VALIDATION METHODS FOR THE ANALYSIS OF HYDROXYPROLINE FROM COLLAGEN UNDENATURED TYPE II COLLAGEN USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FLUORESCENCE
Objective: This study aimed to validate an high-performance liquid chromatography method for analyzing undenatured Type II collagen preparationsusing a fluorescence detector.Methods: Based on the optimum analysis conditions, the compound was detected at an excitation wavelength of 255 nm and an emission wavelengthof 320 nm. The optimum mobile phase was determined to be acetate (pH 4.2) and acetonitrile (60:40) with a flow rate of 1.0 ml/min. Hydroxyprolineis a compound that does not have chromophore moiety; thus, it has to be derivatized first using 9-fluorenylmetoxycarbonyl-chloride.Results: The developed method was validated with linearity and an equation of y=3,249,704 x+141,945,072, with a value of r=0.9994. The detectedrange of hydroxyproline was 4–15 ppm. The limit of detection was determined to be 0.49, with an limit of quantitation of 1.64.Conclusion: Our results indicated that the average level of hydroxyproline was 98.66%, 99.12%, and 99.85%.