The stock solution equivalent to 20 ppm to 80 ppm were prepared, sonicated and filtered through 0.45µ membrane.

Twenty tablets ofNalaxegol were weighed and powdered. The powder equivalent to 500 mg of the active ingredient was accurately weighed and transferred into a 100 mL volumetric flask containing 50 mL mobile phase and sonicated for 15 minutes and the volume was made up to 100 mL with mobile phase and filtered through 0.45micron membrane.

The analytical system consisted of a Binary gradient pump, a manual injector of 20µL capacity, and a diode array detector. As the stationary phase, Inertsil-C18 ODS column,5 μm particle size, 250 mm × 4 mm (Merck, Darmstadt, Germany) was used. The mobile phase contained methanol: acetonitrile; 90:10 v/v. The flow rate of the mobile phase was 1 mL min⁻¹. The wavelength of the detector was set at 250 nm. Separation was performed at 25 °C.

In HPLC method,mobile phase contained methanol: acetonitrile; 90:10 v/v. The detection was carried out at 250 nm. The mobile phase flow rate was 1 mL min⁻¹. Typical retention times of Naloxegol were about 4.23 min (Fig. 1). In pure drug solution, purity peak of Naloxegol was 100 % and peak asymmetry was 1.2.

Figure 2

HPLC method was validated according to the International Conference on Harmonization Guidelines, (ICH Q2B, validation of analytical procedures methodology). The method was validated for parameters such as system suitability, linearity, precision, accuracy, and robustness. 8, 9, 10, 11

A Standard solution was prepared by using Naloxegol working standard as per test method and injected five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD of retention times and peak areas for Naloxegol. The system suitability parameters are given in Table 1.

Linearity was evaluated in the concentration range 40–120 mg L⁻¹. The samples of each solution were injected three times and each series comprised of five experimental points.

The calibration plots were linear in the concentration range of 40–120 mg L⁻¹ (n = 5, r = 0.9992). The calibration curve was described by standard straight line equation; y = (5491604 ± 239226)c.The b value, calculated from equation y = ac + b, was insignificant because it was lower than the critical value t_b = b/S_b. Statistical analysis using Mandel’s fitting test confirmed linearity of the calibration curve (Figure 2).

Figure 3

Precision of the assay was determined in relation to repeatability (intra-day) and intermediate precision (inter-day). In order to evaluate the repeatability of the method, six samples were determined during the same day for three concentrations of Naloxegol. Intermediate precision was studied by comparing the assays performed on two different days.The intra-day andinter-day precision values of measured concentration of Naloxegol were calculated and the results are given in Table 2. The RSD values were 0.05 and 0.02, respectively, demonstrating that the method was precise. Good recoveries were obtained for each concentration, confirming that the method was accurate.

The LOD and LOQ parameters were determined from the regression equation of Naloxegol LOD = 3.3 S_y/m, LOQ = 10 S_y/m, where S_y is a standard error and m is the slope of the corresponding calibration curve. Under applied chromatographic conditions, the LOD of Naloxegol was 2.4 mg L⁻¹ and LOQ of Naloxegol was 7.3 mg L⁻¹.

The robustness study was performed by slight modification in flow rate of themobile phase. Standard solution prepared as per the test method was injected into the HPLC system using flow rates, 0.8 mL/min and1.2 mL/min.The system suitability parameters were evaluated and found to be within the limits for 0.8 mL/min and 1.2 mL/min flow. Naloxegol was resolved from all other peaksand the retention times were comparable with those obtained for mobile phase havingflow rates 1.0 mL/min. The results of robustness study were shown in Table 3.