两步凝胶法限量检查中药注射剂中的大分子蛋白质

目的：建立中药注射剂中大分子蛋白的葡聚糖凝胶色谱法与凝胶高效液相色谱法相结合的两步凝胶限量检查方法，为中药注射剂中大分子蛋白的检测提供方法和实验依据。方法：两步凝胶法第一步为交联葡聚糖凝胶柱色谱分析法，以溶菌酶对照品的出峰时间为临界点，收集临界点之前的中药注射剂洗脱液制备第二步的供试品溶液；第二步为凝胶高效液相色谱法，将制备得到的供试品溶液进样分析。色谱分析条件：色谱柱为TSK-Gel G2000SWXL（7.8 mm×300 mm，5 μm），流动相为0.1 mol·L^-1硫酸钠和0.1 mol·L^-1磷酸缓冲盐等体积混合液（pH 6.7），等度洗脱，流速为0.8 mL·min^-1，进样量100 μL，检测波长280 nm，柱温25℃。结果：溶菌酶对照品进样量在0.05~5 μg（R²=0.999 0）范围内呈良好的线性关系，精密度、稳定性、重现性均良好，平均回收率为88.9%，RSD为4.9%。本实验共检测了8个不同品种共18个批次的中药注射剂，不同中药注射剂品种中均能检出大分子蛋白的存在，虽含量略有差别，但均符合本实验设定的20 μg·mL^-1的检查限量。结论：本实验建立的两步凝胶法准确可靠，解决了中国药典方法的假阳性问题，可用于中药注射剂中大分子蛋白的限量检查，为中药注射剂中大分子蛋白检查提供了更有效的方法，为进一步保证中药注射剂的药品质量和临床用药安全奠定了基础。

Objective:To establish a two-dimensional gel method for the determination of macromolecular protein content in traditional Chinese medicine injections,and to provide a method and experimental basis for the detection of macromolecular proteins in traditional Chinese medicine injections. Methods:The first step is the Sephadex chromatography method,taking the retention time of lysozyme reference substance as the critical point,collecting the eluent of traditional Chinese medicine injection before the critical point as the test solution. In the second step,a TSK-Gel G2000SWXL(7.8 mm×300 mm,5 μm)column was used for the determination of macromolecular protein at 25℃. The injection volume was 100 μL.A gradient was applied,using 0.1 mol·L^-1 sodium sulfate and 0.1 mol·L^-1 phosphate buffer salt in water as mobile phase. The flow rate was 0.8 mL·min^-1 and the detection wavelength was 280 nm.Results:A good linear relationship was showed when injection of lysozyme reference was in the range of 0.05-5 μg(R²=0.999 0).The precision,stability and reproducibility were good.The average recovery were 88.9% and the RSD was 4.9%.A total of 18 batches of 8 different Chinese medicine injections were tested in the experiment.The macromolecular protein was detected in all kinds of Chinese herbal medicine.The contents of macromolecular protein were slightly different,but all of them were under the limit of 20 μg·mL^-1.Conclusion:The two-dimensional gel method established in this experiment was able to avoid the false positives of sulfosalicylic acid turbidity method(described in Chinese pharmacopoeia)and could be used for the determination of macromolecular protein content in traditional Chinese medicine injection.It provides a more effective method for the examination of macromolecular protein in traditional Chinese medicine injections,and lays the foundation for ensuring the quality of Chinese medicine injections.