Liquid Chromatography With Mass-spectrometric Detection (LC-MS) and Biosensors

Gregor Kos and Rudolf Krska

Introduction

Liquid Chromatography with Mass Spectrometric Detection (LC-MS) and Biosensors for mycotoxins represent fairly recent developments in mycotoxin determination. LC-MS techniques are already finding widespread use in mycotoxin analysis despite high costs and the need for experienced personnel. Biosensors, on the other hand, are subject to ongoing research in order to provide suitable and easy to use tools for the determination of fungal infection and toxins.

An introduction to fundamental LC-MS principles and applications is part of the EMAN Training Course on Separation and Detection Techniques. For details on the determination of the toxin content using LC-MS techniques, please refer to the specific fact-sheet.

Liquid Chromatography with Mass Spectrometric Detection (LC-MS)

LC-MS is one of the most advanced techniques available for the detection of mycotoxins. However, methods are time-consuming and require expert knowledge. Extraction and clean-up techniques have to be applied prior to separation and detection in order to enable well-separated peaks without interference from matrix components. Details are available in the internet training course Modern Sample Preparation Techniques

In brief, the HPLC effluent enters an ionisation chamber via a nebuliser. Ionisation is provided by several techniques (e.g. electrospray, thermospray, chemical, and fast atom bombardment) Fragmentation takes place in a collision chamber, and fragments then enter the high vacuum region of the MS where detection takes place. Several set-ups now exist for optimised quantitation and/or identification. Ion trap instruments are generally better suited for identification than triple quadrupole instruments (higher MSn power), whereas triple quadrupole instruments provide better information for quantification (faster scanning, higher sensitivity). Hybrid instruments exist that provide a linear ion trap in a triple quad instrument in an attempt to make the best of both setups.

Determination of Individual Toxins (see also individual fact sheets)

Multimycotoxin Methods

Several methods for the simultaneous determination of mycotoxins have been reported, offering a significant advantage over conventional techniques. Rundberget and Wilkins have determined 13 Penicillium toxins (ochratoxin A [OTA], citrinin, patulin, mycophenolic acid, cyclopiazonic acid, PR-toxin, rubratoxin B, verruculogen, chaetoglobosin B, penitrem A, griseofulvin, roquefortin C, penicillic acid) in standardised matrix samples, which included bread, rice, potatoes, vegetables and fruit. The LC-MS System was an ion trap instrument with electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI).

Driffield et al have developed an HPLC-MS/MS method for OTA (including its metabolite OT, zearalenone (ZON, including metabolites -ZOL and -ZOL), deoxynivalenol (DON) and nivalenol (NIV) on a triple quad instrument with an ESI source from a pork liver matrix. Limits of quantitation were as low as 1ppb.

Berger et al have determined NIV, DON, fusarenon X (FUS-X), 3- and 15- acteylated DON (3-AcDON, 15-AcDON), neosolaniol, diacetoxyscirpenol, HT-2 und T-2 toxins with APCI ionisation.

Trichothecenes

The use of a variety of ionisation methods (e.g. fast atom bombardment (FAB) and atmospheric pressure chemical ionisation (APCI)) has been reported. FAB results in numerous different ions, whereas APCI, which has emerged as the method of choice for the determination of major mycotoxins, provides a suitable spectrum for identification and quantification. Thermospray Ionisation (TSP) is less widely used. Unlike GC-MS, derivatisation is not necessary, unless needed for additional fragments, and detection limits are in the lower ng/g range.

HPLC-TSP MS has also been used as a multimycotoxin method for the determination of several Fusarium mycotoxins together with zearalenone and ochratoxin A in the 1-40 ng/g range.

Zearalenone (ZON)

ZON, which frequently occurs simultaneously with other mycotoxins in Fusarium infected grain samples, has been determined with LC and tandem mass spectroscopy after solid phase extraction (SPE) using immunoaffinity columns (IAC).

Aflatoxins

LC-MS has been used for identification of aflatoxins (B₁, B₂, G₁ and G₂). Derivatisation is not necessary, but enhances sensitivity. In Selected Ion Monitoring (SIM) mode, levels as low as picograms can be detected. The addition of sodium bisulphite as a derivatisation agent leads to the formation of adducts for the B₁ and G₁ metabolites only. Applications of methods to corn, milk and liver samples have been reported.

Fumonisins

ESI and FAB have been used for purity determination, quantitation, confirmation and the detection of structural isomers of partially hydrolysed Fumonisin B₁.

Ochratoxin A (OTA)

HPLC-MS and -MS/MS detection methods have been reported for the determination of OTA. ESI resulted in the detection of sub-ng/g levels of the toxin. Several methods have been presented and compared for the determination of OTA in wine (including electrospray ionisation and MS-MS detection).

Biosensors

Measurement of mycotoxins is necessary in a variety of situations starting in the field (e.g. for monitoring purposes) and ending in controlled laboratory settings (e.g. as part of the production process). Methods are being developed, which can be applied rapidly with minimal technical expertise (e.g. immunoassays and ELISAs, see Rapid Test Kits and Methods and biosensors), to those which can only be carried out by trained personnel. Screening methods include enzyme linked immunosorbent assays (ELISAs) and biosensors using specific monoclonal antibodies. Established chromatographic methods for mycotoxin detection are not replaced, but in these instances are required to confirm results.

In collaboration with instrument producers and end-users, sensors and sensor systems are being developed for food analysis, environmental applications, and for pharmaceutical purposes. A number of sensors are the subject of ongoing research. Immunochemical methods have been most successful as rapid methods in the field of mycotoxin analysis. Details (including the availability of commercially available test kits) can be found in the Rapid Test Kits and Methods.

Other method principles include the use of fluorescence detection (e.g. the B[right]G[reenish]Y[ellow] Fluorescence test) for the detection of Aflatoxins in grain. Other sensor systems that are currently being investigated include opto-chemical sensors (fibre-optic sensors, chemo-luminescence systems) and "electronic noses" for the detection of mycotoxins.

The Biacore System is another commercially available set up, which has also been tested for the determination of mycotoxins. Surface plasmon resonance (SPR)-based biosensors monitor interactions by measuring the mass concentration of biomolecules close to a surface. One of the interacting molecules is attached in order to make the surface specific to the analyte of interest. The sample, which contains the interacting reaction molecule is pumped to the surface, where the sample binds (to the molecules) on the surface. The concentration of the analyte at the surface changes, and this can then be detected and quantified by SPR.

The Biacore uses three key components:

• Surface plasmon resonance detects the mass concentrations at the surface.
• Sensor chips provide the surface for attaching molecules of interest.
• A flow system delivers the (liquid) sample to the surface.

Calibration curves have been set up for Zearalenone, Ochratoxin A and Aflatoxin B₁. Generally the detection limits for these three mycotoxins are between 0.1 and 0.4 microgrammes/kg. Peanut and oat samples were investigated.

Bibliography and References

[1] Berger, U., Oehme, M. and Kuhn, F. (1999) ?Quantitative Determination and Structure Elucidation of Type A- and B-trichothecenes by HPLC/Ion Tap Multiple Mass Spectrometry", J. Agric. and Food Chem. (47) 4240-4245.

[2] Biacore Website: http://www.biacore.com

[3] Driffield, M., Hird, S.J., MacDonald, S.J. (2003), "The Occurrence of a Range of Mycotoxins in Animal Offal Food Products by HPLC-MS/MS", Aspects of Applied Biology (68) 205-210.

[4] Krska, R., Baumgartner, S., Josephs, R. (2001), "The State-of-the-Art in the Analysis of Type A- and Type B-trichothecene Mycotoxins in Cereals", Fresenius Journal of Analytical Chemistry, (371) 285-299.

[5] Langseth, W. and Rundberget, T. (1998) "Instrumental Methods for Determination of Non-macrocyclic Trichothecenes in Cereals, Foodstuffs and Cultures", J. Chrom. A (815) 103-121.

[6] Leitner, A., Zollner, P., Paolillo, A., Stroka, J., Papadopoulou-Bouraoui, A., Jaborek, S., Anklam, E. Lindner, W. (2002) "Comparison of Methods for the Determination of Ochratoxin A in Wine", Analytica Chimica Acta (453) 33-41.

[7] Razzazi-Fazeli, E., Bohm, J., Luf W. (1999) "Determination of Nivalenol and Deoxynivalenol in Wheat using Liquid Chromatography-mass Spectrometry with Negative Ion Atmospheric Pressure Chemical Ionisation", J. of Chrom. A (854) 45-55.

[8] Rosenberg, E., Krska, R., Wissiack, R.,Kmetov, V., Josephs, R., Razzazi, E., Grasserbauer, M. (1998) "High-performance Liquid Chromatography-atmospheric-pressure Chemical Ionisation Mass Spectrometry as a New Tool for the Determination of the Mycotoxin Zearalenone in Food and Feed", J. of Chrom. A (819) 277-88.

[9] Rundberget, T. and Wilkins, A. (2002) "Determination of Penicillium Mycotoxins in Food and Feeds Using LC-MS", J. of Chrom. A (964) 189-197.