Molecularly imprinted polymer coupled to UHPLC-MS/MS for the analysis of phomopsins in lupin samples

The demand for plant-based protein sources in the food industry has significantly increased in recent years, leading to the introduction of legume-based products as meat substitutes. However, concerns regarding food quality have emerged, particularly related to the presence of mycotoxins. This study addresses the need for the sensitive detection of phomopsins (PHOs), a class of peptide-based toxins. A selective extraction method using molecularly imprinted polymer (MIP) coupled with ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) was focused on the most toxic Phomopsin A (PHO-A). A rapid ultrasonochemical synthesis of MIP (5 min) was proposed and its performance was optimized in response to various factors, including the choice of dummy template and the selection of the monomer. The methacrylic acid-vinyl pyridine (MAA-VP) MIP exhibited high selectivity and affinity for PHO-A. The method was tested in lupin samples and the validation, according to SANTE/11312/2021 international guidelines, gave excellent recovery (80-90 %), low matrix effects, and high accuracy and precision. Real samples analysis confirmed the presence of PHO-A in artificially fungal inoculated lupins, with levels ranging from 0.377 to 0.576 mg kg^-1. In order to identify further PHOs, a semi-untargeted approach using multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) was developed. PHO-B, PHO-D, PHO-E and PHO-P, rarely previously reported in lupin matrix, were tentatively identified. This study accounts for the effectiveness of MIP-based extraction coupled with UHPLC-triple quadrupole with linear ionic trap-MS/MS (UHPLC-QqQ-LIT-MS/MS) for quantification of PHO-A and putative detection of other PHOs, offering a promising method for investigating this class of toxins in food.