Simple HPLC Method, with Fluorometric Detection, for Studying the Oral Absorption of Monomeric Catechins in a Small Animal Model

Abstract

A high performance liquid chromatographic (HPLC) method, with fluorometric detection, was developed for studying the oral absorption and ensuing plasma pharmacokinetics of (+)‐catechin and (−)‐epicatechin in a small animal model. A plasma sample was first incubated with buffered β‐glucuronidase plus sulfatase to release the catechins from their conjugated forms, and next deproteinized with methanol‐perchloric acid (3+2) mixture containing an internal standard ((+)‐catechin for the assay of (−)‐epicatechin and the latter for the assay of the former). Analyses were carried out on a Microsorb‐MV C18 column, with methanol‐water‐formic acid (15∶84∶1) flowing at 1 mL/min, and a fluorometric detector set at an excitation wavelength of 280 nm and an emission wavelength of 310 nm. (+)‐Catechin and (−)‐epicatechin eluted at 6.7 min and 13.8 min, respectively. Detector responses were linearly related to concentrations of catechin compound in the range 5–160 nM (r² ≥0.993). The limits of detection were 0.75 nM for (+)‐catechin and 1.5 nM for (−)‐epicatechin. Accuracy and precision were evaluated at three concentrations of each catechin compound. Recoveries of (+)‐catechin and (−)‐epicatechin from spiked rat plasma ranged from 95.0 to 101.8% and from 98.7 to 102.5%, respectively. The RSD values for interday variability were in the range 1.27–4.07% for (+)‐catechin and 0.47–2.04% for (−)‐epicatechin. The method was suitable for assessing the plasma pharmacokinetics of (+)‐catechin and (−)‐epicatechin in rats after their individual oral administration as solutions in either water or milk.

Keywords:

• (+)‐Catechin
• (−)‐Epicatechin
• Oral absorption
• Plasma
• HPLC assay