甘油磷酸胆碱的层析纯化及其稳定性研究

Purification and stability of Glycerophosphatidylcholine

目的对甘油磷酸胆碱粗品进行层析纯化,并对其进行稳定性研究,从而为工业化生产、包装、贮存和制剂的研发提供依据。方法采用三氧化二铝脱色法和硅胶柱层析法相结合的工艺对甘油磷酸胆碱进行层析纯化;设计影响其稳定性试验:分别取三份GPC(批号为100301、100302、100303)光线强度为4 500 LX的强光照射10 d,60℃高温放置10 d,温度25℃、相对湿度75%放置10 d,密闭于40℃、相对湿度75%的器皿中放置12月的加速试验,密闭于25℃、相对湿度75%的器皿中放置12月的长期试验并做重复试验,采用色谱柱为GraceSmart RP C18柱(250 mm×4.6 mm,5μm),流动相为60%乙腈-水溶液,检测其含量变化。结果通过三氧化二铝脱色法和硅胶柱层析法相结合的工艺进行分离纯化,能得到纯度达99.5%以上的甘油磷酸胆碱产品;强光试验含量均数为99.51%(n=9)、RSD=0.40%,高温试验含量均数为99.65%、RSD=0.42%(n=9),高湿试验含量均数为99.30%、RSD=0.47%(n=9),加速试验含量均数为99.61%、RSD=0.57%(n=15),长期试验含量均数为99.12%、RSD=0.50%(n=15),重复试验含量均数为99.68%、RSD=0.43%(n=5)。结论层析纯化度高,工艺简单,可操作性强,适用于工业化生产;甘油磷酸胆碱的稳定性好,且采用的稳定性测定方法灵敏、准确、重现性好。

Objective To isolate and purify the crude product of glycerophosphatidylcholine and conduct a research on its stability, thereby providing a basis for industrialized production,packaging,storage and development of the preparation. Methods A technology of Al 2 O 3 decolorization and isolation by silica gel column was used to isolate and purify glycerophosphatidylcholine,and by the agency of influencing factors,three portions of GPC（ Batch Nos. 100301,100302,100303） were respectively exposed to strong light with light intensity of 4 500 LX for 10 days,laid up at a high temperature of 60℃ for 10 days,placed at 25℃ and in the relative humidity of 75% for 10 days,kept in airtight vessels at 40℃ and in the relative humidity of 75% for 12 months to go through accelerated tests,kept in airtight vessels at 25℃ and in the relative humidity of 75% for 12 months to undergo long-term and repeated tests. And evaporative light scattering detector and HPLC were used to determine the content of glycerophosphatidylcholine under the conditions of severe tests,accelerated tests and long-term storage tests. And we used the chromatographic column GraceSmart RP C 18（ 250 mm ×4. 6 mm,5 μm）, the mobile phase of 60% acetonitrile-water solution,the flow rate 0. 7 mL·min-1,the column temperature（ 25 ± 1） ℃ from N2000 work station of Zhejiang University. The detector is Alltech 2000 evaporative light-scattering detector from the United States,the temperature of the drift tube is 109℃,the carrier gas is air,gas flow rate is 3. 5 mL·min-1. The content change is detected under the condition of the sample size 20 μL. Results The research finding is that by use of Al 2 O 3 decolorization and isolation by silica gel column we have obtained a product of glycerophosphatidylcholine with a purity of more than 99. 5%; the mean content of strong light tests is 99. 51%（ n = 9）,RSD = 0. 40%,the mean content of high temperature tests is 99. 65%,RSD = 0. 42%（ n = 9）,the mean content of high humidity tests is 99. 30%,RSD = 0. 47%（ n = 9）,the mean content of accelerated tests is 99. 61%,RSD = 0. 57%（ n = 15）,the mean content of long-term tests is 99. 12%,RSD = 0. 50%（ n = 15）,and the mean content of repeated tests is 99. 68%,RSD = 0. 43%（ n =5）. Conclusion The obtained chromatographic purification is high,the technology used is simple with strong operability and suitable for industrialized production. Glycerophosphatidylcholine is of good stability,and the stability determination is sensitive,accurate and of good reproducibility.