Determination of deoxynivalenol and deoxynivalenol-3-glucoside in wheat and barley using liquid chromatography coupled to mass spectrometry: On-line clean-up versus conventional sample preparation techniques

• Evaluation of five sample preparation techniques for DON and D3G analysis.
• Inter-laboratory comparison of two analytical methods.
• Automated sample clean-up of DON and its conjugate D3G in wheat and barley flour.
• Critical assessment of on-line clean-up for determination of masked mycotoxins.

In this study, we compared the performance of conventional sample preparation techniques used in mycotoxin analyses against automated on-line sample clean-up for the determination of deoxynivalenol (DON) and its conjugated derivative, deoxynivalenol-3-β-d-glucoside (D3G), in cereal grains. Blank wheat and barley samples were spiked with DON and D3G, extracted with a mixture of acetonitrile:water (84:16, v/v) and processed by one of the following: extract and shoot, MycoSep® 227 clean-up columns, MycoSep 227 with an additional acetonitrile elution step and centrifugal filtration, followed by analysis with liquid chromatography tandem mass spectrometry. Based on method performance characteristics and poor recoveries (<30%) obtained for the polar D3G with some techniques, the extract and shoot approach was chosen for the inter-laboratory method comparison study. Thus, the same spiked samples were analysed in parallel by another laboratory with an in-house validated on-line sample clean-up method, utilising TurboFlow™ chromatography coupled to high resolution mass spectrometry. Method validation was performed by determination of specificity, linearity, recovery, intra-day precision and the limits of detection and quantification. Matrix-matched linearity (R2 > 0.985) was established in the range of 100–1600 and 20–320 μg/kg for DON and D3G, respectively. Average recoveries (%RSD) were acceptable with both methods for wheat and barley, ranging between 73% and 102% (3–12%) for DON and 72% and 98% (1–10%) for D3G. The benefit of using automated sample clean-up in comparison to extract and shoot is the ability to inject directly pure extracts into the mass spectrometer, offering faster analyses and improved sensitivity with minimum system maintenance.