Fluorophotometric Determination of Riboflavin Concentrations in a Human Artificial Anterior Chamber Model

Corneal cross-linking (CXL) requires an adequate corneal riboflavin impregnation, which is clinically assessed by verification of a riboflavin "flare" in the anterior chamber. We set out to replace this subjective assessment with an objective measurement method and evaluated fluorophotometry as an apparatus-based technique for riboflavin detection in the anterior chamber.

In an artificial anterior chamber model using human corneas and a modified Fluorotron fluorophotometer, we determined the detection limits of riboflavin concentrations across native corneas by comparison measurements of the same concentrations in glass cuvettes. Subsequently, standard CXL procedures with corneal application of riboflavin were simulated and the proportions of riboflavin entering the anterior chamber were measured fluorophotometrically.

The measurement results of the riboflavin dilution series in the artificial anterior chamber showed a very high concordance with the results obtained in a glass cuvette (Pitman test P = 0.329). In the CXL simulation, the mean riboflavin concentration measured in the anterior chamber increased within 15 minutes from 5 (±1) to 903 (±204) ng/mL and stood at 1089 (±56) ng/mL after 30 minutes.

Fluorophotometry is able to measure riboflavin in an artificial anterior chamber across human corneas over a wide range of concentrations and it reliably detects the increasing riboflavin signal in simulated CXL procedures.

The replacement of the subjective riboflavin detection by a technically straightforward, objective detection method might increase patient safety and treatment efficiency in CXL.