Isolation and Identification of α-CEHC Sulfate in Rat Urine and an Improved Method for the Determination of Conjugated α-CEHC

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• 作者：Yi-Jen Li ; Sheng-Ching Luo ; Yi-Jing Lee ; Fu-Jung Lin ; Chi-Cheng Cheng ; Yung-Shung Wein ; Yueh-Hsiung Kuo ; Ching-jang Huang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2008
• 出版时间：December 10, 2008
• 年：2008
• 卷：56
• 期：23
• 页码：11105-11113
• 全文大小：293K
• 年卷期：v.56,no.23(December 10, 2008)
• ISSN：1520-5118

文摘

2,5,7,8-Tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), the water-soluble metabolite of α-tocopherol (α-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in human urine and are thought to be produced in significant amount at excess intake of α-TOH. In previous studies, CEHCs in biological specimens were measured by HPLC, GC−MS or LC−MS, preceded by a hydrolysis procedure using either enzyme or methanolic HCl. In an attempt to analyze α-CEHC in rat urine accordingly, we observed that enzyme hydrolysis was relatively inefficient in releasing α-CEHC compared to high concentrations of HCl. The HCl releasable α-CEHC conjugate was isolated and chemically identified as 6-O-sulfated α-CEHC (α-CEHC sulfate). Using the synthetic α-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60 °C in the presence of ascorbate, followed by a one-step ether extraction, not only hydrolyzed the sulfate conjugate completely but also extracted α-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of α-CEHC to α-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of α-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC−ECD was thus established. In conclusion, α-CEHC sulfate was isolated and identified as the HCl-releasable conjugate of α-CEHC in rat urine. A rapid and sensitive method with high reproducibility for the determination of free, conjugated and total α-CEHC is then established.