2,5,7,8-Tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the water-soluble metabolite of alpha-tocopherol (alpha-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 alpha-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 alpha-CEHC in rat urine accordingly, we observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60 degrees C in the presence of ascorbate, followed by a one-step ether extraction, not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. In conclusion, alpha-CEHC sulfate was isolated and identified as the HCl-releasable conjugate of alpha-CEHC in rat urine. A rapid and sensitive method with high reproducibility for the determination of free, conjugated and total alpha-CEHC is then established.