Metal ions can be analyzed by ESI‐MS with chelating agents. However, matrix effect and the marginal difference in atomic weight of the isotopes of some metal ions make the analysis of a metal ion mixture not feasible. In this paper, we present the use of IP‐HPLC in conjunction with a metal ion chelating agent to analyze the metal ion mixtures. Hydrophobic (±)‐trans‐1,2‐cyclohexylenedinitrilotetraacetic acid (CDTA) was used to chelate 16 metal ions. The chelates were then separated by IP‐HPLC followed by ESI‐MS analysis. For IP‐HPLC, different combinations of primary amines and their salts were employed as the ion‐pairing reagent in eluent on a high‐pH resistant C‐18 column under basic conditions. For ESI‐MS, Q‐Exactive Focus system was used. Among the metal ions, the results indicated that Al3+ showed the lowest LOD (3.88 × 10−3 μM) while Zn2+ showed the highest (0.227 μM). The sensitivity was close to that of ICP‐AES. The coefficients of determination (R2) were greater than 0.995 except for Sr2+ (R2 = 0.9899), indicating the high accuracy of the method. Finally, this method was used to analyze the black soybean samples from two different sources. Eight metal ions were successfully identified with simultaneous detection of sugars, organic acids, and nucleotides.

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IntroductionThe reason for the neutral loss of 44 Da in catechin tandem mass spectrometry analysis is not yet clear. Past studies used low‐resolution mass spectrometers, which made isobaric ions easily misjudged or ignored. One of the important fragment ions [(epi)Catechin‐H+‐44]− is usually determined to be derived from the loss of C2H4O (MW = 44.0262) at the C‐ring of catechin anion. However, in the results of high‐resolution mass spectrometry, this ion is m/z = 245.0808, which does not meet the expected theoretical value (m/z = 245.0454). Because this ion should be the parent ion losing CO2 (MW = 43.9898), not C2H4O. Therefore, it is necessary to re‐deduce the structure and generation path of fragment ions.MethodIn this study, high‐resolution mass spectrometry was used to analyze and compare the mass fragmentation differences of phenol, phloroglucinol, and three dihydroxybenzene isomers to establish the source of CO2‐neutral loss of catechins.ResultIt was found that both phloroglucinol and resorcinol, which have the same structure as the A‐ring of catechin, can generate [M‐H+‐CO2]− fragment ions, while phenol, hydroquinone, and catechol are not.ConclusionsIt is demonstrated that the part of catechin anion contributing to CO2 neutral loss is the A‐ring rather than the B‐ring.