Is Accuracy the Price We're Paying for Faster Results?

Is accuracy the price we’re paying for faster results?

Speed or accuracy? Do we have to choose? Applying the right technologies and paying attention to basic sampling guidelines can reduce overall time-to-result without sacrificing accuracy.

By Alois SCHIESSL

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

ISSN: 2414-2042

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Editors:

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Contributors: Michael Zheng, Alois Schiessl, Philipp Gruber

Graphics:

Reinhold Gallbrunner

Research: Kurt Brunner

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Extracting mycotoxins with water –can that work?

In order to make mycotoxins “available” for testing in an assay, they need to be extracted from ground samples. During the extraction process, mycotoxins encased in the grain kernel are transferred into a liquid. This is usually carried out with the help of organic solvents but these are hazardous substances, harmful to both operator and the environment.

By Philipp GRUBER, Product Manager, Romer Labs

Photo: Creativa Images

Photo: heibaihui

Accuracy or speed –the dilemma of the perfect test-kit

According to the latest BIOMIN Mycotoxin Survey Report, up to 84% of all feed grain samples collected from the 2015 harvest tested positive for at least one mycotoxin, and over half were contaminated with more than one mycotoxin.

While climate change and variations in seasonal weather are aggravating the mycotoxin challenge, the good news is that worldwide testing for mycotoxins has increased the availability of more affordable, easy-to-use test kits. Increased testing has also tightened food safety standards.

In light of these developments, some might think that increasing speed and simplicity results in lower performance. Are quick yet accurate testing solutions simply an illusion?

Manufacturers are working hard to prove otherwise. Test performance is improved by conducting matrix validations, demonstrating yet again that science and technology are never static. Today's dreams can often become tomorrow's reality.

Lateral flow devices are one example. Very easy tests to perform, these are suitable for operators with minimal training.

One of the newest and most exciting innovations is water-based extraction. This method applies for most prevalent mycotoxins while proactively supporting customer needs for test procedures that are safe and do not require the use of hazardous and costly substances such as organic solvents.

In this issue of Spot On, we focus on point-of-care testing at grain reception points and explore how mycotoxins can be effectively and safely extracted using water.

We hope you enjoy reading this issue of Spot On!

Michael Zheng Vice President R&D, Romer Labs Singapore

By Alois SCHIESSL

When visiting a grain elevator facility, it is a common sight to see a number of trucks lining up in front of the grain reception point.During harvest season, up to 100 trucks can be lining up over any given day. The reception point has to make a quick decision on whether the trucks are carrying grain that falls below defined mycotoxin contamination limits and can therefore be admitted into the facility’s operations. In harvest years where contamination levels are high, every truck’s payload will be probed and analyzed for mycotoxins before admission into the facility. This has to be done quickly because having results available as soon as possible shortens the waiting time at the back of the queue significantly.

Quick assay turnaround is key but to what extent does it compromise accuracy? These are two important points we shall address next. To provide an answer, we shall first define the right method for such an application.

Every truck is probed and analyzed consecutively; hence a quick, easy-to-use, single sample test method is required. Lateral flow device (LFD) tests, also known as strip tests or dipsticks, are one of the fastest test methods available and a perfect fit for this scenario. Due to

How lateral flow devices work

For a quick LFD test result, a grain sample is ground and extracted with novel water-based extraction solutions, following a subsequent development time of as little as three minutes to form visible test and control lines. In combination with a reader, the lines are then used for quantification. During the three-minute LFD development, the sample extract will flow over the strip and mycotoxin specific antibodies will bind mycotoxins present in the sample. These blocked antibody sites cannot then be used to bind to antibodies coupled to visible gold colloids to the LFD test line, resulting in a lower test line intensity. Therefore, the higher the mycotoxin concentration in a sample, the lower the test line intensity. The control line will bind any excess of antibody gold conjugates, producing a visible line proving that the LFD development was successful. Such a quick and affordable test result cannot be produced by any other commercial test method. Therefore LFD technology shows its unique strength in reception point analysis.

their simplicity, these tests can be performed by almost anyone with basic training and typically produce analytical results in just under ten minutes. This enables quick decisions on whether a truck’s payload can be accepted into the grain elevator’s facility.

What else is out there?

Other mycotoxin rapid test methods, such as enzyme-linked immunosorbent assays (ELISA), are also available on the market. ELISA tests, however, require a calibration curve with each analysis which can take around 20 minutes to produce a final result. Hence, such methods are only feasible when multiple samples are analyzed in parallel using the same calibration cur ve.

Another group of mycotoxin test methods are reference test methods, including high performance liquid chromatography (HPLC) as well as liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). These are typical laboratory methods associated with high accuracy and reproducibility. The drawback of such methods is that they require highly skilled staff and take up to one day to deliver an analytical result. Therefore, they are unsuitable for application in a grain elevator reception point.

The big question of accuracy

The faster an analytical method is, the more often concerns about its accuracy will surface. So how accurate are LFD tests? LFD technology and ELISA tests both use the antibody-antigen reaction detection principle in which a specific antibody detects a unique antigen, in this case a mycotoxin, with a given sensitivity. Both test systems are calibrated against certified reference materials and results are compared to accredited reference methods in thorough validation studies.

Nevertheless, some test methods may not fulfill the operator’s expectations.

What can go wrong?

• Correct sampling (see text box) is the key to producing accurate analysis results. Large lots of grain (e.g. full trucks or barges) must be analyzed for mycotoxins before they are accepted into a facility. It is crucial that representative samples are taken for the analysis.

• One of the most significant factors that can influence an analytical result is the commodity matrix. Ideally, test methods are developed to analyze any kind of food or feed sample for mycotoxin contamination. A limiting factor, however, is that different food or

LFDs are one of the fastest test methods available.

feed samples can have different components which may interfere with the analytical result, depending on their chemical structure and properties. Antibody-based rapid tests are often influenced by certain matrix components which can have major impacts on the antibody-analyte binding reaction, potentially yielding questionable results. Test kit manufacturers face the challenge of reducing or eliminating these interferences through, for example, specific antibody developments and matrix validations.

• Matrix dependent application notes stating the analysis procedure and test performance are usually available and published by the test kit manufacturer. It is crucial that these application notes are followed in order to obtain accurate results, especially when deviating from the standard validation matrix typically associated with testing maize.

• Further sources of error can be environmental factors such as the ambient temperature during tests. The antibody-antigen binding reaction — the detection principle of all immunochemical tests — is temperature dependent. For accurate results, manufacturers have developed test incubators to keep temperatures constant, thereby eliminating interferences caused by fluctuating temperatures.

• Operators of analytical tests must be properly trained in order to administer the tests accurately. Different test methods require different operator know-how. The LFD tests are the easiest mycotoxin tests to perform. Basic training is enough to keep operator errors for such methods at a minimum.

What is the test kit manufacturer’s responsibility?

What can a test kit manufacturer do to ensure that results produced with their test kits are accurate and reliable? First, a test kit manufacturer needs to educate users on the importance of correct sampling, as this is crucial to result accuracy.

Next, it needs to prove the accuracy of its test kit by conducting a validation study that follows USDAGIPSA or AOAC guidelines. The test kit can be submitted to these official bodies for an external third-party evaluation confirming the claimed performance of the analytical method.

For such third-party validations, a test kit manufacturer must include the validation data for claimed sample matrices to ensure that the test kit performs accurately on these different sample matrices. In validation

LOT

Sampling error

The total error of an analytical mycotoxin text result is the sum of sampling, sample preparation and analytical errors. The sampling error contributes the largest portion of error in the final result. This is due to the fact that mycotoxins are unevenly distributed within bulk grain samples, making it essential to take a representative sample before continuing with sample preparation and the actual measurement.

To get a representative sample from a bulk sample lot, it is necessary to take several incremental samples from multiple locations. These incremental samples are then combined to form an aggregate sample and after homogenization and further splitting by a mechanical divider, an analytical sample is taken for subsequent sample preparation. These procedures are described in official sampling plans by authorities, such as the European Commission (EC 401/2006 & EC 519/2014).

The large influence on the final analytical result makes it essential that the sampling error is kept to a minimum. The simplest way to achieve this is by increasing the number of sampling sites as well as the sample size of incremental samples within a sampling plan.

studies, multiple sample sources, e.g. maize samples from different continents, are typically used to show the reliability of the analytical method.

To further improve the robustness of a test system, environmental factors, such as temperature, must also be eliminated or accounted for. This can be done by using an incubator so that the LFD development always occurs at a constant, controlled temperature.

Is accuracy the price grain elevators pay for fast results?

After observing the operations at grain reception points, it is clear that rapid single sample tests are the methods of choice. Lateral flow devices clearly fulfill all criteria and in addition, reduce many sources of handling errors due to their simplicity.

By following clear and basic rules, speed doesn’t have to come at the price of accuracy. Reliable and accurate results can be obtained in just under ten minutes and that is the key to efficiently analyzing and assessing grain from truck to truck at a reception point.

By following clear and basic rules, speed doesn’t have to come at the price of accuracy.

to Whitaker & Dicken, 1974

Photo: aeiddam0853578919

Extracting mycotoxins with water –can that work?

In order to make mycotoxins “available” for testing in an assay, they need to be extracted from ground samples. During the extraction process, mycotoxins encased in the grain kernel are transferred into a liquid, usually with the help of organic solvents that are hazardous substances, harmful to both the operator and the environment.

By Philipp GRUBER, Product Manager, Romer Labs

Why hasn’t water been used from the very beginning?

While fumonisin and deoxynivalenol are easily extracted using water, other mycotoxins like aflatoxins or zearalenone are not very water soluble.

This has led to the use of hazardous organic solvents like chloroform, acetonitrile and methanol in the extraction process, although laboratories are well aware of the downsides of using solvents, such as their high purchase and disposal costs, and issues associated with environment and operator safety.

To utilize water efficiently in an extraction process, some basics should first be understood.

The polarity of substances

The transfer of mycotoxins from grains into a liquid is not as easy as it seems. It depends foremost on the physical properties of the mycotoxin to be extracted, with polarity playing a major role in this context.

As the building blocks of molecules, atoms and how they are arranged can exhibit different electrical charges. Mycotoxins can exhibit positive charges on one side and negative charges on the other. In such cases, the molecule has electrical poles and is called polar.

As such, all substances including mycotoxins, are organized into three major groups:

1) Polar substances like water

2) Nonpolar substances like oil

3) Amphiphilic substances like soap that have both polar and nonpolar properties

Polar molecules are also called “hydrophilic” from the Greek word “water loving”, as they are soluble in water. Their attraction to each other is based on their electrical charges. The negative pole of one molecule is electrostatically attracted to the positive pole of another molecule. This sticking to each other is the reason why they are miscible.

Salt and sugar are examples of solid polar substances. Sugar is perfectly soluble in liquids like coffee, but also attracts moisture from the air. For the same reason, salts are often used as desiccants.

On the other hand, nonpolar molecules are called hydrophobic or “water fearing”. Their attraction to each other is a result of a joint repulsion of water and other polar substances. Amphiphilic molecules have both nonpolar and polar properties. Soap, for example, can attract greasy (nonpolar) stains from skin, hair or clothes and at the same time, bind to water (polar) to wash them off.

The search for a harmless extraction solution

To avoid the use of organic solvents, researchers are on the look-out for safe and harmless ways to extract mycotoxins. Detergents dissolved in water have turned out to be a promising solution. Detergents, like soaps, are amphiphilic substances. Their dual nature facilitates the mixture of nonpolar compounds like aflatoxins with water.

At the molecular level, detergents group themselves around the nonpolar mycotoxin with their nonpolar

How are rapid mycotoxin tests made safer and greener? Perhaps the answer lies in water.

Mycotoxins such as aflatoxins or zearalenone are only slightly soluble in water.

part, while the polar water-soluble part remains in the water. This characteristic allows detergents to literally leach out nonpolar mycotoxins from solid food or feed into water.

A great benefit of such a method is that detergents extract nonpolar substances while the water in which the detergents are dissolved extract polar substances. In this way, one extraction can be used to dissolve various mycotoxins with different physical properties into a liquid.

WATEX - The green solution that works

Due to the different types of mycotoxins and their respective polarities, water alone can’t be the solution. It is the combination of water and detergents (in the form of soluble buffer bags) that is capable of extracting multiple mycotoxins with diverse chemical properties.

With the extraction buffer free of any hazardous substances, operators can conduct safe and fast mycotoxin analyses without harming the environment.

What do washing clothes and extracting mycotoxins have in common?

When clothes are washed, oil droplets that contain dirt particles (a) need to be removed.

By adding a detergent during the wash cycle, soap molecules mix with water and get into contact with the textile fabric (b).

The “oil loving” (hydrophobic) ends of the soap molecules are attached to the oil/dirt while their water loving (hydrophilic) ends remain in the water. When many soap molecules are attached, the oil particle is fully en-

closed and a vesicle forms that is soluble in water (c). The tumbling motion during the wash cycle beats those oil containing vesicles in the wash water and removes them from the textile fabric. Just as hydrophobic dirt particles are removed from textile fabric, hydrophobic mycotoxins are removed from grain particles. The shaking motion during extraction moves the encapsulated mycotoxin vesicles into the extraction liquid, making it accessible for detection.

Source: Beatrice the Biologist

Picture 1. The highly hydrophobic surface of the lotus leaf repels water. The water molecules stick to each other to assume a nearly spherical shape.

hydrogen bond

Figure 1. Electrostatic attraction: The negative part of one molecule is attracted to the positive part of another.

Water alone can't be the solution.