A novel UV-spectrophotometric method as a reliable alternative for the spectrofluorimetric analysis of lucifer yellow

Monir, Hany H.; El-Ashmawy, Ahmed A.; Abdou, Aya R.; Fayad, Walid; Mahmoud, Khaled M.; Emara, Laila H.

doi:10.4103/epj.epj_52_21

A novel UV-spectrophotometric method as a reliable alternative for the spectrofluorimetric analysis of lucifer yellow

Authors

Abstract

Background
Human colon adenocarcinoma cell-line (Caco-2) monolayer is a well-established ex vivo model used for estimation of drug-apparent permeability through the human intestinal barrier. Lucifer yellow (LY) is a paracellular permeability marker used generally for examining the integrity of Caco-2 monolayer using spectrofluorimetric detection. No other spectroscopic methods were reported for analysis of LY.
Objective
To introduce a novel validated simple ultraviolet (UV)-spectrophotometric method, for LY determination, as an alternative to the commonly used spectrofluorimetry, as well as to apply the developed UV method to confirm the monolayer integrity of Caco-2 cell line compared with the spectrofluorimetric detection.
Materials and methods
The measurement was carried out at a wavelength of 277 nm. The linearity range was 1.0–12.5 μg/ml with the regression coefficient ()=0.9992. Validation of the method was carried out following the ICH guidelines concerning linearity, precision, accuracy, robustness, and stability and statistically compared with the conventional spectrofluorimetric one. Measurement of cultured Caco-2 cell monolayer integrity was carried out using LY employing spectrofluorimetric and UV-spectrophotometric methods.
Results and conclusion
The calculated test and values indicated that no significant difference existed between both methods regarding precision and accuracy. The developed method has been successfully applied for checking the monolayer integrity of Caco-2 cell line. Comparing permeability percentage results of LY, both methods resulted in LY-permissible values of 2.354 and 2.303% using spectrofluorimetric and UV-spectrophotometric methods, respectively, indicating Caco-2 cell-line monolayer integrity.

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