FPS_P/N: A two-dimensional mass spectrometry utilization program with precursor ion determination for accurately distinguishing anthocyanin from other flavonoids

Anthocyanins, a unique class of flavonoids with flavylium skeletons, are valued for antioxidant properties. 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 annotations. 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 i) screening candidate pool with molecular networking analysis from MS¹ and secondary MS (MS²), ii) determining precursor ions with FPS-POS/NEG, and iii) 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 to 9.28) and other flavonoids (-2.52 to 2.08). The underlying mechanisms were elucidated by difference in protonated and deprotonated Gibbs free energy and in-source fragmentation (ISF). FPS-POS/NEG strategy was validated across a broad pH range (0.1%-2% formic acid) and demonstrated high alignment accuracy (retention time difference, 0.011 min) and consistency (relative standard deviation (RSD), 0.38%-4.62%). Using blueberry, 20 anthocyanins (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 program 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 complex samples.