Determination of green tea catechins in human plasma using liquid chromatography–electrospray ionization mass spectrometry
Introduction
Green tea catechins, which can be easily infused by hot water, have been extensively reported to possess various biological and pharmacological effects, such as anti-carcinogenic activities [1], anti-oxidant activities [2], lowering of plasma lipid [3] and glucose levels [4], and reducing obesity [5], [6]. Among these, the anti-obesity effect in humans following oral ingestion of greater amounts of green tea catechins [6] has been recently reported, while obesity associated with many health risks (so-called lifestyle-related diseases) is a growing problem in many countries worldwide. Naturally occurring green tea catechins in an infusion of green tea leaves with hot water, which are widely consumed in Asian countries, consist of epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), epigallocatechin (EGC) and epicatechin (EC). On the other hand, Japanese have recently begun to prefer drinking a commercial canned or bottled green tea beverage, which has a different composition of catechins from the hot water infusion. Thus, owing to the epimerization of the four epi-catechins by heat treatment used for retort pasteurization in the manufacturing, four non-epi-forms, gallocatechin-3-gallate (GCG), catehin-3-gallate (CG), gallocatechin (GC) and catechin (C), are produced in the beverage [7], [8]. Therefore, attention has been recently paid to the effects of these non-epi-forms on lipid metabolism [9].
To understand the pharmacokinetics of green tea catechins after oral ingestion of a green tea beverage, all eight green tea catechins must be determined in plasma. Among many methods for the analysis of green tea catechins [10], [11], LC connected to an ultraviolet detector (UVD), a fluorescence detector (FLD), a chemiluminescence detector (CLD) and an electrochemical detector (ECD) have been used to determine the catechins in plasma [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. However, these methods using LC did not determine all eight catechins in the plasma, due to their problems in sensitivity and specificity. While the use of sensitive and specific LC–MS for the analysis of the catechins has been recently increasing [23], [24], [25], LC–MS has not been applied to such analyses of actual plasma after ingestion, although it has been used for the analyses in model plasma with a few spiked catechins [26], [27]. In addition, there are no preparative procedures that can be used for high-throughput analyses with simplicity and practicability, although high throughput performance for the analyses of green tea catechins in many plasma samples obtained from different volunteers at several periods after the ingestion is required for understanding the behaviors of the eight catechins absorbed into human blood among individuals and among populations. Thus, complex procedures including liquid–liquid extraction [12], [17], [21], [22], [26] and solid phase extraction [13], [20] are troublesome and time-consuming. Other simple procedures, such as the addition of either of an acid or an organic solvent [14], [15], [18], [19] and on-line extraction [27], do not permit us to allow the analysis of many plasma samples (intact plasma or prepared plasma solutions) with an autosampler, even at low temperature, due to the instability of the catechins in such non-prepared or prepared plasma samples.
The aim of this study was to establish a method for the sensitive and specific determination of eight green tea catechins in human plasma after oral ingestion of a green tea beverage. We have optimized the LC–ESIMS (liquid chromatography coupled to electrospray ionization mass spectrometry) conditions to simultaneously determine all eight catechins and we have developed a simple preparative procedure for deproteinizing plasma, in which the catechins are stable at 5 °C within 24 h after preparation. This method has been applied to analyze the eight catechins in the plasma after the ingestion of a commercial green tea beverage, in which we succeeded for the first time in detecting and determining all eight catechins in human plasma.
Section snippets
Chemicals
Authentic green tea catechins such as C, EC, GC, EGC, CG, ECG, GCG and EGCG were purchased from Kurita Industrial (Tokyo, Japan). Ultra-pure water was prepared using a Milli-Q purification system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade or of HPLC grade. A commercial green tea beverage, which has been approved as a food for specified health use in the Japanese market by the Ministry of Health, Labour and Welfare in Japan, was orally ingested. This beverage
LC–ESIMS conditions
The LC separation of the eight authentic catechins was examined using two mobile phases of 0.1 M aqueous acetic acid and 0.1 M acetic acid in acetonitrile. Acetic acid was chosen as an additive rather than trifluoroacetic acid or formic acid since it provides the most abundant deprotonated molecular ions [M − H]− characteristic for green tea catechins [29] under negative ESI. An appropriate gradient elution with the two mobile phases as described in Section 2.2 enabled us to mutually separate the
Discussion
In this study, LC–ESIMS conditions were optimized for the sensitive and specific determination of eight green tea catechins. In the optimized LC–ESIMS, the chromatographic separation of the eight catechins is achieved by a reversed phase column Inertsil ODS-2 combined with a gradient elution system of two mobile phases. Mass spectrometric detection is performed with SIM at m/z = [M − H]− for each isomer pair of catechins under negative ESI. Since the LOD in our LC–ESIMS is 0.19–0.25 ng/ml (GC, EGC,
Acknowledgements
We would like to express our cordial gratitude to Dr. Katsumi Kita and Dr. Ichiro Tokimitsu of the Kao Corporation for their invaluable discussion and encouragement of this study. Our sincere thanks are also due to Mr. Tomonori Nagao of the Kao Corporation for his assistance in the collections of human plasma.
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