Analysis of Riboflavin Compounds in the Rabbit Cornea In Vivo

ABSTRACTPurpose: To investigate the composition and concentration of individual riboflavin compounds in the corneal stroma in vivo after soaking with various commercially available riboflavin formulations.Methods: Experiments were performed in 26 rabbit corneas in vivo: 24 corneas were soaked with riboflavin formulations for 30 minutes or with 0.9% NaCl for control (n = 2). After treatment, corneas were excised and prepared for ultra-high-pressure liquid chromatography (UHPLC) analysis. Additionally, computational chemical analysis of riboflavin compounds and keratan sulfate were performed.Results: The amount of riboflavin and riboflavin phosphate isomers in cornea decreased by a factor of 10 to 100, when compared to the amount in riboflavin formulations. In particular, we found an inverse relationship in the ratio of riboflavin to riboflavin phosphate isomer concentration between formulations and cornea. The electronegativity and ionization potential of riboflavin and phosphate isomers are different.Conclusions: The inverse relationship observed might be explained by a stronger electronegativity of the phosphate isomers, leading to a stronger repulsion by corneal proteoglycans. Indicating the individual concentration of riboflavin compounds in formulations is more representative than the total riboflavin concentration. Riboflavin formulations and CXL protocols might be improved considering the differences in diffusion and ionization potentials of the different riboflavin compounds.KEYWORDS: Corneal collagen cross-linkingCXLriboflavinUHPLC AcknowledgmentsWe thank Dr Alessandra Nurisso (PhD) from the pharmacochemistry laboratory of the pharmaceutical sciences of the University of Geneva for the protomer determination with MOE® (MOE 2012.10, CCG Inc., Montreal, Canada). Also, we thank Dr Marc-Olivier Boldi (PhD) from the Research Center for Statistics of the University of Geneva for the statistical analysis counseling.FundingThis study supported by the Swiss National Science Foundation (A.H. and O.R.).Declaration of interestArthur Hammer, None; Serge Rudaz, none; Sylvie Guinchard, none; Sabine Kling, None; Olivier Richoz, named co-inventor of PCT/CH2014/000075 application; Farhad Hafezi, named co-inventor of PCT/CH2014/000075 application.Additional informationFundingThis study supported by the Swiss National Science Foundation (A.H. and O.R.).