A Rapid Size-Exclusion Solid-Phase Extraction Step for Enhanced Sensitivity in Multi-Allergen Determination in Dark Chocolate and Biscuits by Liquid Chromatography–Tandem Mass Spectrometry

Enhanced sensitivity for the simultaneous determination of five nut allergens in biscuit and in dark chocolate complex matrices was obtained by introduction of a rapid size-exclusion solid-phase extraction-based step before liquid chromatography–electrospray ionization-tandem mass spectrometry (LC-ESI-MS2) analysis. A very fast and efficient separation (<12 min) of marker peptides with selected reaction monitoring detection was obtained. Limits of detection in the 0.1–1.3 mg nut/kg and 5–15 mg nut/kg ranges for biscuit and dark chocolate samples as well as high recoveries (84(±6)–106(±4)% for biscuits and 98(±5)–108(±6)% for dark chocolate) proved the excellent capabilities of the exploited sample treatment method combined with the LC-MS2 analysis. Good precision in terms of intra- and inter-day repeatability was calculated, being always lower than 19 % (n=75). Linearity was demonstrated up to four and three orders of magnitude for biscuit and dark chocolate, respectively. Finally, the validated method was successfully applied to the investigation of hidden nut trace allergens in commercially available biscuits and chocolates of different brands aiming to ascertain possible discrepancies between allergen content and food allergen labelling.