**4. Validating food allergen methods**

While mass spectrometry methods are increasingly sensitive, there remains room for improvement. Furthermore, there subsist obstacles to the harmonization of allergen detection methods in food laboratories [85]. In April 2016, the AOAC SMPR 2016.002 guideline 'Standard method requirements for the detection and quantification of selected food allergens' was published. This guideline is the first to specify target limits for sensitivity and range of linearity, target matrices and reference materials for the analysis of allergens (egg, milk, peanut and hazelnut) in food matrices by mass spectrometry (**Table 5**).

To obtain comparable results among laboratories, it is crucial to adopt validation guidelines. The AOAC guideline, however, is not sufficiently detailed, and each laboratory tends to apply its own rules. In what follows, we compare this guideline with published methods in terms of sensitivity, range of linearity, recovery and precision.

**Sensitivity:** In the AOAC guideline, the method quantitation limit (MQL) is defined as MQL = average (blank) + 10 x s0 (blank). Laboratories, however, often use other strategies to determine the limit of quantification (LOQ), such as determining a signal-to-noise (S/N) ratio which should be higher than 10 [56, 60] or estimating an LOD and an LOQ as 3s/slope and 10s/slope, respectively, where s is the standard deviation of the blank signal (n = 10) [57]. On the other hand, the sensitivity can differ from one matrix to another. For example, in a study where cookie, ice cream and sauce were spiked with 0.5 mg milk proteins per kg, the observed S/N ratio was 26 for the cookie matrix, 83 for ice cream and 228 for sauce [85]. This also highlights the importance of a 'fit-for-purpose' description of an analytical method. Moreover, the sensitivities of developed methods should be determined on the same reference materials (MoniQa, LGC) to ensure (1) their capacity to reach the sensitivity set by the AOAC guideline and (2) an appropriate comparison of method performances.

**Linearity:** The range of linearity is set as 0.001 to 0.1% allergen contamination (10 mg to 1000 mg) of allergenic ingredients per kg) and thus does not always include the MQL (e. g. an MLQegg of 5 mg per kg). In the case of high-sensitivity methods, the coefficient of regression is determined using a lower range of concentrations [57, 71].

**Recovery**: Recovery must range from 60 to 120%. Such recovery values are hard to reach for the detection of allergens in processed samples, and recovery can only be determined by spiking food matrices with allergens. Focusing on egg, milk and peanut in spiked and incurred muffin and cereal bars, Parker et al. constructed calibration curves by spiking the matrices with allergen proteins [61]. In the case of spiked muffin, the determined recovery was 98.6% for egg peptide (GGLEPINFQTAADQAR), 87.7% for milk peptide (YLGYLEQLLR) and 100.2% for peanut peptide (SPDIYNPQAGSLK). When the muffins were baked for 48 min at 177°C, the recoveries were dramatically lower: respectively 45.2%, 75.2% and 70.2%.

**Inter- and intra-day coefficients of variation**: According to AOAC SPMR, three unknown samples should be analyzed at least seven times to determine the reproducibility of the method. Lamberti et al. determined an intra-day coefficient of variation between 5 and 20% by performing three independent extractions at two different concentrations and three injections per extract [57].

Guidelines for the validation of mass-spectrometry-based methods for allergen analysis should be more precise, like the guidelines 2002/657/EC 'Validation of residues in products of animal origin' and SANCO/12574/2013 'Residues in products of animal origin method validation procedures for pesticide residues analysis in food and feed'. In SMPR 2016.002, several details are missing:

