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UPLC analysis of stromal riboflavin concentrations in epi-off and epi-on corneal cross-linking

Poster Details

First Author: A.Hammer SWITZERLAND

Co Author(s):    S. Rudaz   S. Guinchard   S. Kling   O. Richoz   D. Tabibian   F. Hafezi     

Abstract Details


To investigate the diffusion of different riboflavin compounds into the corneal stroma after soaking with various commercially available riboflavin formulations for epithelium-off (“epi-off”) and epithelium-on (“epi-on”) corneal cross-linking (CXL).


Laboratory for Ocular Cell Biology University of Geneva, Switzerland


Twenty-eight in-vivo rabbit corneas were used: corneas were either de-epithelialized and soaked with epi-off riboflavin formulations (Mediocross D (n = 6), Mediocross M (n = 6) and Vit. B2 solution (n = 6)) for 30 minutes, or corneas were soaked with an epi-on riboflavin formulation (Mediocross TE, n = 8), while leaving the epithelium intact. Untreated corneas (n = 2) served as control. After treatment, corneas were excised using a manual trepan of 7 mm diameter and prepared for UPLC (Ultra Performance Liquid Chromatography) analysis. Riboflavin and riboflavin phosphate isomers concentrations were determined. Statistical analysis was performed using Xlstat software.


No significant differences in the absolute stromal concentrations of riboflavin and riboflavin phosphate isomers were found for the different formulations. The chemical amount of riboflavin and riboflavin phosphate isomers decreased by 1 to 2 log from the riboflavin formulations to the corneal stroma. In particular we observed an inversion between the riboflavin and riboflavin phosphate isomers concentrations, with riboflavin 5’monophostphate being the major compound in formulations, but the minor compound in the stroma; and riboflavin being the minor compound in formulations but the major in the stroma.


The inversion of riboflavin compounds in the stroma might be explained by a stronger electronegativity of the phosphate isomers leading to a stronger repulsion by the corneal proteoglycans. Further experiments in porcine or human corneas will be needed to confirm the observed effect, which might be used to design more efficient future riboflavin formulations.

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