Overview of mycotoxin methods, present status and future needs

Overview of Mycotoxin Methods, PresentStatus and Future Needs

John Gilbert*Ministry of Agriculture, Fisheries and Food, Central Science Laboratory, Sand Hutton, York YO41 1LZ,

UK

ABSTRACT This article reviews current requirements for the analysis for mycotoxins in foods andidenti®es legislative as well as other factors that are driving development and validation of new methods.New regulatory limits for mycotoxins and analytical quality assurance requirements for laboratories to onlyuse validated methods are seen as major factors driving developments. Three major classes of methods areidenti®ed which serve different purposes and can be categorized as screening, of®cial and research. Ineach case the present status and future needs are assessed. In addition to an overview of trends inanalytical methods, some other areas of analytical quality assurance such as participation in pro®ciencytesting and reference materials are identi®ed. Crown copyright 1999. Reproduced with the permissionof Her Majesty's Stationery Of®ce. Published by John Wiley & Sons, Ltd.

Key words: mycotoxins; screening; of®cial methods; pro®ciency testing; regulations

INTRODUCTION

European regulations (European Commission, 1998) foraflatoxins were implemented on 1 January 1999 andlimits were set for aflatoxin B1 and total aflatoxins of 2and 4 ng gÿ1, respectively, in groundnuts, nuts, driedfruit, cereals (including buckwheat) and processedproducts for human consumption. The regulations set alimit of 0.05 ng mlÿ1 for aflatoxin M1 in milk. Associatedsampling requirements are to be implemented by 31December 2000 although Member States are encouragedto put the stipulated procedures into practice as soon aspossible. Amendment of these regulations to set maxi-mum limits for aflatoxins in spices is under considerationsubject to the findings of an EU-wide co-ordinatedsampling programme. Existing validated methods foraflatoxins do not fulfil European regulatory requirements,either in terms of limits of detection or in terms of thewider range of materials such as dried fruit and spicescovered by European regulations. It is envisaged thatEurope-wide legislation for ochratoxin A will beproduced in the near future as an amendment to theseregulations. To date (European Commission, 1999)discussion has focused on possible levels for legislationof ochratoxin A, with suggested limits of 5 ng gÿ1 incereals not for direct human consumption, and 3 ng gÿ1 ifintended for direct consumption. Additional controls on

ochratoxin A in roasted coffee beans and coffee products(3 ng gÿ1), dried vine fruit (10 ng gÿ1), beer (0.2 ng gÿ1),table wine and grape juice (0.5 ng gÿ1) and cocoa(2 ng gÿ1) are being considered. There are also discus-sions concerning regulation of other mycotoxins such asdeoxynivalenol, and the need for even stricter controllimits to be applied to commodities such as baby food.Whatever levels are ultimately prescribed, in each casefully validated analytical methods will be required tosupport this legislation. Existing official methods such asthose published by the Association of Official AnalyticalChemists (AOAC International) or by the EuropeanCommittee for Standardization (CEN) have neither beenvalidated at these low limits nor have they been tested forthe wide range of different commodities now underconsideration for regulatory control.

Regulation in the area of mycotoxins is thus a majordriver in terms of future requirements for analyticalmethods. This is an influencing factor not only in terms ofdemanding validated methods which are required for

Natural ToxinsNat. Toxins 7: 347±352 (2000)

*Correspondence to: John Gilbert, Ministry of Agriculture,Fisheries and Food, Central Science Laboratory, Sand Hutton, YorkY041 1LZ, UK.E-mail: [email protected]

Crown copyright 1999. Reproduced with the permission of Her Majesty's Stationery Of®ce. Published by John Wiley & Sons, Ltd.

Received 3 November 1999; Accepted 20 July 2000.

RESEARCH ARTICLE

official purposesbut also in terms of the needsformonitoring raw materialsand quality assurancewhererapidscreeningmethodsmaybemoreappropriate.

The secondfactor influencingdevelopmentshasbeenthe formal introduction of analytical quality assurancerequirements.In Europe all food control laboratoriesmustcomply with the requirementsof the FoodControlDirective Additional Measures(EuropeanCommission,1993)which mandatesthat laboratoriesareaccreditedtoEN 45002 (CEN/CENLAC, 1989), take part in profi-ciencytestingandusevalidatedmethods.Theserequire-mentsreinforcetheneedfor additionalmethodvalidationidentifiedaboveand,in addition,meanthat laboratorieshaveto routinely demonstratetheir competencethroughparticipation in proficiency testing. This emphasisonquality is making increaseddemandson traceabilityofanalytical standards,the routine use of control testmaterialsandincreasingthe needfor certifiedreferencematerials.

ANALYSIS OF MYCOTOXINS

Any analytical schemefor mycotoxins falls into fivediscretestages:

. Sampling– essential

. Samplepreparation– essential

. Extraction– essential

. Clean-up– canbeeliminated

. Separation– canbeeliminated

. Determination– canbesimpleyes/no

Thesamplingstageis oneof themostcritical stepsin anyanalysisandthis is particularlythecasewith mycotoxins,where the contamination is known to be extremelyheterogeneous.There has been considerablework onsampling (e.g. Davis et al., 1980; Park and Pohland,1989;Cokeret al., 1995)particularlyfor aflatoxins,andthegeneralruleof largesamplesizesmadeupof multiplesub-samplesis well recognized,although not alwayswidely practised. There does need to be a balancebetweenthe rigour of statistical considerationsbeingappliedto samplingand the practicalconstraintswhichoftengovernwhatcanbecarriedoutin practice(Sharmanet al., 1994). However it is carried out, sampling isinevitably difficult and slow to execute, as is thesubsequentgrindingandmixing of thebulk samplepriorto sub-samplingfor analysis.The samplingstageis notoptional,andis an inevitabletime constraintin carryingout analysis irrespective of how fast the subsequentstagescanbemade.

All methods for the determinationof mycotoxinsrequirepreliminaryextractionof themycotoxinfrom thecommodity into a suitable solvent. This extraction isusually carried out in a high-speedblender or bymechanicalshaking over a period of time. This is a

problematicstagein that it is impossibleto demonstratethat quantitative recovery of a mycotoxin has beenachievedfrom naturally contaminatedmaterial.Perfor-manceof extraction solventsare usually comparedtoone another, which gives relative performance butnot a measure of the ability to extract the ‘true’mycotoxincontentin absoluteterms.Repeatedextractionof the samematrix with the samesolvent also givessome indication of recovery, but only with radio-labelled mycotoxins can the absoluteperformancebedetermined.Recent work on corn screeningsfor theextraction of fumonisins (Bennett and Richard, 1994)has shown that acetonitrile/water (1:1) gives 40%betterextractionthan methanol/water(8:2). For tortillachips(ScottandLawrence,1996)methanol/acetonitrile/water (1:1:2) gave six times better extraction thanmethanol/water(8:2). Thesestudiesand many othersillustratetheneedto rigorouslyevaluatethecompositionof extraction solvents for naturally contaminatedmaterials and not assumethat spikinggives any realindication of extraction performance.Spiking is bestregardedasbeinganindicatorof anylossesatsubsequentstagesof the assayafter effective extractionhas beencarriedout.

Theclean-upstageof theanalysisessentiallyinvolvespreliminaryseparationof the mycotoxin from otherco-extractedsubstances,and an initial concentrationstep.The specificity and sensitivity of the end-measurementfor the analysiswill determinethe extentof subsequentclean-up that is required. The clean-up step can beeliminated by employing very sensitive and highlyspecificscreeningmethods,whereaswith methodssuchasthin layerchromatography(TLC) wheretheseparatingpower is poor andvisualizationmay not be particularlysensitive, extensive clean-up may be required. Mostinstrumentalmethodsrequire the mycotoxin to be in aform freefrom all interferencesprior to themeasurementstep,andthuschromatographyin the form of TLC, gaschromatography(GC) or liquid chromatography(LC) isnormally required.Again with highly specificdetermi-native stepsthis stagecan be eliminated.The develop-ment of any analytical procedureis thus a matter ofbalancingthe relativeperformancesof eachstagein theassay.Thus,the lessspecificthe measurementstep,themoreclean-upandthemorechromatographicseparationis required. Irrespective of the sophisticationof themethod, it is essentialif meaningful resultsare to beobtained to ensure a representativebulk sample isprocuredand this sampleundergoesproperpreparationprior to taking thesub-sample.

In taking an overview of mycotoxin methodsandassessingpresentstatusandfuture needsit is helpful toidentify three broad analytical categories:screeningmethods,validatedofficial methodsand researchmeth-ods.

Crown copyright 1999. Reproduced with the permission of Her Majesty's Stationery Of®ce.Published by John Wiley & Sons, Ltd. Nat. Toxins7: 347–352(1999)

348 GILBERT

SCREENING METHODS FOR MYCOTOXINS

Screening methods are typified as being rapid testmethods.In general they tend to be only qualitativefrequentlygiving ayes/noansweraboveapredeterminedthreshold limit although they may be quantitative orsemi-quantitative.By definition,screeningmethodshavean identified and accepted‘failure’ rate. Low falsepositive ratesare usually acceptableon the basis thatpositive results will need to be confirmed by morerigorous analytical methods. The use of screeningmethodswhich may be prone to false negativesarehowevermore difficult to justify, as critical decisionsmay be irrevocably made basedon the results of thescreening,such as acceptanceof a lot of material.Screeningmethodsare frequently ‘state-of-the-art’ intermsof technologyandarerelativelyquick to introduceinto the market-placeasthey do not necessarilyrequirevalidationor official endorsement.In themycotoxinarea,screeningmethodshaveabounded,rangingfrom thelow-cost Romermini-column (Romeret al., 1978) to morerecentinnovationsbasedonantibodytechnologyvaryingfrom conventionalELISA strips to various cups, testcardsanddip-stick format.

Tremendousadvanceshavebeenmadein termsof theavailability of mycotoxin screeningmethods,with testkits now offering thepossibilitiesof obtainingresultsonasampleextractin amatterof a few minutes.Thereis nodoubtthatthesekits aresimpleto useandanalysiscanbeundertakenin thefield without theneedfor recourseto alaboratoryenvironment.Thedisadvantagesof mycotoxintest kits can be the cost (particularly for developingcountrieswho probablyhave the greatestneedfor thistechnology),the lack of attentionwith regardto sampleextraction conditions and a tendency to disregardsampling requirements.Many test kits require use ofaqueous-basedextraction systemswhich may not beoptimumfor maximumextractionof themycotoxinfromthe matrix of interest.Thereis also a potentialconflictbetweenthe needto take large samplesmadeup of anumber of sub-samplesfor mycotoxin analysisto getrepresentativeresults,which is very timeconsuming,andthemain ‘selling-point’ of testkits which is thespeedofanalysis.The areaof largest impact of immunologicalbased test kits has been the introduction of affinitycolumns (Scott and Trucksess,1997). Not only canaffinity columnsbeusedin a format for rapid testingbuttheycanbeusedto replaceconventionalsampleclean-upfor mycotoxinsin combinationwith aninstrumentalend-point.

In recognitionof thefact thatmanytestkits would notgo through full method validation, that manufacturerswere making performanceclaims, and that usershadindicated they neededQuality Standardsfor test kits(Kitemark), AOAC International establishedPerfor-

manceTestedMethods.Testkits manufacturersrequest-ing AOAC PerformanceTestedMethod statushave tomake a data submissionto AOAC which supportstheproduct performance claims. This is subjected toindependentexpertandgeneralrefereereviewby AOACaswell asindependenttestingby a laboratorycontractedby AOAC to verify the test kit performanceclaims.AOAC Internationalwould normally requireevaluationof the following parametersas appropriate: matrixapplicability; calibration performance(accuracy,preci-sion and recovery);comparisonwith existing methods(for example,benchmarkingagainstofficial methods);cross-reactivity;stability of the test kit components;detectionlimit (limit of quantification);false positive/falsenegativeratesandruggedness.If successfulin theaboveprocessthe test kit is given a CertificationMarkandis saidto haveattainedPerformanceTestedMethodsstatus.The processof certification of test kits must berenewedannually. At presentsomethree test kits arePerformanceTested for the analysis of aflatoxins incereals,cottonseed,raw and roastedpeanutsand fordeoxynivalenolin corn,wheat,barleyandsomederivedprocessedproducts.

In the area of screeningfor mycotoxins, there is aneed to develop tests for monitoring more than onemycotoxinsimultaneously.Thus,in situationswhereit isknown that a certain commodity is likely to becontaminated with more than one mycotoxin, e.g.aflatoxins and fumonisins in corn, or where certainmycotoxinsfrequentlyco-occur,e.g. trichothecenesandzearalenone,it would be useful to carry out a singlescreeningtest for morethanonetoxin. The principle ofbeing able to conducta multi-mycotoxin determinationhasbeendemonstratedin the form of a visual dipstickenzymeimmunoassaytest (Schneideret al., 1995).Thetesthasbeendevelopedfor simultaneouslydeterminingaflatoxinB1, T-2 toxin, 3-acetyldeoxynivalenol,roridinA and zearalenonein a methanol/waterextract of thecommodity,showingbluedotsasnegative,andnocolourdevelopment for positive presence of mycotoxinsagainstassignedpositionson the dipstick comparedtocontrols.

Therelativelyhigh costof affinity columnsmakestheprospectof multiple useattractiveto usersalthoughnotnecessarilyto commercialmanufacturers.Thereis aneedfor multiple use columns which will have a higherantibody loading and ease of regenerationwithoutsubsequentlossof recovery.Sucha column,which canbeusedupto five timesfor aflatoxinanalysis,is currentlybeingvalidatedby AOAC International.Thereis alsoaneedfor multi-analyteaffinity columns,for simultaneousclean-upof co-occurringmycotoxins, to be used in asimilarwayto thedipstickapproachmentionedabove,orfor preparation of sample extracts for subsequentinstrumentalanalysis.


MYCOTOXIN METHODS: PRESENT AND FUTURE 349

OFFICIAL METHODS FOR MYCOTOXINS

Official methodsarethosewhich havebeenvalidatedbyinterlaboratorycollaborativetrial, for whichperformancecharacteristicssuch as repeatabilty(r), reproducibility(R) and limits of detection/quantificationhave beenestablished(IUPAC, 1995). Such methodsare usuallyendorsedby an official body such as AOAC Interna-tional, CEN or the EuropeanCommissionand will beusedas ‘referee’ or ‘reference’methodsparticularly intheeventof disputebetweentwo parties.Theprocessofvalidationof official methodsis rigorousandinevitablyslow, meaning that official methodswhile ‘tried andtested’arerarely‘state-of-the-art’.It mayunderthemostfavourablecircumstancestake 6–9 monthsof work toundertakea collaborative trial and evaluatethe data.Assuming that the resultsare satisfactoryin terms ofacceptabler andR values,thereportof thetrial thenhasto undergoextensivepeerreviewbeforefinal adoptionasan official method.The length and overall cost of thisevaluationprocess,coupled with the fact that not allcollaborativetrials necessarilyyield the desiredresults,goessomeway to explainingthe lag betweenmethodo-logical innovation and adoptionas an official method.Official methods may originate as researchmethodsinitially, and with refinementgain the robustnesstojustify validation.Official methodsarenormallybasedonconventionallaboratoryequipmentandinstrumentation,althoughinstrumentationonceregardedassophisticated(such as combinedgas chromatography/mass spectro-metry– GC/MS) is now consideredasbeingnormal formost laboratoriesand is beginningto featurein officialmethods.

AOAC Internationalofficial methodsareavailableforaflatoxinsB1, B2, G1 and G2 in peanuts,peanutbutter,corn, cottonseed,pistachiosandsoybeans;aflatoxin M1

in milk anddairy products;ochratoxinA in corn,barleyandgreencoffee;sterigmatocystinin barleyandwheat;patulin in apple juice; zearalenonein corn; deoxyniva-lenol in wheatandcorn;andfumonisinB1 in corn(Scott,1995). While many of thesemethodsuse high perfor-manceliquid chromatography(HPLC),anumberarestillTLC-basedandonly a few fully utilize recentdevelop-mentsin antibodytechnology.A recentEuropeanprojecthastackledtheneedfor official methodsvalidatedatnewlow European regulatory limits, and immunoaffinitycolumn methodsfor aflatoxins in peanutbutter, pista-chios,figsandpaprika(Strokaet al., 2000),aflatoxinM1

in liquid milk (Dragacciet al., in press),ochratoxinA inbarleyandroastedcoffee(Entwisleet al., in press)andaflatoxin B1 in baby food (Strokaet al., in press)haverecentlyundergonecollaborativestudies.

Thereis a futureneedfor increasedvalidationof moremycotoxin methodsto cover a wider rangeof matricesandat the demandingregulatorylimits now beingsetin

manycountries.While thesevalidatedmethodsshouldbeup to datein termsof availabletechnology,theneedsofdeveloping countries should not be forgotten. Instru-mentalapproachessuchasLC maynot beanoption,butimproved clean-uptechniques,which may lead to theremoval of most co-extractivescan make TLC as thechromatographicstepmorerobust.Organizationssuchasthe Food and Agriculture Organization(FAO) and theInternational Union for Pure and Applied Chemistry(IUPAC) areactively addressingthis need.

RESEARCH METHODS

Researchmethods are those at the cutting edge ofanalytical science.In the mycotoxinsfield thesewereoften initially employed for the identification andstructural elucidation of novel mycotoxins. Researchmethodsare not limited in accessibilityto instrumenta-tion andthussophisticatedequipmentmaybeemployed.In this categorycombinedliquid chromatography/massspectrometry(LC/MS) hasbeenincreasinglyfeaturedasan importanttool for identification,andis alsothe mostusefulresearchapproachfor unequivocalconfirmationofidentification(Beckeret al., 1998).Rigorousconfirma-tion is now howeverfrequently requiredas a separateconfirmationstepin official methodswhich may requiretheuseof massspectrometry.Researchmethodsarenotformally validated and only require internal qualityassurancemeasuresto beundertaken.

Theidentificationof newmycotoxinshasalwaysbeenproblematicunlessthereis someprior knowledgeof thestructural type of compound to be anticipated. Theliteraturerevealsthat the useof an appropriatebioassaycoupledwith systematicfractionationhasbeenthekey tosuccess,which is then followed by developmentof ananalytical method. In some casesthe availability ofsensitivedetection(suchaselectrosprayandatmosphericpressure chemical ionization – APCI) enables theanalysis of otherwise intractable mycotoxins and anentirelydifferentmethodologicalapproachis requiredforroutineanalysis.In somecasesresearchmethodsenablerapid unequivocalscreeningsuchas with tandemmassspectrometry(MS/MS) (Lukacs et al., 1996) or thepossibility of direct analysisof crude samplematerialwithout prior clean-up,suchaswith fastatombombard-ment (Park et al., 1985). Once a mycotoxin has beenreportedin theliterature,if it is judgedto beof animalorhumanhealthsignificance,thentheneedfor surveillancedatabecomesthe main driver for methoddevelopment.Progressively,by a processof peer-groupconsensus,themorerobustmethodologiesgain generalacceptanceandare eventually validated and adopted with status asofficial methods.


350 GILBERT

CONCLUSIONS

The areasof samplingandsampleextractionhavebeenrelatively neglected in terms of development andimplementation for mycotoxin analysis. Despite thecritical importanceof samplingbeinggenerallyaccepted,thepracticaldifficulty of takingsamplesizesof 20–30kgmadeup of a largenumberof sub-samplesis a practicalconstraint as it is very time consuming and labourintensive. There is a need for sampling plans to bedevelopedto covertherangeof matricesandmycotoxinsfor which regulatorycontrolsarenow in place.Addedtothis, adequateproceduresmust be followed to grind,mince or homogenizesamplesbefore mixing and sub-sampling.Little work hasbeendonein validating theseprocedures(other than in-house) and particularly forofficial methodsthereis aneedto stipulatepreciselyhoweachandeverysamplematrix shouldbehandled.

Quantitativeextractionof mycotoxinsfrom matricesin which they are naturally occurring is difficult toachieve and difficult to demonstratethat it has beenachieved. It is clear that spiking of samples is anecessarystep in demonstratingthe efficiency of aclean-upprocedurebut indicateslittle aboutthe extrac-tion step.Standardreferencematerialsareavailableforaflatoxinsin peanutbutter (Gilbert et al., 1991),peanutmeal and animal feed (van Egmond et al., 1994),aflatoxin M1 in powdered milk (van Egmond andWagstaffe,1986),ochratoxinA in barley (Wood et al.,1997)anddeoxynivalenolin wheatandcorn (Gilbert etal., 1992). Thesematerialsshould be usedto demon-stratetheadequacyof thecompleteanalyticalprocedure.However, it should be rememberedthat the certifiedvaluesarebasedon best-practicein termsof extractionat thetimeof certificationandcannotgiveanyindicationof ‘true’ content other than an assumption thatquantitativeextraction was achieved.There is a needto culturefungi on variousmatricesto generatenaturallyincurredradiolabelledmycotoxins;only in this way canthe ‘true’ values be establishedand the extractionproceduresbe critically evaluated.

In the past few years there has been an increasedemphasisonqualityassuranceof analyticaldata.Thishastakentheform of laboratoryaccreditationwith third partyauditing,useof validatedmethodsandroutineparticipa-tion in proficiencytesting.Arguably it is participationinproficiency testing which is the ultimate test of theadequacyof the quality systemswithin a laboratory,thestaff undertakingtheanalysisandthemethodof analysisbeingemployed.Some650laboratoriesworld-widetakepart in the Food Analysis PerformanceAssessmentScheme(FAPAS)(Key etal., 1997)for whichmycotoxinanalysisis a key element.Foodstuffsand animal feedscontaining aflatoxins, milk containing aflatoxin M1,ochratoxinA in cerealsandcoffeeandpatulin in apple

juice have been successfullyrun by FAPAS over thepast 10 years.For the future the rangeof mycotoxinsand range of matrices will expand, offering theopportunities for most laboratories to find the rightcombination of mycotoxin and matrix to suit theirspecificrequirements.

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Overview of mycotoxin methods, present status and future needs