- Detalles de la revista
- Formato
- Revista
- eISSN
- 1848-6312
- Primera edición
- 26 Mar 2007
- Calendario de la edición
- 4 veces al año
- Idiomas
- Inglés, Slovenian
Reliable analytical methods for mycotoxin determination, preferably employing ultra-high-performance liquid chromatography coupled to (tandem) mass spectrometry (UHP)LC-MS/(MS) for unambiguous detection (1) are nowadays imperative to establish product safety and gather accurate mycotoxin occurrence data for proper assessment of the risk that these contaminants pose to public health.
Numerous methods have been developed over the years, from the enzyme-linked immunosorbent assay (ELISA) to chromatographic methods employing thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC), which use various detection methods and sample preparation procedures (2). The introduction of mass spectrometry (MS) to contaminant analytics brought the opportunity to determine a large number of chemically distinct compounds in a relatively short time and to gain a better insight into the real extent of mycotoxin contamination. In addition to greater reliability compared to the conventional detectors, these new methods have streamlined sample preparation, which saves time and lowers the overall cost of analysis (1, 3).
Preparation of samples containing several mycotoxins for liquid chromatography – tandem mass spectrometry (LC-MS/MS) often relies on the so-called
However, analytes can interact and stick with the filter membrane, which may result in their underestimation. These interactions between the analyte and the filter depend on the analyte’s physicochemical properties, molecular weight, or ionisation state or on membrane structure, hydrophobicity, or hydrophilicity. All these considerations should drive the selection of appropriate filtrate membrane, which, in addition, should take into account sample medium properties and its compatibility with the membrane material (11, 12).
As far as mycotoxins are concerned, several hundred fungal metabolites have been discovered so far (9), yet only a few are regulated by law. In the European Union (EU), maximum permitted levels of mycotoxins are regulated by the Commission Regulation (EC) 1881/2006 (13) for food and Directive 2002/32/EC (14) for animal feed (Figure 1). When it comes to their determination, Malachová et al. (7) have found that filtration is not an option for their analytical procedure, as the loss of certain mycotoxins is too great. Kafouris et al. (8) and Kovač et al. (1), in turn, have reported that filtering solutions before instrumental analysis yields satisfactory results, conforming to method performance criteria.
Figure 1
Chemical structures of EU-regulated mycotoxins (excluding ergot alkaloids)
The aim of this study was, therefore, to investigate how various filter membrane materials affect the recovery of the EU-regulated mycotoxins and to determine the extent of mycotoxin loss to dilute-and-shoot sample preparation procedure.
Certified analytical standards of the following mycotoxins were obtained from Romer Labs Biopure (Romer Labs, Tulln, Austria): AFB1, AFB2, AFG1, and AFG2 (2.0 g/mL for AFB1/AFG1, 0.5 g/mL for AFB2/AFG2), DON (100 g/mL), FB1 and FB2 (50 g/mL), ZEA (100 g/mL), T-2 (100 g/mL), HT-2 (100 g/mL), and OTA (10 g/mL). LC-MS-grade acetonitrile (ACN) and methanol (MeOH) were obtained from J. T. Baker (J. T. Baker, Deventer, The Netherlands). LC-MS-grade formic acid (FA) and LC-MS ammonium formate (AFNH4) were supplied by Sigma-Aldrich (Sigma-Aldrich, Louis, MO, USA). Water (H2O) was purified to the ultrapure grade with a Niro VV (Nirosta d.o.o., Osijek, Croatia) or a Purelab flex system (ELGA LabWater, Woodridge, IL, USA). Syringe filters (13 mm in diameter with a 0.22 µm membrane pore size) were obtained from the following suppliers: nylon (NY) from Kemolab d.o.o. (Veliko Polje, Croatia), polytetrafluoroethylene (PTFE) from Kefo d.o.o. (Sisak, Croatia), while polyethersulphone (PES), mixed cellulose ester (MCE), and cellulose acetate (CA) were obtained from Obrnuta faza d.o.o. (Pazin, Croatia).
The multi-mycotoxin standard solution was prepared by mixing the eleven mycotoxin standards with ACN, and then by diluting this mixture one hundred times with a solvent mixture of ACN/H2O/FA (49.5/49.5/1, v/v/v) as shown in Table 1 to obtain analyte concentrations corresponding to the middle calibration point of the previously developed and validated multi-mycotoxin method by Kovač et al. (1). This solvent mixture composition for standard dilution matches the ratio of solvent components in real dilute-and-shoot without the matrix.
Preparation of the multi-mycotoxin standard solution
| Analyte | Concentration of analytical standard (μ/mL) | Volume of analytical standard for preparation of 1000 μL of combined multi-mycotoxin standard solution* (μL) | Concentration of combined multi-mycotoxin standard solution (ng/mL) | Concentration of 100× diluted combined multi-mycotoxin standard solution (ng/mL) |
|---|---|---|---|---|
| DON | 100 | 50 | 5000 | 50 |
| AFB1 | 2.0 | 25 | 50 | 0.5 |
| AFB2 | 0.5 | 25 | 12.5 | 0.125 |
| AFG1 | 2.0 | 25 | 50 | 0.5 |
| AFG2 | 0.5 | 25 | 12.5 | 0.125 |
| FB1 | 50 | 75 | 3750 | 37.5 |
| FB2 | 50 | 75 | 3750 | 37.5 |
| ZEA | 100 | 7.5 | 750 | 7.5 |
| T-2 | 100 | 2.5 | 250 | 2.5 |
| HT-2 | 100 | 2.5 | 250 | 2.5 |
| OTA | 10 | 2.5 | 25 | 0.25 |
| Volume of combined multi-mycotoxin standard solution for preparation of 1000 μL of 100× diluted combined multi-mycotoxin standard solution** (μL) | 10 | |||
* pure ACN;
** ACN/H2 O/FA (49.5/49.5/1, v/v/v)
The multi-mycotoxin standard solution was microfiltered in such a way that the first three filtrate drops (filtered solution 1) and the following filtrate (filtered solution 2) were collected separately into two different vials and injected (in triplicate) into a UHPLC-MS/MS system. The unfiltered multi-mycotoxin solution was injected into the system as is (Figure 2). The whole procedure was performed in duplicate. To process the obtained data on peak areas for each compound, we took averages of repeated measurements and calculated the relative recovery (expressed with a percentage) for each mycotoxin and filter membrane material by comparing the average area of the filtered solution (1 or 2) with unfiltered standard solution according to Equation 1, as follows:
Figure 2
Microfiltrate preparation. Filtrate solutions (vials) 1 and 2 were injected into the UHPLC column one at a time
For instrumental analysis we used a UHPLC (Acquity H-Class, Waters, Milford, MA, USA) equipped with a quaternary pump system, coupled with a triple quadruple mass spectrometer (XEVO TQ-S micro, Milford, MA, USA) using a ZSpray electrospray interface (ESI). For chromatographic separation of the analytes we used a reverse phase column XBridge Peptide BEH C18 (150×3.0, 2.5 µm) (Waters, Milford, MA, USA) maintained at 50 °C, which we achieved with gradient elution of eluent A (aqueous solution of 5 mmol/L AFNH4+0.02 % FA) and eluent B (MeOH) at a constant flow rate of 0.6 mL/min. The elution started with 75 % of eluent A in gradient, with a hold time of 0.5 min. Then we switched to 10 % of eluent A for 6 min, followed by equilibration to initial elution conditions after 2 min, giving a total run time of 13 min. The sample injection volume was set to 10 µL. Analytes were identified with the ESI-MS/MS working in the multiple-reaction monitoring (MRM) mode in positive and negative polarity, with two MS/MS transitions acquired per analyte (Table 2). The MS and MS/MS parameters used for analysis in both positive and negative ESI were as follows: capillary voltage 3.5 kV (+) and 2.5 kV (–), source temperature 150 °C, desolvation temperature 550 °C, desolvation gas flow 1000 L/h, and cone gas flow 50 L/h (both nitrogen). Argon gas was used for collision at a pressure of about 0.4 Pa in the collision cell. The obtained UHPLC-MS/MS chromatogram of the eleven mycotoxins in the unfiltered solution is shown in Figure 3.
MS/MS parameters for mycotoxin analysis
| Mycotoxin | Precursor ion | Cone voltage | Collision energy | Product ions |
|---|---|---|---|---|
| 313.2 (M+H)+ | 60 | 38 | 241.0 | |
| 23 | 285.3 | |||
| 315.2 (M+H)+ | 60 | 30 | 259.0 | |
| 25 | 287.0 | |||
| 329.2 (M+H)+ | 60 | 28 | 311.0 | |
| 24 | 243.0 | |||
| 331.2 (M+H)+ | 60 | 24 | 313.0 | |
| 28 | 245.1 | |||
| 297.2 (M+H)+ | 25 | 10 | 249.0 | |
| 10 | 203.2 | |||
| 722.4 (M+H)+ | 50 | 40 | 334.3 | |
| 40 | 352.3 | |||
| 706.4 (M+H)+ | 50 | 40 | 336.2 | |
| 40 | 318.2 | |||
| 317.1 (M–H)- | -58 | 30 | 131.0 | |
| 20 | 175.0 | |||
| 484.7 (M+NH4)+ | 25 | 20 | 185.0 | |
| 25 | 215.0 | |||
| 442.6 (M+NH4)+ | 25 | 10 | 263.4 | |
| 15 | 215.3 | |||
| 404.1 (M+H)+ | 30 | 24 | 239.0 | |
| 14 | 358.0 |
Figure 3
UHPLC-MS/MS chromatogram of the selected EU-regulated mycotoxins in the unfiltered multi-mycotoxin standard solution (AFB1/AFG1 0.5 ng/mL, AFB2/AFG2 0.125 ng/mL, DON 50 ng/mL, FB1/FB2 37.5 ng/mL, ZEA 7.5 ng/mL, T-2/HT-2 2.5 ng/mL and OTA 0.25 ng/mL) obtained with an Acquity H-class – Xevo TQ-S micro system
Statistical data analysis was performed using Statistica 13.3. software (TIBCO Software Inc., Palo Alto, CA, USA). Significant difference was set to p<0.05. Before we ran statistical tests, we tested the data for normality of distribution using the Shapiro-Wilk test and for the homogeneity of variance using Levene’s test. All variables that had normal distribution and homogenous variance (AFB1, AFG2, ZEA) were then tested with the
Figure 4 shows mycotoxin recovery by filter membrane material. Mycotoxin loss was generally greater in the first filtrate solution than the second. Cellulose-based materials did not go well with acetonitrile in both filtered solutions, which confirms an earlier report by Carlson and Thompson (11).
Figure 4
Recoveries of mycotoxins with various filter membrane materials in filtrate solution 1 and 2 relative to the recovery (dashed line) from the unfiltered multi-mycotoxin solution. Low recoveries indicating mycotoxin loss during filtration are framed in squares. CA – cellulose acetate; MCE – mixed cellulose ester; NY – nylon; PES – polyethersulphone; PTFE – polytetrafluoroethylene
However, the cellulose-based and polyethersulphone filters yielded unexpectedly high relative recoveries of up to 200 % for several, mainly
What we can see in more detail in Figure 5, is a very high recovery variation for the cellulose-based and polyethersulphone filters, regardless of which part of the filtrate was analysed, and much more even data distribution with the polytetrafluoroethylene and nylon filters. However, statistical analysis showed a significant difference between filtrate solution 1 and 2 recoveries only for AFB2 (p=0.0039) filtered through PES and MCE (49.32 % and 76.15 % in the filtrate solution 1, respectively vs 140.00 % and 97.35 % in the filtrate solution 2) and for HT-2 toxin (p=0.00047) filtered through PES (160.52 % in filtrate solution 1 vs 119.57 % in filtrate solution 2) (Figure 6).
Figure 5
Relative recovery of the 11 mycotoxins in filtrate solutions 1 and 2 by filter membrane material. CA – cellulose acetate; FS1 – filtrate solution 1 (first three drops of the solution); FS2 – filtrate solution 2 (the rest of the solution); MCE – mixed cellulose ester; NY – nylon; PES – polyethersulphone; PTFE – polytetrafluoroethylene
Figure 6
Relative recovery of the “outlier” mycotoxins AFB2 (a) and HT-2 (b) from filtrate solutions 1 and 2 by filter membrane materials. CA – cellulose acetate; FS1 – filtrate solution 1 (first three drops of the solution); FS2 – filtrate solution 2 (the rest of the solution); MCE – mixed cellulose ester; NY – nylon; PES – polyethersulphone; PTFE – polytetrafluoroethylene
On the other hand, recoveries significantly differed between filter types for AFB1 (p=0.000); AFG1 (p=0.0003); AFG2 (p=0.003); DON (p=0.000); FB1 (p=0.000); FB2 (p=0.000); ZEA (p=0.000); T-2 (p=0.001); HT-2 (p=0.000), and OTA (p=0.000), but not for AFB2.
Considering all these findings, PTFE turns out to least affect mycotoxin recovery during microfiltration, and NY material comes as the second best choice. It also turns out that discarding the first few filtrate drops should ensure a more reliable recovery. Other researchers, depending on the configuration of the analytical system, chose either to skip microfiltration or use a specific material of choice. Malachová et al. (7) did not use filtration in their HPLC analysis to avoid losses, whereas Kovač et al. (1), Frenich et al. (15), and de Santis (16) opted for NY and Carballo et al. (17) for PTFE. Some researchers who used microfiltration, such as Kafouris et al. (8), do not mention which type of filter membrane material they used.
Analyte loss to microfiltration following extraction can pose a serious problem to LC-MS quantitation. Although it can be skipped with some analytes and/or systems, there where it is required, the solution to the problem is to choose the membrane material, pore size, and filter size appropriate for specific analyte or solution properties. Furthermore, to ensure the accuracy of the analytical procedure, it may be necessary to discard the initial filtrate drops, as they only serve to saturate the filter membrane. Our study has revealed statistically significant differences in mycotoxin recoveries between filters, and has singled out polytetrafluoroethylene as the material of choice, followed by nylon. It has also pointed to some unexpectedly high recoveries, which deserves further attention.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
MS/MS parameters for mycotoxin analysis
| Mycotoxin | Precursor ion |
Cone voltage |
Collision energy |
Product ions |
|---|---|---|---|---|
| 313.2 (M+H)+ | 60 | 38 | 241.0 | |
| 23 | 285.3 | |||
| 315.2 (M+H)+ | 60 | 30 | 259.0 | |
| 25 | 287.0 | |||
| 329.2 (M+H)+ | 60 | 28 | 311.0 | |
| 24 | 243.0 | |||
| 331.2 (M+H)+ | 60 | 24 | 313.0 | |
| 28 | 245.1 | |||
| 297.2 (M+H)+ | 25 | 10 | 249.0 | |
| 10 | 203.2 | |||
| 722.4 (M+H)+ | 50 | 40 | 334.3 | |
| 40 | 352.3 | |||
| 706.4 (M+H)+ | 50 | 40 | 336.2 | |
| 40 | 318.2 | |||
| 317.1 (M–H)- | -58 | 30 | 131.0 | |
| 20 | 175.0 | |||
| 484.7 (M+NH4)+ | 25 | 20 | 185.0 | |
| 25 | 215.0 | |||
| 442.6 (M+NH4)+ | 25 | 10 | 263.4 | |
| 15 | 215.3 | |||
| 404.1 (M+H)+ | 30 | 24 | 239.0 | |
| 14 | 358.0 |
Preparation of the multi-mycotoxin standard solution
| Analyte | Concentration of analytical standard (μ/mL) | Volume of analytical standard for preparation of 1000 μL of combined multi-mycotoxin standard solution |
Concentration of combined multi-mycotoxin standard solution (ng/mL) | Concentration of 100× diluted combined multi-mycotoxin standard solution (ng/mL) |
|---|---|---|---|---|
| DON | 100 | 50 | 5000 | 50 |
| AFB1 | 2.0 | 25 | 50 | 0.5 |
| AFB2 | 0.5 | 25 | 12.5 | 0.125 |
| AFG1 | 2.0 | 25 | 50 | 0.5 |
| AFG2 | 0.5 | 25 | 12.5 | 0.125 |
| FB1 | 50 | 75 | 3750 | 37.5 |
| FB2 | 50 | 75 | 3750 | 37.5 |
| ZEA | 100 | 7.5 | 750 | 7.5 |
| T-2 | 100 | 2.5 | 250 | 2.5 |
| HT-2 | 100 | 2.5 | 250 | 2.5 |
| OTA | 10 | 2.5 | 25 | 0.25 |
| Volume of combined multi-mycotoxin standard solution for preparation of 1000 μL of 100× diluted combined multi-mycotoxin standard solution |
10 | |||