Abstract
Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.
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Abbreviations
- AAs:
-
Amino acids
- UPLC-QqQ-MS:
-
Ultra-high performance liquid chromatography coupled with triple quadruple mass spectrometry
- MRM:
-
Multiple reaction monitoring
- Fmoc-Cl:
-
9-Fluorenylmethoxycarbonyl chloride
- TCMs:
-
Traditional Chinese medicines
- ChP:
-
Chinese pharmacopoeia
- IS:
-
Internal standard
- ESI:
-
Electrospray ionization
- LOD:
-
Limit of detection
- LOQ:
-
Limit of quantification
- PCA:
-
Principal component analysis
- OPLS-DA:
-
Orthogonal partial least squares discrimination analysis
- CV:
-
Cone voltage
- CE:
-
Collision energy
- Rt:
-
Retention time
- VIP:
-
Variables importance for the projection
- DTT:
-
Dithiothreitol
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Acknowledgements
The project was funded by the National Science Foundation for Young Scientists of China (81803716), the Science Foundation of Youths of Jiangsu Province of China (BK 20170738), the Research Fund for Post Doctorate of Jiangsu Province of China (1701055A), and “the Double First-Class” University Project of China Pharmaceutical University (CPU2018GF05).
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This study involved the chemical analyses of animal-derived traditional Chinese medicine collected from the herb market. No approval of animal use protocols was required.
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Liu, Q., Bi, Q. & Tan, N. Authentication of three main commercial Pheretima based on amino acids analysis. Amino Acids 53, 1729–1738 (2021). https://doi.org/10.1007/s00726-021-03043-2
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DOI: https://doi.org/10.1007/s00726-021-03043-2