192: Comparative Analysis of ELISA and UHPLC-MS/MS for Quantification of Ergot Alkaloids in Wheat: Cross-Reactivity, Matrix Effects, and Analytical Bias

Introduction

Spring wheat and durum wheat are essential crops for Canada, contributing approximately $10.2 billion annually. Predominantly cultivated in Alberta, Saskatchewan, and Manitoba, these grains are critical to global trade, with 65% of wheat and 90% of durum exported. However, ergot contamination, caused by the fungal pathogen Claviceps purpurea, has emerged as a growing concern, particularly in Western Canada over the past decade. Ergot alkaloids (EAs), toxic compounds produced by this fungus, necessitate robust analytical methods to ensure food safety.

Methods

This study compared the performance of a commercially available enzyme-linked immunosorbent assay (ELISA) kit and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for quantifying EAs in wheat. Additionally, it assessed the ELISA kit’s cross-reactivity (CR) towards 12 individual EAs in both solvent and wheat matrix to evaluate the effects of matrix interference. The ELISA kit was validated using standard and in-house reference materials and tested for repeatability, extraction time, test portion mass, and dilution factors. CR was calculated as the ratio of IC50 values of individual alkaloids to ergotamine. Solvent and wheat matrix spiked with 12 alkaloids were analyzed, and ELISA results were compared to UHPLC-MS/MS using regression analysis, Bland-Altman plots and two-way ANOVA.

Results

The ELISA method demonstrated a strong correlation (r=0.8793) with UHPLC-MS/MS for EA. However, there appears to be a potential bias where the ELISA is under-reporting total ergot alkaloid concentrations by a factor of two (p < 0.05). CR values varied widely among alkaloids, with ergometrine showing the highest reactivity in solvent (569 times CR% vs. ergotamine) (p < 0.05) and wheat matrix (18251 times CR% vs. ergotamine) (p < 0.05).

Significance

Matrix effects significantly amplified CR for all alkaloids. While ELISA performance was not affected by test portion mass, extraction time, or dilution factors, the variability in CR values highlights the potential for certain alkaloids to influence total EA results disproportionately. This study emphasizes that while ELISA may offer a rapid and cost-effective screening method, its reliability depends on alkaloid composition and concentration range. Matrix effects, cross-reactivity, and two-fold difference must be considered to ensure accurate EA quantification, complementing the more sensitive UHPLC-MS/MS method for comprehensive grain safety analysis.

Authors: Chamali Kodikara, Dainna Drul, Nandika Bandara, Sheryl A. Tittlemier