rapid methods for mycotoxins screening: the food...

“Rapid Methods for Mycotoxins Screening: The Food Industry Perspective”

Advances in Food Safety Analysis – Separation Science eSeminar – September2013 Michele Suman

Michele Suman, PhD

Barilla SpA – Food Research Labs, Parma, Italy

e-mail: [email protected]

Rapid Methods for Mycotoxins Screening: The Food Industry Perspective



1. Mycotoxins scenario: implications for the food industry

2. Confirmatory/Official Analytical Methods: Pros and Cons

3. Tools for rapid screening: consolidated and emerging strategies

4. Future perspectives and margins for improvement

Talk Overview



MYCOTOXINS

Main contaminants of cereals; acute and chronic toxicity,

thermally stable, ubiquity\ heterogeneous contamination,

very different chemical structures…



Mycotoxins growth:

a challenge worldwide

problem

From field… to table

(more than 20% of the crops affected)

Growth

Harvest Storage

Food

Economic losses estimated at

billions of dollars

Generally produced by species within

Fusarium, Aspergillus & Penicillium

Toxicological risk for

humans and animals

Regulatory levels

to be respected



• Contamination with mycotoxins cannot be completely avoided • Their growth could happen since the pre-harvest and also during

transport\storage of raw-materials • Heterogeneity of contamination and possible elevated levels in

regional hot spots mean that consistent sampling is difficult

• Technological processes could also play an important role: mechanical or thermal energy may cause modification, inducing reactions with macromolecular components such as sugars, proteins or lipids as well as release\degradation of the native forms

• Industrial self-control is therefore unavoidable with particular

emphasis at the raw materials reception stage or at the loading point

• Looking for the best compromise in rapid mycotoxins tests: speed, sensitivity, precision, accuracy, low costs, easy procedures

THE INDUSTRIAL CONTEXT

Deoxynivalenol

http://www.google.it/url?sa=i&source=images&cd=&cad=rja&docid=nJM27rOVyYnRYM&tbnid=rBhWKeiWyoHZJM:&ved=0CAgQjRwwAA&url=http%3A%2F%2Fwww.veryicon.com%2Ficons%2Fbusiness%2Faccounting%2Findustry.html&ei=B_L3UdHDM4qL4ATM8IGQAg&psig=AFQjCNFMP43Q3LdUDmsN2OvyZkentBr38Q&ust=1375290247890223



Source: V. Lattanzio – World Mycotoxin Forum 2012 Rotterdam



A NEW SCENARIO: “MASKED” MYCOTOXINS

It is known that DON can be degraded/detoxified into derivatives by acetylation, oxidation, de-epoxidation or glycosylation

In recent years it emerged that, many structurally related compounds, generated by plant metabolism, can co-exist together with the native toxins: for example, plants are able to convert the relatively apolar trichothecenes in more polar derivatives via conjugation with sugars, amino acids or sulphate groups, in order to compartmentalize them in vacuoles

LC-MS analysis of naturally contaminated wheat and maize samples showed DON-3-glucopyranoside (DON-3G) to be the major form of masked DON. It was also recently found in barley, malt, beer and other cereal-based finished products

Food processing, especially heating or fermentation steps, can potentially alter mycotoxins

Masked mycotoxins are not easy to be extracted\cleaned up. They can escape routine analytical methods and there is also a lack of adequate analytical standard up to now.

Masked mycotoxins can potentially be released after ingestion by hydrolysis in the gastrointestinal tract: preliminary results indicate that DON-3G is resistant to the acid conditions in the stomach of mammals and to most enzymes, while several bacteria in the intestinal tract of humans are able to cleave it into DON.



CONFIRMATORY \ OFFICIAL METHODS: - Sample preparation issues: • Grinding • Extraction • Clean up…

- Time consuming separation phases and detection techniques: • Chromatography (GC, LC,…) • DAD, FD, MS,…detectors

- Skilled operators

- High solvents consumption - Expensive instruments - High sensitivity & selectivity - Good repeatability & reproducibility - Automation - Multitoxins analysis (LC-MS/MS)

Analytical Strategies – Directions…



Analytical Strategies – Directions…



RAPID\SCREENING METHODS (ideas among the reality and the desires): - High productivity (number of samples per die) - Different matrixes potentially analyzable - Future potentialities towards the analysis of more toxins in a «single shoot» - Rapid response - Acceptable sensitivity - Reduced cross-reactivity - Relatively low costs - Application on the field - No skilled technicians necessary - Simple sample preparation - Not expensive instrumentation - False positives\negatives issues - Critical repeatability & reproducibility

Qualitative Test Yes/No answer

Quantitative Test Optical Reader /

Scanner: concentration numerically expressed



Enzyme-Linked Immunosorbent Assay - ELISA The common methods of analysis for mycotoxins are based on the use of ELISA (Enzyme-Linked

Immunosorbent Assay) techniques, mainly adopted for rapid and relatively cheap screening,

exploiting also generally ready to use reagents.

Typically, the specific antibody is linked to a solid phase: sample extract is added, then the enzymatic

conjugated constituted from the mycotoxins tied to a peroxidase is added in excess and goes to occupy

the remained free bounding sites. The detection happens by an indirect way, in fact, the addition of the

substrate and the chromogen determine the appearance of a colorful compound.

The currently tests in commerce generally have an incubation time of 20-60 min and comprise the

simultaneous analysis of tenths of samples; the technique is fast but can be subject to crossed reactivity.



CROSS REACTIVITY?

Cross reactivity of immunoaffinity approaches for mycotoxins detection towards

metabolites and masked mycotoxins: the need to improve the current state of knowledge

Commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites

The DON IACs showed cross-reactivity for nearly all DON derivatives tested The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives



- antibody-colloidal gold particle complex mixed with sample extract

- migration onto a nitrocellulose membrane

- mycotoxin-protein conjugate coated on test zone to capture free antibody-colloidal gold

- photometric reflectance reader for colour reaction

Lateral Flow Immunoassays

They are studied for having a fast answer “on-

site” (attractive also in the places where there is no

laboratory setting available) of the contamination

Possibly portable, cost-effective,…

No expensive equipment\high trained personnel

required

Analytical determination for “semiquantitative”

proposals: yes/no answer at a certain cut off level.

Work is going on in the quantitative screening

direction…looking for adequate levels of accuracy,

repeatability and reproducibility…



Sources: - V. Lippolis et al. The MycoRed Africa 2011 Conference Cape Town, South Africa April,2011 - V. Lattanzio et al. – World Mycotoxin Forum 2012 Rotterdam - Analytica Chimica Acta 2012. Vol.718, Pages 99–108

Multiplex Dipsticks based on LFIA technology

Wheat, oats and maize different cereal matrixes Labelled antibodies freeze-dried within the microwells “Universal” extraction with methanol\water mixture

Screening at levels close to the maximum permitted

established EU legislation Repeatability and false positives still critical factors Surely, additional efforts are necessary to render

them adequately applicable, even if they are promising…



FPIA (Fluorescence Polarisation Immunoassay ) The toxin is marked with a fluorotrope. Marked toxin and free toxin (extracted from the sample)

compete with antibodies for the specific binding sites. If the antibody bind marked toxin, the

increase of the mass of the complex cause change of the polarity of the incident radiation.

The measured change of polarization is therefore inversely proportional to the free toxin

concentration in the sample.

Source: Chris Maragos, Toxins 2009, 1(2), 196-207



+

Automated FPIA System

1 Video FRL



LOD LOQ

0.8 µg/kg 2.0 µg/kg

Accuracy (mean value)

87% (range 3-10 µg/kg)

Precision 6%

Linearity range

2 – 12.5 µg/kg (for conc. >12.5 µg/kg extract dilution

is required)

Time of analysis

20 min

NH

O

O

OH

O OHN

H

O

Cl

O

CH3

H

O

H

O

OH

OTA-FL

Food Analytical Methods 2013 - DOI 10.1007/s12161-013-9627-3



Near-infrared Spectroscopy - (NIR / FT-NIR) Fourier transform near-infrared spectroscopy (FT-NIR) can be used for rapid and non-destructive analysis for

example of deoxynivalenol (DON) in durum and common wheat. The relevance of using ground wheat samples with

a homogeneous particle size distribution to minimize measurement variations and avoid DON segregation.

FT-NIR semi-quantitative model to

discriminate between blank and

naturally contaminated wheat

samples at around 300 µg/kg



SPR (Surface Plasmon Resonance-based immunoassays)

It uses antigen (protein+toxin) in a solid phase that is connected with an optical measurement

device. Specific antibodies are before incubated with the sample to be analyzed, then they are

injected on the solid surface. If the sample is not contaminated, the antibodies incubated,

remained free, are tied to the antigenic substrate causing a change of the optical characteristics of

the matrix analyzed. The optical signal is therefore inversely proportional to the toxin concentration

in the sample. It is a relatively new technique, less sensitive regarding ELISA, but probably more robust.

Source: Matthew A. Cooper Nature Reviews Drug Discovery 1, 515-528 (July 2002)

Rapid Surface Plasmon Resonance Immunoassay for the

Determination of Deoxynivalenol in Wheat, Wheat Products,

and Maize-Based Baby Food - assay has been proven to offer sensitive, accurate and reliable results

- It seems to be potentially applicable also to other cereal matrixes

- no clean-up in the extraction phase

Source: J. Meneely et al. J. Agric. Food Chem 58,

8936-8941 (2010)



- Biosensors technology has grown rapidly in the last decade with emerging applications in a wide range of disciplines

- New generations of biosensors potentially permit to

achieve multiplex testing - Antibodies attached to the surface of the sensor: these

interact with the mycotoxins, producing measurable changes in surface properties by fluorescence, optical fibers, evanescent and planar wave guide (PWG) technologies

BIOSENSORS

Source: Wang et al., Biosensors and Bioelectronics, Volume 34, Issue 1 15 April 2012, Pages 44–50

Source: Bayer BQA Analyzer Brochure 2011



Another approach, with future industrial application perspectives for example for the determination of trichothecenes, is the one based on electrochemical detection, using screen-printed electrodes (ScPEs). - Microelectrode arrays to immobilize antibodies for

different toxins in microchip is nowadays a reality - Electrochemical plate for the fast and sensitive

detection of deoxynivalenol and nivalenol in wheat samples

- Deoxynivalenol and nivalenol are hydrolysed using a

microwave hydrolysis procedure (2 min) which leads to the production of electroactive compounds that can be sensitively detected by the use of cheap screen-printed electrodes.

Source: Ricci et al., World Mycotoxin Journal 2009, 2, 239.

ELECTROCHEMICAL DETECTION



Source: E. Gobbi, M. Falasconi, E. Torelli, G. Sberveglieri; Food Research International 2011, Vol. 44, pp. 992–999

Electronic Noses Electronic nose, based on metal oxide semiconductor chemical sensors arrays, demonstrate ability to diagnose fungal contamination in grains: an example is the detection \prediction of high or low fumonisins content in inoculated maize cultures.

O

OOHO

OH

O

CH3

O

CH3 R2

R1 OH

NH2

OOHO

OH

O

FB1: R1 = OH, R2 = OH

FB2: R1 = OH, R2 = H

FB3: R1 = H, R2 = OH



Source: S. Staiger, M. Reichel, S. Biselli World Mycotoxin Forum 2012 & Rapid Methods Europe 2013 Proceedings

ANALYSIS IN GRAIN DUSTS: PROMISING FOR IMPROVING FUTURE RAPID TEST PROTOCOLS



LC-MS/MS technique is spreading rapidly as successful solution for simultaneous screening, identification and accurate quantitative determination of a large number of mycotoxins: should we have to consider this strategy in some way as “a sort of rapid method”?!??

LC-MS/MS (low and high resolution)



SOME FINAL

CONSIDERATIONS…

Main advantages of rapid methods for mycotoxins screening can be summarized in terms of simplified sampling protocols\procedures, speed, practical aspects, no clean-up/automation in most cases.

The main areas for improvement are in: • increased throughput and sensitivity • reduction of matrix-interferences & antibody cross reactivity, conducing to a limited number of false-positive results • strengthened quantitative performances & robustness • harmonized validation criteria • Batch-to-batch reproducibility • Multitoxins perspectives (from dipsticks up to the «provocative stage» of tandem MS…)

Besides, taking into account of the highly competitive environment, the continuously increasing legal contest & cost-efficient needs, it is clear why rapid methods represent today (and for the next decade…) strategic tools for mycotoxins issues management.

A significant future challenge is represented by derivatives detection (conjugated/masked forms): in this field new analytical approaches are highly demanded for toxicological analysis and wide exposure risk assessment.



Thank you

The Barilla Group

rapid methods for mycotoxins screening: the food...

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