超高效液相色谱-串联四极杆质谱法同时检测鹿角胶饮片中牛皮源、驴皮源及鹿角胶成分

Identification of bovine-hide gelatin,donkey-hide gelatin and deer horn glue of cervi cornus colla by UPLC-MS/MS

目的建立鹿角胶饮片中牛皮源、驴皮源及鹿角胶成分同时检测方法。方法精密称定鹿角胶饮片粉末0.1 g,加1%碳酸氢铵溶液超声溶解、稀释、过滤,用胰蛋白酶酶解12小时,利用超高效液相色谱-串联质谱联用技术(UPLC-MS/MS)和多反应监测模式(MRM)分别检测黄明胶特征肽段离子对(m/z)641.3→726.2,783.3,阿胶特征肽段离子对(m/z)539.8→612.4,924.0,鹿角胶特征肽段离子对(m/z)765.4→554.0,733.0。色谱柱为BEH-C18,以乙腈—0.1%甲酸水溶液为流动相进行梯度洗脱。分别考察上述三种特征肽段离子对的专属性,并用逐级稀释法确定方法检出限。用所建方法检测10批市售鹿角胶饮片。结果黄明胶、阿胶、鹿角胶三种对照药材在所建方法下保留时间分别为3.6分钟、6.2分钟和10.9分钟,无基质干扰,检测限分别为0.1,2.0,1.0 mg/L,对照药材混合溶液进样6次,鹿角胶峰面积RSD为2.1%,黄明胶峰面积RSD为3.7%,阿胶峰面积RSD为2.8%,方法重复性良好。将所建方法应用于10批市售鹿角胶饮片的检测分析,1批结果为不合格(检出牛皮源成分)。结论所建方法专属性强、灵敏度高,实现了鹿角胶饮片中主成分鉴别与非法添加成分检查的同时检测,进一步提高检验效率,可有效控制鹿角胶质量。

Objective To develop an UPLC-MS/MS method for identification of bovine-hide gelatin,donkey-hide gelatin and deer horn glue from cervi cornus colla. Methods 0. 1 g powder of cervi cornus colla was added with 1% ammonium bicarbonate solution to dissolve,dilute and filter,and then dissolved with trypsin for 12 hours. Deer horn glue,donkey-hide gelatin and bovine-hide gelatin were identified by UPLC-MS/MS after hydrolyzed by trypsin. MRM was used and ion pair of bovine-hide gelatin was641. 3（ m/z） → 726. 2,783. 3,ion pair of deer horn glue was 765. 4（ m/z） → 554. 0,733. 0,ion pair of donkey-hide gelatin was 539. 8（ m/z） → 612. 4,924. 0. The column for UPLC analysis was BEH-C18 and eluted with acetonitrile-0. 1% formic acid aqueous phase as mobile phase. Results Retention time of bovine-hide gelatin,donkey-hide gelatin and deer horn glue was 3. 6 min,6. 2 min and 10. 9 min in this method without matrix effect. The detection limits of deer horn glue,donkey-hide gelatin and deer horn glue were 0. 1,2. 0,1. 0 mg/L respectively. RSD of bovine-hide gelatin,donkey-hide gelatin and deer horn glue was 3. 7%,2. 8% and 2. 1% respectively. 10 batches of cervi cornus colla were analyzed by this UPLC-MS/MS method,and 1 batch was unqualified（ bovine-hide gelatin detected）. Conclusion The method was proved to be specific,rapid and sensitive which can be used for simultaneous detection of the main component identification and the illegal addition of component inspection in cervi cornus colla,which is able to effectively control the quality of cervi cornus colla.