Analytical Method of Measuring Tea Catechins in Human Plasma by Solid-Phase Extraction and HPLC with Electrochemical Detection.

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We developed an analytical method for measuring tea catechins in plasma by solid-phase extraction (SPE), followed by HPLC with a coulometric electrochemical detector. The plasma was mixed with an equal volume of acetonitrile to precipitate protein, and cate-chins in the resulting supernatant were extracted by SPE, using a C18 cartridge. To correct the extraction efficiency, ethyl gallate was simultaneously added with acetonitrile as an in-ternal standard. Plasma samples were treated in microtubes, and evaporation and SPE were performed by the use of a vacuum centrifuge and vacuum manifold for SPE. The use of these instruments allowed the handling of a large number of samples simultaneously. In this method, (-)-epicatechin (EC), (-)-epicatechin-3-O-gallate (ECg), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-O-gallate (EGCg), and ethyl gallate could be detected as a sin-gle peak with high sensitivity. For an analysis of the conjugated form of catechins, plasma samples were treated with glucuronidase and sulfatase. Type H-2 β-glucuronidase effec-tively digested the conjugated forms, and the enzyme also converted EGCg and ECg to their nongallated form. When the concentrations of catechins in plasma were analyzed in sub-jects who took a single dose of catechin liquid, the concentration of free EGCg in plasma reached a maximum of 300 nM at 1 h after intake; those of the other free form of catechins increased only slightly after the intake. The concentration of total catechins (free+conju-gated forms) in plasma increased up to 2 h after the intake.

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