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

在药物动力学和药效学研究中，生物体液中对映体化合物的色谱测定是生物医学分析中最重要的课题之一。在各种方法中，液相色谱法（LC）是公认的分离测定外消旋体的有力工具。另一方面，电喷雾电离(ESI) -质谱法在分析具有离子基团的重要生物物质方面正成为越来越有价值的工具。据此，建立了LC/ESI-MS分离测定对映量药物的方法。1)非对映法在灵敏度和通用性方面更有利于生物液体中外消旋药物的测定。由于葡萄糖醛酸分子中含有羧基，是一种天然存在的光学活性化合物，在生理条件下利用固定化β -葡萄糖醛酸基转移酶进行了反衍生化反应，并以普萘洛尔为模型药物进行了更多的研究。在尿嘧啶-5′-二磷酸葡萄糖醛酸（10mM）和Mg^<2+> (5mM)存在下，在37 ^0℃下，用固定酶（300 μ l）进行衍生化反应。在Capcell Pak C_<18>色谱柱上，以50 mM醋酸铵/甲醇（1:2,v/v）为流动相，分离成单个非对映体。在ESI-MS模式下，普萘洛尔葡萄糖醛酸酯的光谱表现为准分子离子[M-H]^-为强峰。2)利用免疫原（1R）和(1S) -bufuralol 1′- o -羧基-甲基肟-牛血清白蛋白偶联物诱导的抗体，建立了对bufuralol及其代谢物的免疫亲和提取方法。所制备的抗体对相应的对极具有低的交叉反应性，对苯段1′位置氧化的主要代谢物具有广泛的亲和谱。固定抗体制备的0.5 ml免疫亲和吸附剂能够分别保留1mug (R)和(S) -bufuralol。吸附物用95%甲醇洗脱，无干扰，定量回收。3)免疫亲和提取和反向衍生化与LC/ESI-MS相结合，在对映药物的异种代谢研究中具有广阔的应用前景。少

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