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

联用质谱分离测定药物对映体方法的建立

• 批准号: 05452336
• 负责人: GOTO Junichi
• 金额: $ 4.61万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05452336/
• 关键词: Enantiomeric drug; Diastereomer; LC; MS; Immobilized antibody; Derivatization; Glucuronosyltransferase; 固体化抗体; ジアステレオマー

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

生物体液中对映体化合物的色谱分析是生物医学分析中最重要的课题之一，与药物动力学和药效学研究密切相关。在各种方法中，液相色谱法（LC）被公认为是分离测定外消旋体的有力工具。另一方面，电喷雾离子化（ESI）-质谱法正在成为分析具有离子基团的生物重要物质的越来越有价值的工具。1）非对映体法在灵敏度和通用性方面更适合于生物体液中外消旋药物的测定。由于分子中具有羧基的葡萄糖醛酸是天然存在的光学活性化合物，因此在生理条件下使用固定化的β-葡萄糖醛酸基转移酶进行反向衍生 ...更多信息 已经使用普拉洛尔作为模型药物进行了研究。在37 ℃下，在尿苷-5 '-二磷酸葡萄糖醛酸（10 mM）和Mg（2+）（5 mM）存在下，与固定化酶（300穆尔）一起孵育，实现衍生化。在Capcell Pak C_柱上<18>，使用50 mM乙酸铵/甲醇（1：2，v/v）作为移动的相，实现对单个非对映体的拆分。普萘洛尔葡萄糖醛酸苷的质谱在ESI-MS模式下显示准分子离子[M-H]^-作为强峰。2）通过使用免疫原（1 R）-和（1 S）-丁呋洛尔1 '-O-羧基-羟甲基肟-牛血清白蛋白缀合物引发的抗体，建立了丁呋洛尔及其代谢物的免疫亲和萃取。产生的抗体对相应的对映体表现出低交叉反应性，对苯部分1'位氧化的主要代谢物表现出宽亲和谱。通过固定抗体制备的免疫亲和吸附剂的0.5 ml等分试样能够分别保留高达1 μ g的（R）-和（S）-丁呋洛尔。吸附物用95%甲醇洗脱后可定量回收，无干扰。3）免疫亲和萃取-反相衍生-LC/ESI-MS联用技术可用于对映体药物的异源代谢研究。少

项目成果

N.Awata, F.Toba, M.Ando, H.Shimada, S.Miyairi, T.Kato, J.Goto, and T.Nambara: "Immunoaffinity extraction of 4-hydroxy-2- (4-methyphenyl) benzothiazole and its metabolites for determination by gas chromatography-mass spectrometry." Biol. Pharm. Bull.17. 84

N.Awata、F.Toba、M.Ando、H.Shimada、S.Miyairi、T.Kato、J.Goto 和 T.Nambara：“4-羟基-2-（4-甲基苯基）苯并噻唑和

Toshifumi Niwa: "Preparation of antiserum for use in the Immunoassay of unconjugated and conjugated 4β-hydroxycholic acid" Anal.Sci.(in press).

Toshifumi Niwa：“用于非缀合和缀合 4β-羟基胆酸免疫测定的抗血清的制备”Anal.Sci.（出版中）。

M.Tanji, J.Goto and I.Makino: "Identification and characterization of dehydrocholic acid reductase system in the cytosol of human red blood cells." J.Gastroenterol.29. 621 (1994)

M.Tanji、J.Goto 和 I.Makino：“人红细胞胞浆中脱氢胆酸还原酶系统的鉴定和表征。”

Shinichi Miyairi: "Flow injection-immunometric assay of 17α-hydroxy-progesterone using fluorescein-labeled Fab fragment." Anal. Sci.10. 109-111 (1994)

Shinichi Miyairi：“使用荧光素标记的 Fab 片段进行 17α-羟基孕酮的流动注射免疫测定。”Anal.109-111。

S.Miyairi, A.Kanamaru, K.Nakajima, T.Kato and J.Goto: "Flow injection-immunometric assay of 17alpha-hydroxyprogesterone using fluoresceinlabeled Fab fragment." Anal. Sci.10. 109 (1994)

S.Miyairi、A.Kanamaru、K.Nakajima、T.Kato 和 J.Goto：“使用荧光素标记的 Fab 片段对 17α-羟基孕酮进行流动注射免疫测定。”