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EDITORIAL Hen's egg (Gallus gallus) is an important food source for humans. Proteins from egg yolk only have minor allergenicity, but many proteins from egg white are known to be allergenic. Besides ovalbumin, ovotransferrin, lysozyme, livetin and ovomucoid represent the most important allergens. They are thermally stable and therefore resist processing like baking. Many processed foods like cookies, chocolate or bakery products may contain egg white. People allergic to egg white must strictly avoid the consumption of food containing egg white. Cross contamination during the production process often occurs and egg white residues in food cannot be excluded. Therefore sensitive detection methods for egg white in food are required. In Europe, the Labelling Directive (Directive 2000/13/EC) and later amendments (2003/89EC and 2007/68/EC) ensure that all consumers are given comprehensive ingredient listing information to make it easier for people with food allergies to identify ingredients they need to avoid. Annex IIIa from the above legislation establishes a list of 14 food allergens including eggs and products thereof, which have to be indicated by reference to the source allergen whenever they, or ingredients made from them, are used at any level in pre-packed foods, including alcoholic drinks. Sonja Steininger

Romer Labs® CheckSample-Survey (CSS) – Egg in a food matrix Introduction There are currently a limited number of providers of proficiency tests for allergens. Therefore, as part of its integral service approach, Romer Labs Diagnostic GmbH has launched a Check-Sample-Survey programme for a range of allergens. The objective of Romer Labs® Check-Sample-Survey is to provide an interlaboratory comparison study of egg analysis in a food matrix. The purpose of this study is to offer a platform for customers to assess the effectiveness and accuracy of their test methods as part of their internal quality management system. The Romer Labs® Check-Sample-Survey aims to industry labs, service labs or manufacturing sites that perform egg analysis on a routine basis. The specific method employed for analysis is up to the participating laboratory (e.g. Lateral Flow Devices, ELISA, PCR or Mass Spectrometry).

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Preparation of test material The test material of this Check-Sample-Survey (A13121E) was prepared by Romer® Labs UK. A blank wheat flour material was spiked with whole egg powder at an appropriate level. The resulting mixed flour sample was homogenized by tumbling and then dispensed into aluminium sachets to create individual sub-samples (approximately 5 g) that were stored in appropriate conditions previous to distribution. Each participant received the flourbased test material together with instructions and a result form. Homogeneity Ten randomly selected test material samples were analyzed in duplicate. The available analytical data of the characterization (20 data point analyses with 10 extractions from the bulk sample - each extract run in duplicate) showed sufficient homogeneity of the test material and was not included into the calculation of the assigned value of the interlaboratory comparison. Initially statistical tests checked the data for any widely discrepant pairs using Cochran’s test. No such pairs were found and no data was removed. Thereafter the remaining data were subject of analysis of variance to estimate the sampling and analytical variances. Distribution The test material of this Check-Sample-Survey (A13121E) has been dispatched in April 2013 to 34 laboratories all over Austria, Estonia, France, Italy, Poland, Slovakia, Spain, the United Kingdom and the USA. Of these, 23 participants returned their results within the time-scale demanded by this Check-Sample-Survey, which makes 68 % (see Figure 1).

food

7

8

research 1  

service lab   n.a.  

7 Figure 2. Participants by industry

Almost 33 % of the participants can be attributed to the Food sector, whereas 38 % are Research Institutes and Service Laboratories (see Figure 2). 16

participants

used

ELISA

(Enzyme

Linked

Immunosorbent Assay), 6 participants used LFD (Lateral Flow Device) and 1 participant used MS (Mass Spectrometry) as method of choice (see Figure 3).

1

ELISA

6

LFD 16  

LC/MS

Figure 3. Methods employed

Austria Estonia   5  

France

9

Italy 1  

Poland

2 2  

1 1   1  

Slovakia

1

Spain United  Kingdom   USA  

Figure 1. Participants by countries

Results Participants were required to report their data in mg/kg before the closing date for this test, May 31th 2013. Results for whole egg and egg white protein were submitted by 23 participants. The quantitative results were statistically analyzed in order to provide an assigned value and z-score for each analyte. The results were separated into sub-sets according to the test kit used.

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Z-scores: The z-score relates the error in the result to the target standard deviation (σp) which is set ahead of the test and reflects ‘best practice’ or fitness for purpose. A z-score between +2 and -2 is considered as satisfactory performance, between +2 and +3 or between -2 and -3 is considered as questionable performance, and anything outside this range (> +3 or < -3) is considered as unsatisfactory (Visser, 2006).

Table 2 - Quantitative results and z-scores, whole egg Whole egg Assigned value 5.02 [mg/kg]

Limit of detection

result [mg/kg]

[mg/kg]

5.37

0.1

0.3

6.25

0.1

1.1

6.145

0.1

1.0

2.3

0.1

-2.5

>2.5

2.5

*

9

---

*

6.6

---

*

presence

---

*

>5

5

*

Calculation of Assigned Value X: The mean of the results was chosen as assigned value (due to the small number of results in the sub-sets no meaningful independent statistical evaluation could be done). Non numerical results, i.e. qualitative or semiquantitative results, were excluded from the calculation of the assigned value as well as results of sub-sets which were too small to permit meaningful statistical assessment. Detailed results of the statistical assessment are shown in Table 3. Quantitative results for whole egg and egg white protein are shown in Table 1 and Table 2. A dot plot showing egg white protein results [mg/kg] for the Romer Labs® – AgraQuant® Egg White ELISA Test Kit and Other Kits/Methods is given in Figure 4.

z-score

positive

2

*

10

negative results

*

8.9

5

*

15.1

---

*

positive

---

*

20

15

*

>5

<5

*

positive

---

*

>5

<5

*

*Resultant sub-sets of results were too small to permit meaningful statistical assessment. This does not negate the accuracy and/or validity of these test results.

Table 1 - Quantitative results and z-scores, egg white protein Egg white protein Limit of detection

Dotplot of  egg  white  protein  against  kit/method  used  for  A13121E  

z-score

result [mg/kg]

[mg/kg]

2.4

0.05

2.3

1.39

0.05

-0.6

1.14

0.05

-1.3

1.03

0.05

-1.6

2.04

0.05

1.3

2.2

0.1

*

0.65

0.25

*

kit/method

Assigned value 1.6 [mg/kg]

Romer Labs®                                                                         AgraQuant®  Egg  White  ELISA  Test  Kit  

Other (egg  white  Protein)  

0

0,5

1

1,5

2

2,5

egg white  protein  [mg/kg]  

Figure 4. Dot plot of egg white protein results against different kits/methods

*Resultant sub-sets of results were too small to permit meaningful statistical assessment. This does not negate the accuracy and/or validity of these test results.

Table 3 - Summary of statistical analysis for egg white protein by Romer Labs® – AgraQuant® Egg White ELISA Test Kit Analyte

Data points, n

Assigned value, [mg/kg]

Uncertainty, u

Target standard deviation, σP

% of scores lzl ≤ 2

egg white protein

5

1.6

0.27

0.4

80

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3


ABOUT THE AUTHOR Name

Position

Education Address

Sonja Steininger, MSc Production Associate at Romer Labs Diagnostic GmbH since 2011 University of Applied Sciences Tulln, Biotechnology Master Thesis: “Development of a LC-MS-Method for the Detection of Pyrrolizidinalkaloids” Romer Labs Diagnostic GmbH, Technopark 1, 3430 Tulln, Austria

Tel: +43 2272 61533, Fax: +43 2272 61533-111

e-mail: sonja.steininger@romerlabs.com

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Newsletter is published by Romer Labs Division Holding GmbH - Austria

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e-Mail: enquiry@romerlabs.com

Fax: +43 2272 61533 13177, e-Mail: marketing@romerlabs.com Editor: Hannes Binder. Publisher: Erich Erber

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