RDA-direct targeted flavonoids profiling strategy combined with fingerprinting and chemometrics for quality evaluation of Changshan Huyou flower

Abstract

The Changshan Huyou flower (HYF) derived from the bud of Citrus changshanhuyou Y. B. Chang is rich in flavonoids and holds significant potential for food development. However, inadequate clarification of the chemical basis and lacked quality control standards hindered its further in-depth utilisation. Herein, a novel Retro Diels-Alder (RDA)-direct strategy was proposed for flavonoid subclass profiling. Through extracting characteristic ^1,3A⁺, ^1,3B⁺ and ^0,2B⁺ ions from MS/MS spectrum, the signal-to-noise ratio can be improved up to 500 times, resulting in higher capacity of identification. Combined with relative retention time, characteristic diagnostic fragment ions, literature data and standard substance comparison, flavonoid subclass skeleton was unambiguously ascertained, with a total of 23 flavonoids (15 first reported) identified from HYF. Furthermore, quality of HYF was evaluated by high-performance liquid chromatography fingerprinting and integrated chemometrics. The similarity (> 0.944) indicated the stable quality of 20 HYF batches. Four potential quality markers including neoeriocitrin, naringin, hesperidin and neohesperidin were screened and quantified with high sensitivity (LOD, 0.039–0.442 μg/mL), satisfying precision and repeatability (RSD < 2.88 %), and pinpoint accuracy (recovery, 97.72 %–100.44 %). These markers exhibited high content levels ranging from 0.133 %–1.479 %, 3.637 %–5.146 %, 0.021 %–1.422 % and 4.978 %–9.756 %, which were closely linked to the quality of HYF that fluctuated at different growth stages. This research may offer a novel strategy and comprehensive guideline for quality evaluating of HYF and other herbal materials containing flavonoids.