Improved Resolution of Mycotoxins from Cereal

Food & BeverageMycotoxins are a diverse group comprised of hundreds of secondary metabolic products of various fungal species. Several show marked toxicity in humans. Contamination of the food supply with mycotoxins is increasingly prevalent, and can occur during growth, harvest, transportation, processing or storage. Techniques to reduce mycotoxin concentration after contamination are expensive, unreliable and sometimes reversible. Therefore, the removal of contaminated products from the food chain is a primary means of eliminating human exposure. The sensitive and accurate detection of very low levels of these compounds is critical to efforts to identify contaminated products. LC-MS/MS is a popular analytical technique for this purpose.ExperimentalA 5 gram cereal sample was spiked with 15 mycotoxins as follows (aflatoxins at 0.25 to 5 ng/mL; ochratoxins at 0.125 to 2.5 ng/mL; all others at 10 to 200 ng/mL). The spiked sample was extracted with 20 mL of acetonitrile:1% formic acid water solution (75:25), shaken for one minute, centrifuged and then filtered through a 0.45 µm syringe filter. LC-MS/MS analysis was performed on the extract with no further processing. Ascentis Express RP-Amide and Ascentis Express F5 HPLC columns were used. Because these columns exhibit alternate selectivity to one another, they produce different elution patterns.Results and DiscussionTable 1 shows the retention times of all 15 mycotoxins on each column. As expected, elution pattern differences were observed. Run time with either column was less than 10 minutes. Ascentis Express columns provide a breakthrough in HPLC column performance. Based on Fused-Core® particle technology, these columns provide the high speed and high efficiency of sub-2 µm particles but with the low backpressure associated with 3 µm particles, allowing faster analysis on any HPLC system.The chromatogram obtained with the Ascentis Express RP-Amide column is shown in Figure 1. This column contains a stationary phase with an embedded polar group (EPG) that is 100% aqueous compatible. Compared to a C18 column, it provides increased selectivity for polar compounds, especially those that can act as a Using Ascentis® Express RP-Amide and Ascentis Express F5 HPLC ColumnsImproved Resolution of Mycotoxins from CerealTable 1. Retention Time Table (min)Peak ID AnalyteAscentis Express RP-AmideAscentis Express F51 Nivalenol (NIV) 1.5 2.02 Deoxynivalenol (DON) 2.3 2.93 15-Acetyldeoxynivalenol 4.0 4.854 3-Acetyldeoxynivalenol 4.0 4.975 Aflatoxin G2 4.8 6.26 Aflatoxin G1 5.1 6.47 Aflatoxin B2 5.2 6.68 Aflatoxin B1 5.5 6.89 Atrazine-d5 6.1 6.510 Fumonisin B1 6.3 8.511 T-2 Toxin 6.5 7.212 Fumonisin B2 7.0 9.013 Ocratoxin A 7.3 7.714 Sterigmatocistin 7.4 8.015 TFF 7.7 7.8hydrogen-bond donor. With the exception of 15-acetyldeoxynivalenol and 3-acetyldeoxynivalenol, it is able to provide great resolution of all analytes tested.Figure 2 shows the chromatogram obtained with the Ascentis Express F5 column. This column employs a pentafluorophenylpropyl stationary phase, able to undergo different analyte-phase interactions than those possible with a traditional C18 phase, or an EPG phase. These differences create an alternate selectivity and result in changes in the elution pattern. The F5 phase also exhibits enhanced shape selectivity, and can provide the separation of closely related compounds. Of particular note is the ability of this column to resolve 15-acetyldeoxynivalenol and 3-acetyldeoxynivalenol, a separation not achievable with traditional reverse phase columns.The combination of modern stationary phase technology with powerful LC-MS/MS detection results in a rapid analysis, without the need for lengthy sample preparation. The utilization of columns with alternate selectivity allowed elution order changes and improved resolution.Food & BeverageOrder/Customer Service (800) 325-3010 • Fax (800) 325-5052 Technical Service (800) 325-5832 • sigma-aldrich.com/techservice Development/Custom Manufacturing Inquiries (800) 244-1173 Safety-related Information sigma-aldrich.com/safetycenterWorld Headquarters 3050 Spruce St. St. Louis, MO 63103 (314) 771-5765 sigma-aldrich.comEnabling Science to Improve the Quality of LifeFeatured and Related ProductsDescription Qty. Cat. No.Ascentis Express HPLC Columns (2.7 µm particles)C18, 10 x 2.1 mm I.D. 1 53823-UC18, 15 x 2.1 mm I.D. 1 53825-URP-Amide, 10 cm x 2.1 mm I.D. 1 53913-URP-Amide, 15 cm x 2.1 mm I.D. 1 53914-UF5, 10 cm x 2.1 mm I.D. 1 53569-UF5, 15 cm x 2.1 mm I.D. 1 53571-UAscentis Express Guard Columns (2.7 µm particles)Holder 1 53500-UC18 Cartridge, 5 mm x 2.1 mm I.D. 3 53501-URP-Amide Cartridge, 5 mm x 2.1 mm I.D. 3 53514-UF5 Cartridge, 5 mm x 2.1 mm I.D. 3 53594-UAnalytical StandardsAflatoxin Mix, 4 analytes at various concentrations in methanol5 mL 46303Aflatoxin B1, 1 µg/mL Aflatoxin G1, 1 µg/mLAflatoxin B2, 0.3 µg/mL Aflatoxin G2, 0.3 µg/mLAflatoxin B2-13C17, at 0.5 µg/mL in acetonitrile 1 mL 32771-1ML3-Acetyldeoxynivalenol, 100 µg/mL in acetonitrile 2 mL 34132-2ML15-Acetyldeoxynivalenol, 100 µg/mL in acetonitrile 2 mL 34133-2MLDeoxynivalenol, 100 µg/mL in acetonitrile 2 mL 34124-2MLDeoxynivalenol-13C15, 25 µg/mL in acetonitrile 1 mL 34128-1MLFumonisin B1, 50 µg/mL in acetonitrile:water 2 mL 34139-2MLFumonisin B2, 50 µg/mL in acetonitrile 2 mL 34142-2MLOchratoxin A, 50 µg/mL in benzene:acetic acid (99:1) 1 mL 46912Patulin, 100 µg/mL in chloroform 1 mL 46914-UAnalytical Reagents and SolventsAcetic acid solution, for HPLC, 50% in water 45754Ammonium acetate, for HPLC, >99.0% 17836Formic acid, for HPLC, 50% in water 09676Methanol, LC-MS CHROMASOLV®, >99.9% 34966Visit our Food and Beverage/Toxins resources sigma-aldrich.com/food-toxinsOXO11546 / T4121331102©2012 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and SUPELCO are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. FLUKA is a registered trademark of Sigma-Aldrich GmbH. Ascentis is a registered trademark of Sigma-Aldrich Co. LLC. CHROMASOLV is a registered trademark of Sigma-Aldrich Laborchemikalien GmbH. Fused-Core is a registered trademark of Advanced Materials Technology, Inc. Solutions within is a trademark of Sigma-Aldrich Co. LLC. Supelco and Fluka brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.Figure 1. Ascentis® Express RP-Amide sample/matrix: 5 grams of cereal spiked with 15 mycotoxins; extract with 20 mL acetonitrile:1% formic acid in water (75:25); shake for 1 min; centrifuge; filter through a 0.45 µm syringe filter column: Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 2.7 µm particles (53913-U) mobile phase: (A) 1 mM ammonium acetate, 0.5% acetic acid in water; (B) 1 mM ammonium acetate, 0.5% acetic acid in methanol gradient: 0 min: 5% B; 0.5 min: 10% B; 12 min: 95% B; 15 min: 95% B flow rate: 400 µL/min column temp.: 40 °C detector: MS/MS, ESI(+) and ESI (-) injection: 2 µL4 5 6 7 8Min3,4567891011121314 153 4 5 6 7 8 9Min2345697811 15 141012131. Nivalenol (NIV)2. Deoxynivalenol (DON)3. 15-Acetyldeoxynivalenol4. 3-Acetyldeoxynivalenol5. Aflatoxin G26. Aflatoxin G17. Aflatoxin B28. Aflatoxin B19. Atrazine-d510. Fumonisin B111. T-2 Toxin12. Fumonisin B213. Ochratoxin A14. Sterigmatocistin15. TFFChromatogram courtesy of Enio Belotti (Water & Life Lab s.r.l., Entratico (BG), Italy)Figure 2. Ascentis Express F5 column: Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 µm particles (53569-U) Peak IDs and all other conditions the same as Figure 1.4 5 6 7 8Min3,4567891011121314 153 4 5 6 7 8 9Min2345697811 15 14101213Chromatogram courtesy of Enio Belotti (Water & Life Lab s.r.l., Entratico (BG), Italy)Including products from