色谱

色谱 ›› 2020, Vol. 38 ›› Issue (10): 1243-1248.DOI: 10.3724/SP.J.1123.2020.07010

• 研究论文 • 上一篇    下一篇

胶束电动色谱柱上酶反应测定低分子量肝素的抗凝血活性

张明瑜, 康经武()   

  1. 生命有机化学国家重点实验室, 中国科学院上海有机化学研究所, 上海 200032
  • 收稿日期:2020-07-10 出版日期:2020-10-08 发布日期:2020-12-11
  • 通讯作者: 康经武
  • 作者简介:康经武.Tel:(021)54925385, E-mail:jingwu.kang@sioc.ac.cn
  • 基金资助:
    国家自然科学基金(21775158);国家自然科学基金(21375140);中国科学院战略先导B(XDB20020200)

Determination of the anticoagulant activity of low molecular weight heparins by micellar electrokinetic chromatography combined with on-column enzymatic reaction

ZHANG Mingyu, KANG Jingwu()   

  1. State Key Laboratory of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
  • Received:2020-07-10 Online:2020-10-08 Published:2020-12-11
  • Contact: KANG Jingwu
  • Supported by:
    National Natural Science Foundation of China(21775158);National Natural Science Foundation of China(21375140);the Strategic Priority Research Program B of the Chinese Academy of Sciences(XDB20020200)

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摘要：

发展了一种基于胶束电动色谱（MEKC）结合柱上酶微反应的方法，用于测定低分子量肝素（LMWHs）的抗凝血活性。肝素与抗凝血酶Ⅲ（ATⅢ）结合后，将ATⅢ抑制凝血因子10（FXa）活性提高了约1000倍。通过测定FXa水解生色肽底物CPS产生的对硝基苯胺（p -NP）就可以测定FXa的活性。因此，通过LMWHs抑制FXa产生的对硝基苯胺的量就可以测定抗凝血活性。该方法将毛细管柱端作为微量的酶微反应器，以呋喃妥英（nitrofurantoin，NF）作为内标，依次将LMWHs溶液、ATⅢ溶液、FXa和CPS溶液导入毛细管柱端，反应物经过分子扩散、横向层流扩散混合和电压混合后反应。反应结束后，采用MEKC分离模式将产物对硝基苯胺与底物以及其他大分子分离，在其最大波长380 nm下测定产生对硝基苯胺的量，从而确定LMWHs的抗凝血活性。该方法具有自动化、重复性好、灵敏度高、样品消耗量少的优点，而且不受其他成分的干扰，可用于各种复杂样品（如血浆）中LMWHs抗FXa活性的监测。

关键词: 胶束电动色谱, 柱上酶反应, 低分子量肝素, 抗凝血活性

Abstract:

Low molecular weight heparins (LMWHs) have largely replaced heparin for the treatment and prevention of thrombosis because of their various advantages over unfractionated heparins (UFHs) such as less bleeding, greater bioavailability, and more predictable anticoagulant effects. For special groups of patients, such as pregnant women, children, and patients with renal failure, it is necessary to monitor the anticoagulant activity of LMWHs in the blood. The traditional method used to determine the anticoagulant activity of heparin is the coagulation test. However, the results are various from different laboratories and different reagents. In contrast, the chromogenic substrate method is more accurate, sensitive and is easy to automate. Here, a method for the determination of the anticoagulant activity of LMWHs was developed by using a capillary-electrophoresis-based substrate chromogenic method.

In this method, micellar electrokinetic chromatography (MEKC) was used in combination with electrophoretically mediated microanalysis to determine the anti-factor Xa (FXa) activity of LMWHs. The inhibition was measured by employing a chromogenic peptide substrate (CPS) with a p -nitroaniline (p -NA) moiety as the chromophore. The injection end of the capillary was used as a microreactor in which solutions of LMWHs, antithrombin Ⅲ (ATⅢ), FXa and CPS were successively injected and mixed by using diffusion, the transverse diffusion of laminar flow profiles and applied voltage. The reaction product p -NA was separated from unreacted CPS and sample matrix by using the MEKC mode with discontinuous background electrolyte system. The produced p -NA was baseline separated from the other components and detected at 380 nm to obtain maximum sensitivity. The amount of p -NA was inversely proportional to the activity of LMWHs in the sample. To improve the accuracy of quantification and the method repeatability of methods, nitrofurantoin (NF) was selected as the internal standard, which was added to the solution of CPS. The method was validated and used to measure a set of samples. The method is characterized by automation, good repeatability, high sensitivity, and cost-effectiveness. Additionally, the method does not interfere by the sample matrix, and thus can be used to monitor the anticoagulant activity of LMWHs in plasma.

Key words: micellar electrokinetic chromatographic (MEKC), on-column enzymatic reaction, low molecular weight heparins (LMWHs), anticoagulant