Objective: Osteoarthritis can be treated by taking oral supplements containing compounds that can nourish bones and joints such as hyaluronic acid, methylsulfonylmethane (MSM), chondroitin, glucosamine, and collagen. This study aimed to develop and validate tests for analyzing two compounds, namely, hyaluronic acid and MSM, simultaneously and to determine both their levels in a mixed sample. Methods: Hyaluronic acid derivatization was carried out using fluorenylmethyloxycarbonyl chloride and then analyzed by liquid chromatography with fluorescence detection, while MSM was analyzed using gas chromatography. After the development of optimal conditions for each separation, system suitability tests were developed and calibration curves used for tests of accuracy and precision as well as for level determination. Hyaluronic was detected at an excitation wavelength of 255 nm and emission wavelength of 330 nm. The mobile phase used was acetonitrile-acetate pH 4.2 (1: 4) with a flow rate of 1.0 mL/min. Results: The developed method was linear (r=0.9983) in the range of 5-50 ppm and the limits of detection (LOD) and quantitation (LOQ) were 3.55 and 11.84 ppm, respectively. The initial column temperature for MSM analysis was 110°C and the mobile phase used was nitrogen gas at a flow rate of 0.8 mL/min. The method was linear (r=0.9998) in the range of 4000-15,000 ppm and the LOD and LOQ were 332.90 and 1109.67 ppm, respectively Conclusion: A simulated sample containing both compounds was assessed to contained 98.63% hyaluronic acid and 99.35% MSM.
- Fluorenylmethyloxycarbonyl chloride
- Gas chromatography
- High-performance liquid chromatography
- Hyaluronic acid