Abstract：
An international collaborative study was run within the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the Commission of the European Union to establish replacement batches for European Pharmacopoeia (Ph. Eur.) Heparin Low-Molecular-Mass (LMM) for calibration Chemical Reference Substance batch 3 (CRS3) used for the characterisation of LMM heparins by high performance size-exclusion chromatography. Two candidate batches (A, cCRS4 and B, cCRS5) were filled using the same material as the existing official calibrants, adopted with either an assigned number-average molecular mass (Mna) or a broad standard table (BST). Fifteen laboratories evaluated the suitability of these candidate batches for use as calibrants with the pharmacopoeial dual refractive index/ultraviolet (RI/UV) detector calibration method, as well as with a modified mobile phase and the BST calibration method. Seven preparations of LMM heparin were tested. The results confirmed that the proposed batches are suitable for use with the same characteristic Mna as CRS3 and with the BST established for the World Health Organization (WHO) 2nd International Standard (IS). The BST calibration method gave comparable results to the RI/UV method, while showing better reproducibility, being easier to perform and requiring no calibrant with UV absorbance. The modified mobile phase had no impact on the calculated values while improving separation between the calibrant and salt peaks. The two candidate batches were adopted as Ph. Eur. Heparin LMM for calibration CRS batches 4 and 5, respectively, with the assigned Mna value of 3800 and a BST. In anticipation of the depletion of the calibrant required for use with the RI/UV method, and taking into account the unlikely procurement of a new lot of suitable starting material, it was recommended to include the BST method in Ph. Eur. monograph 0828, Heparins, low-molecular-mass. In order to improve peak separation, it was also recommended to include the use of ammonium acetate solution as mobile phase in the monograph, both for the Ph. Eur. RI/UV and the proposed BST calibration methods. Further to this study, Ph. Eur. monograph 0828 was revised to replace the RI/UV method by the BST method. This contributed to the harmonisation of methods across regions, thereby facilitating a concerted global action for the development and establishment of the next batches of calibrants for the quality control of LMM heparins.
摘要：
在欧洲委员会和欧盟委员会的生物标准化计划（BSP）的框架内进行了一项国际合作研究，以建立欧洲药典的替代批次（Ph.欧尔.)用于校准的肝素低分子质量(LMM)用于通过高效尺寸排阻色谱法表征LMM肝素的化学参考物质批次3(CRS3)。两个候选批次(A，cCRS4和B，cCRS5)使用与现有官方校准物相同的材料进行填充，采用指定的数均分子量（Mna）或宽标准表（BST）。15个实验室使用药典双折射率/紫外线（RI/UV）检测器校准方法评估了这些候选批次用作校准物的适用性，以及改进的流动相和BST校准方法。测试了7种LMM肝素制剂。结果证实,所提出的批次适用于具有与CRS3相同的特征Mna和针对世界卫生组织(WHO)第二国际标准(IS)建立的BST。BST校准方法给出了与RI/UV方法相当的结果，虽然显示出更好的再现性，更容易执行，不需要校准与UV吸光度。改进的流动相对计算值没有影响,同时改善校准物峰和盐峰之间的分离。这两个候选批次被采纳为Ph。欧尔.分别用于校准CRS批次4和5的肝素LMM，分配的Mna值为3800和BST。预期使用RI/UV方法所需的校准物耗尽，并考虑到不太可能采购新一批合适的起始材料，建议在Ph中包括BST方法。欧尔.专著0828，肝素，低分子质量。为了改善峰分离,还建议在专论中包括使用乙酸铵溶液作为流动相，都是为了Ph.欧尔.RI/UV和提出的BST校准方法。在这项研究的基础上，Ph.欧尔.修订专著0828，以BST方法取代RI/UV方法。这有助于协调各地区的方法，从而促进采取协调一致的全球行动，以开发和建立下一批用于LMM肝素质量控制的校准物。
