摘要Anthocyanins,a unique class of flavonoids with flavylium skeletons,are valued for antioxidant prop-erties.However,distinguishing anthocyanins from co-existing flavonoids using conventional automated tandem mass spectrometry(MS)analysis methods remains challenging.This difficulty arises from low specificity of MS features and confusion of precursor ions,leading to substantial false confidence an-notations.To address it,we have developed the strategy of positive(POS)-to-negative(NEG)primary MS(MS1)intensity ratios detecting with fast polarity switching(FPS),termed FPS-POS/NEG,to determine their specific precursor ions.Moreover,we developed an automated program leveraging FPS-POS/NEG strategy(FPS_P/N)streamlining screening candidate pool with molecular networking analysis from MS1 and secondary MS(MS2),determining precursor ions with FPS-POS/NEG,and annotation with MS2.This program enables simultaneous capture of positive and negative signals in a single run and accurate determination of precursor ions for anthocyanins(5.98-9.28)and other flavonoids(-2.52 to 2.08).The underlying mechanisms were elucidated by difference in protonated and deprotonated Gibbs free en-ergy(△G)and in-source fragmentation(ISF).FPS-POS/NEG strategy was validated across a broad pH range(0.1％-2％formic acid(FA))and demonstrated high alignment accuracy(retention time difference,0.011 min)and consistency(relative standard deviation(RSD),0.38％-4.62％).Using blueberry,20 an-thocyanins(nonacylated and acylated)and 14 additional flavonoids were annotated.With two-dimensional integration of positive and negative MS1 intensities with intensity ratios,FPS_P/N pro-gram provides a novel way to identify anthocyanins from other flavonoids.We anticipate this innovative method will enhance the high-throughput qualification of anthocyanins and other flavonoids in com-plex samples.