Structural characterization of degradation products of phenol red used as zero permeability marker in in-situ rat intestinal permeability studies by LCMS-IT-TOF

In this study, the quantitative determination of phenol red with using HPLC, the permeability marker widely used in gastrointestinal studies, and its stability in various apolar phase solvents were investigated. In addition, the mechanisms of degradation products obtained were tried to be elucidated by using the LCMS-IT-TOF device. The proposed HPLC method utilizes a Perkin Elmer C₁₈, 5 μm, 250 mm × 4.6 mm i.d. column, a mobile phase consisting of phosphate buffer and methanol in the proportion of 50:50 (v/v) with apparent pH adjusted to 3.2 and detected at 430 nm using a photodiode array detector. The method exhibits linearity within the concentration range of 5-200 µg/mL, with a calculated R² of 0.9981. In situ conditions, the recovery is in the range of 89.8-103.6 %. Furthermore, high-resolution mass studies were identified three degradation products that occur when phenol red was dissolved either ethanol or methanol medium. The method has a low cost of consumables and is very easy to apply to the procedure and analyze degradation products while easily applied to phenol red analysis in various mediums. High-resolution mass screening analysis confirmed the proposed structure of the degradation product. The validation and robustness studies were fulfilled to display the performance of the method in various analytical environments. The greenness and blueness evaluations of the developed method were also made in detail. The AGREE score of the method was calculated as 0.64, AGREEprep 0.68, MoGAPI 0.78, and BAGI scale score as 65.0. In short, the method is perfectly green, applicable, and practical. Finally, phenol red concentrations analyzed in samples obtained in in-situ animal studies were conducted to demonstrate the applicability of the developed method.