Rebekah L. Sayers, Lee A. Gethings, Victoria Lee, Anuradha Balasundaram, Philip E. Johnson, Justin A. Marsh, Antonietta Wallace, Helen Brown, Adrian Rogers, James I. Langridge, E. N. Clare Mills

Peanut is an important food allergen but cannot currently be reliably detected and quantified in processed foods at low levels. Three mg protein/Kg is increasingly being used as a reference dose above which precautionary allergen labeling (PAL) is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred at 0, 3, 10 or 50 mg/Kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionisation efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series performed, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimised MRM method. Whilst other peptide targets were detected with three transitions at the 50 mg/Kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could quantify reliably, and only in the chocolate dessert at 10 mg/Kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using simple extraction methodology.