*3.2. Determination of Patient Serum Binding to Egg and Milk Allergens Contained in Biscuits*

The mean total soluble protein recovery was 7.7 mg per gram. Immunoblots did not show reactions between serum of 18 patients and proteins contained in biscuits (Figure 2). In biscuits from patients 3, 6, 12, 15, 18, 20, 22, 23, 25, 26 and 29, non-specific signals were detected accounting for 30.12% of the positive control.

SD: standard deviation; sIgE: specific IgE; c-SPT: commercial skin prick test; ffSPT: fresh-food skin

Five out of the 29 included patients, were not exposed to confirmatory food challenges due to recent anaphylaxis (three in the group under two years, one in the group 5– 13 years and one in the group 13–18 years). The remaining 24 underwent DBPCFCs with baked milk (15 cases), baked egg (five cases), or both (four cases). From these challenges, the mean thresholds of reactivity were 116.3 (± 107.6) and 128.3 (± 96.7) mg protein for

No patient presented any symptoms at any challenge time during OFCs with the tested product. Equally negative were all skin tests: no patient resulted SPT-positive to

The mean total soluble protein recovery was 7.7 mg per gram. Immunoblots did not show reactions between serum of 18 patients and proteins contained in biscuits (Figure 2). In biscuits from patients 3, 6, 12, 15, 18, 20, 22, 23, 25, 26 and 29, non-specific signals

*3.2. Determination of Patient Serum Binding to Egg and Milk Allergens Contained in Biscuits* 

were detected accounting for 30.12% of the positive control.

prick test; n.d.: not determined.

milk and egg, respectively.

Magretti.

**Figure 2.** Representative immunoblots. Proteins extracted from two doses (M1 and M2) of biscuit tested on patients (Pt) 19, 21, and 8, blotted with the corresponding patient serum. Proteins extracted from biscuits containing egg and milk ingredients (BC) were loaded as positive control. MW: protein **Figure 2.** Representative immunoblots. Proteins extracted from two doses (M1 and M2) of biscuit tested on patients (Pt) 19, 21, and 8, blotted with the corresponding patient serum. Proteins extracted from biscuits containing egg and milk ingredients (BC) were loaded as positive control. MW: protein molecular weight standards.

### molecular weight standards. *3.3. Determination of Cow's Milk and Hen's Egg Allergen Levels in Commercial "Milk and Egg Free" Biscuits by Targeted Mass Spectrometry Methods*

*3.3. Determination of Cow's Milk and Hen's Egg Allergen Levels in Commercial "Milk and Egg Free" Biscuits by Targeted Mass Spectrometry Methods*  Using method 2.A, a mean of ~10 mg of protein was extracted from 1 g of biscuits. About 4 mg of each protein extract was subjected to reduction, alkylation and protein digestion. In method 2.B a different sample preparation workflow was used. In both meth-Using method 2.A, a mean of ~10 mg of protein was extracted from 1 g of biscuits. About 4 mg of each protein extract was subjected to reduction, alkylation and protein digestion. In method 2.B a different sample preparation workflow was used. In both methods, peptides from milk αS1-casein and egg ovalbumin allergens were used as marker proteins by MRM analysis.

ods, peptides from milk αS1-casein and egg ovalbumin allergens were used as marker proteins by MRM analysis. To evaluate the sensitivity of the method, matrix-matched calibration curves, obtained by fortifying biscuits with increasing amounts of allergenic ingredients to cover the range 5–50 mg of allergens/kg of matrix, were built up for each milk and egg allergen To evaluate the sensitivity of the method, matrix-matched calibration curves, obtained by fortifying biscuits with increasing amounts of allergenic ingredients to cover the range 5–50 mg of allergens/kg of matrix, were built up for each milk and egg allergen marker selected. By interpolating the data of the matrix matched calibration curve, the linearity over the concentration range investigated for all the markers monitored was verified, with a correlation coefficient always better than 0.98. Finally, limit of detection (LOD) and quantification (LOQ) values were, respectively, calculated as 3 and 10 times the standard deviation of the line intercept divided by the slope of the calibration equation for both methods.

Table 3 reports the calculated LODs for methods 2.A and 2.B depending on the instrumental platform used and the selected transitions monitored. Using method 2.A, we were able to detect traces of milk and egg allergens at the lowest range of 0.6 µg tot prot/g matrix for milk and egg. Method 2.B was able to quantify milk and egg allergen in biscuits at the lowest level 0.1 and of 0.3 µg tot prot/g matrix, respectively.

Once the methods had been optimized, they were applied to Magretti to verify their allergen contamination at the lowest level offered by the method. The two methods showed a good agreement of the results obtained. None of the analyzed samples was found contaminated with milk and egg according to the sensitivity offered by the MS method (Table 3).


**Table 3.** Results of MRM experiments referred to matrix-matched calibration curves produced in fortified biscuits (route 2.A and route 2B) employing synthetic peptides for quantification (in route 2.B).

*m/z*: mass-to-charge ratio of the peptide ion and product; LOD: limit of detection calculated as 3 × SD of the intercept calculated on the matrix matched calibration curve and whose goodness of the linear interpolation is reported by R2.
