The tandem liquid chromatography-mass spectrometry (lc-ms / ms) method for the determination of mycotoxins in infant food and animal feeds
Together with meat, milk, eggs, cereals, nuts and fruits, toxins enter the human body . These compounds have carcinogenic and mutagenic effects in extremely low concentrations. Continuous exposure to mycotoxins can lead to severe damage to the kidneys, liver and cardiovascular system of both humans and animals . They do not decompose during the heat treatment of products. There are five groups of the most common mycotoxins, i.e. deoxynivalenol, zearalenone, ochratoxins, fumonisins and aflatoxins. In many countries including Russia maintenance standards have been introduced for the most dangerous mycotoxins in food, animal feed and respective raw materials. Particular attention is paid to the control of baby food. For this product category, very low mycotoxin content standards are established (Table 1) . The determination of such a concentration level is a difficult analytical problem .
Regulatory documents prescribe the determination of mycotoxins by high-performance liquid, thin-layer and gas chromatography methods. The analysis is preceded by a long sample preparation including extraction, purification of the extracts obtained on sorption columns (including immunoaffinity), evaporation etc. [9–13]. The difficulty in detecting mycotoxins by chromatographic methods is associated with a wide variety of their chemical nature and properties as well as the presence of a large number of unpredictable impurities in plant and animal products. That is why the generally accepted methods for their determination usually concern only one compound.
The tandem liquid chromatography-mass spectrometry (LC-MS/MS) method makes it possible to selectively conduct highly sensitive determination of a large number of compounds at the same time [14, 15]. It was also interesting to choose a single procedure for sample preparation for objects of various nature, in order to determine a wide range of mycotoxins in one analysis. That is why our research was focused on developing a unified approach to identifying the content of the main normalised mycotoxins in samples of the porridge for baby food and dry feed for cats and dogs by using tandem liquid chromatography-mass spectrometry [16, 17].
Ten samples of children’s porridge, six cat feeds and the same amount for dogs were analysed. One of the purposes of our feed research was to determine the amount of toxicants in products of different price categories, both economy class and professional. The content of nine mycotoxins was measured, aflatoxins B1, B2, G1, G2, M1; T-2 toxin; zearalenone; deoxynivalenol; ochratoxin A. All products were purchased randomly in stores in St. Petersburg. Table 2 gives information on the samples studied, Fig.1, 2 provide their photos.
Sample preparation is an important step in the analysis of mycotoxins, which includes the stages of selection, extraction and purification of extracts. It is necessary to take into account that the metabolites of moulds are concentrated in mould formation places unevenly distributed in the object under research. Therefore, it is important to pay special attention to the homogenisation of a sample.
The dry and loose target of research (porridge, feed) was ground with a blender, thoroughly mixed, 100 ml of ACN:H2O (84 : 16 by volume) mixture was added and it was extracted from 25 g of the crushed sample by continuously shaking for an hour. The extracts were held for several minutes before precipitation of the insoluble residue; then 4–5 ml of the top layer of the solution was taken, filtered through a nylon filter with a pore diameter of 0.45 μm by using a syringe and 2 ml was withdrawn. The solution was purified on Supel Tox AflaZea SPE TFE cartridges manufactured by Supelco. The cartridge sorbent does not require any preliminary preparation, which saves time. The purification mechanism is the adsorption removal of impurities from the extract, the mycotoxins pass through the sorbent without being retained. The solution was passed at a rate of about a drop per second.
The procedure was carried out by using a vacuum unit for solid-phase extraction, a 12-position manifold produced by LabTech. For the baby food analysis, 1 ml of eluate was collected and evaporated at 50 °C. under vacuum to dryness in the Smart Evaporator C1 concentration by evaporation system of BioChromato, Japan (Fig.3).
Due to the vortex concentration technology, during vacuum evacuation, the surface area of the contact between the liquid and air (or nitrogen) increases, the solvent evaporation is accelerated, and the samples are concentrated without heating to high temperatures. In addition, the vortex flow blows the solution residue off the vial walls to the bottom by collecting all the analytes at the bottom. The dry residue was redissolved in 500 μl of the ACN: H2O mixture (10 : 90 by volume), filtered through a membrane filter with a pore diameter of 0.2 μm, and subjected to chromatographic analysis.
Since the aflatoxin B1 content standard in feeds for unproductive animals is significantly higher than in children’s food, the stage of concentration with evaporation of the extract and subsequent redissolution have been excluded for these products. 800 μl of water was added to 200 μl of the extract obtained after purification on a Supel Tox AflaZea SPE cartridge, the mixture was filtered through a membrane filter with a pore diameter of 0.2 μm and subjected to chromatographic analysis.
LC-MS/MS analysis conditions
The analysis was performed on an LCMS-8040 triple quadrupole liquid chromatography mass spectrometer (Shimadzu, Japan) in the ESI mode (electrospray ionisation, ionisation with sputtering in an electric field). As the interface settings, the following was chosen: the atomising gas (nitrogen) flow – 2 l/min, the temperature of the drying gas (nitrogen) – 15 l/min and the desolvation temperature – 400 °C. To separate mycotoxins, a Titan C18 column chromatography column was used, 5 cm Ч 3.0 mm; 1.9 μm. As the mobile phase, 5 mM ammonium formate in water and acetonitrile, methanol 1 : 1 (vol. %) were used by using a gradient elution mode. The flow rate of the mobile phase is 0.4 ml/min; column temperature 35 °C; injection volume – 2 μl. MS-detection conditions were also selected, and precursor ions and product ions (MRM transitions) for each compound in (+) and (-) polarity switching modes were optimised. The retention times and MRM transitions of all analytes are given in Table 3. The calibration curve range was 1–50 ng/ml for all mycotoxins.
build the calibration curve, ready-made standard samples were used, the solutions of mycotoxins in acetonitrile, benzene or acetonitrile-benzene mixture Russian made. Aliquots of the standards were evaporated in a nitrogen stream and redissolved in the mobile phase to obtain the original solution. Then a series of calibration solutions were prepared in the mobile phase. The dependencies were constructed by using the external standard method. In Fig.4–6 presented are the chromatogram of the standard solution, the calibration curve and the mass spectrum of aflatoxin B1.
In Fig.7, 8 shows the mass spectra of zearalenone and T-2 toxin, and Fig.9, 10 shows chromatograms of samples of the extracts of porridge No 10 Malyutka (Little Baby) Nutricia and No 8 Sami s Usami for selected ions.
Chromatograms of the sample feed for cats No 15 Monge for selected ions are shown in Fig.11, 12.
The obtained results of the determination of mycotoxins in infant food and feed for unproductive animals are summarised and analysed. Since the threshold allowable concentration of aflatoxin B1, the only one standardised in the diet, is significantly higher than in infant nutrition (10 and 0.15 μg/kg, respectively), we did not calculate the exact concentrations of mycotoxins in feeds if their content did not exceed 0.02 μg/kg. In the infant food samples, precise concentrations were not determined if the content of the component was less than 0.0002 μg/kg, which is by two orders of magnitude lower than the threshold allowable concentration of M1 aflatoxin which has the lowest value of 0.02 μg/kg among the mycotoxins listed.
Excess of mycotoxin content relative to existing standards was detected in not a single sample. However, due to the high sensitivity of the tandem LC-MS/MS method, mycotoxins were found in twelve of the twenty-two samples examined.
Thus, in the following samples of infant food cereals, Nestle Infant Cereal 5 Cereals was foundб zearalenone at a concentration of 0.0006 μg/kg, No 6 Umnitsa Milk Porridge Five Cereals + 13 Vitamins, T-2 in a concentration of 0.005 μg/kg, zearalenone at a concentration of 0.0002 μg/kg, No 8 Sami s Usami Children’s Milk Porridge Five Cereals, T-2 at a concentration of 0.5 μg/kg, No 10 Baby Nutricia Porridge without Dairy Buckwheat, zearalenone at a concentration of 0.07 μg/kg.
Once again, we draw your attention to the fact that the detected concentrations of toxins T-2 and zearalenone are significantly lower than the current threshold allowable concentration.
Our research showed that in the feeds for cats mycotoxins are detected more often than in dog feeds. Thus, in five out of six purchased cat feeds, aflatoxin B1, zearalenone and T-2 toxin were found. An exception was Purina Cat Show Sterilised, in which the content of detectable toxins was at the level of noise. The quantity of standardised aflatoxin B1 was 3 and 4 μg/kg in the Felix samples Felix As Good as it Looks Senior Doubly Delicious Meat and Monge Rich in chicken Sterilised, respectively, which does not contradict the regulated concentration <0.01 mg/kg (<10 μg/kg).
There was no significant difference in the content of mycotoxins in feeds of the economy class and professional class. On the contrary, three of the nine detectable analytes were present in the professional Monge fodder, B1 (4.00 μg/kg), T-2 (5.00 μg/kg), zearalenone (8.00 μg/kg).
The dog food showed a more favourable picture, mycotoxins were found only in three of six. In the Pedigree Vital sample, deoxynivalenol (2.00 μg/kg) and T-2 toxin (6.00 μg/kg) were found, and in the Purina Pro plan and Chappi feed, only zearalenone in concentrations of 7.00 mkg/kg and 6.00 mkg/kg respectively.
All standardised mycotoxins are found in concentrations that do not exceed the maximum allowable limits, both in infant food and feed. In porridges for babies of the non-standardised toxicants (B2, G1, G2) it is not revealed.
Thus, the use of tandem liquid chromatography-mass spectrometry for the determination of mycotoxins has a number of undeniable advantages over HPLC methods with spectrophotometric or fluorimetric detection, enzyme-linked immunosorbent assays and TLC. One analysis is sufficient to obtain information on the quantitative content of a wide variety of mycotoxins due to the exceptional selectivity of ‘triple quadrupole’ detectors in the MRM regime. Sample preparation is greatly simplified, the reliability of results is enhanced, and detection limits are significantly reduced. The latter circumstance makes it possible to detect the target components in objects for which the preceding analytical methods yielded a zero result. Thus, the introduction of highly sensitive instrumental methods of analysis changes our perception of the world around .
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