高效阴离子交换色谱法测定依诺肝素钠1,6-脱水衍生物含量

王悦,李京,李颖颖,范慧红*

中国药学杂志 ›› 2014, Vol. 49 ›› Issue (16) : 1461-1466.

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中国药学杂志 ›› 2014, Vol. 49 ›› Issue (16) : 1461-1466. DOI: 10.11669/cpj.2014.16.021
论著

高效阴离子交换色谱法测定依诺肝素钠1,6-脱水衍生物含量

  • 王悦,李京,李颖颖,范慧红*
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SAX-HPLC Method for Quantification of 1,6-Anhydro Derivatives of Enoxaparin Sodium

  • WANG Yue, LI Jing, LI Ying-ying, FAN Hui-hong*
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摘要

目的 建立依诺肝素钠特异性末端结构1,6-脱水衍生物结构的含量测定方法。方法 肝素酶混合液将依诺肝素钠水解成二糖或四糖单位,水解得到的寡糖峰经硼氢化钠还原后用-高效阴离子交换色谱法方法进行分析。采用Waters Spherisorb S5 SAX(4.0 mm×250 mm,5 μm)色谱柱,以0.28 g·L-1磷酸二氢钠溶液为流动相A含0.28 g·L-1磷酸二氢钠及140 g·L-1高氯酸钠的溶液为流动相B进行梯度洗脱(0~20 min,3%~35%B;20~50 min,35%~100%B;50~60 min,100%B;60~61 min,100%~3%B,61~79 min,3%B),流速0.8 mL·min-1,柱温为50 ℃;检测波长为234 nm,并用归一化面积百分比法计算摩尔百分含量。结果 筛选出了酶解效果达标的国产肝素酶并优化了酶解条件,酶解后1,6-脱水ΔⅠS-ⅠS与1,6-脱水ΔⅠS峰面积比<1.15;优化色谱条件后ΔⅠA和1,6-脱水ΔⅠS的分离度>1.5;硼氢化钠还原后的溶液色谱图中,ΔⅠS和还原ΔⅠS的峰面积比均小于0.02;两家国产依诺肝素钠生产企业样品结果均在15%~25%内。结论 方法可对依诺肝素钠特征还原末端结构进行分离和定量,作为对低分子肝素进行微观结构分析的首个鉴别项目,已收载入新修订依诺肝素钠国家标准草案中。

Abstract

OBJECTIVE To establish an HPLC method to determine the levels of the characteristic 1,6-anhydro derivatives of enoxaparin sodium. METHODS The enoxaparin sodium solution was depolymerized by a mixture of heparinases to obtaine di- or tetra-saccharide units, and the products were reduced by sodium borohydride and then analyzed by SAX-HPLC. The analysis was carried out on a Waters Spherisorb S5 SAX column (4.0 mm×250 mm, 5 μm). The mobile phase was composed of 0.28 g·L-1 monobasic sodium phosphate solution (A) and 140 g·L-1 sodium perchlorate solution (B) (0-20 min, 3%-35% B;20-50 min, 35%-100% B;50-60 min,100% B;60-61 min, 100%-3% B,61-79 min, 3% B)and the flow rate was 0.8 mL·min-1. The column temperature was 50 ℃ and the detection wavelength was set at 234 nm. The normalized area percentage method was used for calculation. RESULTS One domestic heparinase factory was screened out that could pass the depolymerization suitability test, and the ratio of the peak area of 1,6-anhydro-ΔⅠS-ⅠS to that of 1,6-ΔⅠS was not more than 1.15. In the optimal chromatographic condition, the resolution between reduced ΔⅠA and 1,6-anhydro-ΔⅠS was greater than 1.5. The ratio of the peak area of ΔⅠS and reduced ΔⅠS of the reduced solution was less than 0.02. The contents of 1,6-anhydro derivatives in the samples from two domestics factories were between 15%-25%. CONCLUSION This method can be used to separate and quantify the specific reduced end structures of enoxaprin. As the first test to analyze the LMWH’s structure, the method has already been included into the revised national standard draft for enoxaparin.

关键词

依诺肝素钠 / 1 / 6-脱水衍生物 / 高效阴离子交换色谱法 / 肝素酶Ⅰ / 肝素酶Ⅱ / 肝素酶Ⅲ / 国家标准

Key words

enoxaparin / 1,6-anhydro structure / HPLC / heparinase Ⅰ, heparinase Ⅱ / heparinase Ⅲ / national standard

引用本文

导出引用
王悦,李京,李颖颖,范慧红*. 高效阴离子交换色谱法测定依诺肝素钠1,6-脱水衍生物含量[J]. 中国药学杂志, 2014, 49(16): 1461-1466 https://doi.org/10.11669/cpj.2014.16.021
WANG Yue, LI Jing, LI Ying-ying, FAN Hui-hong*. SAX-HPLC Method for Quantification of 1,6-Anhydro Derivatives of Enoxaparin Sodium[J]. Chinese Pharmaceutical Journal, 2014, 49(16): 1461-1466 https://doi.org/10.11669/cpj.2014.16.021
中图分类号: R917   

参考文献

WANG B, LUCINDA F B, ALI A L H, et al. Characterization of currently marketed heparin products: Analysis of heparin digests by RPIP-UHPLC-QTOF-MS . J Pharm Bio Chem Anal, 2012, 67-68:42-50. GIUSEPPE M, MARCO G, GIANGIACOMO T, et al. Characterization of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1, 6-anhydro-β-D-mannosamine residues. Carbohydr Res, 2007, 342(16): 835-842. MARCO G, STEFANO E, DAVIDE G, et al. Effects on molecular conformation and anticoagulant activities of 1,6-anhydrosugars at the reducing terminal of antithrombin-binding octasaccharides isolated from low-molecular-weight heparin enoxaparin .J Med Chem, 2010, 53(22):8030-8040. BIANCHINI P, MASCELLANI G. Few bicyclic acetals at reducing end of low-molecular-weight heparins:Might they restrict specification of pharmacopoeia.Pharmeur Sci Notes,2005,2005(1):1-3. KING J T, DESAI U R.A capillary electrophoretic method for fingerprinting low molecular weight heparins. Anal Biochem, 2008,380(12): 229-234. USP 36 NF31. 2013:141-145. EP 8.1.2014:3749-3750.

基金

国家质量监督检验检疫总局公益性行业专项“双打”中药品检验检测技术方法研究,子课题《低分子肝素标准提高研究》资助项目(2012104008);中国食品药品检定研究院中青年发展研究基金课题《低分子肝素质控对照品、标准试剂的制备及标化》资助项目(2012A5)
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