High Performance Liquid Chromatography (HPLC)

Gregor Kos and Rudolf Krska

Introduction

High Performance Liquid Chromatographic (HPLC) methods in the field of mycotoxins are mainly used for the final separation of matrix components and detection of the analyte of interest. Nowadays, HPLC methods are widespread, because of their superior performance and reliability compared with thin layer chromatography (TLC). However, TLC remains the method of choice for rapid screening purposes and for situations where advanced HPLC equipment is not available. HPLC methods have been developed for almost all major mycotoxins in cereals and other agricultural commodities (The use of gas chromatography is restricted to a limited number, especially trichothecenes). Most methods are reliable and stable. The main challenge today is to provide comparable results: Several EC projects deal with the production of calibrants and (certified) reference materials as well as the organisation of intercomparison studies between different laboratories, a prerequisite for the establishment and implementation of EU guidelines.

This fact sheet deals with the application of liquid chromatographic separation and detection techniques in mycotoxin analysis. An introduction to fundamental HPLC principles and applications is part of the EMAN Training Course on Separation and Detection Techniques for the Determination of Mycotoxins. For details on the determination of the toxin content, please refer to the specific fact-sheet.

Basic Principles of HPLC Determination

Extraction and clean-up techniques have to be applied prior to separation and detection with HPLC (see "Sample Preparation Techniques for the Determination of Mycotoxins").

Reversed Phase (RP) chromatography is most common for the determination of mycotoxins in agricultural samples using, for example, a C₈ or C₁₈-hydrocarbon phase with mixtures of polar solvents (e.g. water:methanol or water:acetonitrile combinations). Detection is mainly performed using Diode Array Detection (DAD), but alternatively, Fluorescence Detection (FLD) that utilises the emission of light from molecules that have been excited to higher energy levels by absorption of electromagnetic radiation is employed. FLD features superior sensitivity, although frequently derivatisation of the analyte has to be performed in order to make the detection possible at all or enhance the sensitivity even further.

Multi-mycotoxin Methods

Several HPLC methods that attempted the simultaneous detection of mycotoxins have been reported, including post column derivatisation. Attempts were especially successful for the detection of B-Trichothecenes. Recent methods focus on the identification and quantification by LC-MS.

Individual Toxins

-Trichothecenes are frequently determined using HPLC methods. Krska et al (2001) have reviewed the most common methods. Most methods employ C₁₈ columns and analytes are detected using UV detection, although in some cases FLD and MS detectors are employed. LODs are in the range between 1 microgramme/kg sample (MS detection) and 500 microgrammes/kg sample (UV detection). Recoveries vary between 60 and 110% with Mycosep™ columns or column chromatography being the clean-up methods of choice.

Fumonisins can also be separated with C₁₈ stationary phase and a mobile phase mixture of acetonitrile/water/acetic acid. Determination requires derivatisation with o-phtaldialdehyde (OPA) or mercaptoethanol (MCE) before fluorescence detection, with limits of detection in the 50 microgramme/kg range.

Aflatoxins: Stationary phases for aflatoxins (B and G derivatives) are mainly C₁₈ materials. For the detection of aflatoxins derivatisation is performed with strong acids or oxidants (e.g. Br₂, I₂, trifluoro-acetic acid [TFA]) resulting in an increase of fluorescence by a factor 20. Fluorescence detection is possible in the microgramme/kg range at 435 nm (excitation at 365 nm).

Ochratoxin A: Determination of ochratoxin A (OTA) in cereals is possible with a C₁₈ column (mobile phase: acetic acid/ acetonitrile/ water). Recoveries of 70-100% were observed after clean-up with SAX columns. Fluorescence detection of OTA in cereals is possible with a limit of detection of 10 microgramme/kg sample.

Zearalenone: The limit of Detection (LOD) for samples containing zearalenone (ZON) analysed with RP-HPLC-FLD after a clean-up with immunoaffinity columns has been reported to be 3-6 microgramme/kg with a mean recovery of 98-100% in the 10-200 microgramme/kg range. Direct fluorescence detection of ZON is possible at 465 nm (excitation at 270 nm). Major metabolites (such as - and -zearalenol) can be detected simultaneously with ZON.

Detection limits for all other major mycotoxins that can be determined with HPLC are usually in the lower microgramme/kg range.

Hyphenated Techniques

LC/MS instruments represent the state-of-the-art in mycotoxin identification and quantification. Atmospheric pressure chemical ionisation (APCI) and electro spray ionisation (ESI) are most widespread after introduction of the HPLC effluent into the MS system. APCI is based on ionisation of sample and solvent molecules with a corona discharge needle, whereas ESI makes use of charged droplets that are produced by forcing the analyte solution through a needle. A potential is used, which is high enough to disperse the emerging solution into a very fine spray of charged droplets. The solvent evaporates away, shrinking the droplet size and increasing the charge concentration at the droplet's surface. Eventually, the droplet's surface tension reaches a point at which the droplet explodes, forming a series of smaller, lower charged droplets. The process is repeated until individually charged analyte ions are formed. Compared to conventional electron impact (EI) mass spectrometery, ESI is a soft ionisation technique and often leads to the formation of molecular ions. ESI is now widely used in the field of bioanalysis.

Commodities such as cereals (Fusarium toxins), peanuts, figs and spices (aflatoxins) are commonly investigated with LC/MS. High selectivity is the major advantage and the reduction of sample preparation steps (injection of raw extracts) is possible in some cases.

Bibliography

Betina V. (1989) "Chromatographic Methods as Tools in the Field of Mycotoxins", Elsevier Science Publishers B.V., Amsterdam, The Netherlands.

Entwisle A.C., Williams A.C., Mann P.J. (2000) "Liquid Chromatographic Method with Immunoaffinity Column Cleanup for Determination of Ochratoxin A in Barley: Collaborative study", Journal of AOAC International, 83 (6) 1377-1383.

Jaimez J. Fente C.A., Vazquez B.I. Franco C.M., Cepeda A., Mahuzier G., Prognon P. (2000) " Review: Application of the Assay of Aflatoxins by Liquid Chromatography with Fluorescence Detection in Food Analysis", Journal of Chromatography A, 882 1-10.

Josephs R., Krska R., MacDonald S., Wilson P. Pettersson H. (2003) Preparation of a Calibrant as Certified Reference Material for Determination of the Fusarium Mycotoxin Zearalenone, Journal of AOAC International, 86, (1) 50-60.

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.

Krska R., Josephs R. (2001) "The State-of-the-Art in the Analysis of Estrogenic Mycotoxins in Cereals" Fresenius Journal of Analytical Chemistry, 369 469-476.

Shepard G.S. (1998) "Review: Chromatographic Determination of the Fumonisins Mycotoxins", Journal of Chromatography A, 815 31-39.