Spot On: Mycotoxin Reference Materials: How Do You Measure Your Measurements?

Issue 12

A Romer LabsÂŽ Publication

Mycotoxin Reference Materials:

How Do You Measure Your Measurements?

7 Things You Need to Know about Reference Materials The Math Behind Mycotoxin Quality Control Materials Certified Reference Materials and Certificates

Photo: Medioimages/Photodisc

Contents

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7 Things You Absolutely Need to Know about Mycotoxin Reference Materials Qualified reference materials are a prerequisite for successful qualification and quantification in analytical chemistry. But how are they made? How can you be sure that they meet the standards of quality that your laboratory needs? Helmut Rost of Romer Labs answers these common questions and more.

By Helmut Rost, Senior Director Analytical Services, Romer Labs

Spot On is a publication of Romer Labs Division Holding GmbH, distributed free-of-charge.

ISSN: 2414-2042

Editors: Joshua Davis, Cristian Ilea

Photo: Martin Barraud

Contributors: Lee Jiuan Chin, Helmut Rost, Martina Bellasio

Graphic: GraphX ERBER AG Research: Kurt Brunner

Publisher: Romer Labs Division Holding GmbH Erber Campus 1 3131 Getzersdorf, Austria Tel: +43 2782 803 0 www.romerlabs.com

ŠCopyright 2020, Romer LabsŽ All rights reserved. No part of this publication may be reproduced in any material form for commercial purposes without the written permission of the copyright holder.

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Certain Uncertainties: The Math behind Mycotoxin Quality Control Materials and How to Use It Here are the basics of calculating uncertainty measurement and how to quantify it in QCMs, in three simple mathematical formulas.

By Martina Bellasio

All photos herein are the property of Romer Labs or used with license.

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Spot On Issue 12

Editorial Keeping your results honest Working in an analytical lab can feel like being a detective: even when you’re not on duty, you’re always turning over some problem or other in your head, worrying.

The reason? A lot is riding on the work that we do at our mycotoxin analytical labs, including here at the Romer Labs APAC Solutions Centre in Singapore, where I serve as laboratory manager. Grain producers, grain traders and feed producers are all depending on us to get them results that they can rely on. These results have real-world consequences for the food and feed supply chains, and must be delivered quickly so that customers can make decisions and move on to the next batches to be tested and processed. There are a host of other considerations to add to the pressure, such as compliance with national and international accreditation authorities and ensuring clear traceability and documentation.

In this issue of Spot On, however, we’re focusing on something more basic that makes our day-to-day work in mycotoxin diagnostics possible: reference materials. While it’s true that results are only as accurate as the devices we use to measure them, that’s only part of the story. How do you measure the accuracy of the measurements coming from, for example, a liquid chromatography/mass spectrometry machine? How can you be sure that you’re compensating for matrix effects to know the true degree of mycotoxin concentration?

To give us some practical insight into these issues, we’ve invited a couple of our mycotoxin experts to share their thoughts. Our senior director of analytical services, Helmut Rost, brings his substantial experience in mycotoxin analytics to discuss seven of the most basic aspects of mycotoxin reference materials, from how they are created to how to interpret the certificate that comes with the highest standardization tool in the discipline, certified reference materials.

Martina Bellasio, product manager at Romer Labs, delves into the math behind these reference materials and emphasizes the advantages of the quantitative approach to measuring uncertainty.

So join me and my fellow mycotoxin detectives for this issue of Spot On. I hope you enjoy it.

Lee Jiuan Chin Laboratory Manager Romer Labs® APAC Solutions Centre

A R moam g ae zr i n L ea bosf® RPoumbel irc a L taibosn®

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Spot On Issue 12

7

Things

You Absolutely

Need to Know about

Mycotoxin Reference Materials Qualified reference materials are a prerequisite for successful qualification and quantification in analytical chemistry. But how are they made? How can you be sure that they meet the standards of quality that your laboratory needs? What are certified reference materials? How do you make sense of the certificate? Helmut Rost of Romer Labs answers these common questions and more.

By Helmut Rost, Senior Director Analytical Services, Romer Labs

A Romer LabsÂŽ Publication

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Think of isotopelabeled reference materials as "super reference materials" for mass spectrometry.

❶ Why do we need reference materials?

The short answer is that regulating and certifying bodies require or recommend that labs use them. The United States and the European Unioni, for example, both regulate several mycotoxins by setting maximum levels for those contaminants in feed and foodstuffs.

Furthermore, laboratories accredited according to ISO 17025 must use qualified and competent reference materials to maintain their accreditation.

The slightly longer answer is that labs use reference materials to increase the reliability of their results.

In recent decades, the analysis of contaminants such as mycotoxins has become more and more important for the characterization and evaluation of food and feed samples. Reference materials are substances with defined properties, such as aflatoxin B1 dissolved in acetonitrile at a concentration of 2 µg/mL. The specific properties of a reference material – and, importantly, its relative purity – provide a benchmark by which lab technicians can confirm accuracy of their own results.

❷ How is a reference material defined?

A reference material is sufficiently homogenous and stable with respect to its specified property; for example, it has a certain percentage of purity of a mycotoxin or mycotoxins as a solid or a certain concentration as a liquid standard. These qualities ensure their fitness to be used in the measurement process. Reference material producers fulfilling the requirements of ISO 17034:2016 (”General requirements for the competence of reference material producers”) are considered to be competent. The text in the ISO norm lists corresponding requirements over several dozens of pages along with further literature.

In summary, candidate material must be well characterized in terms of identity and purity before it can be employed as a starter of any reference material. A production plan ensures correct documentation for all batches that are produced. Long-term and short-term stability tests at different temperatures must prove that the material is stable until its specified expiration date.

A homogeneity test for every batch confirms that the material is evenly distributed throughout the dispatch vessels. All analyses need to fulfill the requirements of ISO 17025; an ISO 17034 accreditation means that the laboratory is accredited according to ISO 17025 with its analytical methods. i

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EC No. 1881/2006.

❸ How are reference materials for mycotoxin analysis produced?

The majority of the mycotoxins are produced by three fungal genera: Aspergillus, Penicillium and Fusarium. A carrier material is inoculated with a fungal suspension and incubated over several weeks. Samples are taken during the fermentation process to confirm that target mycotoxin is being created. Typical yields of the production process are several grams of raw mycotoxin per kg carrier material and can vary significantly depending on the mycotoxin and the fermentation process in question.

Once incubation is completed, the real work begins: the lab engineer purifies the very crude raw extract.

It starts with filtering processes and liquid-liquid extractions to reduce the weight and volume of the extract and to remove the main impurities. The key process of the cleaning consists of (semi-)preparative chromatographic purification steps, in which impurities and by-products are separated from the main product due to different adsorption behavior in the chromatography column. This work demands a high degree skill and experience as each mycotoxin has its own properties and retention behavior. The clean-up concludes with the drying of the product, weighing it and then once again checking its quality.

One specific use of reference materials is as matrix reference materials (or quality control materials), which consist mainly of a food or feed matrix contaminated by naturally incurred mycotoxins. One example is corn with deoxynivalenol. These materials can be employed as a quality check to support the analysis of specific food and feed samples.

❹ Why does it make sense to use isotope-labeled reference materials for mycotoxins?

Analysts employing mass spectrometry attach a great deal of importance to isotope-labeled reference materials – with justification. Think of these as “super reference materials” for mass spectrometry. They can do what non-isotope-labeled materials can do and much more. For example, they compensate for unwanted effects such as diminished extraction efficiency, low recovery or matrix behavior in the analysis of the analytes; consequently, they are essential in setting up any analytical procedure. Isotope-labeled reference materials are produced as described above with one important difference: the 12C atoms are replaced by 13C atoms. Therefore, isotope-labeled products have the Spot On Issue 12

Certified reference materials are the highest level of standardization.

erence material should be accredited according to ISO 17025 (“General requirements for the competence of testing and calibration laboratories”). Since ISO 17025-certified laboratories fulfill the requirement of metrological traceability, their results can be used to specify a certified reference material.

❻ What is a certified reference material? same physical and chemical behavior as the unlabeled analytes. They are added prior to analysis and undergo the same procedure but compensate for any over- or underestimation of unlabeled mycotoxins.

❺ How do you check the quality of reference materials?

The purity, that is, the quality of the product, is of paramount importance and should be as high as possible, or greater than 95%. While recurring products can be compared with the precursor batch to check their quality, a new product must be subjected to far more comprehensive characterization. This kind of characterization for solid materials can involve several highly selective and sensitive methods: structure determination by mass spectrometry, infrared spectroscopy or nuclear magnetic resonance spectroscopy can inform us about any possible inherent impurities.

In any case, the purity must be determined as accurately and precisely as possible. The availability of a certified reference material can be a huge advantage since it makes it possible to trace one’s own reference material directly (see number 6 below). Today, the standard method for determining the quantity of liquid reference materials of mycotoxins (solid materials dissolved in a solvent) is high performance liquid chromatography (HPLC) connected to an appropriate detector such as UV (ultraviolet) or FLD (fluorescence detection).

This method has nearly universal applicability, as most mycotoxins are UV/FLD-sensitive.

In isotope-labeled substances, the proportion of 13C to 12C (also known as the isotopic pattern) can be determined by an enhanced resolution scan with a mass spectrometer; the isotopic purity of 13C should be greater than 98%.

The laboratories determining the property of a refA Romer Labs® Publication

The purity of reference materials must be determined as accurately and precisely as possible.

The availability of a certified reference material is a huge advantage.

Here, we arrive at the highest level of standardization. A certified reference material is characterized by a metrologically valid procedure for its property (such as % purity or concentration) and is accompanied by a certificate that provides the certified value together with the calculated uncertainty of this value. What this means is that there are differences between reference materials and certified reference materials in terms of traceability and uncertainty. In the daily work of a laboratory, reference materials are typically utilized. If a reference material needs to be traced back, the certified reference material with its metrological traceability can be used as a reference. However, certified reference materials currently exist for only a few mycotoxins.

❼ How do you read the certificate?

A qualified certificate provides necessary information about the reference material. It typically includes the description of the product with lot number and expiration date, the supplier with address, the hazard notice, intended use, instructions for correct use, storage conditions and, most importantly, the reference value.

The expiry date applies to the unopened product under the defined storage conditions. For certified reference materials, the certificate documents a few further characteristics: the certified value, the uncertainty budget and the discussion of metrological traceability. The uncertainty is calculated based on factors such as the purity and preparation of the product and – if not negligible – its stability and homogeneity (see the article “Certain Uncertainties” for more about calculating and using uncertainty values). The certified value is confirmed by an analysis in which the calibration is carried out against an independently prepared reference batch of the target analyte.

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Certain Uncertainties:

The Math behind Mycotoxin and How to Use It

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Spot On Issue 12

Quality Control Materials Quality control materials (QCMs) are samples with a known concentration of the analyte of interest. In a service laboratory setting, they are usually analyzed under the same conditions that are used to analyze customer samples. Analyzing QCMs can generate data that labs can use to check how a newly developed method is performing, to troubleshoot any problems with the analysis and to generate quality control charts. In this article, Martina Bellasio guides you through the basics of calculating uncertainty measurement and how to quantify it in QCMs.

By Martina Bellasio

All measurements are subject to uncertainty: no measurement is exact. Hence, uncertainty is a key factor in data interpretation as it gives us information on how exact the assigned value is believed to be.

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hen performing a validation of measurement procedures, analysts compare the value generated by each measurement of the QCM to the certified value stated in its certificate.

Informally, some describe this comparison in a qualitative manner with statements such as, “the measured value agrees well with the certified value of the quality control material.� However, a quantitative approach can compare these two values mathematically, thereby excluding bias and providing a tool to keep track of the results over time. All measurements are subject to uncertainty. Hence, uncertainty is a key factor in scientific data interpretation as it gives us information on how exact the assigned value is believed to be.

Why is a quantitative approach preferable in measuring uncertainty in QCMs? A quantitative approach considers a value measured in a practical application, the value certified by the producer of the QCM, and their respective uncertainties.

A Romer LabsÂŽ Publication

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Quantifying uncertainty goes a long way to ensuring we can rely on our methods and produce more objective results.

Let’s first briefly discuss the concept of uncertainty of measurement and why it is important to consider it.

All measurements are subject to uncertainty: no measurement is exact. Hence, uncertainty is a key factor in scientific data interpretation as it gives us information on how exact the assigned value is believed to be. By quantifying the uncertainty associated with a value, a more complete picture of the results can be communicated, therefore making them more useful.

The “ISO Guide to the Expression of Uncertainty in Measurement (GUM)” and the Eurachem/CITAC guide “Quantifying Uncertainty in Analytical Measurement” are commonly used reference texts for expression of uncertainty.

As previously mentioned, comparing the measured value(s) to the certified value of a quality control material requires calculating the difference between these two values and the so-called expanded uncertainty. In the following paragraphs, we will learn how to calculate these values and how to apply them in a practical example.

How to determine the degree to which the value you measure deviates from the certified value Step 1: Determine the difference between the certified value and the mean of the measured values (∆m).

In this step, we calculate the absolute difference (indicated with the symbol ∆m) between the certified value and the mean of the measured values (Formula 1). We take here the mean of the measured values, as any one sample should be measured more than once to guarantee the accuracy of the result.

Formula 1: Δm = │cm − cQCM │ Δm = absolute difference between the mean of measured and certified values cm = mean of measured values

cQCM = certified value

Step 2: Calculate the combined uncertainty (u∆).

Now, we calculate the combined uncertainty (u∆) (formula 2). To do this, the individual uncertainties (see below) of the certified value and of the measured values must already be known (see box below “Finding the individual uncertainties”). Formula 2 shows the exact mathematical operations.

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Formula 2: u∆ =

um2 + uQCM2

uΔ = combined uncertainty of measured and certified values um = uncertainty of measurement result and

uQCM = uncertainty of certified value

Step 3: Calculate the expanded uncertainty (U∆).

Here, we multiply the combined uncertainty by a coverage factor, usually equal to 2.

Formula 3: U∆ = 2· u∆ U∆ = expanded uncertainty of difference between measured and certified values uΔ = combined uncertainty of measured and certified value

Step 4: Compare the difference between the certified value and the measured values (∆m) with the expanded uncertainty (U∆).

The absolute difference between the mean of the measured values and certified value (Δm) and the expanded uncertainty (U∆) are the two main parameters in determining whether the measured value deviates significantly from the certified value. We define a “significant difference” thusly: if Δm is smaller than or equal to U∆, then there is no significant difference between the measurement result and the certified value.

Comparing the absolute difference between the mean of the measured values and the certified value with the expanded uncertainty allows for a mathematical approach to comparing two values and keeps laboratories in compliance with ISO Guide 98-3:2008 (ISO Guide to the Expression of Uncertainty in Measurement, GUM).

Conclusion: Certain Uncertainty While the concept of the uncertainty of measurement can be somewhat difficult to understand at first glance, it can serve as a statement of the degree of precision of a measuring procedure relative to others. Quantifying this uncertainty goes a long way to ensuring that we can rely on our methods and produce results that are more objective and less reliant on qualitative statements: instead of asserting, for example, that “the measured value agrees with the certified value,” calculating uncertainty allows us to plot and document deviations with greater Spot On Issue 12

and more actionable precision. With quality control materials, laboratories have a powerful tool that can help them continuously improve their methods.

An example to help to elucidate these principles appears below.

Note: Much of this text was adapted from the ERM Application Note “Comparison of a measurement result with the certified value” by Thomas Linsinger.

Example A QCM has an assigned value of 12.9 µg/kg aflatoxin, with an estimated expanded uncertainty U of ± 0.9 µg/ kg and a coverage factor k = 2. UQCM, the uncertainty of the certified value, can be calculated by dividing UQCM by the coverage factor, therefore uQCM = 0.9/2 μg/kg= 0.45 μg/kg.

12 laboratory measurements were performed, yielding an average of 14.3 ± 1.8 μg/kg. The uncertainty of the measured values (um) in this case will be estimated using the standard deviation. The standard deviation is divided by the square root of the number of measurements.

um is roughly calculated as 1.8/ 6 μg/kg = 0.74 μg/kg.

Having both the uncertainty of the certified value and the uncertainty of the measure at hand, we can now evaluate whether there is a significant difference between the measured value and the certified value.

First, the absolute difference between the mean of the measured values and certified value is calculated with formula 1 from above:

QCMs give laboratories a powerful tool that can help them to continuously improve their methods.

Δm = │cm − cQCM │ = │14.3-12.9│ μg/kg = 1.4 μg/kg

Then, the combined (u∆) and expanded (U∆) uncertainties are calculated with formula 2:

u∆ = um2 + uQCM2 = 0.742 + 0.452 μg/kg = 0.87 μg/kg U∆ = 2· u∆ = 2 · 0.87 μg/kg = 1.7 μg/kg

The absolute difference between the mean of the measured values and certified value (∆m = 1.4 μg/kg ) is smaller than the expanded uncertainty (U∆ = 1.7 μg/kg). In this example there is no significant deviation between the measured value and the certified value.

Finding the individual uncertainties Here’s how to find the individual uncertainties. You will need this for step 2 above, “Calculate the combined uncertainty (u∆)”.

• Uncertainty of the certified value: The expanded uncertainty of each certified value (UQCM) is usually provided on the certificate. Using this value, the uncertainty of the certified value can be calculated by dividing it by the coverage factor, which is also indicated in the certificate. In Figure 1, we see an example taken from a BiopureTM certificate. Both the expanded uncertainty and the coverage factor are stated here. The uncertainty of the standard was obtained by dividing the expanded uncertainty by the coverage factor (which is equal to 2).

Figure 1. Expanded uncertainty and other information on a certificate from a Biopure™ QCM of zearalenone in corn.

Zearalenone in Corn Compound Mass concentration a lndicative value b Uncertainty c Zearalenone 366 µg/kg ± 33 µg/kg a Expressed on material as supplied b Unweighted mean of accepted values (n = 20) c Estimated expanded uncertainty U with a coverage factor k = 2

• Uncertainty of the measured values: According to ISO/IEC 17025, the uncertainties of measurement should be known for each measurement performed in the laboratory. However, analysts are not always familiar with these uncertainties. In such cases, a few approaches can help you estimate the uncertainties of measurement. Here are a few common practices:

➤ Quality control charts can be used to determine a rough estimation of the um. ➤ A reproducibility standard deviation from other sources (such as from an interlaboratory comparison) can be used, provided that there is proof that the laboratory’s performance is equivalent to the performance of the participants in the study in question. ➤ The standard deviation of the measurements provides a very rough estimation of their uncertainty. There is, however, an important caveat: this estimation typically underestimates the real uncertainty.

Source: Romer Labs

A Romer Labs® Publication

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The stars of the Biopure™ product line are the fully labeled 13 C internal standards. Available for all regulated mycotoxins, these calibrants rely on a patented technology, assuring the highest standard of analytical accuracy.

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