Authentication of three main commercial Pheretima based on amino acids analysis

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.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics

“…Ultrapure water (18.2 MΩ·cm at 25 °C) used in this study was purified using a Millipore Alpha-Q water purification system (Millipore, Billerica, MA, USA). We purchased 36 batches of Pheretima from a herb market (Bozhou, China), including 11 batches of PA, 12 of PV, and 13 of MM, which were macroscopically and microscopically identified by Dr. De-an Guo from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China), and also examined with DNA barcoding and signature tryptic peptides [ 5 , 7 ]. Detailed information on the study samples is summarized in Table S1 .…”

“…Additionally, there are several sympatrically distributed related species with similar morphological characteristics, making it difficult to distinguish authentic medicines from adulterated species [ 6 ] as well as to identify the source of Pheretima in CPMs. Although much work has been done on the authentication of Pheretima species, such as amino acids [ 7 ], endogenous small molecules [ 1 ], tryptic peptides [ 5 , 8 ], DNA barcoding [ 9 ], and biological source consistency evaluation [ 6 ], a viable approach to identify Pheretima species in CPMs is still lacking.…”

High-Performance Thin-Layer Chromatography Coupled with Single Quadrupole: Application the Identification and Differentiation of Rehmanniae Radix and Its Different Processing Products from Raw Materials to Commercial Products

“…Although methods like HPLC-UV , and TLC , are widely accepted for plant authentication, they face annoying obstacle in throughput and accuracy, owing to the tailored elution program for each plant. Newer techniques like UPLC-Orbitrap MS, , UPLC-Qtof MS, , and UPLC-QqQ MS , have demonstrated their ability to address these issues; however, these technologies not only require expensive instruments and long analysis time but also result in solvent waste and environmental pollution. In our previous studies, UPLC-QDa MS has demonstrated its affordability and wide availability in the authentication of herbal prescriptions, which, however, required respective LC separation for every sample.…”

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as “Guang Dilong”

“…Nevertheless, while the alpha taxonomic classification of Megascolecidae in south China is still ongoing, information on the sympatric distribution of multiple cryptic species 32 and relative taxa abundance is still too limited to inform the selection of suitable collection or breeding sites. On the other hand, although multiple methods were developed for the authentication of Amynthas species 3 , 33 , 34 , 35 , 36 , so far there is no reasonable biological composition assessment approach suitable for batch consistency evaluation of “Guang Dilong”.…”