Development and validation of a robust RP-HPLC method for the quantitative analysis of roxadustat

Abstract Background Roxadustat is a HIF prolyl-hydroxylase inhibitor indicated for the treatment of chronic kidney disease (CKD) associated anemia. Approved in 2018, it has become a drug of great interest to researchers. In addition to its primary indication, roxadustat also exhibits cholesterol-lowering properties, even in patients who are not consuming lipid-lowering agents. The drug inhibits the HIF prolyl-hydroxylase enzyme, which increases iron uptake, reduces hepcidin levels, and promotes erythropoietin production. To facilitate reliable analysis of roxadustat, a validated high-performance liquid chromatography (HPLC) method was developed. This method ensures precision and reproducibility while offering improved temperature control, reducing potential fluctuations in results compared to previously reported methods. Results The HPLC method was developed on the Shimadzu HPLC system, equipped with a CTO-20AC oven and a DGU-20ASR photodiode array detector. The method employed a C-18 column (5 mm × 250 cm with a 0.5 µm particle size) and used a mobile phase consisting of ethanol and water (80:20 v/v). The flow rate was set at 0.8 mL min −1 , and the sample concentration was 150 μg mL −1 . The retention time for roxadustat was 6 min at a detection wavelength of 262 nm. Complete temperature control was ensured by maintaining the column temperature at 28 °C. Validation of the method was conducted by following ICH-Q2 guidelines. A series of tests, including linearity, precision, robustness, reproducibility, repeatability, specificity, and system suitability, were performed. For each test, the mean, standard deviation, percentage recovery, and percentage relative standard deviation (% RSD) were calculated. Additionally, the limit of detection (LOD) and limit of quantitation (LOQ) were evaluated during the validation process. Conclusion The developed HPLC method offers a reliable, precise, and reproducible approach for the qualitative analysis of roxadustat. The improved temperature control minimizes fluctuations in results, addressing limitations noted in previously reported methods. This method provides researchers with a robust tool for analyzing roxadustat with high accuracy and consistency.