VALIDATION OF MULTI-ANALYTE METHODS FOR THE DETERMINATION OF QUINOLIZIDINE ALKALOIDS IN FOOD AND FEED WITH LC-MS/MS – RESULTS OF A COLLABORATIVE METHOD VALIDATION STUDY

Quinolizidine alkaloids (QA) are toxic secondary plant metabolites found in lupine species (Lupinus spp.) that offer protection against competing plants, herbivores, insects or pathogens. The QA containing seeds of certain lupine species (L. albus, L. angustifolius, L. luteus) are used for food and feed production (e. g. flour, grist) and are gaining increasing attention as ingredient in substitute food products (e. g. lupine-based coffee, lupine-based meat substitute, lupine drink). Furthermore, the transfer of QAs from lupine containing feed into cow milk was shown to occur. QAs are toxicologically relevant for animals and humans. Until now, maximum levels for QA in food and feed are not specified by European legislation. In order to protect health of animals and humans and to set maximum levels in the future, more comprehensive data about the occurrence of QA in food and feed is necessary. For this and for future monitoring, reliable and harmonized analytical methods are required. Therefore, following the BfR Opinion on ‘Risk assessment of the occurrence of alkaloids in lupin seeds’ from 2017 and EFSA’s ‘Scientific opinion on the risk for animal and human health related to the presence of quinolizidine alkaloids in feed and food, in particular in lupins and lupin-derived products’ from 2019, the BfR developed two analytical methods to determine 11 QA in (1) food and feed with low water content and (2) food with higher water content. After solid-liquid extraction (1) or liquid-liquid extraction (2), protein precipitation as well as defatting, sample extracts are analysed by reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In an interlaboratory study with 18 laboratories from different European states, the performance characteristics of both multi-analyte method protocols were assessed. The study was designed as a three-step approach: one training part for each of the analytical methods and one main validation part. The validation part was designed as a double-blind study with five ground samples of lupine seed-based feed and food with a low water content (lupine coffee, lupine meal, lupine slices, lupine mixture, lupine feed) and four samples of food with higher water content (milk, yogurt, lupine drink, cream). The assessed within-laboratory precision (relative standard deviation for repeatability, RSDr) was < 20 % for all analyte-matrix combinations. With a few exceptions, the assessed between-laboratory precision (relative standard deviation for reproducibility, RSDR) was < 25 %. The poster presentation will focus on the results of the collaborative method validation study.