An analysis method for flavan-3-ols using high performance liquid chromatography coupled with a fluorescence detector
Liuqing Wang a , Yoko Yamashita a , Akiko Saito b , Hitoshi Ashida a,*
a Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
b Graduate School of Engineering, Osaka Electro-Communication University (OECU), Osaka 572-8530, Japan
Procyanidins belong to a family of flavan-3-ols, which consist of monomers, (þ)-catechin and ()-epicatechin, and their oligomers and polymers, and are distributed in many plantderived foods. Procyanidins are reported to have many beneficial physiological activities, such as antihypertensive and anticancer effects. However, the bioavailability of procyanidins is not well understood owing to a lack of convenient and high-sensitive analysis methods. The aim of this study was to develop an improved method for determining procyanidin content in both food materials and biological samples. High performance liquid chromatography (HPLC) coupled with a fluorescence detector was used in this study. The limits of detection (LODs) of (þ)-catechin, ()-epicatechin, procyanidin B2, procyanidin C1, and cinnamtannin A2 were 3.0 103 ng, 4.0 103 ng, 14.0 103 ng, 18.5 103 ng, and 23.0 103 ng, respectively; the limits of quantification (LOQs) were 10.0 103 ng, 29.0 103 ng, 28.5 103 ng, 54.1 103 ng, and 115.0 103 ng, respectively. The LOD and LOQ values indicated that the sensitivity of the fluorescence detector method was around 1000 times higher than that of conventional HPLC coupled with a UV-detector. We applied the developed method to measure procyanidins in black soybean seed coat extract (BE) prepared from soybeans grown under three different fertilization conditions, namely, conventional farming, basal manure application, and intertillage. The amount of flavan-3- ols in these BEs decreased in the order intertillage > basal manure application > conventional farming. Commercially available BE was orally administered to mice at a dose of 250 mg/kg body weight, and we measured the blood flavan-3-ol content. Data from plasma analysis indicated that up to the tetramer oligomerization, procyanidins were detectable and flavan-3-ols mainly existed in conjugated forms in the plasma. In conclusion, we developed a highly sensitive and convenient analytical method for the analysis of flavan-3- ols, and applied this technique to investigate the bioavailability of flavan-3-ols in biological samples and to measure flavan-3-ol content in food material and plants.
Keywords: bioavailability, epicatechin, fluorescence detection, high performance liquid chromatography, procyanidin