Identification of cultivar-specific markers for Zhangtouhong citri reticulatae pericarpium by integrated GC-MS, electronic nose and HPLC profiling

Abstract: Background: Most quality control frameworks and aging studies on Citri Reticulatae Pericarpium (CRP) are based on mainstream cultivars, particularly Chachi mandarin. Zhangtouhong, a local citrus from Jiangxi Province, has received little systematic attention regarding its cultivar-specific traits and aging-related chemical changes; applying Chachi-derived criteria may thus bias its quality assessment. Objectives: Aging-related changes in key constituents were compared between Zhangtouhong and Chachi CRP to identify shared aging features, cultivar-specific traits and candidate cultivar-specific markers for Zhangtouhong quality evaluation. Methods: Samples of both cultivars aged for 3, 5 and 10 years were analyzed. Five core volatile oil components were quantified by volatile oil determination with GC-MS; holistic odor fingerprints were captured by electronic nose; shared volatile profiles were characterized by HS-GC-MS; and six representative flavonoids were quantified by HPLC. Cultivar, aging year and interaction effects were evaluated using two-factor linear models, Spearman correlation, PCA, PLS-DA, OPLS-DA and PERMANOVA. Candidate cultivar-specific markers were screened through an integrated multi-criteria approach. Results: Volatile oil yields remained relatively stable across aging, with D-limonene dominating in both cultivars, whereas myrcene and γ-terpinene showed divergent aging patterns. Electronic nose fingerprints clearly separated the two cultivars, with eight sensors contributing most. HS-GC-MS identified 40 shared volatiles, with alkenes forming the common aromatic backbone. Integrated screening yielded 16 cultivar-specific volatile markers, among which methyl 2-(methylamino) benzoate, linalool and α-thujene were prioritized. Among flavonoids, hesperidin was strongly associated with aging duration, while nobiletin, 5-demethylnobiletin and sinensetin showed high discriminative power for cultivar differentiation. Conclusion: Zhangtouhong and Chachi CRP exhibit both common aging-related chemical changes and marked cultivar-dependent differences. CRP quality markers should therefore be distinguished as candidate common aging markers and cultivar-specific markers. Systems built solely on Chachi cannot fully capture Zhangtouhong characteristics, underscoring the need for cultivar-adapted quality evaluation methods.