A validated method using RP-HPLC for the simultaneous quantification of creatine, creatinine, cholesterol, and hyaluronic acid across pharmaceutical, food, and biological matrices

Abstract

This study presents a highly efficient, precise, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method that innovatively integrates the concurrent quantification of four important analytes: creatine, creatinine, cholesterol, and sodium hyaluronate into a single analytical procedure. The development of this integrated method marks a significant advancement over traditional approaches, which often require separate methods for each analyte. HPLC separation was achieved using a Hypersil C18 analytical column (150 mm x 4.6 mm, 5 μm) with a mobile phase consisting of 1 % triethanolamine and 80 % acetonitrile, at a flow rate of 1 mL/min and a detection wavelength of 200 nm at room temperature (25 °C). The method exhibited excellent retention times for creatine, creatinine, cholesterol, and sodium hyaluronate (3.155, 5.215, 2.194, and 3.106 min, respectively) and demonstrated a wide linearity range (25 to 80 µg/mL) with correlation coefficients exceeding 0.999 for all analytes. Validation results confirmed that the method meets the acceptance criteria of the ICH Q2 (R1) guidelines. This innovative approach offers a significant improvement in efficiency, reducing both time and resource consumption while maintaining high accuracy and reliability in simultaneous analysis. Hence it can be successfully used for routine analysis of Creatine, Creatinine, Cholesterol, and Hyaluronic acid in pharmaceutical formulations, food, and human body fluids.