Abstract:AIM:To develop a high performance liquid chromatography(HPLC)method for determination of riboflavin in rabbits corneal stroma, and investigate the method's specificity, sensitivity and accuracy to determinate whether it can be or not used to evaluate intrastromal concentrations of riboflavin for the study of corneal cross-linking(CXL).
METHODS:The riboflavin in rabbit's cornea homogenate was extracted on a C18-SPE cartridge after protein precipitated by heating incubation and adding 10% trichloroacetic acid, and analyzed on HPLC system with an ODS-BP column(5μm, 250mm×4.6mm I.D.)and a mobile phase consisted of 40% methanol and 60% pure water. The flow rate was 0.1mL/min. The spectrophoto- fluorometer was set at wavelength of 425nm for excitation and 525nm for emission.
RESULTS: Riboflavin had a good linearity(r2=0.999 584)existed within range of 1.0×10-6 to 1.0×10-4mg/mL. The precision of this method indicated the relative standard deviation(RSD)was 0.86%(n=10), the sample solution stability RSD was 0.51% within 24 hours, and the corneal sample adding standard of the recovery were 99.3%(n=5), RSD=3.7%.
CONCLUSION: This method is specific, accurate and sensitive, and can be used for determination of riboflavin in rabbit's corneal stroma.
