ANALYSIS of MYCOTOXINS Monitoring and control. Wim Traag, Theo de Rijk and Paul Zomer
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1 ANALYSIS of MYCOTOXINS Monitoring and control Wim Traag, Theo de Rijk and Paul Zomer
2 CONTENT Applicable methods Which method should I use?? Methods used at RIKILT Monitoring program Method development
3 ANALYTICAL METHODS Screening methods: TLC ELISA In between methods: HPLC (with IAC) GC-ECD (after deriv.) Confirmatory methods: LC-MS (MS or TOF) GC-MS (after deriv.)
4 Screening methods Elisa kits
5 In between methods
6 Pro s and con s of single compound analyis Pro Low detection limits Simple methods Con Labour intensive Only one mycotoxin detected
7 IN BETWEEN METHODS PRINCIPLE of IAC SAMPLE WASHING ELUTION HPLC-UV Detection
8 IAC, HOW IS IT PERFORMED
9 Single compound, HPLC equipment Switching valve Injection loop Pump HPLC-column UV or Fluorescence detector
10 DEOXYNIVALENOL Reference solution 0.4 µg/ml Negative sample animal feed H 3 C O OH O O OH OH Positive sample animal feed DON (Type B Trichothecene)
11 Single compound, HPLC equipment Switching valve Injection loop Pump HPLC-column Kobra-cel Fluorescence detector
12 Aflatoxin analysis: the Kobra-cell Without Kobra-cell With Kobra-cell
13 G2 G1 B2 B1 AFLATOXINS
14 Chromatogram of spiked maize after cleanup with IAC. Vicam versus R-Biopharm Area 5,00 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 3-Acetyl DON DON 15-Acetyl DON 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 Rt (min) Vicam Chromatogram of spiked maize after cleanup with IAC. R-Biopharm
15 Single compound methods: limits of detection HPLC - IAC analyse Aflatoxin B1: 0.10 µg/kg Aflatoxin M1: µg/kg Ochratoxin: 0.10 µg/kg Zearalenone: 5.0 µg/kg Deoxynivalenol: 0.10 µg/kg
16 Confirmatory methods
17 Mycotoxins, the LC-MS (MS) approach Pro High selectivity General applicable Confirmatory analysis No need for base-line separation Con High training level required Expensive system needed
18 LC-MS versus LC-MS/MS LC-MS techniques generally yield only one molecular ion, little or no fragmentation so structural information from fragmentation is lacking Obtaining structural information requires inducing of fragmentation
19 Fragmentation Why? Fragment ions are characteristic for the molecular structure of the ion and therefore yield information suitable for confirmatory analysis : In order to induce fragmentation LC-MS-MS is required
20 Mass analyzer: triple quadrupole
21 Fragmentation Prod. scan: 236; 10 µg/ml NPAOZ, 5 µl/min, coll. 17 ev Daughters of 236ES+ 3.41e % m/z
22 LC-MSMS, Limits of Detection Aflatoxin B1, B2, G1, G2: 5 µg/kg Fumonisin B1, B2, B3: 0.05 mg/kg T2: 0.10 mg/kg HT2: 0.10 mg/kg Ochratoxin: 0.01 mg/kg Zearalenon: mg/kg Deoxynivalenol: 0.10 mg/kg
23 Example: Mycotoxins in animal feed Reference solution mycotoxins Standaard myc_u1_030131_35 DON FB1 FB2 AB2 AG2 AG1 DAS AB1 HT2 bzl azl T2 OTA ZON STER AcDON % Time
24 WHICH METHOD SHOULD I USE????????
25 Possible approach (1) EIISA kits if interested in one mycotoxin and required answer is negative or suspected If suspected confirmation is needed for legal purposes Result can be quantified (semi) HPLC UV/FLU if interested in one (group) mycotoxin(s) and quantification is required. If above a certain level, result has to be confirmed
26 Possible approach (2) LC MS/MS if interested in limited number of target compounds ( n= < 25) and a quantitative result is required LC TOF- MS if interested in unknown/metabolites, target compounds (unlimited number) can be included
27 Mycotoxins at RIKILT National Plan Animal Feed: 300 samples National Plan Residues Animal Products: 50 samples Private companies :? samples Special projects :? samples NRL mycotoxins in feed Method development
28 Routine Mycotoxin Analysis in Feed IAC HPLC KOBRA-cell Fluorescence (Aflatoxins, validated, accredited) LC-MS-MS (for other mycotoxins) Validated and accredited for FB1/2/3, T2, HT2, OTA, ZON, DON and AFLA B1 (for Afla only screening)
29 IAC-method Extraction with chloroform Aliquot redissolved in MeOH/water IAC cleanup HPLC Kobra-cell + Fluorescence detection Detection Limit: 0.10 μg/kg Expanded Measurement Uncertainty U: 10% at 5 μg/kg, 25% at 1 μg/kg
30 LC-MSMS method Double extraction: - Acidified water + Acetonitril/water (84/16) No cleanup! HPLC MSMS detection: ESI+ (ZON: ESI-) Standard addition quantification - Addition at reporting level - Quantification max x2, otherwise re-analysis
31 Number of samples Total? Other Feed Afla IAC Total? Other Feed Multi
32 Percentage mycotoxins reporting limit Fumonisin DON ZON CORN 48% 48% 49% SOY 21% SILAGE 18% COMPOUND FEED FOR PIGS 34% 13% 15% WHEAT 20% SORGHUM 63%
33 AFLA B1 monitoring result Total number of samples = 1858 Number of samples < 1 ug/kg = 1750 Number of samples > 1 < 5 ug/kg = 85 Number of samples > 5 < 10 ug/kg = 15 Number of samples >10 < 20 ug/kg = 5 Remaining samples have levels of 33; 40 and 140 ug/kg Resultaten Aflatoxine B tm 2007.xls
34 Method development UPLC-MSMS Sharp peaks better s/n ratio = lower LOD s Shorter Rt s, at least factor 2 Stable isotope dillution Multi IAC cleanup Two manufacturers Lowering ionization suppression
35 Wageningen UR Thank you for your attention!
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