Optimization of extraction technology and determination of sodium hyaluronate in solid drinks by ethanol precipitation combined with enzymatic hydrolysis-reversed phase chromatography

Objective: This study aimed to establish the methods for determining the content of sodium hyaluronate in solid drinks by reversed-phase high performance liquid chromatography. Methods: With sodium hyaluronate content as evaluation index, after the sample extract was precipitated by ethanol, hyaluronase was used to hydrolyze the precipitate. The optimal enzymolysis conditions obtained by single factor test and orthogonal test were as follows: enzymolysis, at 34 ℃ and pH 6.5, for 120 min. The determination was performed on Uliumate C18 (4.6 mm×250 mm,5 μm) column with mobile phase: acetonitrile 10 mmol/L sodium octane sulfonate solution (Vacetonitrile∶Voctane sulfonate =10∶90) at flow rate of 0.6 mL/min. The detection wavelength was 232 nm and quantified by external standard method. Results: The correlation coefficient of sodium hyaluronate was 0.999 9. The recoveries of samples were between 96.1%~101%, and the relative standard deviation was 3.16%. The determination of limit was 0.062 g/kg. Conclusion: Ethanol precipitation combined with enzymolysis-reversed phase chromatography has strong specificity, high accuracy, good repeatability and less interference, which is suitable for the determination of sodium hyaluronate in solid drinks.