Journal of AOAC INTERNATIONAL, 105(2), 2022, 442–455

https://doi.org/10.1093/jaoacint/qsab148
Advance Access Publication Date: 16 November 2021
Research Article

FOOD CHEMICAL CONTAMINANTS

Determination of Gliadin as a Measure of Gluten in

R

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Food by R5 Sandwich ELISA RIDASCREENV Gliadin
Matrix Extension: Collaborative Study 2012.01
Markus Lacorn ,1,* Tina Dubois ,1 Thomas Weiss ,1
Lisa Zimmermann ,2 Teresa-Maria Schinabeck ,2
Simone Loos-Theisen ,2 and Katharina Scherf 3
1
R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany, 2Hochschule Geisenheim
University, Department of Food Safety, Von-Lade-Straße 1, 65366 Geisenheim, Germany, 3Karlsruhe Institute
of Technology (KIT), Institute of Applied Biosciences, Department of Bioactive and Functional Food
Chemistry, Adenauerring 20 a, Building 50.41, 76131 Karlsruhe, Germany

*Corresponding author’s e-mail: [email protected]

Abstract
Background: According to Codex Alimentarius, food products containing less than 20 mg/kg gluten can be labeled as “gluten-
free.” Since 2002, the R5 antibody method allowed determination of gluten levels and led to a huge improvement of
products available to celiac disease (CD) patients.
R
Method: The R5-containing test kit RIDASCREENV Gliadin in combination with the cocktail solution was endorsed as Codex
Type 1 Method in 2006 based on a collaborative study with corn-based bread, rice-based dough, wheat starches, rice, and
corn flour. In 2012, the method was approved as First Action Official MethodSM 2012.01 with an “in foods” claim. For Final
Action in 2016, the matrix claim was reduced to rice- and corn-based matrixes.
Objective: Therefore, R-Biopharm decided to start a collaborative study to demonstrate the wide applicability of Official
Method 2012.01 for the quantitative analysis of gliadin in soy, starches, pseudo cereals, legumes, spices, juice, nut nougat
crème, cream cheese, pesto, meat, vegetarian meat alternative, cookies, dessert, cake, fish, bread, candies, and potatoes.
Materials for incurring were the MoniQA wheat flour and the PWG gliadin preparation.
Results: Gliadin levels ranged from 3.4 up to 27.4 mg gliadin per kg. The results of the collaborative study with 14
participating laboratories showed recoveries ranging from 80 to 130%. Relative reproducibility standard deviations for
contaminated samples were between 9.8 and 27.7%.
Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gliadin in
important gluten-free food matrixes.
Highlights: The title and applicability statement of Official Method 2012.01 were changed as proposed.

Received: 10 September 2021; Revised: 15 October 2021; Accepted: 9 November 2021

C AOAC INTERNATIONAL 2021.
V
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecom-
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442
 Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022 | 443

With a prevalence of 0.4 to 1.2% of the population in Europe, communicated by AOAC, laboratories became unsure about the
North America, Australia, and the Middle East (1), CD is consid- applicability of the method for other food matrixes. Some labo-
ered one of the most common food hypersensitivities. CD is an ratories had been using the method according to First Action
immune-mediated inflammatory disease of the upper small in- Official Methods for all kinds of different foods for years and now
testine in genetically predisposed individuals triggered by the questioned the analytical correctness of previous analyses. An
ingestion of dietary gluten (2). In the context of CD, gluten is de- additional reduction of the scope of Official Method 2012.01 at
fined as a protein fraction from wheat, rye, barley, or their Codex Alimentarius would lead to a more confusing situation
crossbred varieties and derivatives thereof, to which some per- for laboratories and finally also for CD patients comparable to
sons are intolerant. This protein fraction is insoluble in water times before 2002. Therefore, R-Biopharm decided to start a new
and 0.5 mol/L NaCl solution, respectively (3). Following a tradi- collaborative study to demonstrate the applicability of Official
tional definition, gluten is composed of prolamins that can be Method 2012.01 for all kinds of different foods and to re-extend
extracted by 40–70% of ethanol, and alcohol-insoluble glutelins the scope for Official Method 2012.01.
that can only be extracted under reducing and disaggregating After prolific consultations with CD patient societies from
conditions at elevated temperatures. The prolamins from the United States and Europe, a list of 19 different matrixes

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wheat, rye, and barley are called gliadins, secalins, and hor- from 16 different food categories was prepared. Four ERP Gluten
deins, respectively. The prolamin content of gluten is generally members approved the collaborative test protocol in October
taken as 50% (3), which will lead to a significant overestimation 2019. Meanwhile, in November 2019, the Codex Committee on
of the gluten content if prolamin is chosen as the analytical ba- Nutrition and Foods for Special Dietary Uses (CCNFSDU) decided
sis (4). that “The Committee noted that it was premature to consider
The only known effective treatment for celiac disease (CD) is a the proposed methods as presented in CX/NFSDU 19/41/2,
lifelong gluten-free diet, which is based on the avoidance of Appendix I, Part C as research is still ongoing to determine the
gluten-containing cereals. The diet should contain less than most appropriate method for determination of gluten. The
20 mg gluten intake per day to prevent a relapse of intestinal dam- Committee agreed to wait for the completion of ring trial tests
age (5). To guarantee the safety of gluten-free products for CD and to consider this matter at a future date when more informa-
patients, a threshold of 20 mg/kg gluten for gluten-free foods is tion became available.” Until finalizing this manuscript no new
recommended by the Codex Alimentarius and legislation, e.g., in discussions either at CCMAS (Codex Committee on Methods of
Europe by the European Commission (6) and in the United States
Analysis and Sampling) or CCNFSDU were perceived.
by the Department of Health and Human Services—Food and
Here, the collaborative study of Official Method 2012.01 for
Drug Administration (7). Therefore, specific and sensitive analyti-
the quantitative analysis of gliadin in soy, starches, pseudo
cal methods are needed for food quality control.
cereals, legumes, spices, juice, nut nougat crème, cream cheese,
Immunochemical methods are currently recommended for the
pesto, meat, vegetarian meat alternative, cookies, dessert, cake,
quantitative and qualitative determination of gluten in foods (3).
fish, bread, candies, and potatoes is presented. Due to the high
The R5 monoclonal antibody raised against x-secalins pri-
number of different matrixes and the total number of samples
marily recognizes the epitope QQPFP, which is present in glia-
for each participant, it was decided to test each matrix at one
dins, secalins, and hordeins and occurs in many peptides that
concentration level and only a few matrixes additionally as
are toxic or immunogenic for CD patients (8–10).
blank matrixes (see Table 1). The different contamination levels
To understand the rationale for the additionally collabora-
were chosen to bracket the gluten threshold level of 20 mg/kg.
tive test, one needs to roll up the history of the R5 method and
More levels were analyzed at the method developer’s site to
Official MethodSM 2012.01 especially. Since its introduction in
cover the measurement range for every matrix (see Tables 1 and
2002, the R5 method allowed determination and control of glu-
2). Katharina Scherf (Department of Bioactive and Functional
ten levels in products with a yet unmet sensitivity (limit of
Food Chemistry at the Karlsruhe Institute of Technology,
quantification of 5 mg/kg gluten), which subsequently led to a
Germany) coordinated the study as the study director.
huge improvement in the quality of products available to CD
patients.
R
The test kit RIDASCREENV Gliadin was endorsed as Codex Experimental
Type 1 Method in 2006 based on a collaborative study performed
Collaborative Study—Study Design
by the Working Group on Prolamin Toxicity and Analysis. In
2011, an additional collaborative study was performed with Following the AOAC guidelines, which are published as
RIDASCREEN Gliadin. In 2012, the system was approved as First Appendix D (11) and Appendix M (12), an international collabo-
Action Official Method 2012.01 with an “in foods” claim. For the rative study was set up to validate the RIDASCREEN Gliadin for
decision on Final Action in 2016, the matrix claim was reduced additional matrixes from important gluten-free food categories
to only rice- and corn-based matrixes despite the fact that data as an extension of AOAC Official Method 2012.01. The experi-
on proficiency test rounds with several additional matrixes ment consisted of 32 different samples (19 different matrixes
were presented. In March 2019, the method developer tried from 16 different food categories) that were analyzed as dupli-
again to include the “proficiency test round matrixes” into the cates in a blinded manner. To allow a uniform calculation of
intended use of the method, but at that time no decision was results later, participants were advised to deliver raw OD data
made. In May 2019, AOAC and AACC proposed at a CCMAS by a data return sheet. The study director did the calculation of
meeting to limit the scope of Official Method 2012.01 to corn- and results using the curve fitting procedure mentioned in the test
rice-based matrixes and to add Official Method 2018.15 as the kit insert of the RIDASCREEN Gliadin (R7001, R-Biopharm).
method for gluten in oats in the Codex Alimentarius.
Already the reduction of the scope of Official Method 2012.01
by AOAC in 2016 led to severe irritations in the analytical com-
Scope of Method
munity, especially for laboratories accredited toward ISO 17025. RIDASCREEN Gliadin is suitable for the quantitative measure-
Since the reasons for this reduction were not clearly ment of intact gliadin as a measure of gluten in unprocessed
 444 | Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022

Table 1. List of gluten-free food categories and the representative sample matrix; all matrixes were incurred using MoniQA wheat flour
(column “Gliadin (gluten)” therein gluten content is given in brackets). Additionally, some matrixes were incurred using PWG gliadin (column
“PWG gliadin”)

Collaborative test, mg/kg In-house testing, mg/kg

Category Sample name Gliadin (gluten) PWG gliadin Gliadin Gluten

Starch Starch 27.36 (39.95) –a Blank, 6.84, 13.69 Blank, 9.99, 19.98
Pseudo cereals Pseudo cereals*b 3.43 (5.00) – Blank, 6.84, 13.69, 27.37 Blank, 9.99, 19.98,
39.96
Legumes Legumes 27.36 (39.95) 20.00 Blank, 6.84, 13.69 Blank, 9.99, 19.98
Soy Soy Blank and 3.43 (5.00) – 6.84, 13.69, 27.37 9.99, 19.98, 39.96
Spices and their mixtures Spices* Blank and 13.70 (20.00) – 6.84, 27.37 9.99, 39.96
Mayonnaise, sauces, vegetables Pesto 6.86 (10.01) 5.00 Blank, 12.08, 24.15 Blank, 17.63, 35.26
Cereals and products thereof Cake* 13.70 (20.00) 10.00 Blank, 6.86, 24.51 Blank, 10.02, 35.78

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Cereals and products thereof Cookies 20.51 (29.95) 15.01 Blank, 6.84, 13.69, 27.38 Blank, 9.99, 19.99,
39.97
Cereals and products thereof Bread 10.27 (14.99) – Blank, 6.84, 13.69, 27.38 Blank, 9.99, 19.99,
39.97
Potatoes and products thereof Gnocchi 10.27 (15.00) – Blank, 6.85, 13.70, 27.39 Blank, 10.00, 20.00,
39.99
Potatoes and products thereof Gnocchi (MW) 10.27 (15.00) – – –
Egg products, ice cream, milk, and Crema Blank and 13.67 (19.96) 10.00 7.01, 28.02 10.23, 40.91
products thereof
Cheese and products thereof Cheese Blank and 20.55 (30.00) – 6.84. 13.69, 27.37 9.99, 19.98, 39.96
Spread Nougat* Blank and 20.55 (30.00) – 6.84. 13.69, 27.37 9.99, 19.98, 39.96
Soft drinks, vinegar, fruits Juice* 6.84 (9.99) 5.00 Blank, 13.69, 27.37 Blank, 19.98, 39.96
Fish and products thereof Cod Blank and 27.75 (40.51) – 6.94, 13.88 10.13, 20.26
Meat and products thereof Meat 27.43 (40.05) – Blank, 7.23, 14.46 Blank, 10.56, 21.11
Vegetarian meat alternatives Burger 13.70 (20.00) 10.00 Blank, 6.84, 27.37 Blank, 9.99, 39.95
Candies Caramel 27.40 (40.00) – Blank, 6.85, 13.69 Blank, 10.00, 19.99

a
– ¼ No gliadin added.
b
Samples with an * had to be extracted with the addition of skim milk powder. In-house testing for additional levels is given in the last two columns on the right (only
MoniQA wheat flour materials were tested). Gnocchi (microwaved, MW) were not analyzed in-house due to the similarity to gnocchi.

and processed matrixes from important gluten-free food cate- important gluten-free food categories according to their view.
gories, including rice- and corn-based products, soy, starches, The result was a list of 16 different food categories (Table 1).
pseudo cereals, legumes, spices, juice, nut nougat crème, cream Afterwards, recipes for 19 different food matrixes were devel-
cheese, pesto, meat, vegetarian meat alternative, cookies, des- oped by the method developer paying special attention to the
sert, cake, fish, bread, candies, and potatoes. The sandwich feasibility of production, achievable sample homogeneity, and
ELISA quantifies intact gliadin from wheat and also intact re- stability. Due to its relevance, the category “cereals and prod-
lated proteins from rye and barley. This method is not accurate ucts thereof” will be represented by three different matrixes
for quantification of fermented or hydrolyzed gluten. (cookies, cake, bread). The 14 recipes were sent to the
Hochschule Geisenheim University for independent production.
Collaborators MoniQA wheat flour (13) with defined gliadin and gluten levels
of 7.26 and 10.60% (personal communication with Katharina
In order to qualify for participation in the collaborative test, all Scherf), respectively, and the PWG gliadin preparation were
laboratories were required to have previous experience with used for incurring the matrixes (14). After preparation, samples
ELISA, and to be familiar with the analytical procedure. It was were aliquoted, blinded, and checked for homogeneity by the
recommended to use a separate room for the collaborative Hochschule Geisenheim University.
study due to the possibility of gluten contamination and the In the following, the ingredients, processing, and contamina-
low detection limit. The laboratories were given the period from tion levels of each matrix at the Hochschule Geisenheim
November 2020 to January 2021 to perform the experiments. University are described. The Hochschule Geisenheim University
Fourteen laboratories (designated A to O, without G) were cho- provided a detailed report on the production of each matrix as a
sen to participate: one each in Austria, Finland, Ireland, Italy, confidential document to the ERP Gluten. The contamination was
two in Canada, four in Germany, and four in the United States. added to the matrixes prior to the processing in all cases. Loss of
One laboratory from the United States obtained sample set G water or other components during the preparation of each matrix
but could not guarantee proper storage over the weekend. It was taken into account by weighing experiments throughout the
was decided to send an additional sample set O to them and to production process. The concentrations in Table 1 were chosen to
discard sample set G. bracket the threshold of 20 mg gluten per kg of food.

Samples and Sample Preparation Methods of Preparation—Food Category

During the preparation of this collaborative test, European and The name of a sample used throughout this document is given
US CD patient societies were asked to deliver the most in brackets after the food category to ease the reading and
 Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022 | 445

Table 2. Results from in-house experiments to characterize additional concentration levels for the matrices used in the collaborative test

Gliadin target, mg/kg Mean, mg/kga SD, mg/kgb CV, %c Recovery, %d

Part A
Starch /f 1.61*e 0.13 8.1
6.84 8.31 0.80 9.6 121
13.69 17.12 2.82 16.5 125
Pseudo cereals / 0.55 0.03 5.6
6.84 7.89 0.83 10.5 115
13.69 13.71 2.83 20.6 100
27.37 26.77 2.48 9.2 98
Legumes / 0.23 0.05 21.1
6.84 9.67 0.61 6.3 141
13.69 18.05 1.23 6.8 132

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Soy 6.84 9.11 1.60 17.5 133
13.69 15.80 0.93 5.9 115
27.37 29.18 2.16 7.4 107
Spices 6.84 6.26 0.73 11.7 92
27.37 22.22 2.32 10.4 81
Pesto / 0.01 0.02
12.08 12.08 0.83 6.9 100
24.15 24.05 2.24 9.3 100
Cake / 0.03 0.01 33.3
6.86 4.47 0.34 7.5 65
24.51 15.52 1.18 7.6 63
Cookies / 0.44 0.16 36.7
6.84 10.57 1.23 11.7 154
13.69 18.95 2.49 13.1 138
27.38 40.10 1.60 4.0 146
Bread / 0.58 0.04 7.0
6.84 9.18 1.07 11.6 134
13.69 17.39 1.92 11.1 127
27.38 31.85 1.86 5.8 116
Part B
Gnocchi / 0.02 0.03
6.85 7.82 1.28 16.4 114
13.7 15.22 1.16 7.6 111
27.39 35.18 4.10 11.6 128
Crema 7.01 7.13 1.24 17.4 102
28.02 34.89 4.14 11.9 125
Cheese 6.84 8.55 0.50 5.9 125
13.69 15.93 1.23 7.7 116
27.37 28.85 3.00 10.4 105
Nougat 6.84 6.09 0.59 9.7 89
13.69 14.57 1.86 12.8 106
27.37 28.26 2.48 8.8 103
Juice / 0.04 0.02
13.69 13.75 1.36 9.9 100
27.37 24.36 3.62 14.9 89
Cod 6.94 8.22 0.68 8.3 118
13.88 15.86 1.11 7.0 114
Meat / 0.09 0.02 16.4
7.23 8.89 0.69 7.8 123
14.46 15.88 1.29 8.2 110
Burger / 0.79 0.04 5.1
6.84 6.51 1.03 15.9 95
27.37 25.49 1.53 6.0 93
Caramel / 0.02 0.07
6.85 6.28 0.94 14.9 92
13.69 12.48 0.34 2.7 91

a
Performance characteristics mean (n ¼ 6 replicates).
b
SD ¼ Standard deviation.
c
CV ¼ Coefficient of variation.
d
Recovery based on gliadin content.
e
In the case of starch, the asterisk indicates a wheat contamination confirmed by PCR (SureFood ALLERGEN 4plex Cereals S7006 together with SureFood PREP
Advanced S1053 for extraction; Congen, Berlin, Germany); the measured values and the recoveries were corrected for the concentration of the nonincurred starch.
f
/ ¼ Assumed to be blank.
 446 | Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022

formatting of tables later on. For concentrations of materials (3) Contaminating material and initial concentration.—
used in the collaborative study and for additional in-house test- MoniQA wheat flour at 38.57 mg gliadin/kg (56.30 mg
ing levels, refer to Table 1. gluten/kg) and 42.77 mg/kg of PWG gliadin.
(i) Cereals and products thereof (bread).—
(a) Starches (starch).—
(1) Ingredients.—500 g gluten-free bread flour mixture
(1) Ingredients.—Mixture of starch from wheat, corn, tapi-
(corn starch, rice flour, millet flour, buckwheat sour-
oca, and potato.
dough powder [buckwheat, quinoa flour, starting cul-
(2) Processing.—Mixing.
ture], thickening agent guar gum), 7 g dry yeast, 5 g
(3) Contaminating material and initial concentration.—
salt, 30 mL olive oil, three eggs, 300 mL water.
MoniQA wheat flour at 27.36 mg gliadin/kg (39.95 mg
(2) Processing.—60 min at 180 C (356 F), dried overnight at
gluten/kg).
40 C (104 F).
(b) Pseudo cereals (pseudo cereals).—
(3) Contaminating material and initial concentration.—
(1) Ingredients.—Mixture of flour from amaranth, quinoa,
MoniQA wheat flour at 34.08 mg gliadin/kg (49.75 mg
and buckwheat.
gluten/kg)
(2) Processing.—Mixing.

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(j) Potatoes and products thereof (gnocchi and gnocchi MW).—
(3) Contaminating material and initial concentration.—
(1) Ingredients.—400 g boiled potatoes, 100 g gluten-free
MoniQA wheat flour at 27.36 mg gliadin/kg (39.96 mg
flour mix (corn starch, rice flour, millet flour, thicken-
gluten/kg). ing agent guar gum), two egg yolks, 0.2 g salt,
(c) Legumes (legumes).— preservatives.
(1) Ingredients.—Mixture of flour from lentils, beans, and (2) Processing.—15 min at 100 C (212 F) and additionally
peas. 2.5 min at 1500 W in a microwave oven (gnocchi MW).
(2) Processing.—Mixing. (3) Contaminating material and initial concentration.—
(3) Contaminating material and initial concentration.— MoniQA wheat flour at 32.09 mg gliadin/kg (46.84 mg
MoniQA wheat flour at 27.36 mg gliadin/kg (39.95 mg gluten/kg) for the boiled gnocchi and 37.22 mg gliadin/
gluten/kg) and 20.00 mg/kg of PWG gliadin. kg (54.33 mg gluten/kg) for the additionally micro-
(d) Soy (soy).— waved (MW) gnocchi.
(1) Ingredients.—Soy flour. (k) Egg products, ice, milk and products thereof (crema).—
(2) Processing.—Mixing. (1) Ingredients.—65 g gluten-free flour mixture (see cake), 5
(3) Contaminating material and initial concentration.— g vanilla sugar, 65 g sugar, one whole egg, two egg
MoniQA wheat flour at 27.36 mg gliadin/kg (39.96 mg yolks, 375 mL milk, 2.25 g gelatin, 40 g butter, 20 g
gluten/kg). grounded gluten-free biscuits (corn starch, butter,
(e) Spices (spices).— corn flour, sugar, soy flour, glucose syrup, salt, rice
(1) Ingredients.—Mixture of paprika, cinnamon, mustard, flour, modified corn starch), preservatives.
pepper, marjoram, and nutmeg. (2) Processing.—10 min at 100 C (212 F).
(2) Processing.—Mixing. (3) Contaminating material and initial concentration.—
(3) Contaminating material and initial concentration.—MoniQA MoniQA wheat flour at 28.02 mg gliadin/kg (40.91 mg
wheat flour at 27.36 mg gliadin/kg (39.96 mg gluten/kg). gluten/kg) and 29.93 mg/kg of PWG gliadin.
(f) Mayonnaise, sauces, vegetables (pesto).— (l) Cheese and products thereof (cheese).—
(1) Ingredients.—200 g dried tomatoes, 200 g sieved toma- (1) Ingredients.—Cream cheese, preservatives.
toes, 100 g tomato paste, 50 g olive oil, two garlic (2) Processing.—Mixing.
cloves, 30 g ground almonds, 5 g salt, 5 g sugar, 30 g (3) Contaminating material and initial concentration.—
dried, finely ground basil, preservatives. MoniQA wheat flour at 20.55 mg gliadin/kg (30.00 mg
(2) Processing.—Boiling for 5 min and stirring at 80 C gluten/kg).
(176 F) for 10 min. (m) Spread (nougat).—
(3) Contaminating material and initial concentration.— (1) Ingredients.—Nut-nougat crème (sugar, palm oil, hazel-
MoniQA wheat flour at 35.50 mg gliadin/kg (51.83 mg nuts, skimmed milk powder, fat reduced cocoa, soy
gluten/kg) and 23.62 mg/kg of PWG gliadin. lecithin, vanillin), preservatives.
(g) Cereals and products thereof (cake).— (2) Processing.—80 C (176 F) for 1 h under stirring.
(1) Ingredients.—150 g butter, 125 g dark chocolate, 100 g (3) Contaminating material and initial concentration.—
gluten-free flour mixture (corn starch, rice flour, millet MoniQA wheat flour at 20.55 mg gliadin/kg (30.00 mg
flour, thickening agents [carob gum, rice whole flour]), gluten/kg).
180 g powdered sugar, 125 g ground almonds, five (n) Soft drinks, vinegar, fruits (juice).—
eggs, 15 g baking powder. (1) Ingredients.—Multivitamin juice (concentrates of juice
(2) Processing.—55 min at 170 C (338 F), dried overnight at from apple, orange, mandarin, pineapple, peach, pas-
40 C (104 F). sion fruit, pear, lime, and lemon), water, fruit concen-
(3) Contaminating material and initial concentration.— trates (banana, guava, mango, papaya) carrot juice,
MoniQA wheat flour at 24.51 mg gliadin/kg (35.78 mg grape juice, vitamins, preservatives.
gluten/kg) and 27.39 mg/kg of PWG gliadin. (2) Processing.—Mixing.
(h) Cereals and products thereof (cookies).— (3) Contaminating material and initial concentration.—
(1) Ingredients.—250 g gluten-free flour mixture (see cake), MoniQA wheat flour at 27.37 mg gliadin/kg (39.96 mg
7.5 g baking powder, 2.5 g salt, 100 g sugar, one egg, gluten/kg) and 20.00 mg/kg of PWG gliadin.
110 g corn oil. (o) Fish and products thereof (cod).—
(2) Processing.—25 min at 150 C (302 F), dried overnight at (1) Ingredients.—500 g cod, 50 g gluten-free flour mixture,
40 C (104 F). 20 mL water, preservatives.
 Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022 | 447

(2) Processing.—100 C (212 F) for 20 min. Method and Measurement of Samples

(3) Contaminating material and initial concentration.—
The method was written in AOAC style and was provided to
MoniQA wheat flour at 27.75 mg gliadin/kg (40.51 mg
each laboratory with the instructions to follow the method as
gluten/kg).
written with no deviations. All OD values obtained had to be
(p) Meat and products thereof (meat).—
recorded in a ready-to-use datasheet. The final data from the
(1) Ingredients.—Ground meat from beef and pork, 20 mL
laboratories were sent to the study director. The participants
water, preservatives.
were advised to analyze sample 1 to 32 in run 1, whereas sam-
(2) Processing.—Fried at 190 C (374 F) for 16 min.
ples coded 33 to 64 were analyzed in run 2. Samples with OD
(3) Contaminating material and initial concentration.—
values higher than calibrator 6 (80 ng gliadin/mL) had to be re-
MoniQA wheat flour at 70.77 mg gliadin/kg (103.32 mg
peated with a 1:2,000 dilution in run 3 (the regular final dilu-
gluten/kg)
(q) Vegetarian meat alternatives (burger).— tion factor is 500). To facilitate the calculation later on, the
(1) Ingredients.—275 g gluten-free commercial vegetarian participants were asked to use a fixed pipetting scheme on
tomato burger mixture (rice flakes, rice flour, millet the microtiter plate. As a check for any contamination at the

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flakes, corn flour, corn extrudate, tomato pieces, to- collaborator’s site, participants were asked to analyze their
mato flakes, salt, basil, onions, parsnip, carrots, cur- skim milk powder (SMP) (added to some samples before ex-
cuma, pepper, olive oil), 5 g gluten-free flour mixture traction; see Table 1) and the sample dilution buffer in
(see gnocchi), 350 mL water, preservatives. addition.
(2) Processing.—Fried at 190 C (374 F) for 20 min, dried AOAC Official Method 2012.01
overnight at 40 C (104 F). Gliadin as a Measure of Gluten in Food
(3) Contaminating material and initial concentration.— by R5 Sandwich ELISA RIDASCREEN Gliadin
MoniQA wheat flour at 53.48 mg gliadin/kg (78.08 mg Based on a Specific Monoclonal Antibody to
Celiac Toxic Amino Acid Prolamin Sequences
gluten/kg) and 55.12 mg/kg of PWG gliadin. First Action 2012
(r) Candies (caramel).— Final Action 2016
(1) Ingredients.—230 g sugar, 80 g water, 110 g butter, 0.2 g
salt, 150 mL cream. [Applicable for the quantitative measurement of intact gliadin
(2) Processing.—100 C for 15 min. as a measure of gluten in unprocessed and processed matrixes
(3) Contaminating material and initial concentration.— from important gluten-free food categories including rice- and
MoniQA wheat flour at 35.57 mg gliadin/kg (51.92 mg corn-based products, soy, starches, pseudo cereals, legumes,
gluten/kg). spices, juice, nut nougat crème, cream cheese, pesto, meat, veg-
etarian meat alternative, cookies, dessert, cake, fish, bread, can-
dies, and potatoes. The sandwich ELISA quantifies intact gliadin
Homogeneity of Samples from wheat and also intact related proteins from rye and barley.
Homogeneity of each material (n ¼ 32) was tested at the This method is not accurate for quantification of fermented or
Hochschule Geisenheim University using the RIDASCREEN hydrolyzed gluten.]
Gliadin. In brief, five vials from each material were randomly
chosen, extracted twice, and analyzed. All samples turned out
to be homogenous since the coefficients of variation (CVs) were Caution: Ethanol is highly flammable and vapor; keep away from
14% or less (results not shown). Only in the case of cookies and heat, hot surfaces, sparks, open flames, and other ignition sour-
juice, CVs were at 18 or 20%, respectively. ces; do not smoke; keep the container tightly closed; store in a
well-ventilated place and keep cool. The cocktail (patented) con-
tains 2-mercaptoethanol, which is toxic; the stop solution con-
Presentation of Samples to Laboratories tains sulfuric acid, which is caustic; work under a chemical
Following the collaborative test guidelines of AOAC fumehood and avoid skin and eye contact and wear protective
INTERNATIONAL, two blinded replicates for each sample were gloves and clothing (see SDS, attached as separate documents
provided to each participating laboratory. The samples were or delivered by the manufacturer in case of ethanol).
marked with a laboratory-specific letter (A to O) and a random- See Tables 2012.01A, 2012.01B, and 2012.01C for the results
ized number (1 to 64). Each laboratory obtained its own coding of the interlaboratory studies supporting acceptance of the
(different randomized numbers for each laboratory). method.

Table 2012.01A. Performance characteristics RSDr, RSDR, and recovery (based on gliadin content); original table taken from (18) and
reformatted

Maize bread (baked) Rice dough Wheat starch Rice flour Wheat starch Maize flour Maize flour

a
mg (PWG) gliadin/kg 168.0 35.0 79.0 0.0 41.0 0.0 147.0 14.0* 13.0* 13.5* <1.5 <1.5
n Labs 19 20 18 20 18 17 20 20 17
Mean, mg gliadin/kg 141.8 36.8 74.1 8.3 34.7 <1.5 126.6 12.5 14.1 13.2 <1.5 <1.5
RSDr, % 20.8 37.7 14.2 32.0 18.3 26.8 26.8 37.4 29.7
RSDR, % 28.6 40.3 32.4 41.5 25.6 35.4 40.7 38.1 52.1
Recovery (%) 84.4 105.0 93.8 84.6 86.1 89.3 108.5 97.8

a
Samples marked with an asterisk were naturally contaminated.
Table 2012.01B. Performance characteristics sr, sR, RSDr, RSDR, and recovery (based on gliadin content)

Soy Soy Starch Pseudo cereals Legumes Legumes Spices Spices Juice Juice Nougat Nougat Cheese Cheese Pesto Pesto
448
|

a
mg gliadin/kg / 3.43 27.36 3.43 27.36 20.00 / 13.70 6.84 5.00 / 20.55 / 20.55 6.86 5.00
n Labs 12 11 13 12 13 13 12 11 12 11 11 11 10 13 13 12
N Replicates 24 22 26 24 26 26 24 22 24 22 22 22 20 26 26 24
Mean, mg gliadin/kg 0.049 4.38 28.6 4.15 29.7 19.8 1.02 11.4 5.49 3.20 0.001 17.7 0.005 25.1 8.20 4.15
sr, mg gliadin/kg 0.0851 0.49 3.41 0.72 5.18 2.54 0.32 1.17 1.77 0.31 0.0543 1.60 0.0492 4.83 1.32 0.35
sR, mg gliadin/kg 0.0981 0.94 4.68 0.92 6.17 3.55 0.35 1.17 2.13 0.38 0.0543 1.74 0.0492 5.73 1.61 0.60
RSDr, % 11.1 11.9 17.4 17.5 12.8 31.3 10.2 32.3 9.8 9.0 19.2 16.1 8.4
RSDR, % 21.4 16.3 22.2 20.8 18.0 34.5 10.2 38.7 12.0 9.8 22.8 19.6 14.4
Recovery (%) / 128 105 121 108 99 / 83 80 64 / 86 / 122 120 83

a
/ ¼ Assumed to be blank.

Table 2012.01C. Performance characteristics sr, sR, RSDr, RSDR, and recovery (based on gliadin content); gnocchi MW is the microwaved version

Meat Burger Burger Cookie Cookie Crema Crema Crema Bread Cake Cake Cod Cod Caramel Gnocchi Gnocchi MW

a
mg gliadin/kg 27.43 13.70 10.00 20.51 15.01 / 13.67 10.00 10.27 13.70 10.00 / 27.75 27.40 10.27 10.27
Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022

n Labs 13 12 12 13 12 11 11 13 11 12 12 10 12 13 13 13
N Replicates 26 24 24 25 24 22 22 26 22 24 24 20 24 25 26 26
Mean, mg gliadin/kg 28.6 15.0 9.60 26.2 16.3 0.034 11.7 8.51 10.7 7.70 5.79 0.002 22.5 31.1 13.4 13.3
sr, mg gliadin/kg 3.37 1.58 0.84 4.09 2.72 0.0419 1.24 0.40 0.48 0.66 0.31 0.0448 5.92 2.98 1.85 1.03
sR, mg gliadin/kg 4.27 1.66 1.62 6.41 3.20 0.0610 1.52 1.25 1.08 1.21 0.81 0.0448 6.24 4.05 2.13 1.49
RSDr, % 11.8 10.6 8.7 15.8 16.6 10.6 4.7 4.5 8.6 5.4 26.3 9.7 13.9 7.7
RSDR, % 14.9 11.1 16.9 24.7 19.6 13.0 14.7 10.1 15.6 14.0 27.7 13.1 16.0 11.2
Recovery, % 104 109 96 128 109 / 85 85 104 56 58 / 81 113 130 129

a
/ ¼ Assumed to be blank.

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 Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022 | 449

A. Principle Prolamin Analysis and Toxicity (14) (PWG gliadin, 97%

highly purified gliadin from 28 different European wheat
The method is based on an enzyme immunoassay format using
varieties).
a monoclonal antibody that can determine gliadin derived from
(e) Substrate.—One vial, 7 mL (urea peroxide).
wheat and related proteins derived from rye and barley. The an-
(f) Chromogen.—One vial, 7 mL (tetramethylbenzidine in
tibody binds to the celiac toxic amino acid sequence QQPFP (8–
10, 16) and to related sequences, which exist as motifs on all methanol).
prolamin proteins. The antibody detects prolamins in unpro- (g) Stop solution.—One vial, 14 mL (1 N H2SO4).
cessed and processed food by using an additional specific ex- (h) Sample dilution buffer.—60 mL, 5 concentrate.
(i) Cocktail (patented).—One vial, 105 mL.
traction method (cocktail patented). No cross-reactivity exists
to oats, maize, rice, millet, teff, buckwheat, quinoa, amaranth, Required but not provided with the test kit:
and 78 other common gluten-free food ingredients. An addi-
(j) Skim milk powder.—Food quality; gluten-free.
tional collaborative study of Official Method 2012.01 was pub-
(k) Ethanol, 80%.—Mix ethanol and water at a ratio of 4 þ1
lished in 2013 (17).
Samples are extracted by using cocktail (patented) contain- parts, e.g., add 120 mL ethanol p.a. to 30 mL distilled water

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ing b-mercaptoethanol and guanidine hydrochloride described and shake well.
by Garcı́a et al. (16), followed by an extraction with 80% ethanol.
After centrifugation, the supernatant is used in a two-step D. General Instructions
sandwich method. The analyte is incubated in monoclonal anti-
body–coated wells forming an antibody–antigen complex. In a (a) Store kit at 2–8 C (35–46 F).
second step, an antibody peroxidase (POD) conjugate reacts (b) Let all kit components warm up to 20–25 C (68–77 F) before
with the complex to form an antibody–analyte–antibody com- use.
plex. A chromogen/substrate reaction with the immobilized (c) Return any unused microwells to their original foil bag,
POD labeled conjugate determines the bound analyte. reseal them together with the desiccant provided, and
Nonimmobilized components are removed by washing steps. store at 2–8 C (35–46 F).
The response of sample extracts is compared with the response (d) Use an uncoated pre-plate if more than six strips are used
observed with calibrators. in one ELISA run.
(e) The colorless chromogen is light-sensitive; therefore, avoid
B. Apparatus exposure to direct light.
(f) Include ready-to-use standards in duplicates to each run of
Apparatus specified has been tested. Equivalent apparatus may
diluted sample extracts in duplicates.
be used.
(g) Do not reuse wells of the plate.
(a) Thermomix TM31.—For sample homogenization (Vorwerk (h) Use separate pipet tips for each standard and each sample
Deutschland Stiftung & Co KG, Wuppertal, Germany). extract to avoid cross-contamination.
(b) Water bath WNB 14.—(Memmert GmbH & Co KG, (i) Use a multistepper pipet for adding the conjugate, sub-
Schwabach, Germany). strate/chromogen, and stop solution. Use a single tip for
(c) Bench-top centrifuge.—Multifuge 3L-R, operating at 2500 rpm each of these components.
(Thermo Electron GmbH, Dreieich, Germany). (j) Components and procedures of the test kit have been stan-
(d) Glass tubes.—10 mL; for extraction (Brand GmbH, dardized for use in this procedure. Do not interchange indi-
Wertheim, Germany). vidual components between kits of different batches (lot
(e) Polystyrol tubes.—5 mL; for sample dilution (Greiner Bio-
numbers).
One, Kremsmünster, Austria).
(k) Do not freeze any of the kit components.
(f) Microtiter plate reader.—With 450 nm filter (Tecan
(l) Carefully dilute the components that are included in the
Deutschland GmbH, Crailsheim, Germany).
kit as concentrates; avoid contaminations by airborne ce-
(g) Micropipettes 20–200 mL and 200–1000 mL.—(Eppendorf AG,
real dust or dirty laboratory equipment.
Hamburg, Germany).
(m) Wear gloves during preparation and performance of the
(h) Glassware.—Wash bottle (1000 mL) and graduated
assay.
cylinders.
(n) Clean surfaces, glass vials, mincers, and other equipment
(i) Roto-Shake Genie.—(Scientific Industries, New York, USA).
with 60% ethanol.
(o) Carry out sample preparation in a room isolated from
C. Reagents ELISA procedure.
Items (a)–(h) are available as a test kit (RIDASCREEN Gliadin, (p) Check for prolamin contaminations of reagents and
R7001, R-Biopharm AG, Darmstadt, Germany); item (i) is avail- equipment.
able separately as cocktail (patented) in two different volumes
(R7006 with 105 mL and R7016 with 1000 mL, R-Biopharm AG,
E. Preparation of Test Samples
Darmstadt, Germany). Refer to kit label for current expiry.

(a) Antibody-coated microwell strips.—Monoclonal antibodies are (a) Grind solid samples to a powder, homogenize pasty-like
coated onto a set of 12 eight-microwell strips. sample with a spatula, and mix liquid samples.
(b) Wash buffer concentrate.—100 mL/bottle, 10 concentrate. (b) Weigh 0.25 g of sample or use 0.25 mL of a liquid sample in
(c) Peroxidase-labeled antibody.—One vial (1.2 mL, 11 a 10 mL glass vial and add 2.5 mL cocktail (patented).
concentrated). (c) If tannin- and polyphenol-containing samples (e.g., choco-
(d) Gliadin standards.—Six vials (1.3 mL each, ready to use). late, chestnut, juice, coffee, cacao, sorghum, spices, or
Calibrated to the gliadin preparation of Working Group on buckwheat) are prepared, add an additional 0.25 g SMP
 450 | Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022

(food quality; gluten-free) to the sample before addition of liquid from the wells. Fill all the wells with 250 mL diluted
cocktail (patented). washing buffer and pour out the liquid again. Repeat two
(d) Close vial and mix well (avoid cross-contamination). more times.
(e) Incubate for 40 min at 50 C (122 F) in a water bath. (g) Add 50 mL substrate and 50 mL chromogen to each well. Mix
(f) Let sample cool down; then mix with 7.5 mL 80% ethanol. gently by shaking the plate manually and incubate for 30
Close vial and shake for 1 h upside down or by a rotator at min at room temperature (20–25 C/68–77 F) in the dark.
room temperature (20–25 C/68–77 F). (h) Positive wells should develop a blue color, indicating the
(g) Centrifuge 10 min at 2500 g at room temperature (20–25 C/ presence of prolamins.
68–77 F) and/or filter the extract. (i) Add 100 mL stop reagent to each well. Mix gently by shaking
(h) Transfer the supernatant (extract) in a screw-top vial and the plate manually. The color of positive prolamin-
keep for testing. containing wells changes to yellow.
(i) Dilute the sample 1:12.5 (1 þ 11.5, 0.08 þ 0.92 mL) with the
prepared sample dilution buffer. H. Reading
(j) Use 100 mL per well in the assay immediately after dilution
Read the results with a microtiter plate reader. Measure the ab-

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(not more than 30 min).
sorbance at 450 nm. Read within 30 min against air after addi-
tion of stop solution.
F. Preparation of Components Delivered With Kit

(a) Sample diluent.—Provided as a concentrate (5-fold). Only the I. Calculations

amount that is actually needed should be diluted 1:5 (1 þ 4)
(a) Determine the gliadin content of each set of duplicate sam-
with distilled water (e.g., 3 mL concentrate þ 12 mL distilled
ple wells by reference to a calibration curve measured by
water, sufficient for the dilution of 15 samples). Make sure
the actual test run.
that the buffer is not contaminated with gliadin.
(b) It is recommended to use the RIDASOFT WIN.NET
(b) Antibody enzyme conjugate.—(Bottle with red cap.) Provided
(Z9996FF, R-Biopharm, Darmstadt, Germany) and a cubic
as a concentrate (11-fold). Since the diluted enzyme conju-
spline curve fitting procedure.
gate solution has a limited stability, only the amount that
(c) The standard calibration curve of the ELISA covers a range
is actually needed should be diluted. Before pipetting, the
from 5 to 80 ng gliadin/mL in the calibrators. Including the
conjugate concentrate should be shaken carefully. For re-
sample dilution factor of 500, this corresponds to 2.5–40 mg
constitution, the conjugate concentrate is diluted 1:11
gliadin/kg sample or 5–80 mg gluten/kg samples (see also
(1 þ 10) with distilled water (e.g., 200 lL concentrate þ
(g) in this chapter for calculation from gliadin to gluten).
2.0 mL distilled water, sufficient for two microtiter strips).
(d) A further dilution and new detection of the sample is rec-
Take care that the water is not contaminated with gliadin. ommended for absorbance values > standard 6. This addi-
(c) Washing buffer.—Provided as a 10-fold concentrate. Before tional dilution factor must be taken into account for
use, the buffer must be diluted 1:10 (1 þ 9) with distilled wa- calculation.
ter (i.e., 100 mL buffer concentrate þ 900 mL distilled water). (e) Convert the units ng gliadin/mL diluted sample to mg glia-
Prior to dilution, dissolve any crystals formed by incubating din/kg sample as follows: Multiply the amount in ng/mL by
the buffer in a water bath at 37 C (99 F). The diluted buffer the dilution factor. Divide the product by 1000 to achieve a
is stable at 20–25 C (68–77 F) for 4 weeks. unit of mg/kg. The dilution factor corresponds to the sam-
ple preparation of at least 500.
G. Determination (f) Absorbance below standard 2 (5 ng gliadin/mL) implies that
the sample assayed is diluted too much or that no gliadin
(a) Bring all reagents to room temperature (20–25 C/68–77 F) or that gliadin below the LOQ is present in the sample.
before use. Do not allow microwells to dry between work- (g) Gluten content of a sample can be calculated from the glia-
ing steps. din value. Gluten values can be expressed in mg/kg by mul-
(b) Insert a sufficient number of wells into the microwell tiplying the gliadin value by 2 as defined by Codex
holder for all standards and samples to be run in duplicate. Alimentarius (3). However, recent research shows a gliadin
Record standard and sample positions. Use an uncoated to gluten conversion factor of 1.5 is more accurate (4, 13).
pre-plate if more than six strips are used in one ELISA run. (h) A measurement result of less than 10 mg gliadin/kg
(c) Add 100 mL of each standard solution or prepared sample to assures a content of gluten below the Codex threshold of
separate wells and incubate for 30 min at room tempera- 20 mg gluten/kg.
ture (20–25 C/68–77 F). (i) For analysis of oat and oat products, AOAC Official Method
(d) Pour the liquid out of the wells and tap the microwell 2018.15 is recommended.
holder upside down vigorously (three times in a row) (j) For analysis of hydrolyzed or fermented gluten, AOAC
against absorbent paper to ensure complete removal of liq- Official Method 2015.05 is recommended.
uid from the wells. Fill all the wells with 250 mL diluted
washing buffer and pour out the liquid again. Repeat two J. Criteria for Acceptance of Standard Curve
more times.
(e) Add 100 mL of the diluted enzyme-labeled conjugate to (a) The course of the calibration curve is shown in the Quality
each well and incubate for 30 min at room temperature Assurance Certificate, enclosed in the test kit.
(20–25 C/68–77 F). (b) Absolute absorbances may vary between different runs
(f) Pour the liquid out of the wells and tap the microwell (e.g., due to different temperatures or analysts). However,
holder upside down vigorously (three times in a row) the shape of the standard curve should be similar to the
against absorbent paper to ensure complete removal of one given in the Quality Assurance Certificate.
 Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022 | 451

(c) OD at 450 nm for standard 6 is higher than 1.2. the OD values of the calibrators also pointed to a severe con-
(d) OD values for standards should continuously increase with tamination problem (Figure 1). With exception of the calibrator
higher concentrations, especially when comparing stan- with highest concentration (80 ng/mL), there was significant dif-
dard 1 and standard 2. ference between results from laboratory L compared to the
(e) An OD value for standard 1 that is much higher than the other labs (at least P < 0.05; P ¼ 0.0013 for standard 1; t-test as-
OD value stated in the certificate could be an indication for suming different variances). It was decided to exclude this data
errors during pipetting or incubation or contamination. set from any further calculation. Nevertheless, Figure 1 also
(f) Indication of instability or deterioration of reagents is depicts the high quality and comparability of results for calibra-
shown by any coloration of the chromogen solution prior tors from the remaining participants. With exception of the
to test implementation. zero calibrator, CVs below 15% were obtained (results not
(g) Coefficients of variation for replicates should be less than shown).
10% for samples within the measurement range. Another participant’s data set (laboratory H) indicated a high
(h) Test controls (e.g., offered by R-Biopharm) should be mea- contamination of the SMP used for extraction of some samples
sured in the reported ranges in each run. (pseudo cereals, spices, juice, nougat, and cake). Laboratory H

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analyzed pure SMP with a mean OD value of 1.055 (n ¼ 4 repli-
Revised: February 2017: Title: Update as part of Final Action cates) compared to a mean OD value of 0.127 6 0.045 from all
(change “foods containing wheat, rye, and barley” to “rice- and the other participants. Comparing the results of the incurred
corn-based foods”) samples analyzed by laboratory H with the results of all the
Revised: March 2017: D(b): Modification of the wash solution other participants, all samples analyzed by laboratory H using
to substitute thimerosal in the washing buffer by the mercury- SMP showed much higher values compared to the other partici-
free preserving agent bronidox L pants (Figure 2; P < 0.001 by paired t-test). These values were ex-
Revised: September 2017: New ELISA plate approved as a re- cluded from any further statistical calculations. All other
placement to the current microtiter plate, which is no longer samples from laboratory H were comparable to the other partic-
available ipants (Figure 2, linear regression; P ¼ 0.91 by paired t-test). It
Posted: February 2017 (Final Action), March 2017 was decided to use the results from those samples for statistical
(Modification), September 2017 (Minor Modification)
analysis.
Data from all other participants showed only two minor
Results and Discussion errors because these participants did not repeat one sample
each, which had OD values higher than calibrator 6 and should
Statistical Analysis of Laboratories
have been analyzed again with a higher dilution (laboratory C
The study director asked 14 laboratories to participate in the with cookie and laboratory M with caramel). These samples
collaborative test. All participants delivered data sets for the 64 were not included for further statistical analyses, as the collabo-
blind coded samples. The data set from one participant (labora- rative study protocol was not followed. Several other laborato-
tory L) showed an undefined contamination in the analytical ries re-analyzed samples additionally in a higher dilution
laboratory (lowest OD values around 1.0, including the OD value although the samples were already within the calibrator range
for the sample dilution buffer and blank samples). Furthermore, in their first ELISA measurements. In this case, only the results

OD value
3.0

2.5

2.0

1.5

1.0

0.5

0.0
0 10 20 30 40 50 60 70 80
ng gliadin/mL
Figure 1. Comparison of calibration data from laboratory L (red triangles) with the mean and standard deviation derived from the other 13 participants (blue circles).
 452 | Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022

Lab H (mg
gliadin/kg)
40

35

30
y = 0.84x + 9.6324
25
y = 1x + 0.0333
20

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15
without SMP
10
with SMP
5

0
0 5 10 15 20 25 30 35

Mean all labs (mg gliadin/kg)

Figure 2. Results from laboratory H (y-axis) were modeled by the mean results from all other participants (x-axis); values (red triangles) and regression in red depict ma-
trixes to which skim milk powder was added for extraction; values (blue circles) and regression in blue depict matrixes to which no skim milk powder was added for
extraction.

from the first ELISA measurements were included in the statis- The collaborative study that led to the approval of Official
tical analyses. Method 2012.01 in 2011 was conducted with PWG gliadin as the
incurring agent and with naturally contaminated samples with
Outlier Detection and Performance Characteristics an unknown source of gluten. Matrixes tested for the first col-
According to AOAC Appendix D (11), data sets from collabora- laborative study were corn-based bread and rice-based dough
tive tests should be checked for outlying values. Twenty-three (contaminated with PWG gliadin), wheat starches, rice flour,
outliers according to Cochran, Grubbs, and double Grubbs were and corn flour. In 2011, an additional collaborative study was
detected with a high proportion in blank samples. In case of the performed with AOAC Official Method 2012.01 using corn bread,
blank cod samples, outlier removal was stopped after detection corn flour, and extruded corn snack as matrixes (17).
of one outlier according to Cochran and one double Grubbs fol- Since most unintended contaminations of supposed gluten-
lowing AOAC guidance (11). In total, 7 out of 13 participants free food occur with flour or products made with flour, this
showed no outliers at all. Seventeen outliers were detected by study was predominantly conducted with matrixes incurred
the Cochran test, indicating higher differences between the two with MoniQA wheat flour with defined concentrations of gliadin
replicates compared to the other labs. Only five outliers accord- and gluten (13, personal communication with Katharina
ing to Grubbs and one so-called double Grubbs were detected. Scherf). In order to enable comparison to the previous two col-
Four participants are responsible for more than 80% of all outlying laborative studies, some matrixes (legumes, juice, pesto, burger,
values. Considering that the participants performed 832 extractions crema, and cake) were additionally incurred with PWG gliadin.
in total, the overall number of outliers is very small (2.8%). For the present collaborative study, typical representative ma-
After elimination of outliers, the performance characteristics trixes from important gluten-free food categories were ana-
precision of repeatability s(r) and precision of reproducibility lyzed. In total, 19 different matrixes from 16 different food
s(R) were calculated using the AOAC Interlaboratory Study categories were prepared independently at the Hochschule
Workbook for Blind (Unpaired) Replicates (version 2.0) and Geisenheim University. Since it was not possible to measure all
cross-checked by an AOAC statistician in February 2021. matrixes at more than one incurred level by participants of the
Table 2012.01A (parts A and B) show the results of these calcu- collaborative study, more levels were analyzed at the method
lations together with calculation of the mean recovery for each developer’s site to cover the measurement range for every ma-
incurred matrix. Table 2012.01 (reformatted) shows the results trix (see Tables 1 and 2).
of the collaborative study that led to Official Method 2012.01 First Additionally, soy, nougat, spices, cheese, crema, and cod
Action in 2011. were analyzed as gluten-free matrixes (Table 1). The spice mix-
ture showed a very low contamination level of about 1 mg glia-
Matrixes din/kg (Table 2012.01B), which was confirmed to be a barley and
This collaborative test was performed to add additional ma- rye contamination by PCR (SureFood ALLERGEN 4plex Cereals
trixes to the already existing Official Method 2012.01 Final S7006 together with SureFood PREP Advanced S1053 for extrac-
Action. tion; Congen, Berlin, Germany). It was therefore excluded from
 Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022 | 453

sR, mg gliadin/kg

6
y = 0.182x - 0.0793
R² = 0.8309
5

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2

0
0 5 10 15 20 25 30 35
mg gliadin/kg
Figure 3. Mean concentrations for each sample (y-axis) were modeled by the reproducibility deviation sR (x-axis) from all participants except laboratory L and SMP-con-
taining samples from laboratory H; data for linear regression are shown.

estimating LOD and LOQ from the data obtained by 13 different shown). A possible explanation is the high level of polyphenols
participants. Following the procedure described in AOAC and pectins in many fruits. These components tend to bind to
Appendix M (12), LOD and LOQ can be estimated in two different proteins and may thereby mask gliadin proteins present in the
ways. The first one is the so-called basic approach using the juice. By adding SMP, other proteins are added in high excess,
mean of each matrix specific sR of all blank samples (0.061 mg/ so that the binding of the polyphenols to gliadin proteins is re-
kg) multiplied by a factor of 3.3 and 10, respectively. Following duced. Since the MoniQA flour was never completely solved in
this approach, LOD and LOQ are 0.20 mg gliadin/kg or 0.61 mg the juice, aggregation and sedimentation could have taken
gliadin/kg, respectively. For the second one, sR is modeled by place, which could also explain the high variation of results
mean concentration, and slope and intercept of the linear re- among participants.
gression are calculated to 0.182 and 0.0793 mg/kg, respectively Matrixes with a moderate heat treatment were nougat (80 C
(Figure 3). Using these data together with mean concentration for an hour) and pesto (100 C for 5 min and 80 C for 10 min),
from all blank samples (0.015 mg/kg), LOD and LOQ are calcu- both with a quite high fat content. As can be seen in
lated to 0.40 mg gliadin/kg and 1.19 mg gliadin/kg, respectively, Table 2012.01B, these matrixes showed excellent relative stan-
by using the equation dard deviations of reproducibility of less than 20%, and in the
case of nougat it is less than 10%. Recoveries were between 83
LOD ¼ ðmean concentration þ 3:3  interceptÞ=ð1–1:65  slopeÞ and 120%.
Gnocchi and cod were cooked at 100 C in a boiling water
The LOQ by this method is 3 times the LOD. Since the second bath for 15 or 20 min, respectively. Gnocchi were also analyzed
approach is dependent on all other samples analyzed for this with additional microwave treatment at 1500 W for 2.5 min due
collaborative test, we prefer the basic approach described above. to a report stating that the R5 antibody is not able to detect
Nevertheless, the LOD and LOQ stated in the instruction for use microwaved gluten (15). It is quite clear from the results in
are 0.5 mg gliadin and 2.5 mg gliadin/kg, respectively. Table 2012.01C that neither cooking nor microwaving has an
Seven different matrices (soy, starches, pseudo cereals, important effect on recovery. RSDR was at or below 16% and
legumes, spices, juice, and cheese) were incurred by mixing therefore excellent for gnocchi and 27.7% for cod. This higher
with MoniQA wheat flour or PWG gliadin (Table 1) without any RSDR is probably due to the difficult homogenization of this ma-
further treatment such as heat. With exception of juice spiked trix. Tendencies toward higher recoveries in gnocchi may be
with wheat flour, none of these matrixes showed RSDR values due to the high water content. In contrast to most other ma-
higher than 23%. The range of recoveries for these matrixes was trixes, the gnocchi were not dried additionally after preparation,
between 83% for spices and 128% for soy (Table 2012.01B). Juice so that the remaining moisture was quite high. In this case, wa-
spiked with PWG gliadin had lower recoveries of around 64 and ter from the sample may have evaporated and (re-)frozen on
80% spiked with MoniQA wheat flour (Table 2012.01B). Without the inner lid of the vial, resulting in an increase of analyte
addition of SMP, recovery for both MoniQA wheat flour and concentration.
PWG gliadin is nearly zero, and even neutralizing the low pH Two matrixes (caramel and crema; Table 2012.01C) were
value of the juice did not help to obtain higher recoveries also cooked, but due to their higher fat and sugar content com-
(results are not part of this collaborative study and are not pared to cod and gnocchi, the cooking temperature should be
 454 | Lacorn et al.: Journal of AOAC INTERNATIONAL Vol. 105, No. 2, 2022

higher than 100 C, especially for caramel. Again, recoveries quantify gliadin from matrixes representing important gluten-
were between 85 and 113%, and precision was excellent (RSDR free food categories.
better than 15%). It should be noted that caramel was delivered The study director Katharina Scherf together with the
as chunks to the participants, which had to be heated in a 50 C method developers from R-Biopharm and the provider of test
water bath before weighing. samples from Hochschule Geisenheim University recommend
All highly heated matrixes (meat, burger, cake, cookie, and that the title and applicability statement of the Official Methods
bread) showed precision measures below 25% (Table 2012.01C). of AnalysisSM 2012.01 Final Action of AOAC INTERNATIONAL is
Both fried matrixes and the cake showed even lower values. changed as proposed.
Recoveries were very good, with values between 96 and 128%.
The chocolate cake with almonds was an exception because re- Acknowledgments
coveries were unexpectedly low irrespective of the incurring
We would like to thank Laura Allred and Hertha Deutsch for
material. Since the nougat also contained high levels of choco-
their important input on gluten-free food categories and their
late and the bread had similar processing as the cake but
showed better recoveries, the lower recovery for the cake proba- ongoing activities to protect their patient groups. We also thank

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bly comes from the combination of a high level of cocoa and a Patricia Meinhardt for proofreading of the manuscript and for
high level of processing. her helpful contribution to this successful study. Furthermore,
Supplementary to levels tested in the collaborative study, we thank Sophie Ballmann, Lukas Kraft, and Niklas Weber for
additional levels were analyzed by an in-house study at the their helping hands in all kinds of preparations for the collabo-
method developer’s site. These results (Table 2, part A and B) rative study as well as their help during additional level testing
underpin that each matrix shows comparable recoveries at dif- at the method developer’s site.
ferent levels. In addition, the CVs were comparable at different The participation of the following laboratories in the collabo-
levels and matrixes, as also observed in the collaborative study rative study is greatly acknowledged: Joe Baumert, FARRP
(see Figure 3). (University of Nebraska, Lincoln, NE), Sharon Crowe (Public
Analyst’s Laboratory, Galway City, Ireland), Tina Dubois (R-
Biopharm AG, Darmstadt, Germany), Harrison Feldkamp
Implications
(General Mills, Golden Valley, MN), Rupert Hochegger (AGES,
The results of this comprehensive collaborative study are even Wien, Austria), Peter Koehler (biotask AG, Esslingen am Neckar,
better than the results of the two preceding collaborative stud- Germany), Terry Koerner (Health Canada, Ottawa, Canada),
ies (17, 18), especially regarding the complexity of all incurred Roberto Lattanzio (Eurofins, Hamburg, Germany), Rakhi Panda
matrixes and their closeness to commercial samples. Taken (U.S. Food and Drug Administration, Laurel, MD), Katharina
these two facts together, the results clearly show that Official Scherf (Karlsruhe Institute of Technology, Karlsruhe, Germany),
Method 2012.01 is suitable for all these additional matrices from Tuula Sontag-Strohm (University of Helsinki, Helsinki, Finland),
16 different food categories (see also Figure 3). These matrixes John Szpylka (Food Safety Net Services, San Antonio, TX),
included all relevant ingredients for all kinds of foods in various Jeremie Theolier (Université Laval, Quebec, Canada), Alessandro
combinations and different processing levels. These included Urbani (Nestlé Quality Assurance Center, Padova, Italy).
very high levels of potentially difficult ingredients such as
spices, cocoa, fat, and sugar, as well as very high processing
such as baking at high temperatures for almost an hour or fry-
Funding
ing at very high temperatures. The completeness of this study There was no funding except the fact that we supplied the
is to our knowledge unprecedented. participants of the collaborative test with test kits for free.
Official Method 2012.01 is calibrated toward PWG gliadin since R-Biopharm paid for the whole study.
2002 and showed excellent recoveries of all matrixes incurred
with PWG gliadin in the present collaborative study.
Furthermore, the MoniQA wheat flour used for incurring all ma- Conflict of Interest
trixes also showed excellent recoveries of the independently de- All authors declare no conflict of interest.
termined gliadin content. Official Method 2012.01 is in general
also able to determine wheat gluten with a similarly high accu-
racy using an appropriate conversion factor for gliadin to gluten
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