CHIRAL ANALYSIS OF 3-METHOXY-4-HYDROXYPHENYLGLYCOL IN HUMAN URINE

Since 3-methoxy-4-hydroxyphenylglycol (MHPG) is a neutral metabolite f rom norepinephrine, it will be a diagnostic marker for mental diseases such as depression, For the development of an immunoassay, the natura l enantiomer of MHPG would be required to prepare its antigen and to e xamine the specificity of the antibody. A natural enantiomer synthesiz ed, however, has not been obtained so far. In this paper. we attempted to enantioseparate synthetic DL-MHPG and to assign D-enantiomer from the optical rotation of MHPG purified from human urine: because endoge nous norepinephrine occurs as D-enantiomer which should metabolically generate D-MHPG. Enantioseparation conditions were rested using a Cera mospher Chiral RU-1 column (4.6 x 250 mm) at a flow rate of 0.5 ml/min . The resolution was adequate for the analysis and purification of syn thetic DL- and the urinary MHPGs using methanol as a mobile phase and the column temperature at 0 degrees C, where DL-MHPG was detected as t wo peaks. The earlier peak (peak 1) showed (-) optical rotation, while the latter gave (+) optical rotation. After being treated with beta-g lucuronidase, the normal human urine was extracted with ethyl acetate and then evaporated to dryness. The residue was suspended in water and the supernatant was analyzed and purified by a reversed phase column with a multi channel detector. A peak corresponding to MHPG was collec ted and concentrated to dryness. The pooled residues were dissolved in methanol and enantioseparated on the chiral HPLC. The urinary MHPG ap peared as a single peak which was corresponded to the earlier peak of DL-MPHG and showing (-) optical rotation. Thus, the urinary MHPG was f ound to be D-(-)-MHPG. Then the absolute configuration of enantiosepar ated MHPGs were assigned to each optical rotation: judging from the ch emical data and the metabolic pathway of the urinary D-MHPG. These ena ntiomers will be useful for studying on biochemistry and immunoassay. (C) 1997 Elsevier Science B.V.