Application of a GPC-LC-MS/MS method for the determination ......Gel permeation chromatography with...

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C. Gottschalk 1 , J. Barthel 2 , U. Aulwurm 3 , G. Engelhardt 2 , J. Bauer 1 , K. Meyer 1 1 TU München, Chair of Animal Hygiene, Weihenstephaner Berg 3, D-85354 Freising 2 Bavarian Health and Food Safety Authority, Veterinärstraße 2, D-85764 Oberschleißheim 3 LCTech GmbH, Bahnweg 41, D-84405 Dorfen Application of a GPC-LC-MS/MS method for the determination of various mycotoxins in edible oils – results of the method development Influence of pH Also the variation of the pH of the eluent (0.2, 1.0 and 2.0% formic acid) influenced the retention of fumonisins, but not of the other toxins (fig. 4). Best results were obtained with 1% formic acid (fig. 4b); a higher acid content resulted in a better peak symmetry, but FB1/FB2 coeluted with the oil fraction (fig. 4c). References: [1] Hetmanski, M. T., Scudamore, K. A., 1989. A simple quantitative HPLC method for determination of aflatoxins in cereals and animal feedstuffs using gel permeation chromatography clean-up. Food Add Contam, 6, 35- 48. [2] Ranfft, K., Gerstl, R., Mayer, G., 1990. Determination of occurrence of zearalenone in cereals and mixed feeds. Z Lebensm Unters Forsch, 191, 449-453. [3] Dunne, C., Meaney, M., Smyth, M., Tuinstra, L. G., 1993. Multimycotoxin detection and clean-up method for aflatoxins, ochratoxin and zearalenone in animal feed ingredients using high-performance liquid chromatography and gel permeation chromatography. J Chromatogr, 629, 229-235. [4] Kappenstein, O., Klaffke, H., Mehlitz, I., Tiebach, R., Weber, R., Lepschy, J., Wittkowski, R., 2005. Determination of zearalenone in edible oils with SEC and LC-ESI-MS/MS. Mycotoxin Research, 21, 3-6. [5] Kocher, U., 2006. Multimethode zur Bestimmung von Mykotoxinen in Speiseölen mittels LC-MS/MS. Conference transcript 28th Mycotoxin-Workshop, Bydgoszcz, Poland, May 29-31. Figure 1: GPC-MS/MS coupling for method development The new GPC-column allows a proper separation of the oil and the analytes (fig. 2). The final parameters and steps of the method are shown in the following scheme. The collected fraction was neutralized as it was noticed that certain analytes were destroyed by a low or high pH during evaporation. The fraction was evaporated to about 500 μl and quantitatively transferred into a volumetric flask. The use of gel permeation chromatography (GPC) for sample clean-up in mycotoxin analysis was first reported in 1989 by Hetmanski and Scudamore [1]. They developed a method for the determination of aflatoxins in cereals and animal feed. Later, GPC-methods for zearalenone (ZEA), ochratoxin A (OTA) and other mycotoxins in cereals and feed were published [2, 3]. For the clean-up of mycotoxins from edible oils and fatty matrices GPC is currently regarded to be a promising approach. Kappenstein et al. [4] developed a method for ZEA in corn oils and reported levels up to 920 μg/kg. Also traces of T-2 toxin have been reported to occur in this matrix [5]. All these GPC- methods were based on commonly used BioBeads SX-3 which appeared to be suitable for ZEA and aflatoxins as well as trichothecenes. But the selectivity of this material is not adequate to separate fumonisins (FB1/FB2) from the oil fraction. However, in the first version of commissions regulation (EC) 1881/2006 from 19 december 2006, a maximum level of 1000 μg/kg had been proposed for these toxins in maize oil. Here a new-developed GPC-column is presented which has been designed by LCTech for the simultaneous clean-up of zearalenone, fumonisins, trichothecenes (type A, B and D), aflatoxins, ochratoxin A and other mycotoxins from edible oils. Introduction Primarily, different column materials and compositions have been tested for their suitability to separate the analytes from the oil fraction. Therefore, columns useable in HPLC-scale were produced. Simultaneously, influencing factors like the eluent composition (tetrahydrofuran/water), pH, temperature, and the column loading capacity had to be studied. Subsequently, the selected material with the optimized parameters has been transferred to GPC-scale and tested by a direct Method development Weigh in 1 g of oil, fill up to 10 ml with THF Gel permeation chromatography with MykoClean™ column - Eluent: THF/water/formic acid 89.5/10/0.5 (v/v/v) - Flow rate: 3 ml/min - Injection volume: 500 μl Discard fraction 0-18 min, collect 18-32 min Adjust pH to 7 with NH 3 in methanol Evaporate and fill up to 1 ml with methanol/water 30/70 (v/v) Measure with LC-MS/MS Final GPC-LC-MS/MS method and parameters Influence of water content Variation of the water content of the eluent had a remarkable influence on the retention times of the fumonisins (fig. 3), while the retention of the other toxins was hardly modified. With 10% water, the separation was satisfactory (fig. 3b); with 20% (fig. 3c), the toxins coeluted with the oil fraction (ref. fig. 2). 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 0 100 Intensity (%) 50 40 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 Intensity (%) 50 FB2 FB1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 0 100 Intensity (%) 50 40 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 Intensity (%) 50 FB2 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 0 100 Intensity (%) 50 40 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 Intensity (%) 50 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 FB2 FB1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 FB2 FB1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 Time (min) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time (min) 40 0 100 Intensity (%) 50 Time (min) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 100 Intensity (%) 50 Time (min) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 100 Intensity (%) 50 FB1/FB2 Figure 4a: Multi-mycotoxin mix with eluent THF/water/formic acid 89.8/10/0.2 (v/v/v) Figure 4b: Multi-mycotoxin mix with eluent THF/water/formic acid 89/10/1 (v/v/v) Figure 4c: Multi-mycotoxin mix with eluent THF/water/formic acid 88/10/2 (v/v/v) Figure 2: Chromatogram (direct GPC-MS/MS coupling) of maize oil (0.1 g/1 ml THF) spiked with a multi-mycotoxin mix Figure 3a: FB1 and FB2 with eluent THF/formic acid 99/1 (v/v) Figure 3b: Multi-mycotoxin mix with eluent THF/water/formic acid 89/10/1 (v/v/v) Figure 3c: Multi-mycotoxin mix with eluent THF/water/formic acid 79/20/1 (v/v/v) 0 100 Intensity (%) 50 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Time (min) FB2 FB1 0 100 Intensity (%) 50 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Time (min) FB2 FB1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 FB2 FB1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) 50 FB2 FB1 GPC-MS/MS coupling (fig. 1). The separation of the oil from the analytes was satisfactory up to an oil concentration of 0.1 g/ml THF (fig. 2). Influences of the water content of the eluent and of the pH are shown exemplary in the following MRM-chromatograms (LC-MS/MS). Results and discussion The toxins were measured by a LC-MS/MS multi- mycotoxin method with ESI+/- ionization. The recoveries obtained with the combined GPC-LC- MS/MS method ranged between 74 and 104 % (fig. 5). For some analytes, low matrix suppression effects were observed. The limits of quanitification complied with the maximum levels for analytes regulated by the EC (1881/2006). 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) oil fraction 50 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Time (min) 0 100 Intensity (%) oil fraction 50 Figure 5: Recoveries from spiked corn oil for different groups of mycotoxins obtained with the final GPC-LC-MS/MS method 0 70 80 90 100 110 T-2 HT-2 T-2 triol T-2 tetraol DAS MAS NEO VOL DacVOL DON DON-3-Gluc NIV 3acDON 15acDON FX SG SH SF RA RE RL-2 VJ Afla B1 Afla B2 Afla G1 Afla G2 SMC FUM B1 FUM B2 FUM B3 PHFB1a+b HFB-1 OTA OTB MPA ZEA Type A trichothecenes Type B trichothecenes Fumonisins Others Type D trichothecenes Aflatoxins Recovery (%) 0 70 80 90 100 110 T-2 HT-2 T-2 triol T-2 tetraol DAS MAS NEO VOL DacVOL DON DON-3-Gluc NIV 3acDON 15acDON FX SG SH SF RA RE RL-2 VJ Afla B1 Afla B2 Afla G1 Afla G2 SMC FUM B1 FUM B2 FUM B3 PHFB1a+b HFB-1 OTA OTB MPA ZEA Type A trichothecenes Type B trichothecenes Fumonisins Others Type D trichothecenes Aflatoxins Recovery (%)

Transcript of Application of a GPC-LC-MS/MS method for the determination ......Gel permeation chromatography with...

Page 1: Application of a GPC-LC-MS/MS method for the determination ......Gel permeation chromatography with MykoClean column - Eluent: THF/water/formic acid 89.5/10/0.5 (v/v/v) - Flow rate:

C. Gottschalk1, J. Barthel2, U. Aulwurm3, G. Engelhardt2, J. Bauer1, K. Meyer1

1 TU München, Chair of Animal Hygiene, Weihenstephaner Berg 3, D-85354 Freising2 Bavarian Health and Food Safety Authority, Veterinärstraße 2, D-85764 Oberschleißheim

3 LCTech GmbH, Bahnweg 41, D-84405 Dorfen

Application of a GPC-LC-MS/MS method for the determination of various mycotoxins in edible oils

– results of the method development

Influence of pHAlso the variation of the pH of the eluent (0.2, 1.0 and 2.0% formic acid) influenced the retention of fumonisins, but not of the other toxins (fig. 4). Best results were obtained with 1% formic acid (fig. 4b); a higher acid content resulted in a better peak symmetry, but FB1/FB2 coeluted with the oil fraction (fig. 4c).

References: [1] Hetmanski, M. T., Scudamore, K. A., 1989. A simple quantitative HPLC method for determination of aflatoxins in cereals and animal feedstuffs using gel permeation chromatography clean-up. Food Add Contam, 6, 35-48. [2] Ranfft, K., Gerstl, R., Mayer, G., 1990. Determination of occurrence of zearalenone in cereals and mixed feeds. Z Lebensm Unters Forsch, 191, 449-453. [3] Dunne, C., Meaney, M., Smyth, M., Tuinstra, L. G., 1993. Multimycotoxin detection and clean-up method for aflatoxins, ochratoxin and zearalenone in animal feed ingredients using high-performance liquid chromatography and gel permeation chromatography. J Chromatogr, 629, 229-235. [4] Kappenstein, O., Klaffke, H., Mehlitz, I., Tiebach, R., Weber, R., Lepschy, J., Wittkowski, R., 2005. Determination of zearalenone in edible oils with SEC and LC-ESI-MS/MS. Mycotoxin Research, 21, 3-6. [5] Kocher, U., 2006. Multimethode zur Bestimmung von Mykotoxinen in Speiseölen mittels LC-MS/MS. Conference transcript 28th Mycotoxin-Workshop, Bydgoszcz, Poland, May 29-31.

Figure 1: GPC-MS/MS coupling for method development

The new GPC-column allows a proper separation of the oil and the analytes (fig. 2). The final parameters and steps of the method are shown in the following scheme. The collected fraction was neutralized as it was noticed that certain analytes were destroyed by a low or high pH during evaporation. The fraction was evaporated to about 500 µl and quantitatively transferred into a volumetric flask.

The use of gel permeation chromatography (GPC) for sample clean-up in mycotoxin analysis was first reported in 1989 by Hetmanski and Scudamore [1]. They developed a method for the determination of aflatoxins in cereals and animal feed. Later, GPC-methods for zearalenone (ZEA), ochratoxin A (OTA) and other mycotoxins in cereals and feed were published [2, 3]. For the clean-up of mycotoxins from edible oils and fatty matrices GPC is currently regarded to be a promising approach. Kappenstein et al. [4] developed a method for ZEA in corn oils and reported levels up to 920 µg/kg. Also traces of T-2 toxin have been reported to occur in this matrix [5]. All these GPC-methods were based on commonly used BioBeads SX-3 which appeared to be suitable for ZEA and aflatoxins as well as trichothecenes. But the selectivity of this material is not adequate to separate fumonisins (FB1/FB2) from the oil fraction. However, in the first version of commissions regulation (EC) 1881/2006 from 19 december 2006, a maximum level of 1000 µg/kg had been proposed for these toxins in maize oil. Here a new-developed GPC-column is presented which has been designed by LCTech for the simultaneous clean-up of zearalenone, fumonisins, trichothecenes (type A, B and D), aflatoxins, ochratoxin A and other mycotoxins from edible oils.

Introduction

Primarily, different column materials and compositions have been tested for their suitability to separate the analytes from the oil fraction. Therefore, columns useable in HPLC-scale were produced. Simultaneously, influencing factors like the eluent composition (tetrahydrofuran/water), pH, temperature, and the column loading capacity had to be studied. Subsequently, the selected material with the optimized parameters has been transferred to GPC-scale and tested by a direct

Method development

Weigh in 1 g of oil, fill up to 10 ml with THF

Gel permeation chromatography with MykoClean™ column- Eluent: THF/water/formic acid 89.5/10/0.5 (v/v/v)

- Flow rate: 3 ml/min- Injection volume: 500 µl

Discard fraction 0-18 min, collect 18-32 min

Adjust pH to 7 with NH3 in methanol

Evaporate and fill up to 1 ml with methanol/water 30/70 (v/v)

Measure with LC-MS/MS

Final GPC-LC-MS/MS method and parameters

Influence of water contentVariation of the water content of the eluent had a remarkable influence on the retention times of the fumonisins (fig. 3), while the retention of the other toxins was hardly modified. With 10% water, the separation was satisfactory (fig. 3b); with 20% (fig. 3c), the toxins coeluted with the oil fraction (ref. fig. 2).

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Figure 4a: Multi-mycotoxin mix with eluent THF/water/formic acid 89.8/10/0.2 (v/v/v)

Figure 4b: Multi-mycotoxin mix with eluent THF/water/formic acid 89/10/1 (v/v/v)

Figure 4c: Multi-mycotoxin mix with eluent THF/water/formic acid 88/10/2 (v/v/v)

Figure 2: Chromatogram (direct GPC-MS/MS coupling) of maize oil (0.1 g/1 ml THF) spiked with a multi-mycotoxin mix

Figure 3a: FB1 and FB2 with eluent THF/formicacid 99/1 (v/v)

Figure 3b: Multi-mycotoxin mix with eluent THF/water/formic acid 89/10/1 (v/v/v)

Figure 3c: Multi-mycotoxin mix with eluent THF/water/formic acid 79/20/1 (v/v/v)

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GPC-MS/MS coupling (fig. 1). The separation of the oil from the analytes was satisfactory up to an oil concentration of 0.1 g/ml THF (fig. 2). Influences of the water content of the eluent and of the pH are shown exemplary in the following MRM-chromatograms (LC-MS/MS).

Results and discussionThe toxins were measured by a LC-MS/MS multi-mycotoxin method with ESI+/- ionization. The recoveries obtained with the combined GPC-LC-MS/MS method ranged between 74 and 104 % (fig. 5). For some analytes, low matrix suppression effects were observed. The limits of quanitification complied with the maximum levels for analytes regulated by the EC (1881/2006).

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Figure 5: Recoveries from spiked corn oil for different groups of mycotoxins obtained with the final GPC-LC-MS/MS method

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