An analytical workflow including mass spectral library, generic sample preparation, chromatographic separation, and analysis by high-resolution mass spectrometry (HRMS) was developed to gain insight into the occurrence of plant toxins, mycotoxins and phytoestrogens in plant-based food. This workflow was applied to 156 compounds including 90 plant toxins (pyrrolizidine alkaloids, tropane alkaloids, glycoalkaloids, isoquinoline alkaloids and aristolochic acids), 54 mycotoxins (including ergot alkaloids and Alternaria toxins) and 12 phytoestrogens (including isoflavones, lignans and coumestan) in plant-based protein ingredients, cereal and pseudo-cereal products. A mass spectral library was built based on fragmentation spectra collected at 10 different collision energies in both positive and negative ionisation modes for each toxin. Emphasis was put on a generic QuEChERS-like sample preparation followed by ultra-high-pressure liquid chromatography using alkaline mobile phase allowing the separation of more than 50 toxic pyrrolizidine alkaloids. HRMS acquisition comprised a full-scan event for toxins detection followed by data-dependent MS2 for toxin identification against mass spectrum. Method performance was evaluated using fortified samples in terms of sensitivity, repeatability, reproducibility and recovery. All toxins were positively identified at levels ranging from 1 µg kg⁻¹ to 100 µg kg⁻¹. Quantitative results obtained by a standard addition approach met SANTE/12682/2019 criteria for 132 out of 156 toxins. Such a workflow using generic, sensitive and selective multi-residue method allows a better insight into the occurrence of regulated and non-regulated toxins in plant-based foods and to conduct safety evaluation and risk assessments when needed. Graphical Abstract

The consequences of climate change along with diverse food regulations and agricultural practices worldwide are complexifying the occurrence and management of chemical contaminants in food. In this context, we present an ultra-high-performance liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach for the simultaneous identification and quantitation of over 1100 pesticide residues, mycotoxins, and plant toxins in cereals and fruits and vegetables. Analytical conditions were optimized to maximize the scope of the targeted molecules, the reliability of compound identification, and quantification performance within a single method. The method was further transferred and validated in another laboratory to assess its ruggedness. Validation according to the SANTE 11312/2021v2 guidelines showed that 92% and 98% of the molecules fulfill the quantification criteria at the lowest validated level in the cereals and fruits and vegetables groups, respectively. Analysis of fifteen certified reference materials led to a 96% satisfactory rate of z-scores confirming method’s competitiveness. Furthermore, the occurrence of these contaminants was studied in 205 cereals and grains samples collected worldwide. The low µg/kg quantification limits make this LC-HRMS method a valuable tool to ensure compliance toward regulations and to screen for non-regulated substances for which occurrence data are crucial for an appropriate risk evaluation.

Two methods based on a modified QuEChERS sample preparation and either LC coupled to atmospheric pressure ionisation and high-resolution MS or GC coupled to electron ionisation and tripled quadrupole MS have been assessed for the quantification of folpet and phthalimide in tea and other dry herbal infusions. Both methods have been fully validated in green tea and further checked in black tea, verbena and rooibos, and they performed according to the SANTE/11813/2017 criteria at the target LOQ concentration level (50 µg/kg). These methods allow the accurate quantification of folpet in the selected matrices according to the new EU residue definition, which includes phthalimide. Phthalimide is the main metabolite and degradation product of folpet, although according to recent studies, it could be generated from different sources than folpet breakdown, such as food processing or analysis by GC.

Indole-3-acetic acid (IAA) is a natural plant hormone which can also be used as a plant growth regulator. However, its usage on crops is not permitted in the EU, with a maximum residue limit (MRL) currently set at 0.1 mg kg^-1 for all food commodities. As available data on the occurrence of IAA in food commodities are limited, this work aims at evaluating the amount of IAA in important food commodities such as coffee and cereals, but also selected processed ingredients such as cocoa and malt. In total, 133 samples representing 18 different food commodities were analysed for the occurrence of free IAA using a liquid chromatography high resolution mass spectrometry method. IAA was detected in all samples except malt powders and tomatoes. The analysed crops were either grown under conventional, organic, or strictly controlled (i.e. excluding the use of IAA as pesticide/plant growth regulator) farming conditions. No significant differences in the amounts of IAA were found in crops grown under the three different farming conditions. A high percentage of the samples (63%) showed levels of free IAA above the EU MRL. We therefore conclude that the natural abundance of IAA was not properly assessed prior to the establishment of some current EU MRLs. Based on the natural occurrence of IAA in a wide range of foods; we question the need for an MRL for IAA in plant-based foods.