**3. Quantifying food allergens**

Detecting hidden allergens in food products is essential to protecting the food-allergic population. For full transparency of allergen labelling, laboratories should also be able to quantify allergens in order to help food manufacturers manage cross-contamination during food production [70]. However, significant signal suppressions have been observed in various food matrices, and the level of suppression depends on the matrix considered. In one study, for example, high-proteincontent food products showed greater suppression of the peptide signal than ones with a low protein content: the determined LOQ values were 20 mg skim milk powder per kg for high-protein foods and 5 mg skim milk powder per kg for low-protein foods [71]. The food protein content is not the only parameter to be considered in relation to suppression of the peptide signal obtained by mass spectrometry: factors such as the type of process, the fat content and the presence of tannins also have an important influence on food allergen detection and must be taken into account.

While detecting allergens in various food products is difficult, quantifying them is even worse. In recent years, mass spectrometry techniques have been used for quantitation in proteomic analysis. Two approaches have emerged as the most relevant for food allergen quantification: labelfree quantification and the use of stable-isotope-labelled peptides or proteins [70, 72, 73]. The two strategies are compared in **Table 4** (target peptides, internal standards and calibration curves) and discussed in relation to the AOAC guideline 2016.002 method performance requirements for the quantification of allergens in food products, specifying a recovery between 60 and 120% and intra-day and inter-day coefficients of variation lower than 20 and 30%, respectively [74] (**Table 5**).


the samples (**Figure 4** of Ref. [69]). While mass spectrometry and ELISAs show comparable sensitivities when applied to unbaked products, mass spectrometry seems to be the method

**Figure 4.** Analytical results for 1000 mg spray-dried whole egg powder (National Institute of Standards and Technology RM 8445) per kg incurred cookies, obtained with the different enzyme-linked immunosorbent assay test kits for egg

Detecting hidden allergens in food products is essential to protecting the food-allergic population. For full transparency of allergen labelling, laboratories should also be able to quantify allergens in order to help food manufacturers manage cross-contamination during food production [70]. However, significant signal suppressions have been observed in various food matrices, and the level of suppression depends on the matrix considered. In one study, for example, high-proteincontent food products showed greater suppression of the peptide signal than ones with a low protein content: the determined LOQ values were 20 mg skim milk powder per kg for high-protein foods and 5 mg skim milk powder per kg for low-protein foods [71]. The food protein content is not the only parameter to be considered in relation to suppression of the peptide signal obtained by mass spectrometry: factors such as the type of process, the fat content and the presence of tannins also have an important influence on food allergen detection and must be taken into account. While detecting allergens in various food products is difficult, quantifying them is even worse. In recent years, mass spectrometry techniques have been used for quantitation in proteomic analysis. Two approaches have emerged as the most relevant for food allergen quantification: labelfree quantification and the use of stable-isotope-labelled peptides or proteins [70, 72, 73]. The two strategies are compared in **Table 4** (target peptides, internal standards and calibration curves) and discussed in relation to the AOAC guideline 2016.002 method performance requirements for the quantification of allergens in food products, specifying a recovery between 60 and 120% and intra-day and inter-day coefficients of variation lower than 20 and 30%, respectively [74] (**Table 5**).

of choice for the analysis of allergens in baked food products.

**3. Quantifying food allergens**

detection (A–E) (from Ref. [69]).

22 Allergen




**Authors**

**Matrix**

**Allergen** Pistachio

Pis v 5

AMISPLAGSTSVLR

ITSLNSLNLPILK

GFESEEESEYER

Walnut

Jug r 2

Jug r4

Mattarozzi

Pasta,

Lupin

β-conglutin

et al. [77]

Zhang et

Infant

Milk

α-lactalbumin

VGINYWLAHK

Xevo TQ triple

KILDKVGINNYWLAHKALCSE

Matrices were

spiked with

synthetic peptide

VGINYWLAHK

Standard addition

of mustard in

sauces and salty

biscuits

quadrupole

(Waters)

formulas

and whey

proteins

Posada-Ayala et al.

Sauces

Mustard

Sin a1

ACQQWLHK IYQTATHLPK

EFQQAQHLR

6460 triple

Purified protein Sin a 1

quadrupole

(Agilent

technologies)

Quantification of food allergens in different food products by mass spectrometry using label-free quantification with an (1) external calibration curve [75–79, 88],

and salty

biscuit

[79]

**Table 4a.** (2) unlabelled modified synthetic peptide [78], and (3) standard addition [79].

al. [78]

biscuit

FFDQQEQR

ATLTLVSQETR

ALPEEVLATAFQIPR

IVEFQSKPNTLILPK

LTQ XL linear

No internal standard

Pasta and biscuits

were fortified

with lupin

proteins

ion trap

(Thermo)

**Protein**

**Peptide**

**Mass** 

**Internal standard**

**Calibration curve**

24 Allergen

**spectrometer**

#### Food Allergen Analysis: Detection, Quantification and Validation by Mass Spectrometry http://dx.doi.org/10.5772/intechopen.69361 25


**Table 4b.** Quantification of food allergens in different food products by mass spectrometry using stable isotope labelling quantification with an (1) isotope-labelled protein [82], (2) isotope-labelled peptide [59, 61, 71, 75, 84, 89, 90] or (3) long isotope-labelled peptide [83].


Reported as ppm of the target allergen in food commodity i.e. 25 ppm of 'whole egg' in cookies.

**Table 5.** Method performance requirements from the AOAC guideline SMPR 2016.002 for egg, milk, peanut and hazelnut allergens in terms of analytical range, method quantification limit, recovery and intra-day and inter-day coefficients of variation (table from Paez et al. [74]).

### **3.1. Label-free quantification**

**Authors** Yi-Shun

Beer, wine,

Gluten

α-glyadin γ-Hordein

QQCCQQLANINEQSR

LWQIPEQSR

> et

[84]

Ippoushi

Sweet cherry

Cherry

Pru av2

TGCCAMSTDASGK

Xevo TQD

Zspray ion source

(Waters)

et al. [89] Rahman

/

Shrimp

Tropomyosine

Arginine kinase

QQLVDDHFLFVSGDR

SEEEVFGLQK

Micro mass

SEEEV[D8]VFGLQK

QQLV[D8]VDDHFLFV[D8]SGDR

VL[13C6, 15N]PV[13C5, 15N]PQK (IS1)

Solvent were

spiked with milk

proteins

QSVLSLSQSKVL[13C6, 15N]PV[13C5, 15N]PQKAVPYPQRQ (IS2)

Human β-casein (IS3)

Quantification of food allergens in different food products by mass spectrometry using stable isotope labelling quantification with an (1) isotope-labelled protein

[82], (2) isotope-labelled peptide [59, 61, 71, 75, 84, 89, 90] or (3) long isotope-labelled peptide [83].

Quattro Ultima

(Waters)

TOF-MS Synapt

G2 HDMS

(Waters)

et [90]

Chen et al.

Baked food

Milk

β-casein

VLPVPQR

(170°C, 25 min)

(2014) [83]

**Table 4b.**

al. (2012)

fruit

al. (2017)

chips, flour,

cookies…

**Matrix**

**Allergen**

**Protein**

**Peptide**

**Mass** 

**Internal standard**

**Calibration** 

26 Allergen

**curve**

Matrices were

spiked with

gluten proteins

**spectrometer**

6490 triple quad

LWQIPEQSR[13C, 15N]

QQCCQQLANINEQSR [13C, 15N]

TGCCAMSTDASGK[13C6,15N2]

Sweet cherry

fruit were spiekd

with isotope

labelled peptides

Solvent were

spiked with

shrimp proteins

(Agilent)

The label-free quantification strategy is based on comparing the peptide signal intensities of different samples (**Table 4a**). Three label-free quantification possibilities are described below.

**External calibration:** Monaci et al. used this approach to quantify milk proteins in fruit juice. Using a calibration curve obtained by spiking fruit juice with extracted milk proteins, they found recoveries between 68 and 79% [75]. This strategy was also used to quantify peanut proteins in rice crispy/chocolate snacks [76]. A significant suppression effect, ranging from 30 to 50%, was observed for the Ara h2 peptide signal, while suppression of the Ara h3/4 peptide signal was less than 10%. A more recent study by Mattarozzi et al. obtained recoveries between 95 and 118% for lupin β-conglutin peptide in spiked biscuits [77]. Although less expensive than other approaches, this approach requires a calibration curve for each matrix.

**Modified synthetic peptide approach:** Zhang et al. introduced an internal standard peptide (KILDKVGINNYWLAHKALCSE) with an added asparagine residue (N) in the β-casein peptide VGINYWLAHK. They obtained recoveries between 98.8 and 100.6% [78]. The use of an internal standard allows better recovery, but adding an amino acid can change the retention time and modify the ionization of target peptides.

**Standard addition**: This label-free quantification strategy consists in adding standards to the matrices. It was used by Posada-Ayala et al. for the quantification of commercial food products [79]. This approach consists in adding different known quantities of extracted allergen proteins directly to the sample to be analysed before digestion and in quantifying the target allergens with the resulting calibration curve. The recovery was not specified, but this approach allows correcting at least for digestion and matrix effects. However, the theoretical level of contamination in the samples must be known in order to adapt the quantities of standards to be added.

#### **3.2. Stable isotope labelling quantification**

This strategy is based on the use of isotope-labelled (13C-, 15N-, D-labelled) peptides or proteins [80] (**Table 4b**). It is recommend to use a 6-Da mass difference with respect to the amino acid for doubly charged precursors and an 8–10-Da mass difference for triply charged precursors [52]. Although more expensive than the strategies described above, this approach has the advantage that the unlabelled and isotope-labelled peptides show similar ionization and similar mass spectrometry response signals. For allergen quantification, three kinds of isotope-labelled standards exist [81]: proteins [82], concatemers [83] (or long isotope-labelled peptides) and Aqua peptides [61, 71, 75, 84] (isotope-labelled peptides) (**Figure 5**).

**Isotope-labelled proteins:** The principle of this approach is to add a labelled protein to the sample before extraction. Newsome et al. studied the recovery of the milk allergen α-S1 casein in baked cookies using a labelled internal α-S1 casein, and obtained recoveries ranging from 60 to 80% [82]. Use of an internal standard allows correcting for the matrix effect and for effects linked to different steps in the sample preparation protocol (protein extraction and enzymatic digestion). It thus allows accurate determination of the recovery and precision for processed samples. This 'gold standard' approach is really expensive, however, making its use unrealistic for the vast majority of routine laboratories.

**Isotope-labelled peptides**: The principle is to add labelled peptides to the sample after digestion and before the purification steps. This approach is less expensive than the use of isotope-labelled proteins, and synthetic labelled peptides can easily be commercially obtained. Huschek et al. used isotope-labelled peptides to quantify soy, lupin and sesame allergens [59]. They determined the recovery of their method by spiking wheat, cookie and bread with the labelled peptides and obtained results between 69.4 and 112.9%. One could argue, however, that very similar matrices were used in this study (wheat-based products) and that this type of study should be extended to other matrices in order to validate the ability of the isotopelabelled peptide to correct for matrix effects.

Lutter et al. quantified milk proteins in baby food, infant cereals, breakfast cereals and rinsing water, using a calibration curve obtained by spiking 0.1% formic acid with milk protein. The estimated recovery rates were between 16 and 66% [71] Lutter et al. In this study, the isotope-labelled peptides were used to correct for effects related to different steps of the analysis. While using a single calibration curve can be useful in the routine laboratory context, the relatively low recoveries obtained in this study reveal the inability of an isotope-labelled peptide to correct for sample-preparation-related effects. We have compared the areas of milk, egg, peanut and soy peptide peaks for three matrices with and without isotope labelled peptides. Our results clearly show that an isotope-labelled peptide is able to correct for matrix effects but not for effects linked to the extraction and digestion steps [85] planque et al.

**Isotope-labelled concatemers/long isotope-labelled peptides**: The isotope-labelled concatemer used in this technique is a chimeric protein containing all the labelled target peptides. Food Allergen Analysis: Detection, Quantification and Validation by Mass Spectrometry http://dx.doi.org/10.5772/intechopen.69361 29

**3.2. Stable isotope labelling quantification**

use unrealistic for the vast majority of routine laboratories.

labelled peptide to correct for matrix effects.

digestion steps [85] planque et al.

(**Figure 5**).

28 Allergen

This strategy is based on the use of isotope-labelled (13C-, 15N-, D-labelled) peptides or proteins [80] (**Table 4b**). It is recommend to use a 6-Da mass difference with respect to the amino acid for doubly charged precursors and an 8–10-Da mass difference for triply charged precursors [52]. Although more expensive than the strategies described above, this approach has the advantage that the unlabelled and isotope-labelled peptides show similar ionization and similar mass spectrometry response signals. For allergen quantification, three kinds of isotope-labelled standards exist [81]: proteins [82], concatemers [83] (or long isotope-labelled peptides) and Aqua peptides [61, 71, 75, 84] (isotope-labelled peptides)

**Isotope-labelled proteins:** The principle of this approach is to add a labelled protein to the sample before extraction. Newsome et al. studied the recovery of the milk allergen α-S1 casein in baked cookies using a labelled internal α-S1 casein, and obtained recoveries ranging from 60 to 80% [82]. Use of an internal standard allows correcting for the matrix effect and for effects linked to different steps in the sample preparation protocol (protein extraction and enzymatic digestion). It thus allows accurate determination of the recovery and precision for processed samples. This 'gold standard' approach is really expensive, however, making its

**Isotope-labelled peptides**: The principle is to add labelled peptides to the sample after digestion and before the purification steps. This approach is less expensive than the use of isotope-labelled proteins, and synthetic labelled peptides can easily be commercially obtained. Huschek et al. used isotope-labelled peptides to quantify soy, lupin and sesame allergens [59]. They determined the recovery of their method by spiking wheat, cookie and bread with the labelled peptides and obtained results between 69.4 and 112.9%. One could argue, however, that very similar matrices were used in this study (wheat-based products) and that this type of study should be extended to other matrices in order to validate the ability of the isotope-

Lutter et al. quantified milk proteins in baby food, infant cereals, breakfast cereals and rinsing water, using a calibration curve obtained by spiking 0.1% formic acid with milk protein. The estimated recovery rates were between 16 and 66% [71] Lutter et al. In this study, the isotope-labelled peptides were used to correct for effects related to different steps of the analysis. While using a single calibration curve can be useful in the routine laboratory context, the relatively low recoveries obtained in this study reveal the inability of an isotope-labelled peptide to correct for sample-preparation-related effects. We have compared the areas of milk, egg, peanut and soy peptide peaks for three matrices with and without isotope labelled peptides. Our results clearly show that an isotope-labelled peptide is able to correct for matrix effects but not for effects linked to the extraction and

**Isotope-labelled concatemers/long isotope-labelled peptides**: The isotope-labelled concatemer used in this technique is a chimeric protein containing all the labelled target peptides.

**Figure 5.** Three types of internal standards are used for the quantification of proteins by mass spectrometry (1) isotopelabelled protein (2) Isotope-labelled concatemers or long isotope-labelled peptides (3) isotope-labelled peptide (from Ref. [81]).

This internal standard is added to the sample before enzymatic digestion. The advantage of this method is that a single concatemer can contain peptides belonging to different proteins or allergens. This strategy has been used in proteomics, but it is not yet used for food allergen quantification [86]. An emerging alternative to use of a concatemer is use of a so-called 'long isotope-labelled peptide'. Chen et al. compared the use of three types of internal standard: human β-casein, isotope-labelled peptide VL [13C6 , 15N] PV[13C5 , 15N]PQK and a long isotopelabelled peptide QSVLSLSQSKVL[13C6 , <sup>15</sup>N] PV[13C5 , 15N]PQKAVPYPQRQ [83]. The long isotope-labelled peptide provided better recovery, due to correction for digestion-step-related effects. The recovery based on spiked materials was between 98.8 and 106.7%. In 2016, it was 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 guideline requiring a recovery between 60 and 120%.

Future studies should thus still be done to improve the quantification of allergens from a single calibration curve with a good recovery.
