UHPLC Analysis of Multiple Mycotoxins Using Sub-2 µm Porous Particles

Transcription

1 UHPLC Analysis of Multiple Mycotoxins Using Sub-2 µm Porous Particles Gaurang Parmar, Dave Bell, Craig Aurand, and Emily Barrey Supelco, Div. of Sigma-Aldrich Bellefonte, PA USA T sigma-aldrich.com/analytical 2014 Sigma-Aldrich Co. All rights reserved.

2 Introduction Mycotoxins are toxic secondary metabolites produced by fungi, which can exist in food as a result of fungal infection of crops. Their strong resistance to decomposition and digestion cause mycotoxins to remain in the food chain. The analysis of mycotoxins in food and animal feed has been a challenge mainly due to the complexity of food matrices and desired low detection limits. In recent years, significant advances in the analytical techniques have been applied to detection of mycotoxins. There has been an increasing need for a method to detect multiple mycotoxins with a single sample preparation and analysis method. 2

3 Traditional Mycotoxin Sample Preparation Involves immunoaffinity SPE cleanup Multi-step extraction LC-UV/fluorescence detection Analyte specific Numerous assays for multiple mycotoxins 3

4 Experimental Aflatoxin Analysis HPLC Conditions column: Ascentis Express C18, 10 cm x 2.1 mm, 2.7 µm mobile phase: (A) water; (B) acetonitrile; (C) methanol; (74:13:13, A:B:C) with g potassium bromide and 230 µl nitric acid flow rate: 0.40 ml/min column temp.: 35 ºC det.: FLD, ex 360, em 440 nm FL, KOBRA cell injection: 40 µl 4

5 Spiked Wheat Matrix B1 & B2 (4 ppb) G1 & G2 (16 ppb) LU Aflatoxin G2 2. Aflatoxin G1 3. Aflatoxin B2 4. Aflatoxin B1 Spiked Peanut Paste B1 & B2 (4 ppb) G1 & G2 (16 ppb) 4 LU

6 B-Trichothecenes Analysis column: Ascentis Express C18, 10 cm x 3.0 mm, 2.7 µm mobile phase: (A) 92:4:4, water:acetonitrile:methanol; (B) acetonitrile flow rate: 0.8 ml/min column temp.: 35 ºC det.: UV 220 nm, 16; ref 360 nm, 100 injection: 40 µl gradient: Min %A %B

7 DON and B-Trichothecenes in Corn Matrix, 2 ppm Spike 1. Nivalenol 2. DON AcetylDON 4. 3-AcetylDON Time (min) 7

8 Recent Mycotoxin Sample Preparation Involves QuEChERS cleanup Simplified extraction for various matrices Multicomponent assays LC-MS and LC-MS/MS detection Single assays for multiple mycotoxins 8

9 UHPLC Mycotoxin Analysis Titan UHPLC columns are the outcome of the Ecoporous process, a patent pending, silica manufacturing process. Titan UHPLC columns provide all of the performance of leading UHPLC columns at about half the cost. Narrow particle size distribution may create more stable, uniform column beds than particles having broader size distribution. 9

10 Ecoporous Technology Particle Size Distribution Comparison for Different Silica 10

11 UHPLC Mycotoxin Analysis HPLC Conditions column: Titan C18, 5 cm x 3.0 mm, 1.9 µm mobile phase: (A) 5mm ammonium formate with 0.1% formic acid in water; (B) 5mm ammonium formate with 0.1% formic acid in methanol flow rate: 0.40 ml/min column temp.: 35 ºC det.: LC-MS/MS Shimadzu 8030 with UHPLC injection: 2 µl gradient: Min %A %B

12 Mycotoxin Mix Standard in Solvent DON 2. 3-AcetylDON & 15-AcetylDON 3. Aflatoxin G2 4. Aflatoxin M1 5. Aflatoxin G1 6. Aflatoxin B2 7. Aflatoxin B1 8. Fumonisin B3 9. Ochratoxin A 10. Nivalenol

13 Conclusion Using Titan UHPLC columns, efficient separation was achieved in 6 minutes for 10 mycotoxins from 4 different classes. Utilizing the benefits of smaller particle size, HPLC run to separate 10 mycotoxins took one-half of the time in comparison to the traditional LC-UV and fluorescene methods that only separated 4 compounds. Fast and efficient analytical methods may be developed by combining the QuEChERS sample preparation technique with the benefits of the UHPLC Titan column. Ascentis is a registered trademark of Sigma-Aldrich Co. LLC Ecoporous and Titan are trademarks of Sigma-Aldrich Co. LLC.. Fused-Core is a registered trademark of Advanced Materials Technology, Inc. 13