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United States Department of Agriculture

Agricultural Research Service

Title: Quantitation of Clovamide-Type Phenylpropenic Acid Amides in Cells and Plasma Using High Performance Liquid Chromatography with a Coulometric Electrochemical Detector

Author
item Park, Jae

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 12, 2005
Publication Date: September 16, 2005
Citation: Park, J.B. 2005. Quantitation of clovamide-type phenylpropenic acid amides in cells and plasma using high performance liquid chromatography with a coulometric electrochemical detector. Journal of Agricultural and Food Chemistry. 53:8135-8140.

Interpretive Summary: Clovamide-type phenylpropenic acid amides are phytochemicals found in numerous plants such as Capsicum spp. and Cocoa. These phytochemicals are believed to have significant effects on human chronic diseases such as obesity, pulmonary and cardiovascular disease, and cancers. Although there is always a great need for quantifying clovamide-type phenylpropenic acid amides in biological samples (cells and plasma), currently no method is available for the purpose. Therefore, a high-performance liquid chromatography (HPLC) method was developed in order to determine concentrations of clovamide-type phenylpropenic acid amides (N-caffeoyltyramine and N-caffeoyldopamine) in biological samples (cells and plasma). Because this developed HPLC method provides great peak resolutions of clovamide-type phenylpropenic acid amides with high sensitivity and reliability, this method can be used for analyzing not only N-caffeoyltyramine and N-caffeoyldopamine, but also other clovamide-type phenylpropenic acid. The outcomes of this study will provide researchers in nutrition, molecular biology, and medicinal fields with a powerful new method to measure N-caffeoyltyramine and N-caffeoyldopamine which are potent dietary factors for reducing risks of human chronic diseases such as pulmonary, cardio-vascular, and other diseases.

Technical Abstract: A high-performance liquid chromatography (HPLC) method was developed for measuring the concentrations of clovamide-type phenylpropenic acid amides (N-caffeoyldopamine and N-caffeoyltyramine) in cell and plasma samples. The HPLC separation of N-caffeoyldopamine and N-caffeoyltyramine was performed on a Nova-Pak C18 column (stationary phase) using an isocratic buffer (mobile phase) with a coulometric electrochemical detector with four electrode channels. Using the HPLC method, N-caffeoyldopamine and N-caffeoyltyramine could be detected with fine peak resolutions at respective retention times (4.5 and 6.7 min). The calibration curves for N-caffeoyldopamine and N-caffeoyltyramine were linear over the ranges 0.1 and 100 microM, and their lower limit of detection was as little as 100 fmol. For quantifying N-caffeoyldopamine and N-caffeoyltyramine in cell and plasma samples, the samples were extracted with methods providing more than 95% recoveries. After the extractions, N-caffeoyldopamine and N-caffeoyltyramine were analyzed with same sensitivity, peak resolutions, and retention times using the HPLC method. This HPLC method also enabled determination of the concentrations of N-caffeoyltyramine in mice plasma samples collected at 15, 30, 45, 60, and 70 min after the oral administration of N-caffeoyltyramine (0.5 mg and 2 mg/30 g body weight). This HPLC method, with an electrochemical detector, provides a sensitive and reproducible method for quantifying N-caffeoyldopamine and N-caffeoyltyramine in biological samples with superb detection limits, fine peak resolutions, and discrete retention times.

Last Modified: 10/21/2014
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