肝素钠杂质总己糖胺中半乳糖胺的方法学研究

A methodological study of limit test of galactosamine for total hexosamine in heparin sodium

目的建立离子色谱法测定肝素钠样品水解后的半乳糖胺含量。方法通过计算半乳糖胺和葡萄糖胺的响应值的比值从而得到半乳糖胺含量。采用戴安离子色谱仪,色谱柱为氨基酸捕获柱（30 mm×3 mm）,串联保护柱（30 mm×3 mm）和Carbo Pac PA20分析柱（150 mm×3 mm）进行分析,脉冲安培检测器检测。流动相为14 m M氢氧化钾溶液,流速为0.4 m L/min,柱温30℃,进样体积10μL。结果盐酸葡萄糖胺在1.013~16.211μg/m L范围内,葡萄糖胺色谱峰面积与其浓度成良好线性关系（Y=2.303 4X＋0.824 2,r=0.998 3）,回收率为92.7%,RSD为3.2%（n=9）,最低检测限为0.1013μg/m L,定量限为0.3377μg/m L。盐酸半乳糖胺在0.0102~0.1625μg/m L范围内,半乳糖胺色谱峰面积与其浓度成良好线性关系（Y=31.157X-0.114 4,r=0.999 3）,回收率为102.1%,RSD为2.4%（n=9）,最低检测限为0.001 0μg/m L,定量限为0.003 4μg/m L。3批肝素钠原料中半乳糖胺检测结果分别为未检出,（0.02±2.1）%,（0.03±1.5）%,均小于美国药典规定的限值1%。结论经方法学验证,本文所建立的离子色谱法可用于肝素钠中半乳糖胺含量测定,可为肝素钠质量标准的提高提供参考。

Objective To establish an ion chromatography method to determine the content of galactosamine in heparin sodium sample.Methods The content of galactosamine was determined by the ratio of response value of galactosamine and glucosamine. The determination was performed on an Dionex ICS,and the separation was carried out on a Amino acids capture column（ 30 mm × 3 mm）,Series protect column（ 30 mm ×3 mm） and analytical column Carbo Pac PA20（ 150 mm × 3 mm）. The mobile phase was 14 m M potassium hydroxide solution at a flow rate of 0. 4 m L /min; the column tempertature was at 30 ℃; the injection volume was 10 μL. Results Glucosamine hydrochloride had good linearity within the range of1. 013-16. 211 μg / m L（ Y = 2. 303 4X ＋ 0. 824 2,r = 0. 998 3）,the average accuracy was 92. 7%,and RSD was 3. 2%（ n = 9）,the limit of detection was 0. 101 3 μg / m L,and the limit of quantitation was 0. 337 7 μg / m L. D-Galactosamine hydrochloride had good linearity within the range of 0. 010 2-0. 162 5 g / m L,（ Y = 31. 157X-0. 114 4,r = 0. 999 3）. The accuracy was 102. 1%,RSD was 2. 4%（ n = 9）. The limit of detection was 0. 001 0 μg /m L,and the limit of quantitation was 0. 003 4 μg / m L. The determination of galactosamine in 3 batches of heparin sodium raw material was not detected,（ 0. 02 ± 2. 1） %,（ 0. 03 ± 1. 5） %,respectively,which were all lower than the limit value（ 1%） of United States Pharmacopeia regulation. Conclusion The method for the determination of galactosamine in total hexose amine is successfully developed,which could be used as reference for improvement of the quality standard of heparin sodium.