Comprehensive identification of Vitex trifolia fruit and its five adulterants by comparison of micromorphological, microscopic characteristics, and chemical profiles

Abstract Prevention against the adulteration of traditional Chinese medicine in an accurate way has been long exploring. Vitex trifolia fruit (VTF), as a widely used analgesic in East Asia, has frequently been found to be adulterated with five adulterants, namely Vitex cannabifolia fruit (VCF) (Fam. Verbenaceae), Vitex negundo fruit (VNF) (Fam. Verbenaceae), Piper cubeba fruit (PCF) (Fam. Lauraceae), Euphorbia lathyris seed (ELS) (Fam. Euphorbiaceae), and Vaccinium bracteatum fruit (VBF) (Fam. Ericaceae). In this study, the methods of micromorphological identification, microscopic identification, and chemical analysis were combined to distinguish VTF from its five adulterants comprehensively. As a result, the micromorphological features in terms of fruit or seed epidermis were photographed by stereomicroscopy firstly. Secondly, the microscopic characteristics of various herb powders were captured under light microscopy. Thirdly, 33 nonvolatile components and 124 volatile components in VTF were identified by ultra‐performance liquid chromatography coupled with Orbitrap mass spectrometry (UPLC‐Orbitrap‐MS) and comprehensive two‐dimensional gas chromatography hyphenated with mass spectrometry (GC × GC–MS), respectively. Furthermore, betulinic acid, persicogenin, and the volatile 4‐(2,2,6‐trimethyl‐bicyclo[4.1.0]hept‐1‐yl)‐butan‐2‐one were screened out to be the specific markers of VTF distinctive from the adulterants. Collectively, VTF and its five adulterants were distinguished successfully by the comparison of micromorphological, microscopic characteristics, and chemical profiles.