Experimental Study Of Determinating The Concentrations Of Riboflavin In Corneal Intrastromal By A HPLC Method

Objective To develop a high performance liquid chromatography (HPLC) method fordetermination of riboflavin in rabbits corneal stroma, and investigate the method’sspecificity, sensitivity and accuracy to determinate whether it can be used to evaluateintrastromal concentrations of riboflavin for the study of corneal cross-linking (CXL).Methods The riboflavin in rabbits cornea homogenate were extracted on a C18-SPEcartridge after protein recipitated by heating incubation and adding10%trichloroaceticacid, and analyzed on HPLC system with an ODS-BP column (5μm,250mm×4.6mmI.D.) and a mobile phase consisted of40%methanol and60%pure water(40:60, v/v).The flow rate was0.1mL/min. The spectro-photofluorimeter was set at wavelength of425nm for excitation and525nm for emission.Results Riboflavin had a good linearity (r2=0.999584) existed within range of1.0×10-6to1.0×10-4mg/mL,and the detection limit of the method was2×10-8mg/mL.The precision of this method indicated the relative standard deviation (RSD) was0.86%(n=10), the sample solution stability RSD is0.51%within24h, and the corneal sampleadding standard of the recovery is (n=5) were99.3%, RSD=3.7%.Conclusion This HPLC method is specific, accurate and sensitive, and can be usedfor determination of riboflavin in rabbits corneal stroma. Objective To determine the concentrations of riboflavin in corneal intrastromal underdifferent interventions using high-performance liquid chromatography (HPLC). Toensure the efficacy and safety of0.5%EDTA in Riboflavin penetrating to corneal stromaby the transepithelial procedures.Methods Fifteen New Zealand white rabbits were divided into three groups, all thecorneal epithelium of five rabbits were debrided, corneal epithelium of another fiverabbits were left in situ and0.5%EDTANa2(ethylenediaminetetraacetic acid disodiumsalt) was topically applied for1h，and the corneal epithelium of last five rabbits werejust left in situ， after those procedures0.1%riboflavin+20%dextran was appliedtopically to those corneas for30min(1drop every3minutes). Then,all the epithelium inthe corneas of EDTA and epi-situ group were carefully removed before all the corneaswere punched with an8.5mm diameter blade and homogenized subsequently.Ultimately, the riboflavin concentrations of those corneal homogenate were determinedby a designed HPLC.Results After treated with Riboflavin for30min, the mean concentration of riboflavinin the epi-removed group was23.54±1.61μg/g tissue, the EDTA group was2.04±0.25μg/g tissue and the epi-situ group was1.44±0.06μg/g tissue. Epi-removedgroup, EDTA group, epi-situ group, respectively, showing significant differences amongthem(F=1792.839， P=0.000). There were significant differences statistically incomparison between any two groups(t=41.780, t=7.484,t=43.408, p<0.05). However, excepted for epi-removed group the riboflavin concentration of other two groups werefailed to meet the theoretical value15μg/g tissue.Conclusions The diffusion of riboflavin to the corneal stroma can be fully enhancedby removing the epithelium preoperatively, which is conducive to the smoothdevelopment of the collagen crosslinking. Penetration enhancer EDTA can effectivelypromote the penetration of riboflavin to the cornea, which may provide an experimentalbasis for the study of transepithelium collagen cross-linking by EDTA, but it still cannot replace the role of removing epithelium on the penetration of riboflavin to thecornea.