Fluorescence-Coupled Techniques for Determining Rose Bengal in Dermatological Formulations and Their Application to Ex Vivo Skin Deposition Studies

Rose Bengal (RB) is a fluorescent dye with several potential biomedical applications, particularly in dermatology. Due to RB's poor physicochemical properties, several advanced delivery systems have been developed as a potential tool to promote its permeation across the skin. Nevertheless, no validated quantitative method to analyse RB within the skin is described in the literature. Considering RB exhibits a conjugated ring system, the current investigation proposes fluorescence-based techniques beneficial for qualitatively and quantitatively determining RB delivered to the skin. Notably, the development and validation of a fluorescence-coupled HPLC method to quantify RB within the skin matrix are herein described for the first time. The method was validated based on the ICH, FDA and EMA guidelines, and the validated parameters included specificity, linearity, LOD, LLOQ, accuracy and precision, and carry-over and dilution integrity. Finally, the method was applied to evaluate RB's ex vivo permeation and deposition profiles when loaded into dermatological formulations. Concerning qualitative determination, multiphoton microscopy was used to track the RB distribution within the skin strata, and fluorescence emission spectra were investigated to evaluate RB's behaviour when interacting with different environments. The analytical method proved specific, precise, accurate and sensitive to analyse RB in the skin. In addition, qualitative side-analytical techniques were revealed to play an essential role in evaluating the performance of RB's dermatological formulation.

Automated summary

Rose Bengal (RB) is a fluorescent dye used in biomedical applications, especially in dermatology. Advanced delivery systems have been developed to enhance its permeation across the skin. However, there is currently no validated method for quantitatively analyzing RB in the skin. This study proposes fluorescence-based techniques and describes the development and validation of a fluorescence-coupled HPLC method to quantify RB in the skin matrix for the first time. Qualitative analysis using multiphoton microscopy and fluorescence emission spectra provides additional insight into RB's distribution and behavior in different environments.