基于UPLC-MS/MS的特征多肽识别技术鉴别中成药中的阿胶

Qualitative Analysis of Donkey-hide Gelatin in Chinese Traditional Patient Medicines by UPLC-MS/MS Based on Collagen Marker Peptides Recognition Technology

目的建立基于超高效液相色谱-三重四级杆质谱(UPLC-MS/MS)的特征多肽识别技术检测中成药中阿胶成分的专属鉴别方法。方法将中成药供试品进行提取及胰蛋白酶酶解,以阿胶特有的特征多肽离子对m/z 539.8(双电荷)→612.4和m/z 539.8(双电荷)→923.8作为鉴别离子对,采用UPLC-MS/MS液质联用仪、BEH C_(18)柱(50 mm×2.1 mm,1.7μm),以乙腈(A)-0.1%甲酸(B)为流动相进行梯度洗脱,总运行时间为15 min。同时对方法学进行考察,主要包括专属性、检出限、重复性等。结果阴性样品及其他常见胶类药材(龟甲胶、鹿角胶、黄明胶等)对阿胶的测定不产生干扰,方法检出限为含0.5%阿胶的供试品,10批市售的口服液类和颗粒类中成药中均检出阿胶的特征多肽离子对。结论方法专属性强、普适性好、灵敏度高、分析时间短,可用于口服液和颗粒类中成药中阿胶的鉴别。

OBJECTIVE An ultra-performance liquid chromatography coupled to triple quadruple mass spectrometry(UPLC-MS/MS) method based on marker polypeptide recognition technology was developed for qualitative analysis of donkey-hide gelatin in Chinese traditional patient medicines.METHODS A BEH C_(18) column(50 mm×2.1 mm, 1.7 μm) was used and eluted with acetonitril and 0.1% formic acid solution in gradient mode.The samples were extracted and enzymolysis treatment used trypsin.The characteristic molecular peaks of donkey-hide gelatin m/z 539.8(double-charge)→612.4 and m/z 539.8(double-charge)→923.8 were detected by UPLC-MS/MS with multiple reaction monitoring(MRM).The total elution time were 15 minutes.Meanwhile, the methodology were studied.RESULTS The method validation data showed that the donkey-hide gelatin was not interfered with the negative gelatins and other gelatins such as tortoise shell gelatin, deer-horn gelatin and bovine-hide gelatin.The detection limit of the method were both 0.5% donkey-hide gelatin in two matrix medicines.CONCLUSION The established method was specific, precise and reproducible.It can be used for the qualitative analysis of donkey-hide gelatin in Chinese traditional patient medicines.