application of a gpc-lc-ms/ms method for the determination ......gel permeation chromatography with...

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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Time (min)40

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Time (min)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38400

FB1/FB2

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FB1/FB2

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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/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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