CN112816566A - 一种那屈肝素钙末端结构确认方法 - Google Patents

一种那屈肝素钙末端结构确认方法 Download PDF

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CN112816566A
CN112816566A CN201911146201.1A CN201911146201A CN112816566A CN 112816566 A CN112816566 A CN 112816566A CN 201911146201 A CN201911146201 A CN 201911146201A CN 112816566 A CN112816566 A CN 112816566A
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heparinase
nadroparin calcium
disaccharide
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田立华
贾风燕
刘遂库
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Yantai Dongcheng Pharmaceutical Group Co ltd
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Abstract

本发明公布了一种那屈肝素钙末端结构确认方法，包括：(1)用分子排阻色谱‑高分辨质谱联用(SEC‑ESI‑MS)分析那屈肝素钙的完整糖链指纹图谱，确定那屈肝素钙的末端结构(还原末端：6‑O‑磺基‑2，5‑脱水‑D‑甘露醇)。(2)那屈肝素钙样品经过肝素酶I、II、III混合酶降解，通过强阴离子交换色谱分析二糖谱，验证那屈肝素钙二糖中含有末端结构二糖。(3)通过13C核磁共振图谱，确认那屈肝素钙结构中的末端结构。

Description

一种那屈肝素钙末端结构确认方法
技术领域
本发明涉及一种那屈肝素钙末端结构分析方法，属于肝素类生化原料药分析技术领域。
背景技术
低分子量肝素(low-molecular-weight heparin，LMWH)是未分级肝素(unfractionated heparin，UFH)中具有较低分子量的组分或片段，具有与UFH相同的母体结构。
LMWH的结构复杂，其中组成LMWH的单糖有10余种，各种单糖的糖环上还有硫酸基、乙酰基等不同的取代基，各种单糖之间的连接方式不同，及取代基的不同或者取代基但取代的位置不同，导致LMWH具有不同的生物活性。不同的LMWH品种，由于生产工艺的不同，其分子量分布、生物活性、抗Xa/抗II活性比、硫酸化程度和末端结构等方面存在差异。
那屈肝素钙是由来源于猪小肠粘膜的肝素经亚硝酸降解后，再经分级选择性去除大部分分子量低于2000道尔顿物质而获得的一种低分子量肝素钙盐。多数组分在其主链的非还原性末端含有个2-O-磺基-α-L-吡喃艾杜糖醛酸的结构，还原末端含有6-O-磺基-2，5-脱水-D- 甘露醇结构。本内容中的末端结构指还原末端(6-O-磺基-2，5-脱水-D-甘露醇)。
结构鉴定对于仿制药的研发、生产控制和保障药品安全是必不可少的。末端结构鉴定是其中比较重要的一种鉴定。
发明内容
本发明的目的在于明确那屈肝素钙末端结构确定方法，从而建立一套比较完善的那屈肝素钙末端结构分析手段。
本发明的目的可以通过下述技术方案实现：1)使用分子排阻色谱-高分辨质谱联用 (SEC-ESI-MS)分析那屈肝素钙的完整糖链指纹图谱，比较主要糖链分布和糖链结构信息，确定那屈肝素钙的末端结构(还原末端：6-O-磺基-2，5-脱水-D-甘露醇)(见附图1)。2)那屈肝素钙样品经过肝素酶I、II、III混合酶降解，通过强阴离子交换色谱分析二糖谱，验证那屈肝素钙二糖中含有末端结构二糖(见附图2)。3)通过13C核磁共振图谱，确认那屈肝素钙结构中的末端结构(还原末端：6-O-磺基-2，5-脱水-D-甘露醇)(见附图3)。
本发明的测定方法如下步骤所示：
1.1分子排阻色谱与高分辨质谱联用(SEC-ESI-MS)
液相条件：
色谱柱：BEH 200(4.6mm*300mm)串BEH 125(4.6mm*300mm)
流动相：50mM乙酸铵水溶液，含20％甲醇；
流速：0.2mL/min，等度洗脱；
质谱条件：
Capillary：-2.3kV；Sampling Cone：10；Source offset：80；Source120℃；Desolvation 400℃；
Cone Gas：20I/h；Desolvation gas：600L/h。
1.2肝素酶I、II、III混合酶降解，通过强阴离子交换色谱分析二糖谱
肝素酶I、II、III完全酶解步骤：
20μl样品+70μl乙酸钙溶液(pH＝7.0)+100μl肝素酶I、II、III混合溶液(pH 7.0磷酸二氢钠溶液溶解)，混匀，置于25℃水浴中反应48h，灭活，过滤后进样。
酶解后液相分析条件：
色谱柱：Waters Spherisorb S5 SAX(4.0*250mm)
流动相：A---NaH2PO4(0.028％)；B---NaH2PO4(0.028％)+NaClO4(14％)
流速：0.45ml/min
柱温：35℃
进样体积：10μl
检测器：紫外检测器
波长：232nm
流动相梯度如下：
时间(min) 流动相A(％) 流动相B(％)
0 97 3
20 65 35
50 0 100
60 0 100
60.1 97 3
80 97 3
1.3 13C核磁共振
13C核磁共振分析条件：
磁场强度：125MHz
扫描次数(NS)：不低于10240次
温度(TE)：40℃
采样时间(AQ)：不少于1.0S
延迟采集(D1)：不少于2.0S
采样中心(O1P)：100.0ppm
去偶中心(O2P)：4.0ppm
谱宽(SWH)：220～240ppm
脉冲(PUL)：30°～90°之间
LB：1.0Hz
核磁管直径：5mm
附图说明
图1：那屈肝素钙完整糖链SEC-MS图
图2：那屈肝素钙二糖对比图(黑色圆圈标记为末端结构)
图3：那屈肝素钙13C核磁共振图(黑色圆圈标记为末端结构)
具体实施方式
本发明是一种那屈肝素钙末端结构分析方法，分为三种不同的方式确认末端结构：
a.采用分子排阻色谱与高分辨质谱联用(SEC-ESI-MS)；
b.采用肝素酶I、II、III混合酶降解，强阴离子交换色谱；
c.采用13C核磁共振。
实施例
首先称取那屈肝素钙20.08mg于1.5ml EP管中，加入1ml流动相(50mM乙酸铵水溶液，含20％甲醇)中，过0.22μm微孔滤膜，进样检测。得到完整糖链SEC-MS图，取图中33.71min的峰进行详细质谱解析。
依据欧洲药典上那屈肝素钙的结构，如结构式1，结合根据质核比推测各碎片结构。
Figure BSA0000195435530000031
解析质核比m/z＝537.4897：带两个电荷，推测是Dp4 5S 0AC(带有5个磺酸基0乙酰基的四糖)，见结构式2。
Figure BSA0000195435530000041
解析质核比m/z＝550.3123：带三个电荷，推测是Dp6 8S 0AC(带有8个磺酸基0乙酰基的六糖)，如结构式3。
Figure BSA0000195435530000042
确定那屈肝素钙的末端结构是
Figure 1
。

Claims (4)

1.一种进行那屈肝素钙末端结构确认的分析方法，其特征在于包含使用分子排阻色谱-高分辨质谱联用分析完整糖链指纹图谱；经肝素酶I、II、III混合酶降解，强阴离子交换色谱分析二糖谱；13C核磁共振图谱确认末端。
2.如权利要求1所述的那屈肝素钙末端结构确认的分析方法，其特征在于使用分子排阻色谱与高分辨质谱联用，液相分析条件：色谱柱为BEH 200(4.6mm*300mm)串联BEH 125(4.6mm*300mm)，流动相为50mM乙酸铵水溶液，含20％甲醇，流速为0.2mL/min，等度洗脱；质谱分析条件：Capillary：-2.3kV；Sampling Cone：10；Source offset：80；Source120℃；Desolvation 400℃；Cone Gas：20L/h；Desolvation gas：600L/h。
3.如权利要求1所述的那屈肝素钙末端结构确认的分析方法，其特征在于经肝素酶I、II、III混合酶降解，强阴离子交换色谱分析二糖谱：用肝素酶I、II、III混合酶进行完全酶解，得到二糖，酶解步骤：20μl样品+70μl乙酸钙溶液(pH＝7.0)+100μl肝素酶I、II、II溶液(pH 7.0磷酸二氢钠溶液溶解)，酶解温度为25℃，酶解时间为48h，检测条件：色谱柱为Waters Spherisorb S5 SAX(4.0*250mm)，流动相A为0.028％的NaH2PO4溶液，流动相B为0.028％的NaH2PO4-14％的NaClO4溶液，流速为0.45ml/min，柱温为35℃，进样体积为10μl，检测器为紫外检测器，检测波长为232nm，采用如下梯度洗脱方式，0～20min，97％A；20～50min，65～0％A；50～60min，0％A；60～80min，97％A。
4.如权利要求1所述的那屈肝素钙末端结构确认的分析方法，其特征在于13C核磁共振确认末端，检测条件：样品溶解适量D2O，加入内标氘代甲醇，磁场强度为125MHz，扫描次数(NS)不低于10240次，温度(TE)为40℃，采样时间不少于1.0S，延迟采集不少于2.0S，采样中心(O1P)为100.0ppm，去偶中心(O2P)为4.0ppm，谱宽(SWH)为220～240ppm，脉冲(PUL)为30°～90°之间，LB为1.0Hz，核磁管直径为5mm。
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