Separation, identification, and quantification of dicyclohexylformyl oxyphenisatin, an illegal additive, from a fruit and vegetable plum

• Dicyclohexylformyl oxyphenisatin was detected in a fruit and vegetable plum. • Qualitative confirmation of this analog was performed by HRMS and NMR. • An UPLC quantitative method was developed for the analysis of this analog. This study is to develop an effective method for separating, identifying, and quantifying suspected illegal additives from fruit and vegetable plums. Ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was employed to perform non-targeted screening of illegal additives from more than 10 batches of fruit and vegetable plums. A diesterified oxyphenisatin analog never detected before in foodstuffs was identified from a batch of fruit and vegetable plum. This analog was separated and purified by column chromatography, and its molecular structure was analyzed and identified using one dimension and two dimension-nuclear magnetic resonance spectra (1D and 2D NMR). After obtaining the synthesized reference standard, an UPLC-PDA detection method for the quantitative analysis of this analog in real samples was established. The results showed that the characteristic fragment ions with mass/charge ratio (m/z) of 224.07 and 196.07 appeared in the MS 2 spectrum, indicating that the analog had the skeleton of oxyphenisatin. Combining the fitted molecular formula of suspected compound, its degree of unsaturation, and the fragment ions with mass/charge ratios of 428.18, 334.14, 83.08 in the secondary mass spectrum, we speculated that the anhydride of esterified oxyphenisatin might be cyclohexanecarboxylic anhydride, which was in good consistent with NMR spectroscopic analysis. Based on these findings, the analog was eventually named dicyclohexylformyl oxyphenisatin. UPLC-PDA test results showed that the content of this analog in a batch of fruit and vegetable plum was 614.0 mg/kg. As far as we know, dicyclohexylformyl oxyphenisatin is not a food additive. Structurally, it is the product from dicyclohexylformylation of oxyphenisatin. The illegal addition of this analog to food will cause unknown harm to uninformed consumers and requires the attention of relevant departments.