg/ml), and PAR (4, 10, and 22 µg/ml) using the UHPLC method on the same day. Similarly, the same concentrations were analyzed on three different days to evaluate inter-daily precision, as shown in (Table 1).

For the drug spiked human plasma analysis, the precision of the suggested method was examined intra-daily and inter-daily using three different concentrations (low, medium, and high). Intra-day precision was evaluated by analyzing three different concentrations of drug spiked plasma, including SOF (10, 20, and 35 µg/ml) and LDV (6, 10, and 20 µg/ml), on the same day using the UHPLC method with PAR as the internal standard (at a concentration of 10 µg/ml). The same concentrations were analyzed on three different days to assess inter-daily precision.

To evaluate the specificity of the proposed techniques, laboratory-made combinations of SOF, LDV, and PAR were prepared at different concentrations and ratios. These combinations were tested to ensure that there was no interference observed in the presence of each other, as depicted in Fig. 4. Additionally, the techniques were assessed for any interference with plasma content, as shown in Fig. 5. The results showed no significant interferences, confirming the specificity of the method.

UHPLC chromatogram of standard solutions of: SOF 10 µg/mL, Rt = 3.28 min, LDV 10 µg/mL, Rt = 2.28 min and PAR 10 µg/mL, Rt = 1.70 min

A Chromatogram of blank human plasma showed no drug peak interference. B UHPLC chromatogram of analytes in drug spiked human plasma: SOF (35 µg/mL; Rt = 3.2 min), LDV (20 µg/mL; Rt = 2.27 min) using PAR (10 µg/mL; Rt = 1.66 min) as internal standard

Furthermore, the mean recoveries obtained from the analysis were found to be acceptable, indicating the accuracy of the suggested method. Additionally, the method demonstrated good resolution, as demonstrated in Table 4.

Robustness of the suggested UHPLC method was assessed by examining the impact of small variations in flow rate (0.35 and 0.45 ml/min), the ratio of the mobile phase (40:60 and 44:56 v/v acetonitrile: 0.1 orthophosphoric acid), and temperature (30 ± 2 °C). These variations were evaluated to determine their effect on the peak areas. The results obtained demonstrated that the peak areas exhibited low %RSD (Relative standard deviation) values, indicating the robustness of the method. These findings are presented in Table 5.

The system suitability parameters, such as retention time (min), capacity factor (k’), selectivity (α), resolution (Rs), tailing factor (T), number of theoretical plates (N), and height equivalent to theoretical plates (HETP), were examined and assessed in accordance with the guidelines of US Pharmacopeia [28] (Table 6).