Mycotoxins in Food and Feed

Transcription

1 Mycotoxins in Food and Feed a sum up of 6 years LC-MS/MS in routine analysis, pros and cons Scarlett Biselli, Simone Staiger 27/04/2010

2 Challenges for a Laboratory today Monitoring mycotoxins and quality control of food and feed is still of high relevance Processing chains and international trade requires overall fast responses at low price level Goods are contaminated with several toxins in parallel, a lot of toxins are officially regulated Altogether, we still not know which group or kind of toxin might be harmful for human consumption, therefore research and identification of relevant toxins is still under investigation A lot of different commodities including processed foods have to be considered 27/04/2010 Dr.Scarlett Biselli 2

3 LC-MS/MS versus Fluorescence The use of mass pectrometric detection is recently more and more the first choice in routine analysis However, the questions arises: Is LC-MS/MS the better technique compared to existing conventional methods?! What are main benefits and disadvantages? Newest Developments/Applications/Trends 27/04/2010 Dr.Scarlett Biselli 3

4 Pros One instrument could be used for the detection of several toxins and in addition for other non mycotoxin- applications Investment for only one instrument, instead of 2 or 3 FD or UV There are a lot of possibilities for fast chromatography to reduce the time of analysis High selectivity and more or less sensitivity Due to high selectivity, often time consuming cleanup procedures could be neglected Non-target screening and structure identification is an option in combination with other spectrometry techniques Very often toxins-matrix combinations were identified, which are previously unknown 27/04/2010 Dr.Scarlett Biselli 4

5 Cons I with regard to quantitative analysis High investigation costs Mostly operators needs to be specifically trained and educated to serve the instruments and develop methods Chromatographic methods are mostly time consuming, because gradient programs are needed to minimize by separation matrix effects Its recommended for more or less all matrices to perform standard additions or to use of isotope labelled internal standards to cover the ionisation effects. Often the working range of the detector is quite narrow, this requires a lot of sample dilutions and replicate analysis 27/04/2010 Dr.Scarlett Biselli 5

6 Cons II with regard to quantitative analysis Discrimination of single analytes by using one extraction solvent only Method validation is on a large-scale, validation results in terms of precision still not reach the level of fluorescence or UV applications Collaborative studies on LC-MS/MS still in the starting phase, no reference method up to now At the ppt level the technique is still not sensitive and selective enough to omit enrichment and sample preparation, this results in cost efficient analysis 27/04/2010 Dr.Scarlett Biselli 6

7 3 Issues Accuracy and Trueness (Results from PT-Studies) Precision of the instruments Signal to noise ratios and level of sensitivity 27/04/2010 Dr.Scarlett Biselli 7

8 Portions of LC-MS/MS technique within PT-Studies 2% Animal feed 3% Peanuts 5% Almonds 20% 15% 10% 5% 0% Hazelnut 3% Babyfood 8% Ochratoxin A satisfactory z-scores MS/MS Satisfactory negative Z-score (MS/MS) average 77% 25% 33% sd 29% 39% 31% 6% Maize satisfactory z-scores Pistachios 8% Aflatoxin B1 MS/MS Satisfactory Curry Powder 5% negative Z-score (MS/MS) average 86% 69% 65% sd 4% 30% 34% 3% 5% 0% 6% Barley Dried wine fruits Green Coffee Babyfood animal feed 10% 8% 6% 4% 2% 0% Paprika 9% 9% roasted coffee oats wine 5% 8% 4% Instant Coffee 27/04/2010 Dr.Scarlett Biselli 8

9 Fusarientoxins 17% Maize 2 21% 11% Maize 1 20% 15% 10% 5% satisfactory of all Breaksfast Cereals 19% ZON negative Z- score (MS/MS) Babyfood 0% 89% 100% 50% Babyfood 16% animal feed 85% 83% negative 57% Z- 13% 20% Breaksfast Cereals satisfactory 88% MS/MS 79% score 36% animal feed Babyfood wheat 89% 15% of all Satisfactory 100% (MS/MS) 83% animal feed DON 92% 100% 10% 27% Maize 1 82% animal feed 63% 50% 16% MS/MS Satisfactory 14% Babyfood 5% Breaksfast Cereals 77% 60% 70% 0% animal feed 69% 86% 71% wheat 84% 79% 32% Maize 2 78% 64% 55% 11% animal feed Breaksfast Cereals 14% 27/04/2010 Dr.Scarlett Biselli 9

10 Results DON in wheat Results DON FAPAS wheat june 2008 Robust AV: 817 ppb statistics: AV: SD: 244 Other ppb 836 ppb detection mode (95 SD: % UV) RSD: 30 % 188 ppb RSD: 22 % 1 AV: 880 ppb MS-detection SD: 339 ppb RSD: 38 % 27/04/2010 Dr.Scarlett Biselli 10

11 Patulin and Fumonisins 20% Infant drink apple juice 115% 20% 15% 10% 5% 0% 10% apple puree 1 satisfactory of all MS/MS Satisfactory negative Z- score (MS/MS) Maize 87% 90,91% 55% Maize 89% 75% 38% 10% apple juice 3 Patulin 9% apple juice 2 satisfactory MS/MS negative Z- score of all Satisfactory (MS/MS) average 89 % 94% 47% sd 6 % 8% 13% 30% 20% 10% 0% Fumonisins 31% 23% Maize Maize Mai 08 Jun 07 27/04/2010 Dr.Scarlett Biselli 11

12 Are MS-MS methods the better ones? Interesting Aspects: MS-Application Overall matrices and toxins at least 10 % up to 30 % of the applied methods using mass spectrometric detection Highest application level was found for Fusarium toxins, Patulin and Fumonisins Matrix effects With regard to matrix effects, results of the PT studies could be interpreted that DON in wheat shows more often results above the average level (positive z-scores) when MS detection is used While, on the other hand more often negative z-score levels could be observed for ZON in Breakfast Cereals and in average for OTA over all commodities 27/04/2010 Dr.Scarlett Biselli 12

13 What do we know about precision? Comparison of OTA and Aflatoxin spiked samples, analysed by FD and MS/MS detection Aflatoxin B1 at a Level of 1 ppb Ochratoxin A at a Level of 5,4 ppb LC-MS/MS Matrix FD CV % CV % Ratio FD CV % LC-MS CV % Ratio Rice 2,78 6,06 2,18 1,31 3,41 2,60 Figs 3,55 3,83 1,08 1,4 1,79 1,28 Almonds 1,22 11,02 9,03 0,93 4,31 4,63 Peanuts 1,83 1,79 0,98 1,51 1,79 1,19 green coffee 0,21 1,41 6,71 27/04/2010 Dr.Scarlett Biselli 13

14 Signal to Noise ratios Intensity, cps XIC of +MRM (22 pairs): Period 2, 313.0/241.0 amu XIC of +MRM (22 pairs): Period 2, 313.0/241.0 amu Intensity, cps 700 Signal 650 to Noise to ,023ng/ml Signal to Noise 1 to Time, min Time, min Aflatoxin B1 at a Level of 0,02 ppb 1.MRM for Quantification 1/42 Aflatoxin B1 at a Level of 0,02 ppb 2.MRM for Verification 1/3 27/04/2010 Dr.Scarlett Biselli 14

15 HPLC-FD 20 times lower concentrated Aflatoxins at a Level of 0,001 ng/ml (1 ppt) 27/04/2010 Dr.Scarlett Biselli 15

16 Ochratoxin A Intensity, cps XIC of +MRM (22 pairs): Period 2, 404.0/239.0 amu Max cps. XIC of +MRM (22 pairs): Period 2, 404.0/239.0 amu OTA 0,02ng/ml 280 Intensity, cps Ochratoxin A Ochratoxin A (Qualifier) at 400 a level of 0.02 ng/ml (20 ppt) At a level of 0,02 ng/ml (20 ppt) Signal to Noise 1: 4,8 OTA 0,02ng/ml Max cps Signal to Noise Time, min Time, min 1:4 OTA 0,02 µg/l, 1.MRM Ratio 1:5 OTA 0,02 µg/l, 2.MRM Ratio 1:4 27/04/2010 Dr.Scarlett Biselli 16

17 HPLC-FD Ochratoxin A at a level of 0,02 µg/l Signal to Noise: 1:2,5 Ochratoxin A 27/04/2010 Dr.Scarlett Biselli 17

18 Third Issue How about co elution of Signals in fluorescence detection even after Immunoaffinity clean-up?! 27/04/2010 Dr.Scarlett Biselli 18

19 Verification of false positives Sample extract from herbs 27/04/2010 Dr.Scarlett Biselli 19

20 LC-MS/MS Chromatogramm RT 7:20 Aflatoxin B1 313/241 amu No Aflatoxin B1 detectable The FD fakes the high contamination level Verification by LC-MS/MS shows false positive, although a selective cleanup was carried out 27/04/2010 Dr.Scarlett Biselli 20

21 Positive fluorescence detection of Aflatoxins µv Standard ,17 Aflatoxin G2 Aflatoxin G1 Aflatoxin B1 Aflatoxin B2 9, Intensity, cps , , RT [min] 8,15 8, /04/2010 Dr.Scarlett Biselli Cocoa beans Cocoa beans 7,64 10,63 11,42 8,77 9,03 10,00 Aflatoxin G ,33 10,87 12, ,16 10,49 13,51 11,15 11,6711,80 13,06 Aflatoxin B2 9 Aflatoxin B RT [min] 13

22 Examples for a positive Verification µv Fluorescence Chromatogram incl. post column derivatisation: OTA in tobacco ~ 20 ppb 11,5 OTA _Ochra1_O_ O4_22.DATA RT [min] 27/04/2010 Dr.Scarlett Biselli 22

23 MS Verification 1,4e4 6,48 Max. 1,4e4 cps. Intensity, cps 1,3e4 1,2e4 1,1e4 1,0e4 9000,0 8000,0 7000,0 6000,0 5000,0 4000,0 3000,0 2000,0 1000,0 0,0 XIC of +MRM (24 pairs): Period 2, 404,0/239,0 Da from Sample 1 ( O5) of O5.wiff (Turbo Spray) Positive Verification of OTA Content ~ 20 ppb 6,3 6,5 6,7 6,9 7,1 7,3 7,5 Time, min 27/04/2010 Dr.Scarlett Biselli 23

24 Trends Recent developements and Trends in terms of quantitative mycotoxin analysis? How to reach more sensitivity and minor matrix effects? 27/04/2010 Dr.Scarlett Biselli 24

25 Recent developements / Trends There is still a need for clean-up of samples in terms of quantitative analysis Immunoaffinity Chromatography is the by far most selective technique, but as well time consuming and expensive Alternative solutions could be specific SPE Clean-ups to decrease matrix effects for MS detection Online-SPE might be a good solution to get better Signal to noise ratios and to decrease Limit of Quantification of the method 27/04/2010 Dr.Scarlett Biselli 25

26 Offline and online clean-up Status quo: Status Online SPE-MS or FD Detection: Extraction 45 min Extraction 45 min Offline Clean-up with liquid handler 30 min Online Clean-up + HPLC 15 min HPLC-run 20 samples: 30 h 15 min 90 min 20 samples: 20 h Further advantages: 60 min -New materials are under development which connects antibodies covalently on a solid phase - => clean-up using high flow rates possible -Clean-up could be carried out automatically over night 27/04/2010 Dr.Scarlett Biselli 26

27 ONLINE-SPE-LC-MS/MS Feed Sample NIV Intensity, cps Standard Chromatogramm of 13 Toxins (0.1 to 50 ppb) ,5 5,0 5, DON 250 ppb DON ESI negative 6,69 Aflatoxins + OTA I-STD 50 ppb Max. 7,6e4 cps. 6,3 6,6 7,0 7,3 7,5 7,7 7,9 8,1 8,3 8,5 Time, min 6,1 6,2 6,3 6,4 6,5 6,6 6,7 6,8 6,9 7,0 7,1 7,2 7,3 7,4 7,5 7,6 Time, min T2-Toxin HT2 + T2 5 ppb HT2 8,48 ESI positive 9,01 ZAN 8,99 ZON I-STD + ZON 30 ppb 9,0 9,5 10,0 8,5 9,0 9,5 10,0 10,5 Time, min 27/04/2010 Dr.Scarlett Biselli 27

28 To summarize.. PT Study reports demonstrate that LC-MS/MS methods are widespread present overall commodities to detect every regulated toxin Level of successful participation mostly lower compared to the total value With regard to mass fractions, precision of the instrument full fills criteria approaches, but variance is higher compared to fluorescence Mass Spectrometry is a fast and easy way to analyse various toxins in parallel, in addition its powerful tool to verify critical fluorescence results Comparison of solvent standards demonstrates at least comparable sensitivity and signal to noise ratios For sample extracts it has to be considered that matrix influence could decrease signals by 80 to 90% Mass Spectroscopy combined with online cleanup facilities might be a good solution to improve quantification methods in terms of precision, selectivity and sensitivity 27/04/2010 Dr.Scarlett Biselli 28

29 The better methods. Still, Yes and No! With regard to science and developments, this is the technique to move forward Quantitative analysis is less expensive and precise by performing conventional methods 27/04/2010 Dr.Scarlett Biselli 29

30 Thank you for your attention! Competencecenter for Contaminant and Vet Drug Residue Analysis 27/04/2010 Dr.Scarlett Biselli 30