Objective  To search for specific peptides which can distinguish between turtle-shell gelatin and tortoise-shell gelatin, and to establish a detection method for turtle-shell gelatin mixed in tortoise-shell gelatin.

Methods  Using trypsin to cleave samples of turtle-shell gelatin and tortoise-shell gelatin, the peptide chromatogram of turtle-shell gelatin and tortoise-shell gelatin were determined using ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QQQ/MS). The peptide chromatogram were compared and analyzed to find out the molecular ions with strong response in turtle-shell gelatin, but no response or very weak response in tortoise-shell gelatin, and these molecular ions were detected by mass spectrometry multi-reaction monitoring technology, and two specific peptides of turtle-shell gelatin were obtained through specificity verification, and they were used as identification features of turtle-shell gelatin to determine the tortoise-shell gelatin sample.

Results  Two new specific peptides of turtle-shell gelatin were detected in the samples of turtle-shell gelatin, but they were not detected in the samples of tortoise-shell gelatin, deer-antler gelatin, deer-hide gelatin, ass-hide gelatin, pig-hide gelatin, cattle-hide gelatin, horse-hide gelatin, sheep-hide gelatin and camel-hide gelatin. The detection limit of this method was for tortoise-shell gelatin samples containing 0.5% turtle-shell gelatin. Eleven batches of tortoise-shell gelatin samples were tested, and turtle-shell specific peptides were detected in only one batch of samples.

Conclusion  The established method has strong specificity and high sensitivity, and can be used for the identification of turtle-shell gelatin and the detection of turtle-shell gelatin mixed in tortoise-shell gelatin.

1.中国药典2020年版. 一部[S]. 2020: 187-189, 197-198, 402.

2.卫生部颁布药品标准(中药材第一册). WS3-B-3719-98)[S]. 1998: 103.

3.蔡朔, 韩疏影, 夏浩然, 等. 基于特征肽发现与应用的胶类中药质量控制[J]. 中草药, 2022, 53(21): 6888-6899. [Cai  S, Han  SY, Xia HR, et al. Research progress on quality control of gelatinous traditional Chinese medicine based on discovery and application of specific peptides[J]. Chinese Traditional and Herbal Drugs, 2022, 53(21): 6888-6899.] DOI: 10.7501/j.issn.0253-2670.2022.21.028.

4.程显隆, 李明华, 郭晓晗, 等. 胶类药材质量问题变化情况及标准研究对策[J]. 中国现代中药, 2020, 22(7): 996-999. [Cheng  XL, Li MH, Guo XH, et al. Change of quality problem of glue crude drugs and trategy of quality standards research[J]. Modern Chinese Medicine, 2020, 22(7): 996-999.] DOI: 10.13313/j.issn.1673-4890.20200316010.

5.国家食品药品监督管理总局. 药品检验补充检验方法和检验项目批准件. 龟甲胶中牛皮源、驴皮源成分检查(2014013)[S]. 2014.

6.国家药品监督管理局. 龟甲胶中猪皮源成分检查项补充检验方法. BJY201916[S]. 2019.

7.杭宝建, 田晨颖, 陈晓, 等. 超高效液相色谱-串联质谱法测定阿胶中马、牛、羊、猪、骆驼、鹿皮源成分[J]. 色谱, 2018, 36(4): 408-412. [Hang BJ, Tian CY, Chen X, et al. Determination of the ingredients of horse, ox, sheep, pig, camel and deer skin in colla corii asini by ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2018, 36(4): 408-412.] DOI: 10.3724/SP.J.1123.2017.11002.

8.赵艳霞, 巩丽萍, 石峰, 等. 基于特征肽测定阿胶食品中的5种杂皮源成分[J]. 现代食品科技, 2022, 38(4): 234-240, 281. [Zhao YX, Gong LP, Shi F, et al. Determination of five skin ingredients in asini corii colla foods based on marker peptide[J]. Modern Food Science and Technology, 2022, 38(4): 234-240, 281.] DOI: 10.13982/j.mfst.1673-9078.2022.4.0676.

9.潘志文, 黄婉锋, 钱鑫, 等. 基于超高效液相色谱串联质谱法特征肽检测技术的阿胶鉴别与6种动物异源性成分检查[J]. 中国药业, 2024, 33(11): 79-83. [Pan ZW, Huang WF, Qian X, et al. Identification of colla corii asini and examination of six animal heterologous components based on UPLC-MS/MS characteristic peptide detection technology[J]. China Pharmaceuticals, 2024, 33(11): 79-83.] DOI: 10.3969/j.issn.1006-4931.2024.11.018.

10.程显隆, 李文杰, 张小龙, 等. UPLC-QTOF-MS结合主成分分析法用于龟甲胶、鹿角胶中添加牛皮源成分的检测研究 [J]. 药物分析杂志, 2012, 32(6): 931-935. [Cheng XL, Li WJ, Zhang  XL  ,et al. Identification of bovine-hide gelatin from glue of tortoise shell and deer-horn gelatin by UPLC-QTOF-MS and principal component analysis[J]. Chin J Pharm Anal, 2012, 32(6): 931-935.] DOI: 10.16155/j.0254-1793.2012.06.004.

11.中国药典2015年版.一部[S]. 2015: 189-190.

12.王忠合, 李晓婷, 胡文梅, 等. 超高效液相色谱-高分辨质谱法测定肉类特征肽[J]. 食品与发酵工业, 2021, 47(18): 258-266. [Wang ZH, Li XT, Hu WM, et al. Determination of peptide markers of meat species by ultra-high pressure liquid chromatography coupled with high resolution mass spectrometry[J]. Food and Fermentation Industries, 2021, 47(18): 258-266.] DOI: 10.13995/j.cnki.11-1802/ts.028030.

13.张淑霞, 田亚, 邢荣花, 等. 基于特征肽段的液相色谱质谱联用技术对核桃、杏仁露进行掺假鉴别[J]. 食品与发酵工业, 2020, 46(21): 215-222. [Zhang SX, Tian Y, Xing RH, et al. Identification the adulteration of walnut and almond drink based on marker peptides with liquid chromatography mass spectrometry[J]. Food and Fermentation Industries, 2020, 46(21): 215-222.] DOI: 10.13995/j.cnki.11-1802/ts.024604.

14.石峰, 杭宝建, 迟连利, 等. 驴皮特征肽的发现及其在阿胶鉴别中的应用[J]. 药物分析杂志, 2017, 37(12): 2272-2278. [Shi  F, Hang BJ, Chi LL, et al. Discovery of marker peptide of donkey for identification of donkey-hide gelatin[J]. Chinese Journal of Pharmaceutical Analysis, 2017, 37(12): 2272-2278.] DOI: 10.16155/j.0254-1793.2017.12.23.

15.巩丽萍, 杭宝建, 迟连利,等. 马皮特征肽的发现及其在阿胶中马皮源成分检测中的应用[J]. 药物分析杂志, 2018, 38(2): 364-369. [Gong LP, Hang BJ, Chi LL et al. Identification of the horse skin marker peptide for test of horse-hide gelatin in donkey-hide gelatin[J]. Chinese Journal of Pharmaceutical Analysis, 2018, 38(2): 364-369.] DOI: 10.16155/j.0254-1793.2018.02.27.

16.程显隆, 陈佳, 李明华, 等. 特征肽段检测技术用于胶类药材专属性鉴别方法研究[J]. 中国药学杂志, 2015, 50(2): 104-108. [Cheng XL, Chen J, Li MH, et al. Identification study of glue medicines[J]. Chinese Pharmaceutical Journal, 2015, 50(2): 104-108.] DOI: 10.11669/cpj.2015.02.003.

17.苏雪媚,赖林城,尹淑华,等. 基于UPLC-MS技术和代谢组学对胶类中药的比较分析[J]. 中国药师, 2021, 24(6): 1025-1029. [Su XM, Lai LC, Yin SH, et al. Comparative analysis of gum traditional Chinese medicine based on UPLC-MS and metabonomics[J]. China Pharmacist, 2021, 24(6): 1025-1029.] DOI: 10.3969/j.issn.1008-049X.2021.06.007.