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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Title:	Isolation and Identification of α-CEHC Sulfate in Rat Urine and an Improved Method for the Determination of Conjugated α-CEHC
Authors:	(Yi-Jen Li);(Sheng-Ching Luo);(Yi-Jing Lee);(Fu-Jung Lin);(Chi-Cheng Cheng);(Yung-Shung Wein);郭悅雄;(Ching-jang Huang)
Contributors:	藥學院中國藥學研究所;中國附醫院長室
Date:	2008-11
Issue Date:	2009-08-26 16:19:54 (UTC+8)
Abstract:	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.
Relation:	JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 56(23)11105~11113
Appears in Collections:	[Graduate Institute of Chinese Pharmaceutical Science] Journal articles

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