Last Month's Most Accessed Feature: Multiple mycotoxin monitoring: Dried blood spots and dried serum spots

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  • Published: Jul 3, 2017
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Food mycotoxins

Multiple mycotoxins can be measured in blood spots and serum spots by LC/MS say European scientists, providing a more accurate way to estimate individual dietary exposure from contaminated food.

We all love our food but one thing that is guaranteed to turn our stomachs is the discovery of mould, except on some cheeses, of course, where it can be desirable. Mould has an undesirable effect on the appearance of food items but there are also more sinister consequences. The presence of mould invariably leads to the production of toxic metabolites called mycotoxins which induce many harmful effects such as cancer, DNA damage, kidney damage and suppression of the immune system.

Mycotoxins can be found in crops in the field but can also form during storage. Cereals and cereal products such as bread and biscuits, fruits, spices, nuts, and wine are all susceptible but the levels can be reduced during processing. The maximum permitted levels take into account the individual mycotoxin, the food treatment procedure and the contribution of that food to the typical consumer daily dietary intake.

There are a number of established methods for estimating the concentrations of mycotoxins in foods, whether fresh or processed. However, these do not necessarily equate to individual exposure levels due to variations in personal and regional diet and culture. A more accurate personal indicator of exposure is a person's blood or urine levels and several methods exist for measuring these. Now, as an alternative, one group of European researchers has devised a method to measure a wide range of mycotoxins in dried blood spots (DBS) or serum spots (DSS).

Dried blood and serum spots

Hans-Ulrich Humpf, Bernd Osteresch and Susana Viegas from Westfälische Wilhelms-Universität Münster and Benedikt Cramer from both the Polytechnic Institute of Lisbon and the Universidade NOVA of Lisbon have been working with DBS in the mycotoxin field. In 2015, they published a successful method for measuring ochratoxin A and 2'R-ochratoxin A in dried blood spots from coffee drinkers.

In new work, they have extended that procedure to 27 toxicologically relevant mycotoxins and metabolites which have been measured previously in breast milk, urine, blood, serum and plasma. The target compounds included aflatoxins, Alternaria toxins, trichothecenes, ochratoxins, cyclic hexadepsipeptides, enniatins, fumonisin B1, zearalenone and zearalanone. The reasons for moving to dried blood and serum include easier collection by pricking, simplified storage and transport and simple sample preparation.

Healthy volunteers supplied 100 µL of blood that was spotted onto filter paper cards. Serum was prepared by centrifuging whole blood, collecting the supernatant and spotting onto protein saver cards. Both sets of cards were dried for storage. When it was time for analysis, the spots were extracted with an aqueous mixture of acetone and acetonitrile for analysis by LC/MS.

The C18 column that was selected gave the best retention behaviour and peak shape from four that were tested, while reducing the levels of any interfering compounds from the blood matrix that eluted near any of the mycotoxins. The column was connected to a mass spectrometer for electrospray ionisation operating in both positive- and negative-ion modes. Using scheduled multiple reaction monitoring, fewer transitions were monitored at the same time to allow the number of data points to be increased for each analyte while increasing the dwell times and reducing noise levels.

Mycotoxins in coffee drinkers

The detection limits for the analytes ranged from 0.001-0.006 ng/mL for the enniatins up to 1.4 ng/mL for HT-2 toxin and deoxynivalenol 3-glucuronide. One major effect that the research team observed was high signal suppression caused by other blood components that were coextracted with the mycotoxins. In some cases, they reduced the signal to 13% of that from a standard solution. Although it could cause problems with the assay, these matrix effects were highly reproducible so were countered using matrix-matched calibration standards.

One further issue was the stability of the mycotoxin spots during storage, which had not been investigated previously for physiological samples such as DBS and DSS. Significant degradation occurred at room temperature over ten weeks for most compounds and was also notable at 4°C. So, storage at -18°C in sealed containers in the absence of light was recommended.

The 50 samples had already been tested to measure the two aforementioned ochratoxins in dried blood spots from coffee drinkers and they were confirmed in the new extended test. As expected, all positive findings were confirmed. The only other mycotoxin found from the group of 27 was enniatin B at a mean value of 0.0367 ng/mL.

The results from DBS and DSS were equivalent, demonstrating that both methods are suitable for the detection of mycotoxins. This procedure should be used to supplement urine testing, since some mycotoxins are found mainly in urine and others in blood. The ease of sample collection and multidetection capabilities make it a suitable method for biomonitoring and biomarker studies of mycotoxins.

Related Links

Analytical and Bioanalytical Chemistry 2017, 409, 3369-3382: "Multi-mycotoxin analysis using dried blood spots and dried serum spots"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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