The efficiency of mycotoxin binding by sorbents in the in vitro model using a naturally contaminated animal feed

Standards of 99.6% pure AFL B1, 99.0% pure DON, 98.6% pure T-2, 98.6% pure HT-2, 99.0% pure OTA, 98.0% pure FB1, 99.8% pure FB2 and 99.7% pure ZEN were obtained from Sigma-Aldrich (Schnelldorf, Germany). Standard solutions of DON, ZEN, T-2 and HT-2 were prepared in acetonitrile, and those of AFL B1 and OTA were prepared in methanol. Solutions of FB1 and FB2 were prepared in 50% acetonitrile. A standard solution containing eight mycotoxin analytes was used in the analysis in concentrations corresponding to the maximum levels of mycotoxins in pig feeds set by the European Parliament and Council and by the Commission (Table 1) (7, 11). In addition, a mixture of seven internal standards was used in a chromatographic analysis. The reason why seven standards were visualised in chromatography instead of eight is that the internal standard FB1 was used for the analysis not only of FB1 but also of FB2. This is justified by the separation of both molecules, i.e. FB1 and FB2, in liquid chromatography–tandem mass spectrometry (LC-MS/MS) being similar to that of an internal standard FB1 molecule.

Acetonitrile (LC/MS grade), methanol (LC/MS grade) and acetic acid were purchased from J.T. Baker (Deventer, the Netherlands). Sodium chloride (p.a. grade), potassium chloride (p.a. grade), phosphoric acid (p.a. grade) and ammonium acetate (p.a. grade) came from Sigma-Aldrich (Darmstadt, Germany). The final reagent, disodium hydrogen phosphate (p.a. grade), was obtained from Chempur (Piekary Śląskie, Poland).

The phosphate-buffered saline (PBS) for the analysis of mycotoxin binding by sorbents consisted of 4 g of sodium chloride, 1.8 g of disodium hydrogen phosphate and 0.1 g of potassium chloride, in a 500 mL volume of distilled water and had its pH adjusted to 3.5 or 7 with phosphoric acid. The ammonium acetate solution for the LC-MS/MS phases consisted of 400 mg ammonium acetate and 500 μL of acetic acid in a 500 mL volume of distilled water. The phases for LC-MS/MS analysis were prepared according to the scheme of phase A (ammonium acetate: methanol 95 : 5) 380 mL of ammonium acetate solution and 20 mL of methanol and phase B (ammonium acetate : methanol 5 : 95) 20 mL of ammonium acetate solution and 380 mL of methanol.

The investigated sorbents were bentonite, calcium bentonite, zeolite, calcium lignosulphonate, activated charcoal, sepiolite, attapulgite and ground attapulgite, and silica. Samples of these were supplied by Certech (Niedomice, Poland).

The feed used for the tests was prepared from naturally contaminated grain. The grain became contaminated because the plants were cultivated in inappropriate conditions. The feed samples were prepared and investigated by Tkaczyk et al. (26) and comprised contaminated feed with DON at the level of 1,126 ± 91.43 μg/kg, ZEN at 34.7 ± 4.34 μg/kg and OTA at 226 ± 21.8 μg/kg.

It was assumed that the sorbent would bind mycotoxins at a pH corresponding to the gastric phase. The stability of toxin binding and possible desorption was tested at intestinal pH. Each experimental sample contained sorbent, buffer at the appropriate pH and mycotoxin-standard solution (Fig. 1).

Fig. 1.

In the gastric phase (sorption at pH 3.5), duplicate samples with 100 mg of a sorbent were incubated and shaken with 5 mL of pH 3.5 PBS for 2 h at 39°C. The temperature of 39°C reflected the swine body temperature. A control sample was prepared by mixing 10 μL of the pH-adjusted PBS solution with eight mycotoxin standards. After centrifugation at 3,500 rpm for 15 min, 4.5 mL of extract was taken from each sample into new test tubes, and 10 mL of distilled water was added. The extract prepared was applied to solid-phase extraction (SPE) columns with Oasis hydrophilic-lipophilic balance columns (60 mg; Waters, Milford, MA, USA). The columns had been conditioned with 2 mL of MeOH and 2 mL of H₂O. Then, after the extract had passed through, it was washed with 2 mL of H₂O and dried for 1 min. Elution was carried out with 3 mL of MeOH. The extract was evaporated under N₂ at 45°C, and the dry residue was dissolved in 500 μL of phase A and 500 μL of phase B and transferred to vials for LC-MS/MS analysis.

In the intestinal phase (desorption at pH 7.0), the residual sorption precipitate was washed three times with 10 mL of distilled water, the water was poured off, and the pellet was incubated with 5 mL of PBS at pH 7.0 for 2 h at 39°C. The remaining steps of the procedure were of the same as for sorption.

Based on the results presented in Table 2 regarding the mycotoxin-binding ability of sorbents in a pH 3.5 buffer, sepiolite and calcium lignosulphonate were selected for further study as the two sorbents binding greater than 70% of the tested mycotoxins. Mixtures of the two sorbents were made in different proportions (Table 4) to assess which proportion would bind mycotoxins with the best effectiveness. The procedure for testing the sorbent mixtures in mycotoxin binding was the same as described above.

These two sorbents were assessed in combinations for their binding of DON, ZEN and OTA in contaminated feed. Each sample contained naturally contaminated feed, a sorbent mixture and the buffer at the appropriate pH (Fig. 1).

Each experimental sample contained 10 mg of sorbent mixture and 1 g of feed. The 1% proportion of sorbent in the feed mass simulated the percentage of sorbent potentially administered in future practice. The control sample contained only 1 g of feed and PBS. Duplicate samples with 10 mg of a sorbent and feed were incubated and shaken with 30 mL of PBS at pH 3.5 for 2 h at 39°C. After centrifugation at 3,500 rpm for 15 min, 15 mL of extract was taken from each sample into new test tubes, and 10 mL of distilled water was added. The extract prepared this way was applied to the SPE Oasis HLB columns. Conditioning and sample preparation for LC-MS/MS analysis were performed similarly to how they were performed when mycotoxin standards devoid of feed mycotoxin were bound by sorbents.

The residual sorption sediment was washed three times with 10 mL of distilled water, the water was poured off, and the sediment was incubated with 30 mL of PBS at pH 7.0 for 2 h at 39°C. The remaining steps of the procedure were performed as in the case of sorption.

Detailed conditions for detection and LC-MS/MS analysis are described in the publication by Jedziniak et al. (14). Analysis was performed on an LCMS-8050 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) operating in positive and negative ionisation modes. Chromatographic separation was undertaken at 40°C with a constant flow of 0.3 mL/min in a Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm) (Phenomenex, Torrance, CA, USA). Separation was performed using a gradient elution of mobile phase A and phase B as described earlier in this section. The volume of the test sample injected was 5 μL, and the chromatographic analysis of one test sample had a 16 min runtime.

Lab Solutions software (version 5.60 SP2) (Shimadzu) was used for data processing. The percentage of mycotoxin binding by sorbents was calculated by comparing the results for the tested samples with those for the control samples.

The results were calculated by comparing test sample results with those for a control sample containing a pH 3.5 buffer and a mycotoxin standard solution. The higher the percentage of mycotoxin binding was, the more effectively the sorbent bound a toxin. In gastric conditions (pH 3.5), activated charcoal bound all tested mycotoxins with high efficiency; the other sorbent with similar efficiency was sepiolite, although it bound DON much less well than activated charcoal (Table 2). No sorbent besides activated charcoal could bind all eight mycotoxins simultaneously, and with the exception of sepiolite, they bound effectively to three or fewer mycotoxins. All sorbents except calcium lignosulphonate showed binding of AFL B1 at 100%. The most difficult mycotoxin to bind was DON; only calcium lignosulphonate (87%) and activated charcoal (70%) could bind this mycotoxin. Bentonite bound fumonisins at a low level of 21–33%; however, calcium bentonite bound these toxins much more effectively at 91–93% (Table 2).

The binding results in the intestinal phase (mycotoxin desorption at pH 7.0) indicated to what extent the mycotoxin-sorbent binding was stable in the later phase of the digestive process. The lower the percentage was, the less the sorbent released mycotoxin to the external environment and the more stable the binding was (Table 3). The results were calculated by comparing test sample results with those for a control sample containing PBS at pH 7.0 and the mycotoxin standard solution. The sorbents that showed the greatest binding stability in the intestinal phase (pH 7.0) were activated charcoal and sepiolite. In the cases of seven of the tested mycotoxins, very good durability of the mycotoxin–activated charcoal bond was observed, DON proving an exception with lower stability at 59% of mycotoxin released. Stable binding to most of the tested mycotoxins at a satisfactory level was also maintained by sepiolite, once again except for binding to DON and HT-2. Calcium lignosulphonate, which bound DON well at pH 3.5, did not show good stability of binding to this mycotoxin in the intestinal phase, and this also related to the other mycotoxins tested. Aflotoxin B1 was a toxin that passed through the conditions in the gastrointestinal tract without its bond with most sorbents being broken. Fairly unstable bonds to ZEN by attapulgite and ground attapulgite were noted at 74% and 72% toxin release, respectively. Fumonisins were mycotoxins to which most of the sorbents bound stably, calcium lignosulphonate being the worst exception. Where the sorbent had not bound to mycotoxin in the gastric phase, the stability of the mycotoxin-sorbent complex in the intestinal phase could not be evaluated.

In the experiment using a mixture of sorbents in various proportions, the highest effectiveness was shown by a mixture of sepiolite and calcium lignosulphonate in the proportions of 80 : 20; this mixture bound to proportions of DON, OTA and ZEN in a 62–82% range (Table 4). The mixture in the proportions of 40:60 sepiolite : calcium lignosulphonate was the most effective in binding DON. The mixture in the proportions of 20 : 80 was the least able to bind the three tested mycotoxins, sequestering only an average of 46%. The raising of the calcium lignosulphonate proportion mixed with sepiolite improved the ability of the mixture to bind DON. The higher the content of this sorbent, the greater the percentage of DON binding. A similar dependence could be observed in the case of OTA and ZEN: as the proportion of sepiolite in the mixture of the two sorbents increased, the binding capacity also increased.

Examining the binding by sorbents of three mycotoxins in naturally contaminated feed samples demonstrated low mycotoxin sorption in the case of two of them, namely DON and ZEN (Table 5). Therefore, no desorption investigation could be performed. All tested sorbents bound OTA in the gastric phase at a level in the range of 30–55%. The mycotoxin-sorbent complex was not stable enough to confirm the good sorption capacity of the sorbents for mycotoxins or the stability of the mycotoxin bond.

In the experiment using contaminated feed in the pH 3.5 phase, there was a significant decrease in the mycotoxin-binding capacity of the tested sorbents compared to the experiment using mycotoxin standards. Calcium lignosulphonate in the buffer with mycotoxin standards bound DON at a satisfactory level of 87%, while when feed was added to the sample, its binding capacity decreased to 11%. In the first step of the experiment modelling sorption, activated charcoal and sepiolite bound OTA and ZEN at very high levels ranging 94–99%. Adding feed resulted in a dramatic decrease in binding. In the case of activated charcoal, the effectiveness in binding OTA decreased to 39% and that of binding ZEN to merely 4%. A similar decline was observed for sepiolite; for OTA the percentage in binding decreased to 53% and for ZEN to as low as 6%.