Determination of deoxynivalenol and its major conjugates in cereals using an organic solvent-free extraction and IAC clean-up coupled in-line with HPLC-PCD-FLD.

A method for the determination of deoxynivalenol (DON) and its major conjugates in cereals was developed including an immunoaffinity column (IAC) clean-up coupled in-line with high-performance liquid chromatography, post-column derivatisation and fluorescence detection. An IAC for DON with cross-reactivity to 15-AcDON, 3-AcDON and DON-3-G enabled this approach. The isolated analytes were introduced into the chromatographic system without aliquotation employing the hot water elution technique, resulting in the desired low LOQ values for monitoring these analytes in cereals. The absence of any organic solvent during sample preparation in combination with an in-line IAC clean-up renders the method simple, fast, and environmentally friendly. Special attention was paid to inherent IACs properties such as cross-reactivity, analytes' competition and capacity. The method was applied to determine DON and its major conjugates in barley, wheat and maize in the range of 10-1000 µg kg-1 of DON, 10-300 µg kg-1 of DON-3-G and 15-AcDON and 10-100 µg kg-1 of 3-AcDON. The apparent recoveries varied from 87% to 110% (average of 98%) and the intermediate precision was below 13.5% RSD (except for DON-3-G in wheat). Fifteen maize, wheat and barley samples were analysed revealing levels of DON conjugates that accounted from 9% to 60% of the "total DON" content (m/m). In general, the frequency and the measured mass fractions decreased in the following order: DON>DON-3-G>15-AcDON>3-AcDON.