WO2017197950A1 - 一种低分子肝素基本组成单元的亲水相互作用色谱多反应监测二级质谱联用检测方法 - Google Patents

一种低分子肝素基本组成单元的亲水相互作用色谱多反应监测二级质谱联用检测方法 Download PDF

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WO2017197950A1
WO2017197950A1 PCT/CN2017/074966 CN2017074966W WO2017197950A1 WO 2017197950 A1 WO2017197950 A1 WO 2017197950A1 CN 2017074966 W CN2017074966 W CN 2017074966W WO 2017197950 A1 WO2017197950 A1 WO 2017197950A1
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molecular weight
low molecular
mobile phase
weight heparin
interaction chromatography
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French (fr)
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迟连利
孙晓君
盛安然
刘欣悦
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山东大学
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Priority to US15/735,139 priority Critical patent/US10416132B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/004—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/26—Mass spectrometers or separator tubes
    • H01J49/34—Dynamic spectrometers
    • H01J49/42—Stability-of-path spectrometers, e.g. monopole, quadrupole, multipole, farvitrons
    • H01J49/4205—Device types
    • H01J49/421—Mass filters, i.e. deviating unwanted ions without trapping
    • H01J49/4215—Quadrupole mass filters

Definitions

  • the invention relates to a hydrophilic interaction chromatography and a multi-reaction monitoring secondary mass spectrometry detection method for a complete degradation product of low molecular weight heparin, belonging to the technical field of medicine, raw material medicine and raw material detection.
  • Low molecular weight heparin is a class of glycosaminoglycans that have anticoagulant effects and have been used clinically as anticoagulant drugs. In order to reduce the risk of side effects such as hemorrhage, osteoporosis and thrombocytopenia, and improve its bioavailability, low molecular weight heparin is widely used as a new anticoagulant drug. Low molecular weight heparin is prepared by enzymatic degradation or chemical degradation, and each low molecular weight heparin has its special structure depending on the process. Enoxaparin sodium is a class of low molecular weight heparin obtained by alkaline degradation of heparin benzyl ester derivatives.
  • non-reducing ends are composed of chemically modified unsaturated structures, and there are saturated structures derived from raw heparin and amino sugars.
  • the reducing end structure is mainly amino sugar, and 15%-25% of reducing ends are 1 , 6-endoether structure, and a small amount of uronic acid structure and linking domain.
  • Dalteparin sodium is a class of low molecular weight heparin obtained by nitrous acid degradation. Its main non-reducing end is a saturated uronic acid structure, and the main reducing end is a mannitol structure and a small amount of a linking domain.
  • the structural changes caused by the process also increase the difficulty in analyzing the low molecular weight heparin constituent units.
  • heparinase is used to decompose low molecular weight heparin into constituent units, and is analyzed by capillary electrophoresis, high performance liquid chromatography and liquid chromatography-mass spectrometry.
  • the above methods generally only identify 8 common disaccharides and some special structures, and cannot perform comprehensive qualitative and quantitative analysis on all basic constituent units, and cannot be used for low molecular weight heparin, such as enoxaparin sodium, the original reducing end of the sugar chain and The non-reducing end was identified.
  • the identification of these lower basic special elemental unit structures is essential for the structural characterization of low molecular weight heparin.
  • the basic constituents of low molecular weight heparin are 3-O-sulfate tetrasaccharide associated with anticoagulant action, trisaccharide produced by exfoliation reaction, saturated non-reducing end, and free amino disaccharide.
  • galacturonic disaccharide, disaccharide and epoxy ether structures in which sulfuric acid is directly attached to carbon atoms and specific structures present in different low molecular weight heparins, such as the structure of 1,6-endoether in enoxaparin sodium and Its original non-reducing end and reducing end, the 2,5-anhydromannitol structure in the sodium heparin sodium.
  • the present invention provides a hydrophilic interaction chromatography and multi-reverse of a complete degradation product of low molecular weight heparin.
  • the secondary mass spectrometry detection method should be monitored;
  • the invention identifies the original reducing end and the non-reducing end of enoxaparin sodium by reducing the reducing end of enoxaparin sodium and hydrolyzing with hydrogen peroxide.
  • Low-molecular-weight heparin was characterized by quantitative analysis of all constituent elements by hydrophilic interaction chromatography and multiple reaction monitoring secondary mass spectrometry, especially for the specific structure with low content.
  • a hydrophilic interaction chromatography and multi-reaction monitoring two-stage mass spectrometry detection method for a complete degradation product of low molecular weight heparin the steps are as follows:
  • the hydrolyzate of the sample of the enzymatic hydrolysate is configured to a concentration of 1 to 10 ⁇ g/ ⁇ L of the solution to be tested, and the step (4) is entered; if it is not necessary to distinguish the end structure of the low molecular weight heparin, 10 to 50 ⁇ g of the low molecular weight heparin added to the internal standard
  • the enzymatic product sample is directly prepared into a solution having a concentration of 1 to 10 ⁇ g / ⁇ L, and proceeds to step (4);
  • the mass spectrum in the step (4) adopts a triple quadrupole mass spectrometer (TSQ), and the setting parameters are: positive ion mode spray voltage: +4.0 kV; negative ion mode spray voltage: -3.2 kV; sheath flow gas: 20-30 arb ; tubular lens voltage: ⁇ 50 ⁇ 150V; collision energy: 20-50.
  • TSQ triple quadrupole mass spectrometer
  • the sample of the low molecular weight heparin hydrolysate added to the internal standard in the step (3) is dried under vacuum at 30 to 60 ° C for 1 to 3 hours.
  • the centrifugation in the step (4) is carried out at room temperature of 10,000 to 15,000 rpm for 5 to 15 minutes.
  • the centrifugation in the step (4) is carried out by centrifugation at room temperature 12000 rpm for 15 min.
  • the invention can simultaneously detect all the reported end-modifying structures and special structures of the basic constituent units of low molecular weight heparin except 8 common heparin disaccharides, and find three new structures, including trisaccharides with two amino sugars.
  • uronic acid produces 3-O-sulfur
  • the acidified tetrasaccharide and the trisaccharide with uronic acid as the reducing end, and the relative quantification of all the low molecular weight heparin basic constituent units can be simultaneously determined by the internal standard, and only 8 kinds of common heparin disaccharides can be detected in the prior art. And some special structural problems have great practical value for the research and development, production control and drug safety of low molecular weight heparin.
  • Example 1 is an extracted ion flow diagram of the basic constituent units of enzymatic degradation and reductive hydrolysis degradation of enoxaparin sodium standard in Example 1;
  • Example 2 is a second-order mass spectrum of the enzymatic degradation of the basic constituent unit components 30, 31 and the reducing end portion 5 of the reducing hydrolyzed basic constituent unit of the enoxaparin sodium standard in Example 1;
  • a is a secondary mass spectrum of the enzymatic degradation of the basic constituent unit component 30 of the enoxaparin sodium standard in Example 1;
  • b is a secondary mass spectrum of the enzymatic degradation of the basic constituent unit component 31 of the enoxaparin sodium standard in Example 1;
  • c is a secondary mass spectrum of the reducing end 5 of the basic constituent unit of the enzymatic reduction of the enoxaparin sodium standard in Example 1;
  • Fig. 3 is a diagram showing the extracted ion flow of the basic constituent unit of the enzymatic degradation of the dalteparin sodium standard in Example 2.
  • the liquid chromatograph is an Agilent 1100 liquid chromatograph, the workstation is ChemStation; the mass spectrometer is the Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer, and the workstation is Xcalibur.
  • a hydrophilic interaction chromatography and multi-reaction monitoring two-stage mass spectrometry detection method for a complete degradation product of low molecular weight heparin the steps are as follows:
  • Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer uses the Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer to perform multi-reaction monitoring in negative ion mode, and obtain the extracted ion flow diagram of each component.
  • the set parameters are: spray voltage: -3.2kV; sheath gas: 20 to 30 arb; tubular lens voltage: -75 V, collision energy: 35.
  • ⁇ UA means unsaturated uronic acid
  • Hex means uronic acid
  • GlcA means glucuronic acid
  • IdoA means iduronic acid
  • GlcN means glucosamine
  • Ac means An acetyl group
  • S represents a sulfate group
  • NS represents an N-sulfate group.
  • NRE non-reducing end
  • RE reducing end
  • -ol sugar alcohol
  • the ion chromatogram of the basic unit of enoxaparin sodium enzymatic hydrolysis was extracted as shown in Fig. 1.
  • the ion chromatogram of the basic unit of enoxaparin sodium reduction hydrolysis was extracted as shown in Fig. 2.
  • the enoxaparin sodium standard was successfully detected by this method. All possible basic constituent units except the eight common disaccharides in the basic constituent unit.
  • a hydrophilic interaction chromatography and multi-reaction monitoring two-stage mass spectrometry detection method for a complete degradation product of low molecular weight heparin the steps are as follows:
  • heparin sodium standard was degraded by heparinase I, II and III (both purchased from Beijing Aidehaoke International Technology Co., Ltd.) at 25 ° C for 48 h, and the internal standard degradation product was added with Millipore 30KDa.
  • the ultrafiltration membrane is filtered and dried under vacuum under reduced pressure;
  • Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer uses the Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer to perform multi-reaction monitoring in negative ion mode, and obtain the extracted ion flow diagram of each component.
  • the set parameters are: spray voltage: -3.2kV; sheath gas: 20 to 30 arb; tubular lens voltage: -75 V, collision energy: 35.
  • ⁇ UA means unsaturated uronic acid
  • Hex means uronic acid
  • GlcA means glucuronic acid
  • IdoA means iduronic acid
  • GlcN means glucosamine
  • Ac means An acetyl group
  • S represents a sulfate group
  • NS represents an N-sulfate group
  • NRE a non-reducing end
  • RE reducing end

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Abstract

一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法。通过对依诺肝素钠的还原端进行还原，并利用双氧水进行水解的方法对依诺肝素钠的原还原端及非还原端进行鉴定。利用亲水相互作用色谱与多反应监测二级质谱对所有组成单元进行定量分析，特别是对含量较低的特殊结构进行定量，对低分子肝素进行表征。

Description

一种低分子肝素基本组成单元的亲水相互作用色谱多反应监测二级质谱联用检测方法 技术领域
本发明涉及一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，属于药物、原料药、原料检测技术领域。
背景技术
肝素及其衍生物低分子肝素是一类糖胺聚糖，具有抗凝血作用，在临床上一直被用作抗凝药物。为了降低肝素所引起的出血、骨质疏松及血小板减少等副作用的风险，并提高其生物利用率，低分子肝素作为新型抗凝血药物被广泛应用。低分子肝素通过酶降解法或化学降解法进行制备，根据工艺的不同，每种低分子肝素有其特殊的结构。依诺肝素钠是通过碱降解肝素苄基酯衍生物得到的一类低分子肝素。其大部分非还原端是经过化学修饰的不饱和结构构成，同时存在来源于原料肝素的饱和的结构及氨基糖，其还原端结构主要为氨基糖，有15％-25％的还原端为1,6-内醚结构，并存在少量的糖醛酸结构和连接结构域。达肝素钠是通过亚硝酸降解得到的一类低分子肝素。其主要的非还原端为饱和的糖醛酸结构，主要的还原端为甘露醇结构及少量的连接结构域。除了以上特殊的末端结构，由于工艺造成的主链结构变化，也增加了对低分子肝素组成单元的分析的困难。目前分析肝素的方法有自上而下与自下而上两种策略。对组成单元的分析主要采用自下而上的策略，首先使用肝素酶将低分子肝素降解为组成单元，利用毛细管电泳、高效液相色谱法以及液质联用技术进行分析。上述方法一般仅对8种常见二糖和部分特殊结构进行鉴定，不能对所有基本组成单元进行全面的定性定量分析，且不能对低分子肝素，如依诺肝素钠，糖链的原还原端及非还原端进行鉴定。而且对这些含量较低的特殊基本组成单元结构的鉴定对低分子肝素的结构表征是必不可少的。
低分子肝素的基本组成单元除8种肝素二糖外，还有与抗凝血作用相关的3-O-硫酸四糖、剥落反应产生的三糖、饱和的非还原端、自由氨基的二糖、半乳糖醛酸二糖、硫酸直接连接于碳原子的二糖和环氧醚结构，以及存在于不同低分子肝素中的特殊结构，如依诺肝素钠中的1,6-内醚结构及其原非还原端和还原端，达肝素钠中的2,5-脱水甘露醇结构。已经报道过的方法没有对所有基本组成单元进行鉴定，而对末端修饰结构和特殊结构的分析鉴定对低分子肝素仿制药的研发、生产控制和保障药品安全是必不可少的。依诺肝素钠的非还原端及还原端与经过酶解新生成的末端相同，因此使用原有的策略不能对其进行全面表征。
发明内容
针对现有技术的不足，本发明提供了一种低分子肝素完全降解产物的亲水相互作用色谱与多反 应监测二级质谱联用检测方法；
本发明通过对依诺肝素钠的还原端进行还原，并利用双氧水进行水解的方法对依诺肝素钠的原还原端及非还原端进行鉴定。利用亲水相互作用色谱与多反应监测二级质谱对所有组成单元进行定量分析，特别是对含量较低的特殊结构进行定量，对低分子肝素进行表征。
本发明的技术方案为：
一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，步骤如下：
(1)将醋酸铵试剂溶解于去离子水，制得醋酸铵浓度为3～10mM的流动相A；
(2)将醋酸铵试剂溶解于去离子水，并加入乙腈，制得流动相B，流动相B中，醋酸铵的浓度为3～10mM，乙腈的体积百分比为90～98％；
(3)如果需要区分低分子肝素末端结构，则将10～50μg加入内标的低分子肝素酶解产物样品经硼氢化钠还原10～12h，并使用双氧水进行水解，将加入内标的低分子肝素酶解产物样品的水解产物配置成浓度为1～10μg/μL的待测溶液，进入步骤(4)；如果不需要区分低分子肝素末端结构，则将10～50μg加入内标的低分子肝素酶解产物样品直接配制为浓度为1～10μg/μL的待测溶液，进入步骤(4)；
(4)对步骤(3)得到的待测溶液离心处理，离心后，使用亲水相互作用色谱柱进行分离：流速0.1～0.5mL/min，洗脱梯度如下，均为体积百分比：
0～5min，5％流动相A，95％流动相B；5～107min，5～23％流动相A，95～77％流动相B；107～112min，23～50％流动相A，77～50％流动相B；112～125min，50％流动相A，50％流动相B；
(5)在正离子模式或负离子模式下用多反应监测二级质谱仪进行检测；
所述步骤(4)中的质谱采用三重四极杆质谱仪(TSQ)，设定参数为：正离子模式喷雾电压：+4.0kV；负离子模式喷雾电压：-3.2kV；鞘流气：20～30arb；管状透镜电压：±50～150V；碰撞能：20-50。
根据本发明优选的，所述步骤(3)中的加入内标的低分子肝素酶解产物样品在30～60℃条件下真空减压干燥1～3h。
根据本发明优选的，所述步骤(4)中的离心，条件为室温10000～15000rpm离心5～15min。
进一步优选的，所述步骤(4)中的离心，条件为室温12000rpm离心15min。
本发明的有益效果为：
本发明可以同时检测低分子肝素基本组成单元中除8种常见肝素二糖外的所有已报道的末端修饰结构和特殊结构，而且发现了三种新结构，包括还有两个氨基糖的三糖，糖醛酸发生3-O-硫 酸化的四糖和以糖醛酸作为还原端的三糖，并且可以通过内标同时对所有低分子肝素基本组成单元组分进行相对定量，解决了现有技术中仅能检测8种常见肝素二糖及部分特殊结构的问题，对于低分子肝素仿制药的研发、生产控制和保障药品安全具有极大的实用价值。
附图说明
图1为实施例1中依诺肝素钠标准品酶法降解与还原水解降解基本组成单元的提取离子流图；
图2为实施例1中依诺肝素钠标准品酶法降解基本组成单元组分30、31以及还原水解基本组成单元组分还原端5的二级质谱图；
图2中，a为实施例1中依诺肝素钠标准品酶法降解基本组成单元组分30的二级质谱图；
b为实施例1中依诺肝素钠标准品酶法降解基本组成单元组分31的二级质谱图；
c为实施例1中依诺肝素钠标准品酶法还原水解基本组成单元组分还原端5的二级质谱图；
图3为实施例2中达肝素钠标准品酶法降解基本组成单元的提取离子流图。
具体实施方式
下面结合说明书附图和实施例对本发明作进一步限定，但不限于此。
液相色谱仪为Agilent1100液相色谱仪，工作站为ChemStation；质谱为赛默飞TSQ Quantum Ultra型三重四极杆质谱，工作站为Xcalibur。
实施例1
一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，步骤如下：
(1)将醋酸铵试剂溶解于去离子水，制得醋酸铵浓度为5mM的流动相A；
(2)将醋酸铵试剂溶解于去离子水，并加入乙腈，制得流动相B，流动相B中，醋酸铵的浓度为5mM，乙腈的体积百分比为95％；
(3)将依诺肝素钠标准品用肝素酶I、II和III(均购自北京艾德豪克国际技术有限公司)在25℃条件下降解48h，将加入内标的降解产物用Millipore 30KDa的超滤膜过滤后真空减压干燥；
(4)则将50μg加入内标的低分子肝素酶解产物样品经硼氢化钠还原12h，并使用双氧水进行水解，将加入内标的低分子肝素酶解产物样品的水解产物配置成浓度为10μg/μL的待测溶液，进入步骤(5)；
(5)对步骤(3)得到的待测溶液离心处理：离心后，使用填料粒径为
Figure PCTCN2017074966-appb-000001
色谱柱内径为2.0mm、色谱柱长度为150mm的亲水相互作用色谱柱进行分离；流速0.15mL/min，洗脱梯度如下，均为体积百分比：
0～5min，5％流动相A，95％流动相B；5～107min，5～23％流动相A，95～77％流动相B；107～112min，23～50％流动相A，77～50％流动相B；112～125min，50％流动相A，50％流动相B；
(6)使用赛默飞TSQ Quantum Ultra型三重四极杆质谱在负离子模式下进行多反应监测，得到各组分的提取离子流图，设定参数为：喷雾电压：-3.2kV；鞘流气：20～30arb；管状透镜电压：-75V，碰撞能：35。
(7)根据步骤(6)中获得的各组分提取离子流图中获得的峰面积A以及已知浓度cIP的内标的峰面积AIP，经如下公式计算该组分的浓度c：
c＝cIP×(A/AIP)
(8)用步骤(7)中得到的对依诺肝素钠样品基本组成单元进行组成成分分析，酶解基本组成单元各组分含量信息如表1所示，还原水解基本组成单元各组分含量信息如表2所示。
表1
Figure PCTCN2017074966-appb-000002
Figure PCTCN2017074966-appb-000003
表2
Figure PCTCN2017074966-appb-000004
注：“△UA”表示不饱和糖醛酸，“Hex”表示糖醛酸，“GlcA”表示葡萄糖醛酸，“IdoA”表示艾杜糖醛酸，“GlcN”表示葡萄糖胺，“Ac”表示乙酰基团，“S”表示硫酸基团，“NS”表示N-硫酸基 团，“NRE”非还原端，“RE”还原端，“-ol”糖醇。
依诺肝素钠酶解基本单元的质谱分析提取离子流图如图1，依诺肝素钠还原水解基本单元的质谱分析提取离子流图如图2，通过本方法成功检测到依诺肝素钠标准品基本组成单元中除8种常见二糖以外的所有可能基本组成单元。
实施例2
一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，步骤如下：
(1)将醋酸铵试剂溶解于去离子水，制得醋酸铵浓度为5mM的流动相A；
(2)将醋酸铵试剂溶解于去离子水，并加入乙腈，制得流动相B，流动相B中，醋酸铵的浓度为5mM，乙腈的体积百分比为95％；
(3)将达肝素钠标准品用肝素酶I、II和III(均购自北京艾德豪克国际技术有限公司)在25℃条件下降解48h，将加入内标的降解产物用Millipore 30KDa的超滤膜过滤后真空减压干燥；
(4)将50ug达肝素钠完全酶解样品配制成浓度为10μg/μL的待测溶液；
(5)对步骤(4)得到的待测溶液离心处理：离心后，使用填料粒径为
Figure PCTCN2017074966-appb-000005
色谱柱内径为2.0mm、色谱柱长度为150mm的亲水相互作用色谱柱进行分离；流速为0.15mL/min，洗脱梯度如下，均为体积百分比：
0～5min，5％流动相A，95％流动相B；5～107min，5～23％流动相A，95～77％流动相B；107～112min，23～50％流动相A，77～50％流动相B；112～125min，50％流动相A，50％流动相B；
(6)使用赛默飞TSQ Quantum Ultra型三重四极杆质谱在负离子模式下进行多反应监测，得到各组分的提取离子流图，设定参数为：喷雾电压：-3.2kV；鞘流气：20～30arb；管状透镜电压：-75V，碰撞能：35。
(7)根据步骤(6)中获得的各组分提取离子流图中获得的峰面积A以及已知浓度cIP的内标的峰面积AIP，经如下公式计算该组分的浓度c：
c＝cIP×(A/AIP)
(8)用步骤(7)中得到的对依诺肝素钠样品基本组成单元进行组成成分分析，酶解基本组成单元各组分含量信息如表3所示。
表3
Figure PCTCN2017074966-appb-000006
Figure PCTCN2017074966-appb-000007
注：“△UA”表示不饱和糖醛酸，“Hex”表示糖醛酸，“GlcA”表示葡萄糖醛酸，“IdoA”表示艾杜糖醛酸，“GlcN”表示葡萄糖胺，“Ac”表示乙酰基团，“S”表示硫酸基团，“NS”表示N-硫酸基团，“NRE”非还原端，“RE”还原端，“-ol”糖醇。
达肝素钠酶解基本单元的质谱分析提取离子流图如图3，通过本方法成功检测到达肝素钠标准品基本组成单元中除8种常见二糖以外的所有可能基本组成单元。

Claims (4)

  1. 一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，其特征在于，步骤如下：
    (1)将醋酸铵试剂溶解于去离子水，制得醋酸铵浓度为3～10mM的流动相A；
    (2)将醋酸铵试剂溶解于去离子水，并加入乙腈，制得流动相B，流动相B中，醋酸铵的浓度为3～10mM，乙腈的体积百分比为90～98％；
    (3)如果需要区分低分子肝素末端结构，则将10～50μg加入内标的低分子肝素酶解产物样品经硼氢化钠还原10～12h，并使用双氧水进行水解，将加入内标的低分子肝素酶解产物样品的水解产物配置成浓度为1～10μg/μL的待测溶液，进入步骤(4)；如果不需要区分低分子肝素末端结构，则将10～50μg加入内标的低分子肝素酶解产物样品直接配制为浓度为1～10μg/μL的待测溶液，进入步骤(4)；
    (4)对步骤(3)得到的待测溶液离心处理，离心后，使用亲水相互作用色谱柱进行分离：流速0.1～0.5mL/min，洗脱梯度如下，均为体积百分比：
    0～5min，5％流动相A，95％流动相B；5～107min，5～23％流动相A，95～77％流动相B；107～112min，23～50％流动相A，77～50％流动相B；112～125min，50％流动相A，50％流动相B；
    (5)在正离子模式或负离子模式下用多反应监测二级质谱仪进行检测；
    所述步骤(4)中的质谱采用三重四极杆质谱仪(TSQ)，设定参数为：正离子模式喷雾电压：+4.0kV；负离子模式喷雾电压：-3.2kV；鞘流气：20～30arb；管状透镜电压：±50～150V；碰撞能：20-50。
  2. 根据权利要求1所述的一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，其特征在于，所述步骤(3)中的加入内标的低分子肝素酶解产物样品在30～60℃条件下真空减压干燥1～3h。
  3. 根据权利要求1所述的一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，其特征在于，所述步骤(4)中的离心，条件为室温10000～15000rpm离心5～15min。
  4. 根据权利要求1-3任一所述的一种低分子肝素完全降解产物的亲水相互作用色谱与多反应监测二级质谱联用检测方法，其特征在于，所述步骤(4)中的离心，条件为室温12000rpm离心15min。
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