"Precise-Practical-Rapid" strategy for discriminating closely related cultivars: insights from chrysanthemum varieties.

The Chrysanthemum genus comprises six predominant varieties (Chuju, Boju, Gongju, Hangju, Huaiju, Jinsihuangju). However, their highly similar morphological and chemical characteristics make accurate discrimination challenging. To address this, a 'Precise-Practical-Rapid' integrated strategy for origin discrimination was developed, incorporating LCHRMS metabolomics (precise), polyphenol-specific HPLC fingerprinting and quantitative analysis (practical), and direct-infusion mass spectrometry and infrared spectroscopy methods (rapid). LC-QTOF MS-based metabolomics identified 221 compounds across six varieties, successfully distinguished all geographical origins, and revealed 13 key discriminatory markers. HPLC-based polyphenol-specific fingerprints and quantitative profiles achieved clear origin clustering, with a minimal four-marker panel (luteolin, rutin, 3-caffeoylquinic acid [3-CQA], and 5-CQA) retaining full discriminatory power. DI-QDa MS and FT‑IR spectroscopy enabled rapid (2 min per sample) high-throughput screening, with t‑SNE, PLS‑DA, and hierarchical cluster analysis achieving successful discrimination. Collectively, these platforms provided complementary resolution tiers: LC-QTOF MS for discovery and traceability in complex matrices, HPLC for routine quality control with reference-standard quantification, and DI-QDa or FT‑IR for on-site screening. This multi-platform comparison establishes a practical decision framework for Chrysanthemum authentication and offers a transferable pipeline for origin verification of other botanicals.