Determination of Protein in Medical Cross-Linked Sodium Hyaluronate Gel by the Method of Bradford after De-Crosslinking with Sulfuric Acid

The medical cross-linked sodium hyaluronate gel was insoluble in water and could not be directly measured by current standard methods such as Lowry method and Bradford method. The determined results of medical cross-linked sodium hyaluronate gel obtained by the 2 methods of Lowry and Bradford after de-crosslinked by sulfuric acid solution were compared. It was shown that the degradation products of cross-linked sodium hyaluronate (D-glucuronic acid and N-acetyl-D-glucosamide), the hydrolysate of cross-linking agent 1,4-butanediol diglycidyl ether and additive of lidocaine hydrochloride could seriously affect the determined result of Lowry method, based on this fact, the method of Bradford was selected for determination. The medical cross-linked sodium hyaluronate gel of 0.5 g was taken, and 0.5 mol·L^-1 sulfuric acid solution (de-crosslinking agent) of 0.25 mL was added according to the ratio of sample mass to de-crosslinking agent volume at 2：1 (g：mL). After sealing, the sample was heated in the boiling water bath for 10 min. Then 1 mol·L^-1 sodium hydroxide solution equal in volume to the above-mentioned sulfuric acid solution was added. After mixing well, 5.0 mL of 0.10 g·L^-1 Coomassie brilliant blue G-250 solution was added, and the mixed solution was placed for 10 min. The absorbance of the above system at 595 nm was measured by UV-Vis spectrophotometer with blank standard solution of bovine serum albumin as reference, and the standard curve was made with bovine serum albumin standard solution series. As found by results, linear relationship between values of absorbance and mass concentration of bovine serum albumin was kept in the range of 2.04-10.22 mg·L^-1, with detection limit (3.3s/k) of 1.353 mg·L^-1. Test for the spiked recovery was made at three concentration levels with medical cross-linked sodium hyaluronate gel as matrix, giving recoveries in the range of 99.3%-100%, and RSDs (n=6) of the determined values were in the range of 1.3%-3.5%.