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Many accredited labs use reference materials to show traceability and reliability of their analysis results. What’s less well-known is how reference materials are manufactured.

By Anna LILEK, Quality Manager, Romer Labs®

Some toxins have favorable molecular structures, and can be crystallized from a supersaturated solution of polar or apolar organic solvents.

Reference materials, or calibrants describe substances or objects with one or more defined characteristic property value(s) that are used as a measure or as a benchmark for measurement methods. Given the importance of consumer safety for the food and feed industry, mycotoxin testing involves the use of reference materials in order to obtain accurate and reliable results.

Ever-present danger

Mycotoxins are naturally-occurring secondary fungal metabolites toxic to animals and humans. Mold fungi grow on the field and during storage. Found in almost all agricultural commodities worldwide, more than 380 mycotoxins have been identified and the toxicity of each substance varies greatly. The predetermined maximum permitted concentrations of various mycotoxins in vegetable raw materials such as grain, wheat, corn – to name just a few – have forced commodity producers to examine their samples carefully in an analytical laboratory to be sure about the quality of their products.

Gravimetric preparation

Frequently, a fungus that grows under optimal conditions – sufficiently warm temperatures, high humidity and a suitable substrate – can produce mycotoxins. In the lab, the story of a reference material begins with the attempt to adjust artificial growth conditions in order to obtain optimal mycotoxin yields. This includes the use of a suitable fungal genus – as every fungus produces its own characteristic pattern of metabolites that can number into the several hundreds. The maintenance of fungal strains for production is crucial. Their vitality and functionality are constantly being monitored, since this forms the basis of all activities for the reference material production processes. Molds are living organisms that might mutate over time, or even degenerate and result in decreasing mycotoxin yields. New metabolites may form after a certain storage period, which can also influence the isolation process immensely. Strains must be renewed regularly in order to counteract mutations, impurities or other undesirable characteristics.

The first step, fermentation, hinges on making the

Challenge Solution

Mutations of fungal strains (formation of other metabolites, decreases/increases in yields, formation of impurities)

Standardization of fermentation and isolation processes

Regular review of the strains. Check for productivity and vitality. Renew strains as needed.

Use state-of-the-art techniques, materials and equipment. Ensure a robust clean up strategy. Undetected impurities by superimposing

Seek other solutions for the identification of the substance to be isolated –

Finding external certified reference materials for process and quality controls e.g. by analyses of accredited service laboratories that can perform an identification and traceable purity determination (quantitative NMR, LC-MS / MS, elemental analysis, etc.) lab environment literally as tasty as possible for the fungus to promote its growth. The optimal media also vary from strain to strain, so components such as salts and minerals are provided as a source of nutrients. The mold is allowed to grow for a certain time – anywhere from a few days to a few weeks – during which time the fungus metabolizes its medium.

After completion of the fermentation and a careful control of the process the mycotoxin is extracted from the culture material using a suitable organic solvent. Depending on the molecular structure, these can be polar or non-polar organic solvents. During fermentation, molds mostly produce impurities in addition to the toxin of interest, e.g. other metabolites, colorings, oils, etc.

The resulting crude extract often contains many impurities. During isolation or purification, the mycotoxin is brought step-by-step closer to the target purity of >98% through various chromatographic and preparative applications with different selectivity.

Some toxins have favorable molecular structures, and can be crystallized from a supersaturated solution of polar or apolar organic solvents. This happens, for example, by cooling the solution, by evaporating the crystallization solvent or by mixing several solvents of different polarity. Other toxins may be rendered in crystalline, powdery form by freeze-drying. By crystallization the purity of the toxin is thereby increased again until the target purity is obtained.

Gravimetric Preparation

• Tempering of solid mycotoxin to room temp.

• Weighing

• Dissolving

• Homogenizing

Quality Control

• HPLC

• UV Photometer

• HPLC-MS

Bottling and CoA

• Filling to separate units

• Certificate of Analysis (CoA)

• Shipping to customer

Quality control

HPLC, UV Photometer and HPLC-MS are used to determine/confirm the purity of the raw material produced. Depending on the toxin, this can be done for example by High Performance Liquid Chromatography in combination with Diode Array Detection, Fluorescence detection or similar, with UV Photometer (qualitative and quantitative analysis of the compound) or by High Performance liquid Chromatography combined with mass spectrometry detection. MS is particularly required for the above described determination of the isotopic purity (e.g. > 98% 13C atoms) of 13C isotope-labeled mycotoxins. Mycotoxin reference materials have a niche position on the market. Therefore, it is sometimes difficult to find comparative substances that are commercially available and can be used for process control during production and quality controls. Table 1 illustrates a number of challenges that can occur during the production and QC stages, along with solutions implemented at Romer Labs.

Delivery

After passing the final quality control, the solid mycotoxin is liquefied for use as a liquid mycotoxin reference material. The liquid calibrant solution is then bottled and a certificate of analysis is created, stating the property value and its uncertainty, which accompanies every single calibrant.