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Arthur Hammer, Serges Rudaz, Sylvie Guinchard, Olivier Richoz, David Tabibian, Sabine Kling, Farhad Hafezi; Determing stromal riboflavin concentrations for epi-off and epi-on formulations using UPLC (Ultra Performance Liquid Chromatography). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1139.
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To investigate on the concentration of different riboflavin compounds in the corneal stroma after epi-off and epi-on instillation
Various riboflavin solutions were tested using UPLC analysis to determine riboflavin and riboflavin phosphate isomers concentrations. Solutions were either tested as formulation, or after a 30 minutes in vivo instillation into the stroma of rabbits, followed by excision of the cornea using a manual trepan (7mm diameter) and extraction. Untreated corneas served as controls. Prior to in vivo riboflavin instillation, either an 8 mm abrasion was performed, or the epithelium was exposed to one drop of tetracaine 1% collyre per minute for five minutes.
The concentrations of riboflavin and riboflavin phosphate isomers did not show significant differences for the different formulations when tested as is. However, when comparing the concentrations of riboflavin and riboflavin phosphate isomers between the formulations and their respective corneal concentrations after 30 minutes of instillation, we observed a decrease of 2 to 3 log units. In particular, we observed an “inversion” between the riboflavin and riboflavin phosphate isomers concentrations, with riboflavin 5’monophosphate being the compound with the highest concentration in the formulations, but the lowest in the cornea, and the non-phosphated riboflavin being the compound with the lowest concentration in the formulations, but the highest in the cornea.
Statistical analysis showed no significant differences between the stromal concentrations of the various riboflavin compounds using different solutions and epi-on and epi-off protocols. This lack of significance could be due to the thinner epithelium in the rabbit cornea when compared to the human cornea, allowing the epi-on formulations to penetrate the stroma more efficiently.<br /> The observed and unexpected “inversion” of the compound concentrations from the formulations to the corneas could be explained by the higher electronegativity of the phosphated isoforms, hindering stromal penetration. Modification of the riboflavin vs phosphate isoforms ratio might influence the efficacy of current epi-off protocols
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