驴皮特征肽的发现及其在阿胶鉴别中的应用

目的：建立阿胶中驴皮源成分的鉴别方法。方法：通过序列比对发现理论的驴皮特征肽，并利用纳升液相色谱-高分辨质谱进行确证阿胶特征肽GPTGEPGKPGDK。利用胰蛋白酶对阿胶样品进行酶解，利用超高效液相色谱-三重四极杆质谱（UPLC-QQQ MS）对阿胶的专属性特征肽段进行检测。色谱柱为ACQUITY UPLC BEH C₁₈（2.1 mm×150 mm，1.7 μm），流动相为0.1%甲酸溶液（A）-乙腈（B），梯度洗脱（0~3 min，96% A；3~8 min，96% A→92% A；8~10 min，92% A→50% A；10~12 min，50% A；12~12.1 min，50% A→96% A；12.1~15 min，96% A），流速0.3 mL·min^-1，柱温40℃，进样量5 μL；离子化模式为ESI+，进行多反应监测，选择阿胶特征分子离子峰m/z 380.71→374.82，m/z 380.71→493.56作为检测离子对。结果：10批阿胶样品均检出驴皮特征肽。结论：所建立的方法专属性强，可用于阿胶中驴皮源成分的鉴别。

Objective: To develop a method for identification of donkey-hide gelatin.Methods: Species-specific peptide GPTGEPGKPGDK of donkey skin was predicted by sequence comparison and was verified by NanoLC coupled with Orbitrap mass spectrometry.Samples were digested by trypsin.Ultra-high performance liquid chromatography(UPLC) coupled with triple quadruple mass spectrometry(QQQ MS) was used to detect the specific marker peptides in donkey-hide gelatins.Determination was carried out on an ACQUITY UPLC BEH C₁₈(2.1 mm×150 mm,1.7 μm) column at temperature of 40℃.The mobile phase was composed of 0.1% formic acid(A) and acetonitrile(B) with gradient elution(0-3 min,96%A;3-8 min,96%A→92%A;8-10 min,92%A→50%A;10-12 min,50%A;12-12.1 min,50%A→96%A;12.1-15 min,96%A) at a flow rate of 0.3 mL·min^-1.The electrospray ionization(ESI) source was performed in multiple reaction monitoring(MRM) mode with the transitions of m/z 380.71→374.82 and 493.56.Results: The sequence of marker peptide was GPTGEPGKPGDK.The marker peptide could be detected in all samples by this method.Conclusion: The established method is specific,which is suitable for the identification of donkey-hide gelatin.