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.

1. 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 Union¹, 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 characteristics, such as aflatoxin B1 dissolved in acetonitrile at a concentration of 2 µg/mL. The specific characteristics of a reference material – and, importantly, its relative purity – provide a benchmark by which lab technicians can confirm accuracy of their own results.

2. 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.

3. 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.

4. 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 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.

5. 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 reference 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.

6. What is a certified reference material?

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.

7. 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.

 ¹ EU 2023/915, EU 2024/1022, EU 2024/1038