DEVELOPEMENT OF THE METHOD FOR SEPARATORY DETERMINATION OF ENANTIOMETRIC DRUGS BY HYPHENATED MASS SPECTROMETRY

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In connection with pharmacokinetic and pharmacodynamic studies, chromatographic determination of enantiomeric compounds in biological fluids is one of the most important subjects in the biomedical analysis. Among various methods, liquid chromatography (LC) is well recognized as a powerful tool for the separatory determination of racemates. On the other hand, electrospray ionization (ESI) -mass spectrometry is becoming increasingly valuable tool for the analysis of biologically important substances having ionic groups. Accordingly, the method for separatory determination of enantiometric drugs by LC/ESI-MS have been developed.1) The diastereometric method is more favorable for the determination of racemic drugs in biological fluids with respect to sensitivity and versatility. Since glucuronic acid having carboxyl group in the molecule is a naturally occuring optical active compound, reversed derivatization employing immobilized beta-glucuronosyltransferase under physiological conditions … More has been studied using proplanolol as a model drug. The derivatization was achieved by incubating with immobilized enzyme (300 mul) in the presence of uridine-5'-diphosphoglucuronic acid (10mM) and Mg^<2+> (5mM) at 37 ^0C.The resolution into individual diastereomer was achieved on a Capcell Pak C_<18> column using 50 mM ammonium acetate/methanol (1 : 2, v/v) as a mobile phase. The spectrum of propranolol glucuronide exhibited a quasimolecular ion [M-H]^- as an intense peak under the ESI-MS mode.2) Immunoaffinity extraction of bufuralol and its metabolites has been developed by the use of the antibodies elicited from immunogens, (1R) -and (1S) -bufuralol 1'-O-carboxy-methyloxime-bovine serum albumin conjugates. The antibodies produced exhibited low cross reactivities toward the corresponding antipode and a broad affinity spectrum for major metabolites oxidized at the 1' position on the benzene moiety. A 0.5 ml aliquot of the immunoaffinity adsorbent prepared by immobilization of the antibody was capapable retaining up to 1mug of (R) -and (S) -bufuralol, respectively. The adsorbates were recovered quantitatively by elution with 95% methanol without any interfering.3) The combination of immunoaffinity extraction and reversed derivatization with LC/ESI-MS is promising for the xenobiotic metabolic study of enantiometric drugs. Less