Detection of Relevant Mycotoxins in Wheat Beer ... - ACS Publications

Chapter 12

Detection of Relevant Mycotoxins in Wheat Beer and Sake by LC-MS/MS Using Prototype Immunoaffinity Column Clean-Up: A Preliminary Study Downloaded by UNIV OF GUELPH LIBRARY on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch012

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Mahalakshmi Rudrabhatla , John E. GeorgeIII ,Norma R. Hill , and Darsa P. Siantar * 2,

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Applications Laboratory of Varian, Inc. and 2Compliance Laboratory of Alcohol and Tobacco Tax and Trade Bureau (TTB), 490 North Wiget Lane, Walnut Creek, CA 94598

All of 12 relevant mycotoxins, namely deoxynivalenol (DON), aflatoxins B 1 , B 2, G and G2, ochratoxin A (OTA), fumonisins B , B and B , T-2 toxin (T-2), HT-2 toxin (HT-2), and zearalenone (ZON) present in complex wheat beer and sake matrices were extracted using multifunctional mixed bed immunoaffinity columns. They were detected using a LC -MS/MS method that achieved good separation and detection. Recoveries of all the toxins from the immunoaffinity columns ranged from 64-127%. The LC separation was performed using a reverse phase C18 column. MS measurements were done in both the positive and negative ESI modes. Multiple reaction monitoring (MRM) was carried out for each com pound. 1

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© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by UNIV OF GUELPH LIBRARY on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch012

242 Mycotoxins are toxic metabolites produced in food and feed by field and storage fungi. The occurrence of mycotoxins in a variety of foods has become a great concern as they are often associated with acute and chronic toxicity in humans and animals. The most important mycotoxins are aflatoxins, ochratoxin A (OTA), trichothecenes, fumonisins, and zearalenone (ZON). Because of their acute and chronic toxicity, guidelines and tolerance levels have been set for food and feed worldwide. Contamination of fermented alcoholic beverages such as wheat beer or sake is possible if contaminated wheat or rice are used in the manufacturing process. Several mycotoxins have been found in beers of different countries (7). It is therefore very important to monitor for mycotoxins in beverages before they are released for public use. Several analytical methods such as GC-MS and LC with UV, fluorescence, and MS detection have been used for the analysis of mycotoxins (2-4). LCMS/MS has broad applicability and is highly selective. Matrix effects are generally observed while analyzing the mycotoxins present in complex matrices. Sample preparation that helps minimize the ion suppression or enhancement is required prior to the LC-MS/MS analysis. Immunoaffinity clean-up has been extensively used for the extraction of mycotoxinsfromcomplex matrices and the extracted compounds were generally analysed by LC-fluorescence or LC-UV and LC-MS analysis (J-P). Presently available immunoaffinity catridges extract one, two or three groups of mycotoxins at a time. In the present study we have evaluated two different mixed bed multifunctional cartridges, one that retains four mycotoxins and the other that retains 12 mycotoxins simultaneously. Determination was by an LC-MS/MS method.

Materials and Methods Materials Aflatoxins B B , Gj and G , deoxynivalenol, T-2, HT-2, ochratoxin A, zearalenone, zearalanone (internal standard), fumonisins B and B standards were purchased from Sigma-Aldrich Co, St. Louis, MO. Fumonisin B was from PROMEC, the Medical Research Council in Tygerberg, South Africa. Acetic acid (analytical-reagent grade) and HPLC grade methanol were purchased from Fisher Chemicals (Boston, MA). Water (conductivity of 18 Mil" ) was purified by a Picotech Hydro UltraPure Water System (Garfield, NJ, USA). b

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Instruments A Varían 320-MS LC-MS/MS equipped with an ESI source coupled to Varian Prostar 210™ solvent delivery system and Varian Prostar 430™ auto-


243 sampler were used for this work. Chromatographic separation was achieved on a 150 mm x 3.0 mm id RP-C18 column. M R M and atmospheric ionization (API) conditions given in our previously published multicomponent LC-MS/MS method (70) were used to analyze the wheat beer and sake samples that were cleaned-up using the prototype multifunctional immunoaffinity columns. Details of the LC-MS/MS method are as follows. M R M conditions are given in Table 1.


LC Conditions Column: Polaris C-18A 5\M 150 mm x 3.0 mm id (Varían part No A2000150x030) Buffer A: 5 mM ammonium acetate, 1% CH COOH in 10% methanol Buffer B: 5 mM ammonium acetate, 1% CH COOH in 100% methanol LC Gradient: 15% to 60% buffer B in 40 min at 0.3 mL/min 9

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API Conditions Ionization Mode: ESI (positive and negative) Collision Gas: Argon, 1.8 mTorr API Drying Gas: 30 psi at 250 °C API Nebulizing Gas: 50 psi Needle: 4500V Detector: 1900V

Sample Preparation Two immunoaffinity columns, one provided by VICAM and the other by RBiopharm Rhône, were used for this study. VICAM's "all-in-one" prototype immunoaffinity column has antibodies for the simultaneous purification of 12 mycotoxins namely DON, ZON, T-2, HT-2, aflatoxins B B , G G , fumonisins B i , B , B , and ochratoxin A. R-Biopharm Rhone's prototype immunoaffmity column has antibodies for DON, ZON, T-2, and HT-2. Entire sample preparation was performed using Zymark's automated SPE work station. Stock solutions of individual mycotoxin standards were prepared by mixing pure standards in methanol and storing at 4 °C until use. These standards were used to make mixed standard solutions of all 12 analytes. Dealcoholized wheat beer and sake samples were spiked with the mixed toxin standard at known concentrations. The internal standard zearalanone spike concentration was 70 ng/g. The above samples were degassed by ultrasonication for 15 min and filtered through Whatman No.l filter paper. Three mL of these samples were mixed with three mL of 10% phosphate buffered saline (PBS) aqueous solution. b

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Deoxynivalenol Aflatoxin G Aflatoxin Gi Aflatoxin B Aflatoxin B Fumonisin B Fumonisin B Fumonisin B T-2 OTA Zearaianone ZON HT-2

Mycotoxin

Collision Precursor Product Energy 1 Ion (m/z) Ion 1 (m/z) (V) 59.2 25.0 355.1 331.0 16.5 245.0 329.0 283.0 25.5 315.0 287.0 21.5 313.0 16.5 245.0 33.0 720.9 157.0 706.3 332.2 33.0 704.6 33.0 546.0 484.0 305.0 18.5 403.9 25.0 238.9 319.0 136.0 26.5 317.0 24.5 185.0 447.0 11.5 265.0

Collision Product Energy 2 Ion 2 (m/z) (V) 265.0 11.5 275.0 11.5 243.0 25.5 259.0 10.0 241.0 22.0 563.0 24.0 318.1 40.0 159.0 33.0 117.0 13.5 357.9 25.0 187.0 28.5 187.0 18.5 345.0 13.5

Ta ble 1. Multitoxin method - MRM parameters


0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

Dwell (sec)

245 The solution was then passed through VICAM's or R-Biopharm's prototype immunoaffinity column. The column was washed with 5 mL of deionized water. Mycotoxins were eluted using 3 mL methanol. This solution was further dried under nitrogen, reconstituted to a known volume with LC buffer and then analyzed by LC-MS/MS.


Results and Discussion Wheat beer or sake spiked with mixed toxin standard at medium level (DON, ZON, T-2, HT-2, fiimonisins B B , and B at 350 ng/g, aflatoxins B,, B , Gi and G at 35 ng/g, and ochratoxin A at 175 ng/g) were extracted using the VICAM mixed bed multifunctional immunoaffinity cartridge that simultaneously retains all the 12 mycotoxins. Mixed toxin standard spiked in 10% PBS (with no alcoholic beverage matrix) loaded into the multifunctional column using the above procedure was used as the control sample. Unspiked sake or wheat beer passed through the mixed bed immunoaffinity column served as the blank. The eluate from the cartridge was collected and analyzed using an LC-MS/MS method that achieved good separation and detection for all the 12 mycotoxins. The LC-MS/MS total ion chromatogram (TIC) of the multi-toxin spiked sake is given in Figure 1; good separation and peak shapes were obtained for all 12 mycotoxins. The M R M chromatogram of the 12 mycotoxins with zearalanone (ZAN) as an internal standard is given in Figure 2. Triplicate recoveries of the mycotoxins were calculated (Table 2). The recoveries were acceptable for all 12 mycotoxins indicating the high specificity of monoclonal antibodies and ease of use of immunoaffinity column clean-up. The RSD's were very low indicating excellent selectivity and robust clean-up of the immunoaffinity column for mycotoxins from wheat beer and sake. In the next step, triplicate recoveries of the four mycotoxins namely, DON, ZON, T-2 and HT-2 at low spike concentration (35 ng/g) extracted from a complex wheat beer matrix using R-Biopharm and VICAM's mixed bed columns were compared (Table 3). The recoveries obtained from both the columns were above 95% and were comparable. Well-resolved peak shapes were obtained indicating the suitability of immunoaffinity column clean-up for quantitation by LC-MS/MS at low levels (Figure 2). Recoveries met the EU requirements for the recovery rates of mycotoxins from complex matrices (2005/3S7EG) (11). In the guideline 2005/3 S7EG, the recovery limits are given as follows: 60 to 120% for DON/ZON and 60 to 130% for T-2/HT-2. Recently, recovery rates of 100 ± 2.1 to 3.3% were reported using RP CI8 columns for ZON, a-zearalenol and p-zearalenol from 23 beer samples (12). Dalí' Asta and co-workers demonstrated that sample clean-up may be omitted for the direct injection of wine samples on LC-MS and LC-fluorescence systems (13). Although, good recoveries were achieved, it remains doubtful that a similar h

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Figure 1. Total ion chromatogram (TIC) of the 12 mycotoxins purifiedfrom sake using VICAM'S mixed bed multifunctional immunoaffinity column. [NOTE: Aflatoxins and Fumonisins are abbreviated as Afla and FBJ


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Table 2. Mycotoxin Recoveries Obtained Using a Mixed Bed Multifunctional Immunoaffinity Column (n=3)

Mycotoxin DON Aflatoxin B, Aflatoxin B Aflatoxin Gj Aflatoxin G T-2 HT-2 OTA Fumonisin B, Fumonisin B Fumonisin B ZON 2

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VICAM Recovery %± RSD% at medium spike level Wheat beer Sake 98.1 ± 1.4 92.8 ± 1.5 100.0 ±3.2 93.1 ± 1.7 81.7 ±0.7 92.5 ±2.3 92.5 ±1.2 93.1 ±3.1 91.7±2.1 81.8 ±5.3 99.9 ± 1.7 100.7 ± 1.2 74.0 ±1.0 64.0 ± 3.9 111 ±0.8 102.4 ±3.9 107.9 ±2.0 107.0 ±5,9 95.7 ± 6.5 107.8 ±13.8 127.0 ±6.9 116.4 ±2.8 101.6 ±4.3 101.6±3.1

Table 3. Comparison of VICAM's and R-Biopharm's Mixed Bed Multifunctional Immunoaffinity Columns for the Recovery of DON, ZON, T-2 and HT-2 (n=3)

Mycotoxin DON

Recovery % ± RSD % in Wheat Beer at Low Spike Levelfor Four Mycotoxins VICAM R-Biopharm 101.8±2.8 95.5 ±2.3

ZON

115.8 ±1.3

113 ± 1.0

T-2

110 ±4.5

96 ± 1.4

HT-2

107.6 ± 1.39

101.9 ± 1.4



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Figure 3. LC-MS/MS chromatograms of T-2 and HT-2 toxins extracted from wheat beer using VICAM and R-Biopharm's cartridges at 35 ng/g.

method can be successfully applied without any matrix interferences especially at low mycotoxin concentrations. In such cases, immunoaffinity columns containing immobilized antibodies that selectively retain mycotoxins, that produce cleaner extracts with a minimum level of interfering matrix components, and that have excellent signal to noise ratios compared to polymeric SPE sorbent materials would be an ideal choice.

Conclusion Recoveries of 12 mycotoxins from the the prototype mixed bed multifunctional immunoaffinity columns are all in acceptable ranges. Mixed bed immunoaffinity purification of mycotoxins coupled with LC-MS/MS analysis is an excellent solution to the simultaneous detection of several mycotoxins present in complex matrices.


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6. 7. 8. 9.

10. 11. 12. 13.

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Detection of Relevant Mycotoxins in Wheat Beer ... - ACS Publications

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