Fitness for purpose – Ochratoxin A analytical developments

Abstract

Laboratory accreditation involving third party auditing, the use of validated methods and participation in laboratory proficiency testing are essential elements for laboratory quality assurance in relation to ochratoxin A (OTA) analysis. A number of methods, mainly based on liquid chromatography (LC) with fluorescence detection (FD), coupled with immunoaffinity column or solid phase extraction cleanup, have been collaboratively validated and adopted as official standards for OTA determination in a variety of foods, including cereals, coffee, wine and beer. Enzyme-linked immunosorbent assays (ELISA) kits are widely used as screening methods for the occurrence of OTA in food. Novel technologies using anti-OTA antibodies (electrochemical immunosensors, fluorescence polarisation, lateral flow devices, enzyme-based flow through membranes, and surface plasmon resonance biosensors) have been proposed for rapid analysis of OTA in food and beverages, and may be applied for in situ measurements. Validation of these immunochemical methods and commercial kits is required. Liquid chromatography-mass spectrometry represents an adequate alternative to LC-FD particularly in the area of multi-mycotoxin analysis. OTA specific molecularly imprinted polymers are currently considered for cleanup as a potential and cheaper alternative to immunoaffinity or solid-phase extraction sorbents.

Keywords:

• Ochratoxin A
• immunochemical methods
• proficiency testing
• biosensors
• food analysis
• molecularly imprinted polymers

Introduction

Ochratoxin A (OTA) is produced by several fungi of the genera Aspergillus and Penicillium, and can be frequently found in a variety of foods and beverages, including cereals, coffee, cocoa, spices, beer, wine, grape juice, dried fruits, blood derived meat products, etc.

Based on risk assessment performed by the Joint FAO/WHO Experts Committee on Food Additives (JECFA), cereals and cereal products contribute more than 50% of human OTA exposure, followed by wine, grape juice and coffee (JECFA 2001). Pending the results of studies on the mechanism of nephrotoxicity and carcinogenicity, the JECFA Committee retained the previous provisional tolerable weekly intake (PTWI) of 100 ng/kg of body weight (b.w.)/week, corresponding to approximately 14 ng/kg b.w./day. Tolerable Daily Intakes (TDIs) of 1.2–5.7 and 5 ng/kg of b.w. have been proposed by the Canadian and the Nordic expert groups on food toxicology, respectively. The European Scientific Committee for Food (SCF) advised that it would be prudent to reduce exposure to OTA as much as possible, ensuring that exposures are toward the lower end of the range of TDI of 1.2–14 ng/kg b.w./day, e.g., below 5 ng/kg b.w./day. Based on the results of a recent SCOOP project (Scientific Cooperation Task 3.2.7) to assess dietary intakes of OTA by the population of EU Member States, the exposure seems to be in most cases below the value indicated by the SCF (SCOOP 2002). Maximum limits have been established by the European Commission at 10.0 µg/kg for dried vine fruits and soluble coffee, 5.0 µg/kg for raw cereal grains and roasted coffee, 3.0 µg/kg for cereals intended for human consumption, 2.0 µg/l for wine and grape juice and 0.5 µg/kg for baby foods and cereal-based foods intended for infants and young children (European Commission 2002, 2004, 2005).

The reliability of analytical results is essential when data are used in food surveillance studies to assess mycotoxin intake, or for monitoring standards for trading purposes. The use of validated methods is generally recommended, although it is no guarantee of accurate results. Laboratories that supply these analytical data should have been accredited by a recognized body to ensure that they are applying a system of analytical quality assurance. Such a system should include the use of certified reference materials, regular participation in proficiency or inter-laboratory studies and application of statistical evaluation. At present only two certified reference materials (CRM) are available for OTA from the Institute for Reference Materials and Measurements of the EU Joint Research Centre (Geel, Belgium), i.e., a blank and a contaminated wheat flour material containing an OTA mass fraction <0.6 µg/kg (CRM 471) and 8.2 µg/kg (CRM 472), respectively (Wood et al. 1997).

Validated and official methods

Several methods have been developed and validated for the analysis of OTA in a variety of food commodities including cereals (barley, maize, wheat bran and flour), coffee, cocoa, wine, beer and dried fruits. Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. It requires the testing of the method on the target food matrix by several laboratories participating in “ad hoc” collaborative studies. Most validated methods have been adopted as Official International Methods or as European Standards by bodies such as the Association of Official Analytical Chemists International (AOAC) or the European Committee for Standardisation (CEN). A list of validated and official methods for OTA determination in several food commodities is described in Table I.

Table I. Official or validated methods for OTA analysis in different food matrices.

Matrix (LOQ^a, μg/kg)	Solvent extraction	Cleanup/detection^b	Official method^c (reference)
Barley, maize, wheat bran (≥25.0)	CHCl3-aq. H3PO4	Basic diatomaceous heart/TLC	AOAC 973.37 (Nesheim et al. 1973)
Green coffee (≥41.0)	CHCl3	Basic diatomaceous heart/TLC	AOAC 975.38 (Levi 1975)
Barley, maize (≥10.0)	CHCl3-aq. H3PO4; aq. NaHCO3 partition	C18/LC-FD	AOAC 991.44 (Nesheim et al. 1992)
Barley, maize, wheat bran (≥3.0)	CHCl3-aq. H3PO4; aq. NaHCO3 partition	C18/LC-FD	CEN 15141-2 (EN 15141-2 1998)
Barley, rye, wheat bran (≥2.0)	CHCl3-aq. H3PO4; aq. NaHCO3 partition	C18/LC-FD	NMKL 143 (Larsson and Moller 1996)
Wheat whole meal (≥0.4)	Toluene-HCl-MgCl2	Silica gel/LC-FD	CEN 15141-1 (Majerus et al. 1994)
Barley (≥1.3)	CH3CN-H2O	IMA/LC-FD	CEN 14132; AOAC 2000.03 (Entwisle et al. 2000)
Roasted coffee (≥1.2)	CH3OH-aq. NaHCO3	Phenylsilane + IMA/LC-FD	CEN 14132; AOAC 2000.09 (Entwisle et al. 2001)
White wine (≥0.1); beer, red wine (≥0.2)	Addn. PEG-aq. NaHCO3	IMA/LC-FD	CEN 14133; OIV 16/2001; AOAC 2001.01  (Visconti et al. 2001)
Cocoa powder (≥0.1)	aq. NaHCO3	IMA/LC-FD	CEN submitted (Brera et al. 2005)
Dried fruits (≥0.1 − ≥10.0)	CH3OH-aq. H3PO4	IMA/LC-FD	CEN submitted (MacDonald et al. 2003)
Peanut butter, maize matrices (≥0.1)	CH3CN-H2O	IMA^d/LC-FD	(Chan et al. 2004)
Cocoa beans (≥0.1)	CH3OH-aq. NaHCO3	IMA/LC-FD	(Amezqueta et al. 2004)
^aLOQ, Limit of Quantification; ^bTLC, Thin Layer Chromatography; IMA, Immunoaffinity Cleanup; LC, Liquid Chromatography; FD, Fluorescence Detector; ^cCEN, European Committee for Standardization; AOAC, Association of Analytical Chemists; NMKL, Nordic Committee on Food Analysis; OIV, Organization Internationale de la Vigne et du Vin; ^dAflaOchra™ immunoaffinity columns (Vicam Ltd, Watertown, MA, USA), suitable for simultaneous detection of aflatoxins and OTA.

The first two methods, adopted by the AOAC as official method 973.37 (Nesheim et al. 1973) and 975.38 (Levi 1975), are based on thin layer chromatography (TLC) analysis and do not fulfill EU regulatory requirements. The official method AOAC 991.44 (Nesheim et al. 1992), based on liquid chromatography (LC) with fluorescence detection (FD) and effective for OTA determination in maize and barley at levels ≥10.0 µg/kg, was further validated by the Nordic Committee on Food Analysis (NMKL) at OTA levels ≥2 µg/kg in cereals and cereal products (Larsson and Moller 1996), and successively adopted as CEN standard (EN 15141-2).

All the remaining methods reported in Table I are based on immunoaffinity cleanup (IMA) and LC with FD. These methods show good performances in terms of precision, accuracy, specificity, sensitivity and reproducibility, and are suitable for food control and monitoring programmes. Some of the OTA validated methods have been adopted as standard by both CEN and AOAC, namely EN 14132/AOAC 2000.03 for barley, EN 14132/AOAC 2000.09 for roasted coffee, and EN 14133/AOAC 2001.01 for wine and beer. The latter has also been adopted as standard by the Organization Internationale de la Vigne et du Vin (OIV 2001).

Besides the standard methods, the CEN Committee for mycotoxins (CEN 275 WG5) has established general performance criteria that a method for OTA analysis should meet to be recognized for the purposes of enforcement or international trade. In particular, depending on OTA levels in the food matrix, the method should have recoveries in the range of 50–120% with a repeatability (RSD_r) and reproducibility (RSD_R) values ≤20 and ≤30%, respectively (CEN 1999).

Proficiency testing programmes

Laboratory proficiency testing is an essential element of laboratory quality assurance and together with laboratory accreditation and the use of validated methods is an important requirement of the EU Additional Measures Directive 93/99/EEC (European Commission 1993). Unlike those from collaborative trials, proficiency testing is a comparison of a laboratory's reported result for the analyte in question with the best estimate of the “true” value of the analyte, and all participants are free to use the analytical method to which they are accustomed (Gilbert 1999).

In proficiency testing schemes performance of a laboratory is given by z-scores calculated on the basis of the “true” value of the analyte (X) and the standard deviation (σ) expected at that concentration (x) from collaborative trial data or the Horwitz curve [z = (x − X)/σ]. A z-score in the range of +2 and −2 is deemed satisfactory, whereas a z-score outside this range is deemed questionable.

Results of proficiency testing are used by laboratory accreditation bodies as part of the process to assess the ability of laboratories to perform analytical tests for which accreditation is required. In the food sector the largest and most comprehensive proficiency scheme is organized in the United Kingdom by Central Science Laboratory (CSL) and is called Food Analysis Performance Assessment Scheme (FAPAS). FAPAS scheme includes a wide range of analytes in food, feed and drink, and organizes proficiency tests for OTA several times a year. Matrices commonly included for OTA analysis by the FAPAS scheme include dried vine fruits, cereals, green coffee, wine, roasted coffee, cocoa, spices, baby food and animal feed.

Recent FAPAS studies (FAPAS 2004–2005) performed on cereals, wine, dried vine fruits and spices at levels ranging from 0.89–56.10 µg/kg and involving 36–95 laboratories (scores), showed that satisfactory scores ranged from 78–98% for various test materials (see Table II).

Table II. FAPAS results of OTA analysis in different food matrices^a.

Report number	Matrix	Assigned OTA value (μg/kg)^b	Total number of scores^c	Number of satisfactory scores (%)^d
1731	Dried vine fruit	10.50	71	62 (87%)
1732	Barley	5.60	95	75 (79%)
1733	Green coffee	9.60	56	48 (86%)
1734	Baby food	0.89	36	33 (96%)
1735	Spices	56.10	46	36 (78%)
1736	Wine	1.80	43	42 (98%)
1737	Wheat	24.50	79	66 (84%)
1738	Roasted coffee	3.09	72	62 (86%)
^aRetrieved from FAPAS Series 17 Rounds 31–38, Reports 1731–1738 (FAPAS 2004–2005); ^bAssigned value, calculated from the most appropriate measure of central tendency of participants’ results; ^cTotal number of scores, number of participants laboratories; ^dNumber of satisfactory scores, number of laboratories returning results with a z-score within the range of ±2.

The results indicate that participating laboratories generally had their methods of analysis for OTA well under control. These studies also showed that IMA cleanup, in combination with LC and FD was, by far, the most often used procedure to determine OTA. Only incidentally determination were performed with TLC or ELISA and that mass spectrometry (MS) and ultraviolet detectors were sporadically used.

Immunochemical methods

Methods using LC are laborious, time-consuming, require sophisticated equipment and/or trained personnel, and can not be used in situ. Enzyme-linked immunosorbent assays (ELISA), based on antigen-antibody reactions, have been developed for fast, specific and inexpensive screening of OTA in foodstuffs. Moreover, due to the increasing demand from the industry for quick and reliable screening tests for field uses, a variety of methods using antibody-based biosensors have been developed. All these methods are based on competitive immunochemical assays and have the potential for rapid qualitative or semi-quantitative in-situ measurements, representing a promising alternative to ELISA assays. Although immunochemical methods have become very popular in recent years, they require a stable source of antibodies, with relatively high costs and limited reusability. The state of the art of immunochemical methods for OTA analysis is summarized below.

Enzyme-linked immunosorbent assays (ELISA)

Different monoclonal and polyclonal anti-OTA antibodies have been developed, and various ELISA formats have been described for determination of OTA in barley, wheat, maize based foods, pig kidney, chicken meat, human and animal blood, and extracts of OTA producing fungi (Morgan et al. 1983; Lee and Chu 1984; Kawamura et al. 1989; Ueno et al. 1991; Barna-Vetrò et al. 1996; Solti et al. 1997; Park et al. 2002).

Nevertheless only a few of them are used in competitive ELISA microtitre plates test kits, suitable for screening large numbers of samples. In particular, ELISA test kits for OTA analysis have been applied to cereals, cereal products, dried fruits, coffee, cocoa, wine and tea in the range 1.0–50.0 µg/kg (www.tepnel.com; Zheng et al. 2005); beans, potatoes, maize, wheat, flour in the range 5.0–25.0 µg/kg (Abouzied et al. 2002); and barley/malt and beer with detection limits of 0.4 µg/kg and 0.08 µg/l, respectively (Gumus et al. 2004).

Although some ELISA test kits show detection limits comparable to those of LC methods, the possibility of false positive results, due to the antibody cross-reaction with matrix components or other ochratoxins (OTB, OTC, OT-α, OTA methyl ester), and false negative results, due to inadequate sensitivity, necessitates confirmatory LC tests using IMA cleanup or MS detection.

Lateral flow devices

A lateral flow device (LFD), based on carrier membrane with immobilized antibodies, was developed for competitive immunoassay detection of OTA in fungal cultures. The test was validated on a wide array of Penicillium and Aspergillus species by 13 European laboratories (Danks et al. 2003). After extraction of fungal culture plates the test allowed a visual identification of OTA producing and non-producing fungi with an accuracy of 94% and 96%, respectively. The LFD format, in combination with IMA cleanup and a quantitative reader, showed a potential application for detection of OTA in grains and other matrices at European legislative levels (Danks et al. 2003).

Flow-through enzyme immunoassay

A membrane-based flow-through enzyme portable immunoassay was developed to visually screen roasted and green coffee samples for OTA levels. The test allowed OTA detection to achieve a visual cut-off point of 4.0 and 8.0 µg/kg in spiked roasted and green coffee, respectively (Sibanda et al. 2001, 2002). Results obtained with this method showed a good correlation with those obtained by LC. The presence of cross reacting compounds with the anti-OTA antibodies in roasted coffee samples necessitated the introduction of a solid-phase (aminopropyl) cleanup step. Without the aminopropyl cleanup step, cross reacting compounds resulted in 100% false positive for both flow-through enzyme immunoassay and LC methods (Sibanda et al. 2002). Test kits based on this immunoassay were collaboratively validated to rapidly screen wheat, rye, maize and barley for OTA presence (De Saeger et al. 2002).

Array biosensors

Ngundi et al. (2005) reported the application of an array biosensor for detection of OTA in cereals and beverages with sensitivity similar to ELISA and other immunoassay methods. The array biosensor utilized a competitive immunoassay format and consisted of an immobilized OTA derivative that competed with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. The competition was quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The method, consisting of simple methanol extraction without sample cleanup, allowed OTA detection limits of 14.0, 3.8, 25.0 and 100.0 µg/kg in wheat pasta, cornmeal, cornflakes and pasta, respectively, and of 7.0 and 38.0 µg/l in coffee and wine, respectively. Detection limits could be improved by introducing a cleanup or pre-concentration step prior to immunoassay analysis.

Fluorescence polarization immunoassay

A fluorescence polarization competitive immunoassay method was developed for the determination of OTA in standard solution in the concentration range of 5.0–200.0 µg/l, with a detection limit of 3.0 µg/l (Shim et al. 2004). When the method was applied to barley samples spiked with 50.0–500.0 µg/kg OTA and compared to indirect competitive ELISA, recoveries higher than 90% were observed with both techniques. However, the analysis of naturally contaminated barley samples showed some disagreements between the results obtained with the two techniques due to a stronger matrix effect observed with ELISA (Shim et al. 2004). On the basis of preliminary results fluorescent polarisation immunoassay was suggested as a potential method for OTA screening of food samples without cleanup. However, additional research is required to validate the procedure on real samples.

Screen printed electrode immunoassay

Recently, a rapid and selective immunochemical method, combining the high selectivity of a competitive immunoassay with the sensitivity of electrochemical screen-printed carbon electrodes, was developed for OTA determination in wheat samples (Alarcon et al. 2005). Samples were extracted with aqueous acetonitrile and the extracts were directly analyzed by the assay without cleanup. The I₅₀ in the extracts was 0.2 µg/l corresponding to 1.6 µg/kg in the wheat samples with a detection limit of 0.4 µg/kg. Results obtained with OTA spiked wheat samples assayed with the optimized electrochemical immunosensor were good correlated with those obtained by analysing the same extracts by LC after IMA cleanup (Alarcon et al. submitted).

Other immunochemical biosensors

Immunochemical competitive biosensors based on surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) technologies were developed for rapid OTA determination (without a cleanup step) in cereals and beverages (water and juices), respectively. These immunoassay based methods may be appropriate for low cost and portable equipments (van der Gaag et al. 2003; Hauck et al. 1998).

Other analytical methods

A good alternative to LC-FD for OTA determination in food is represented by LC-MS particularly in the area of multi-mycotoxin analysis (OTA, ochratoxin α, aflatoxin B₁, zearalenone, α-zearalenol, β-zearalenol, nivalenol and deoxynivalenol) and for OTA confirmatory tests in pig liver (Driffield et al. 2003). Multi-mycotoxin analysis by using LC-MS and LC-MS-MS technology was also reported by Rundberget and Wilkins (2002) for determination of several Penicillium mycotoxins, including OTA, in food mixtures. LC/MS/MS was also applied to the OTA analysis of contaminated coffee samples, using a method developed for OTA determination in human plasma, and gave results comparable to those obtained with a conventional LC-FD method (Lau et al. 2000). Jorgensen and Vahl (1999) reported a LC-electrospray ionization (ESI)-MS-MS method for OTA quantification in pig kidneys with a detection limit of 0.02 µg/kg, using OTA methyl ester as the internal standard. Other applications of LC-ESI-MS-MS were reported on pig kidneys and tissues by De Saeger et al. (2004) and Losito et al. (2004) with quantification limits of 2.5 and 1.5 µg/kg, respectively.

Capillary zone electrophoresis with laser induced fluorescence (CZE-LIF) was used for quantification of OTA in roasted coffee, maize and sorghum, after tandem cleanup on silica and IMA columns (Corneli and Maragos 1998). The method had a sensitivity comparable to LC and proved excellent separation of OTA from interferences. Mean recovery values for samples spiked in the range of 0.2–10.0 µg/kg were 86%, 99% and 91% for roasted coffee, maize and sorghum samples, respectively. The use of small volumes of samples and less expensive and versatile capillaries, together with the absence of organic solvents during the determinative step, make of this method an adequate alternative to LC-FD (Corneli and Maragos 1998).

Molecular imprinted polymers

Mimicking antibodies is the basic idea of molecularly imprinted polymers (MIPs) technology and are generated by polymerization of a functional monomer in presence of cross-linker, radical initiator and template (analyte) molecules. After polymerization, removal of the template produces cavities with specific binding sites for the template molecules. A functional polymer material with high affinity for OTA was prepared by using N-phenylacrylamide (PAM) as functional monomer and trimethylolpropane trimethacrylate (TRIM) as cross linker (Zhou and Lai 2004). The MIP was used to prepare microcolumns for selective solid-phase extraction (MISPE) with fluorescence detection of OTA in wheat extracts spiked with 100 µg/l OTA. The detection limit was 5.0 µg/l, whereas mean recovery was 103 ± 3%, demonstrating an excellent affinity strength of the MIP to bind OTA during MISPE; similar recoveries were also obtained by using the control polymer prepared without OTA template (Zhou et al. 2004).

Polymeric stationary phases obtained by molecular imprinting could be a valid alternative to the immunoaffinity or SPE phases, as they often exhibit very selective analyte retention, and do not suffer from storage limitations and stability problems regarding organic solvents (Jodlbauer et al. 2002). Nevertheless, the preparation of an OTA imprinted polymer may pose some problems in terms of cost and safety when using OTA as template. The use of a template that mimics the structure of OTA acting as imprinting molecule has been reported in literature (Baggiani et al. 2002; Jodlbauer et al. 2002; Maier et al. 2004).

Conclusions

There are a number of official/validated methods available for OTA analysis in several food matrices. With the exception of two TLC methods, all validated methods are based on LC with FD detection. Most of them use IMA cleanup that provides good precision, accuracy, specificity, sensitivity and reproducibility, and are suitable for food control and monitoring programmes. Laboratory proficiency testing for OTA is currently running on a variety of food matrices with a satisfactory level of quality assurance for all participating laboratories. ELISA test kits, having also an acceptable sensitivity, are available for a variety of foodstuffs, and can be used for screening purposes, but LC analysis is often required for confirmation of the results. Novel technologies combined with immunochemical assay have been proposed for rapid qualitative or semi-quantitative analysis of OTA in food and beverages, and can be assembled in portable devices for in situ measurements. They include lateral flow devices, flow-through enzyme immunoassay, array biosensors, fluorescence polarisation immunoassay, screen printed electrode immunoassay, surface plasmon resonance, and quartz crystal microbalance.

Alternative technologies to LC-FD for OTA determination in food are offered by LC-MS-MS, particularly useful for multi-mycotoxin analysis, and capillary electrophoresis. Molecular imprinted polymer is a novel technology under investigation as a potential alternative to IMA cleanup.

Further investigations are recommended for the implementation and validation of ELISA test kits and novel immunochemical biosensors on different food matrices. Additional OTA certified reference materials are needed at levels close to OTA regulatory limits, and regular participation in proficiency testing programmes is encouraged.