**5. Conclusion**

shown that long isotope-labelled peptides allow recoveries of 97.2–102.5% for α-lactalbumin and 99.5–100.3% for β-casein in the quantification of human milk [87]. This strategy is a good compromise between isotope-labelled proteins and peptides. It allows correcting both for the matrix effect and for digestion-step effects, unlike the use of isotope-labelled peptides.

In conclusion, these studies show that using an isotope-labelled protein or a long isotopelabelled peptide provides better recovery than the isotope-labelled peptide approach. As explained below in the section devoted to result validation, the recovery must be determined with allergen-spiked samples and processed matrices in order to meet AOAC specifications. Published methods, however, do not always meet the AOAC requirements, even with spiked samples. For instance, Careri et al. [76] observed a suppression effect between 30 and 50% for the Ara h2 peptide signal, and Monaci et al. [75] obtained recoveries ranging from 68 to 79% for α-lactalbumin and β lactoglobulin. Altogether, these works show that internal standards are needed for the quantification of allergens in food matrices. Currently, furthermore, the use of a calibration curve for each type of sample is the best way to respect the AOAC guide-

Future studies should thus still be done to improve the quantification of allergens from a

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

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:** In the AOAC guideline, the method quantitation limit (MQL) is defined as

gies 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

(blank). Laboratories, however, often use other strate-

line requiring a recovery between 60 and 120%.

30 Allergen

single calibration curve with a good recovery.

**4. Validating food allergen methods**

hazelnut) in food matrices by mass spectrometry (**Table 5**).

sensitivity, range of linearity, recovery and precision.

MQL = average (blank) + 10 x s0

The major increase of the allergic population has prompted the development of numerous allergen detection methods. Over the past few years, improvements in the detection of allergens by mass spectrometry have been impressive, allowing detection of processed allergens with high sensitivity (a few mg of proteins per kg of food). Optimization of extraction and purification steps has notably played a key role in the improvement of analytical methods. Allergen quantification is performed mainly with labelled internal standards. The best approach involves the use of labelled proteins, allowing correction for effects occurring throughout the sample preparation protocol. The high cost of labelled proteins, however, has promoted the use of other strategies, such as methods based on long isotopelabelled peptides and standard addition of allergens.

The validation of qualitative and quantitative MS-based methods for routine detection of allergens is still very recent. The AOAC guideline is a first step towards the development of methods that will allow procedure harmonization, making it possible to compare results between laboratories. These methods should be both improved and extended to other allergens in order to demonstrate their validity and robustness.
