Development of a RP-HPLC method for simultaneous determination of reference markers used for in-situ rat intestinal...

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Biopharmaceutical Sciences

Background:

• The single-pass intestinal perfusion (SPIP) method is crucial for evaluating drug intestinal absorption.
• Metoprolol tartrate (MT) and Phenol red (PR) are commonly used in SPIP studies.
• Accurate quantification of these compounds in perfusion media is essential for reliable results.

Purpose of the Study:

• To develop and validate a simultaneous RP-HPLC method for MT and PR.
• To enable precise determination of drug concentrations in SPIP experiments.
• To support in-situ intestinal permeability studies.

Main Methods:

• Reverse-phase high-performance liquid chromatography (RP-HPLC) with UV detection.
• Utilized a Restek Raptor C18 column with a mobile phase of Methanol and Phosphate buffer (pH 3.0).
• Validated the method according to ICH Q2(R1) guidelines for linearity and other parameters.

Main Results:

• Achieved simultaneous determination of MT and PR with retention times of 2.89 and 3.80 min, respectively.
• The method demonstrated linearity in the ranges of 2-50 μg/mL for PR and 10-75 μg/mL for MT.
• Successfully applied the validated RP-HPLC method to quantify MT and PR in perfusion medium.

Conclusions:

• A robust and validated RP-HPLC method for simultaneous MT and PR analysis in SPIP studies was successfully developed.
• This method provides a reliable tool for researchers conducting in-situ rat intestinal permeability studies.
• The developed method is adaptable for future research and modifications.