毛细管电泳法测定肝素和低分子量肝素平均硫酸化程度

肝素和低分子量肝素(LMWHs)作为临床上常用的抗凝血药物,其抗凝血活性与硫酸化程度(SD)密切相关。然而,肝素类药物的生产需经历一系列复杂的工艺过程,在制备和储存过程中,肝素的硫酸基团容易水解丢失,影响抗凝血活性,这将直接影响肝素药物的使用安全性。为保证产品质量,需要发展一种快速检测肝素硫酸化程度的技术,以监测原料质量和工艺条件的稳定性。虽然已有一些测定肝素硫酸化程度的报道,但这些方法均有局限性,不适用于肝素生产的质量控制。为此,开发了一种基于毛细管电泳技术(CE)检测肝素和低分子量肝素的平均硫酸化程度的方法。首先,用肝素酶混合液彻底消化未分级肝素(UHF)和低分子量肝素,然后用毛细管电泳分离酶解得到的所有寡糖和二糖构建模块,并对它们进行定性和定量分析。随后,根据每种寡糖和二糖的峰面积及其硫酸酯基团的数量,便可计算出每个构成肝素二糖单元硫酸化程度的平均值。使用该方法对来自两个生产商各4个批次依诺肝素(低分子量肝素)和5个批次肝素原料进行检测,并计算了各批次样品的相对标准偏差(RSD),对不同厂家生产的依诺肝素平均硫酸化程度进行了比较,验证了该方法的实用性。该方法灵敏度高,准确可靠,分...

Determination of sulfation degree of heparin and low molecular weight heparins by capillary electrophoresis

Heparin is composed of a highly sulfated linear saccharide and is widely used as an anticoagulant. Low molecular weight heparins (LMWHs) are derived from the unfractionated heparin (UFH) by enzymatic or chemical degradation. LMWHs have largely replaced heparin as an anticoagulant for treatment and prevention of thrombosis because of the advantages of less bleeding, greater bioavailability, and more predictable anticoagulant effects in comparison to heparin. Enoxaparin, produced by the alkaline degradation of UFH through β-eliminative cleavage, represents the most commonly used LMWH. The structural characteristics of LMWHs differ from their parent heparin not only in terms of molecular weight but also in the sulfation degree as a result of losing the sulfate ester groups during the manufacturing process. The resulting compositional variation directly leads to a fluctuation in anticoagulant activity. In vitro functional assays showed that there is a wide variation in anticoagulant activity among the various LMWHs from different manufacturers owing to slight differences in the manufacturing process. This will directly affect heparin drug safety. In order to ensure the stability of product quality, it is necessary to develop a method for detecting the degree of heparin sulfation to monitor the stability of UFH and processing conditions. During the last two decades, various analytical methods based on chromatography or NMR have been developed for structural characterization of UFH and LMWHs. However, the reported methods require expensive equipment and professional data processing. These limitations make it difficult to apply the current methods to quality control via sulfation degree determination. Herein, we report a simple and robust method for the detection of the sulfation degree of UFH and LMWHs. The determination is based on the separation of building blocks of heparin obtained by exhaustive digestion of UFH and LMWHs in a mixture of heparinases. A mixed solution of heparinase Ⅰ, Ⅱ, and Ⅲ was prepared to give a final content of 0.13 IU/mL for each enzyme. The digestion of enoxaparin and heparin samples was performed at 25 ℃ for 48 h. By using a capillary electrophoresis (CE) method, a total of 18 oligosaccharides building blocks of heparin, including ten disaccharides, one trisaccharide, three tetrasaccharides, and four 1, 6-anhydro derivatives, can be baseline separated. Then, the compositions of enoxaparin and UFH can be precisely determined. Based on the assumption that the molar extinction coefficient of each oligosaccharide at UV 232 nm is the same, the concentration of each oligosaccharide can be conveniently replaced by their peak area, and the accurate number of sulfate ester groups in each disaccharide unit can be determined, hence the average sulfation degree (SD). The developed method allows us to compare the sulfation degree data between the enoxaparin batches from the different manufacturers to evaluate the composition similarity. Herein, eight batches of commercially available enoxaparin from two manufacturers and four batches of UFH source materials were measured. Each sample was measured in triplicate, and the average values as well as the relative standard deviations (RSD) were calculated. The total sulfation degree (T-SD), the individual degree of N-sulfation (N-SD) and O-sulfation (O-SD) data were also determined and compared. A significant difference was observed in the SD of the products from the different manufacturers, which indicated that our method can be used as one of the quantitative compositional analysis methods for quality control of LMWHs and UFH. The variation in terms of the sulfation degree of enoxaparin products from different manufacturers can be precisely identified using this method. This allows us to determine the detailed compositional differences between products from the different manufacturers. The obtained satisfactory data show that high fluctuation in the sulfation degree of UFH could transmit to the final enoxaparin products. The consistency of the products can also be evaluated by using these methods. The CE method has several advantages for quantitative compositional analysis of LMWHs, such as high separation efficiency, high sensitivity, automation, short analysis time and low consumption of both sample and reagents. It has a good application potential in the quality control heparin production.