ISSUE FOCUS 44 FEED & ADDITIVE MAGAZINE May 2023 the ELISA test was validated by Randox Food Diagnostics, WFSR and Nutrilab as an independent third party. The collaboration resulted in a useful ELISA test for detecting Ergot Alkaloids in rye, barley, wheat, oats and spelt. RESEARCH DESIGN The project was divided into two phases. In phase 1 (2021, see figure 1 above), the grain traders supplied a total of 70 samples for five different grains. These samples were sanitised by manually removing all sclerotia present, following thereafter, the cereals were ground into a powder. EAs were added up to the maximum permitted limit concentrations according to EU 2021/1399. The EA levels were measured with a validated LC-MS/MS method and this data was used for the first validation of the newly developed ELISA. New grain samples were delivered in phase 2 (2022). These samples were ground without cleaning and divided into three portions. Aliquots were analysed independently for the presence of EAs. In this phase, the ELISA was performed by the kit developer (Randox Food Diagnostics) and by an independent laboratory (Nutrilab). RESULTS Figure 2 below shows results from phase 1. The squares represent the EA content of the cleaned blanks as measured by the LC-MS/MS method and the ELISA. The ELISA gives a slightly higher background than the LC-MS/MS. The dots represent the results of the grain samples spiked with Grain samples Collected in 2021 Sclerotia removal Milling Spike with EAs Quantitative analysis with LC-MS/MS Quantitative analysis with LC-MS/MS ELISA by independent lab Grain samples Collected in 2022 Milling First validation ELISA Second validation ELISA Rye Wheat, oats, barley, spelt Validated at 250 µg/kg Validated at 150 µg/kg (ash content > 900 mg/100 g) EA concentration (µg/kg) EA concentration (µg/kg) 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Samples 300 200 100 0 200 150 100 50 0 Samples LC-MS/MS Blanks ELISA Spikes ELISA Spikes LC-MS/MS Spikes ELISA Blanks ELISA Blanks LC-MS/MS Blanks LC-MS/MS Spikes 1 2 3 4 5 6 7 8 910 11 121314151617181920 Figure 2 Figure 1
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