LC-MS-based metabolomics combined with chemometrics for revealing chemical markers used to discriminate Arnebia euchroma from Arnebia benthamii.

Arnebia euchroma (AE) is broadly recognized as the dominating botanical source of Arnebiae Radix, a widely-used Chinese herbal medicine. However, it is frequently adulterated with the morphologically similar but pharmacologically and economically inferior Arnebia benthamii (AB). Such adulteration poses significant risks to both the therapeutic efficacy and safety in AE applications. Although our previous study employed near-infrared spectroscopy to differentiate AE from AB, the underlying disparities in their chemical constituents remain insufficiently elucidated, thereby impeding the development of robust quality evaluation criteria. To bridge this gap, the present study adopted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, with an integrated use of chemometric analysis, aiming to identify discriminatory chemical markers, and further, establish a quantitative method for precise discrimination between AE and AB. A total of 14 batches of AE and 7 batches of AB samples were collected, whose metabolite profiles were then comprehensively characterized using ultra-high-performance liquid chromatography coupled with quadrupole-exactive orbitrap mass spectrometry (UHPLC-QE-Orbitrap-MS). In the wake of a procedure where the chemical constituents in both species were tentatively identified, unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to determine the discriminatory chemical markers, followed by a validated high-performance liquid chromatography (HPLC) method for quantifying these markers. The results revealed that a total of 93 chemical compounds were identified in AE and AB samples, comprising 51 naphthoquinones, 13 phenols, 5 flavonoids, 22 organic acids, and 2 other compounds. No evident differences were observed in the distribution of the compound classes between the two species. Through chemometric analysis, six chemical markers were eventually identified, and further confirmed based on the corresponding authentic standards, as the discriminators for differentiating AE from AB samples - β-hydroxyisovalerylshikonin, acetylshikonin, β-acetoxyisovalerylalkannin, isobutyrylshikonin, β,β'-dimethylacrylalkannin, and isovalerylshikonin. Their contents in AE ranged 0.01-0.07 %, 0.05-1.50 %, 0.13-0.59 %, 0.08-0.79 %, 0.42-1.84 %, and 1.28-3.82 %, respectively (total summed content: 3.20-6.93 %), while those in AB were determined as 0.01-0.03 %, 0.03-0.14 %, 0.06-0.26 %, 0.03-0.15 %, 0.04-0.94 %, and 0.18-1.10 % (total summed content: 0.36-2.43 %). The markedly higher cumulative content of these chemical markers in AE effectively distinguished it from AB. This study successfully establishes validated chemical markers and a robust quantitative analytical methodology for the precise differentiation of AE from its adulterant, thereby facilitating the improvement of AE quality assurance and the standardization of its quality assessment.