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Ochratoxin A in dried vine fruit: methoddevelopment and surveySusan MacDonald , Pete Wilson , Karen Barnes , Andrew Damant , Rob Massey ,Eileen Mortby & Martin J. ShepherdPublished online: 10 Nov 2010.

To cite this article: Susan MacDonald , Pete Wilson , Karen Barnes , Andrew Damant , Rob Massey , EileenMortby & Martin J. Shepherd (1999) Ochratoxin A in dried vine fruit: method development and survey, FoodAdditives & Contaminants, 16:6, 253-260, DOI: 10.1080/026520399284019

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Ochratoxin A in dried vine fruit: method development andsurvey

Susan MacDonald² , Pete Wilson ² , Karen Barnes ² ,Andrew Damant² , Rob Massey ² , Eileen Mortby ³and Martin J. Shepherd² *² CSL Food Science L aboratory, Norwich Research Park, ColneyLane, Norwich, NR4 7UQ, UK; ³ Joint Food Safety and StandardsGroup, Ergon House, Smith Square, L ondon, SW1P 3JR, UK

(Received 15 July 1998; revised 17 November 1998; accepted29 January 1999)

A method is described for the determination of con-centrations of the mycotoxin ochratoxin A in dried vinefruits (currants, raisins and sultanas) using acidicmethanolic extraction, immunoa� nity chromatographyclean-up and HPLC determination. The limit ofdetection was estimated as 0.2 ¹g/kg, and recoveriesof 63± 77% were achieved at 5 ¹g/kg. HPLC± massspectrometric con® rmation of the identity of ochratoxinwas obtained. Ochratoxin A and a¯ atoxins were de-termined in 60 samples of retail dried vine fruitspurchased in the United Kingdom. Ochratoxin A wasfound in excess of 0.2 ¹g/kg in 19 of 20 currant, 17 of20 sultana and 17 of 20 raisin samples examined, anoverall incidence of 88% . The maximum level foundwas 53.6 ¹g/kg. No a¯ atoxin was found in any sampleanalysed, using a method with a detection limit of0.2 ¹g/kg for each of a¯ atoxin B1, B2, G1 and G2.

Keywords : mycotoxin, ochratoxin A, currants,raisins, sultanas, vine fruit

Introduction

The UK Committees on Toxicity of Chemicals inFood, Consumer Products and the Environment(COT) and on Carcinogenicity of Chemicals in Food,Consumer Products and the Environment (COM)have advised that ochratoxin A is a genotoxic carci-

nogen (Ministry of Agriculture, Fisheries and Food[MAFF] 1993). It also has e� ects on fetal develop-ment and can cause immune system e� ects (MAFF1993). The COT and COM recommended that itwould be prudent to reduce the level of ochratoxinA contamination to the lowest level that is technolo-gically feasible (MAFF 1993).

Permissible concentrations of a¯ atoxins in nuts, dried® gs, and their products are controlled in the UK andmany other countries (van Egmond and Dekker1995). Anticipated European Community (EC) legis-lation will harmonize Community legislation in thisarea, while EC legislation to control ochratoxin Aconcentrations in food is also being considered.Although certain commodities are known to be par-ticularly susceptible to mycotoxin contamination andare therefore regularly monitored (e.g. a¯ atoxins innuts and dried ® gs; ochratoxin A in cereals and greenco� ee), there are reports in the literature of otherfoodstu� s occasionally containing these toxins(MAFF 1993, van Egmond and Speijers 1995,Doster et al. 1996, Jorgensen and Bilde 1996, Patelet al. 1996, 1997, Thellmann and Weber 1997).

MAFF has a programme of surveillance for myco-toxins (MAFF 1993). As part of this continuingprogramme, commodities where there may be thepotential for contamination by ochratoxin A or a¯ a-toxins have been monitored. We report here thedetermination of these mycotoxins in retail samplesof currants, raisins and sultanas.

Experimental

Materials

Samples. Retail food samples were obtained mainlyby the British Marketing Research Bureau, employ-ing a detailed sampling scheme devised by MAFF,to ensure coverage of the whole of the UK both

Food Additives and Contaminants , 1999, Vol. 16, No. 6, 253 ± 260

* To whom correspondence should be addressed.

Food Additives and Contaminants ISSN 0265± 203X print/ISSN 1464± 5122 online Ñ 1999 Crown copyrighthttp://www.tandf.co.uk/JNLS/fac.htm

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geographically and in terms of type of retail outletÐsupermarket, small shop, etc. A number ofadditional purchases were made from a range ofshops in Norwich. In all cases a 1 kg sample wasobtained, typically as a single 1 kg bag, but in somecases multiple retail packs were purchased to providea total of 1 kg dried fruit. All unopened sampleswere stored at room temperature. Prepared samples(homogenates with added water) were stored frozenat ­ 20 C.

Immunoa� nity columns. Ochratoxin A and a¯ atoxinimmunoa� nity columns were obtained from Rhoà neDiagnostic Technologies Ltd (Glasgow).

Reagents. All solvents employed were glass distilledor HPLC grade. All reagents were Analar or similarquality. Ochratoxin A, a¯ atoxins, and borontri¯ uoride± methanol were purchased from Sigma.Phosphate bu� ered saline (PBS) was prepared byadding potassium chloride (0.2 g), potassiumdihydrogen phosphate (0.2g), anhydrous disodiumhydrogen phosphate (1.16 g) and sodium chloride(8.0 g) to water (900 ml). The pH was adjusted to7.4 0.1 with 0.1 m HCl or 0.1 m NaOH asappropriate, and the volume made to 1 l with water.Pyridine hydrobromide perbromide (Sigma) solution(50 mg/l in water) was prepared fresh when required.

Methods

General procedure. Samples were prepared andextracted as described below. Extracts (50 ml) wereloaded onto an ASPEC-401 system with Version 712or 715 HPLC control software (Gilson, Luton) forseparate automated immunoa� nity column clean-upand HPLC analysis of ochratoxin A (Sharman et al.1992) and a¯ atoxins (Sharman and Gilbert 1991).The methanol or acetonitrile eluent from theimmunoa� nity column was split in two, and onepart was analysed by HPLC while the other wasreserved either for re-analysis, con® rmation bymethyl ester formation and chromatography, orcon® rmation by mass spectrometry.

Sample preparation. The entire sample was passedthrough an Omega TE22 mincer with 4.5 mm screen(Wright, Oldham) into a pre-weighed bowl. Thesample was weighed and for every ® ve parts driedvine fruit, four parts tap water were added and the

sample mixed for ca 10 min using a Chef mixer(Kenwood, Havant), then blended for a further ca10 min using a mixer± emulsi® er with a 9 mm screen(Silverson, Chesham). This slurry was used for bothochratoxin A and a¯ atoxin analyses.

Ochratoxin A

Sample extraction. Sample (45 g, equivalent to 25 gdried vine fruit and 20 g water), was mixed withmethanol (50ml) and 0.1 m phosphoric acid (5 ml),and homogenized for 2 min using an Ultra-Turrax.This mixture was ® ltered through Whatman No. 1paper for 40 min, and the ® ltrate collected in ameasuring cylinder. Filtrate (12.5 ml) was diluted to100 ml with PBS. The diluted extract was analysedusing an ASPEC 401 system.

Immunoa� nity puri® cation. The immunoa� nity col-umn was washed with PBS (20 ml), loaded withsample, washed with water (10 ml), and eluted with2% acetic acid in methanol (2 ml). One aliquot ofeluate (1 ml) was retained in a glass vial, and theremainder diluted with 2% aqueous acetic acid(2 ml). An injection (400 m l) of the diluted eluate wasmade onto the HPLC system.

HPLC analysis. A Spherisorb Excel ODS2(250 4.6 mm; 5 m m) column was employed with amobile phase of acetonitrile± water± acetic acid(99:99:2, v/v/v) at a ¯ ow rate of 1.00 ml/min.Detection was via a Perkin Elmer LC420¯ uorescence detector, with excitation at 333 nm andemission at 477 nm. Ochratoxin A and its methylester elute with retention times of ca 11 and 23 minrespectively.

A¯ atoxins

Sample extraction. Sample (90 g, equivalent to 50 gfruit and 40 g water) was mixed with acetonitrile:water (87.5 + 22.5 v/v; 110 ml) and homogenized for2 min using an Ultra-Turrax. The mixture was® ltered through Whatman No. 1 paper for 40 min,and the ® ltrate collected in a measuring cylinder.Filtrate (10 ml) was diluted to 150 ml with PBS. Thediluted extract was analysed using an ASPEC 401system.

254 S. MacDonald et al.

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Immunoa� nity puri® cation. The immunoa� nity col-umn was washed with PBS (10 ml), loaded withsample, washed with water (10ml), and eluted withacetonitril (1.5 ml). One aliquot of eluate (0.5 ml)was retained in a glass vial, and the remainderdiluted with water (2 ml). An injection (400 m l) of thedilute eluate was made onto the HPLC system.

HPLC analysis. A Spherisorb Excel ODS1(250 4.6 mm; 5 m m) column was employed with aguard column (25 4.6 mm i.d.). The mobile phasewas water± methanol± acetonitrile (56:14:30, v/v/v) ata ¯ ow rate of 0.86 ml/min. Detection was via aPerkin Elmer LC420 ¯ uorescence detector, withexcitation at 364 nm and emission at 440 nm. Post-column derivatization was achieved with a zero deadvolume T-piece and 30 cm 0.3 mm i.d. PTFEreaction tube. Pyridine hydrobromide perbromidereagent was added at 0.3 ml/min. A¯ atoxins elute inthe order G2, G1, B2, B1 with retention times ofapproximately 10, 12, 13 and 15 min respectively.

Con® rmation of ochratoxin A

Methyl ester formation. Methanolic eluent from theimmunoa� nity column was evaporated to dryness.Boron tri¯ uoride± methanol (500 m l) was added andthe mixture heated at 60 C for 20 min. The reagentwas evaporated at 55 C, and the residue dissolved inmethanol (1 ml) and diluted with 2% acetic acid inwater (2 ml). This solution was used for HPLCanalysis.

Mass spectrometry. Mass spectra were obtained on aMicromass Platform benchtop mass spectrometer.The instrument was calibrated in the positiveionization mode on a mixture of PEG 300, 600 and10000. The instrument was operated in positiveionization nebulizer assisted electrospray mode withcapillary voltage 2.6 kV, source temperature 140 C,cone voltage 10 V, drying gas 300 l/h, and nebulizinggas 10 l/h. The ions monitored were the protonatedmolecule [M + H]+ at m/ z 404 and the chlorineisotope at m/ z 406. A dwell time of 0.1 s was usedthroughout.

HPLC was performed throughout using a WatersS5ODS2 (150 1 mm) column coupled to a HewlettPackard HP1100 pump, Gilson 231 XL autosampler

and Universal valve switching module. A pre-injectorsplit (ca 10:1) was installed such that the ¯ ow throughthe column was ca 50m l/min. Mobile phase was® ltered through 10 m m ® lters and degassed. Followinginjection (400 m l), ochratoxin A was retained on a pre-concentration cartridge (ODS2, 10 4.6 mm), beforebeing transferred to the analytical column. Mobilephase A was 0.04 m ammonium acetate adjusted topH 4.5 with glacial acetic acid. Mobile phase B wasacetonitrile. The gradient through the analytical col-umn was such that at times 0, 2, 12, 20 and 20.1 minthe percentage acetonitrile was 20, 20, 90, 90 and20%. Under these conditions the retention time ofochratoxin A was approximately 8.8 min.

Calibration graphs were constructed by plottingochratoxin A peak areas (m/ z 404) versus concentra-tion (ng/ml) for a selection of standard solutions (1±15 ng/ml). Concentrations of ochratoxin A in sampleextracts were determined by interpolation of resultingpeak areas from the calibration graphs. Con® rmationof identity was a� orded by comparing the 404:406peak area ratios of ochratoxin A in samples withstandard solutions. Identity was con® rmed if thesample ochratoxin A peak area ratio fell within

20% of the mean value found for spiked samples.

Method performance

Validation. The method for ochratoxin A determi-nation in currants, raisins and sultanas was validatedinitially by analysis of a batch of four to six replicateblank samples for each matrix spiked by addition ofochratoxin A at 5.0 m g/kg. Samples were allowed toequilibrate for 30 min before extraction. In addition,matrix blanks were taken through each procedureand the absence of ochratoxin A demonstrated. Therecoveries and within-batch repeatability of thevalidation batches are given in table 1. The between-batch reproducibility data available are also given intable 1.

Calibration. Fresh standard solutions were prepareddaily, and 4-point calibration graphs were obtainedin each analytical batch by including a set ofstandards both at the beginning and end of each runand additionally by interspersing standards betweeneach four samples. Calibration curves were drawnusing the average response for each standardconcentration employed, and always gave acorrelation coe� cient of 0.995 or better.

255Ochratoxin A in dried vine fruit

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L imits of quanti® cation. These were 0.2 m g/kg forochratoxin A and each a¯ atoxin, based on a signal-to-noise ratio 3 : 1.

Accreditation and performance assessment. TheLaboratory is United Kingdom AccreditationService (UKAS)-accredited for a¯ atoxins in nuts,dried ® gs and their products and is currently seekingaccreditation for a wider range of mycotoxinanalyses. It has participated in all a¯ atoxin andochratoxin Food Analysis Performance AssessmentScheme (FAPAS) rounds to date using the methodsemployed here and on each occasion achieved asatisfactory z-score.

Replicate analysis and con® rmation procedures. Eachbatch of samples included a blank sample (i.e.< 0.2 m g/kg per individual toxin, and the same blankspiked at 5.0 m g/kg if ochratoxin A; or if a¯ atoxins,1 m g/kg of each of the four toxins. One sample ineach batch was analysed in duplicate. Recoveries of> 55% were accepted and all results given arecorrected for recovery. Recoveries were typically 60±80%. Each sample was analysed singly. Thosecontaining > 4 m g/kg ochratoxin A were sub-sequently repeated in duplicate, and con® rmed byboth methyl ester preparation and by massspectrometry.

Results and discussion

The method developed for ochratoxin A in dried vinefruit was similar to that previously described forcereals and animal products and subsequently appliedin this Laboratory to other dried fruits (unpublished).The previously developed extraction procedure washowever found to be inadequate for dried vine fruits,giving low and variable recoveries. Use of methanolwas found to be crucial in obtaining acceptable

recoveries. It is speculated that methanol, a hydro-gen-bond forming solvent, solvates the sugars andother polar components of dried vine fruits betterthan acetonitrile. Recoveries were typically 60± 80%,which compares reasonably well with the manufac-turer’s quality control data for the immunoa� nitycolumns of 89± 96% for standard ochratoxin A inbu� er.

One signi® cant problem with the analysis of driedvine fruit is preparation of a suitably homogenizedsample. The initial method employed, mincing,proved inadequate as shown by replicate analysis ofaliquots from the same sample, and it was foundnecessary to mince and then to blend the sample withwater to ensure a relatively mobile paste. The ® nalmaterial obtained could be seen to contain pieces ofskin no greater than 0.5 mm in any one dimension.

Taking into account the blanks, spikes and duplicateanalyses carried out with each batch, a single analystcould carry out 16 ochratoxin A determinations perday. The ASPEC system is limited to 20 samples in asingle run. Validation data for a¯ atoxins are notpresented in detail, but that method is robust anda� ords recoveries typically of 80± 90% at 1 m g/kg.

Details of the samples and the results of ochratoxin Aanalyses after correction for recovery are given intables 2a, 2b and 2c. Figure 1 shows chromatogramsof ochratoxin A in dried vine fruits. Results arecorrected for recovery throughout. No a¯ atoxinswere found in any of the samples analysed. Thedetection limit was 0.8 m g/kg for total a¯ atoxins(0.2 m g/kg for each of the four individual a¯ atoxins,B1, B2, G1 and G2).

Con® rmation of ochratoxin A was achieved initiallyby re-chromatography after derivatization of the ex-tract to a� ord the methyl ester, which has a retentiontime of 23± 24 min compared with ca 11 min for theparent compound. In each case con® rmation wasachieved, although derivatization was not alwayscomplete.

256 S. MacDonald et al.

Table 1. Ochratoxin A validation batch recoveries, within-batch CVs, and between-batch CVs.

MatrixValidation recovery

(%)Within-batch

CV (%) Within-batch naBetween-batch

CV (%) Between-batch na

Currants 63.1 5.5 6 14.4 4Raisins 76.7 11.2 6 4.3 3Sultanas 71.4 2.8 4 7.8 3a Number of replicates (within-batch) or number of batches (between-batch).

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257Ochratoxin A in dried vine fruit

Table 2a. Dried vine fruit ochratoxin A concentrations (currants), corrected for recovery.

Country oforigina

Ochratoxin Ainitial analysis

( m g/kg)Recovery

%

Ochratoxin Areplicate analysisb

( m g/kg)Recoveryb

%

Ochratoxin Amean value

( m g/ kg)

Ochratoxin AMSc valueb

( m g/ kg)Greece 9.1 82.7 12.8, 12.0 58.8 11.3 8.7Greece 8.3 73.3 3.3, 5.2 66.8 5.6 6.8Greece 4.5 82.7 5.6, 5.1 58.8 5.1 4.7Greece 10.5 82.7 13.7, 11.6 58.8 11.9 8.8Greece 5.5 82.7 8.0, 7.6 58.8 7.0 5.2Greece 4.1 82.7 4.3, 4.3 58.8 4.2 2.9Greece 3.6 82.7 ND ND 3.6 NDGreece 2.1 82.7 ND ND 2.1 NDGreece 2.6 82.7 ND ND 2.6 NDGreece > 23.6 82.7 28.5, 33.0 73.3 30.8 21.0Greece 4.1 82.7 4.7, 4.3 58.8 4.4 2.9Greece 3.7 82.7 ND ND 3.7 NDGreece 5.1 82.7 6.4, 5.7 58.8 5.7 3.8Greece 2.9 82.7 ND ND 2.9 NDGreece 8.5 82.7 9.0, 8.7 58.8 8.7 6.4Greece 70.9 73.3 46.7, 43.7 66.8 53.6 74.7Greece 13.5 82.7 12.1, 11.8 73.3 12.5 7.9Greece 1.0 73.3 ND ND 1.0 NDGreece < 0.2 82.7 ND ND < 0.2 NDGreece 6.8 82.7 6.6, 7.8 58.8 7.1 5.2a As stated on product packaging.b ND, not done.c MS, mass spectrometry.

Table 2b. Dried vine fruit ochratoxin A concentrations (raisins), corrected for recovery.

Country of origina

Ochratoxin Ainitial analysis

( m g/kg)Recovery

%

Ochratoxin Areplicateanalysisb

( m g/ kg)Recoveryb

%

Ochratoxin Amean value

( m g/ kg)

Ochratoxin AMSc valueb

( m g/kg)Greece 8.5 73.1 8.3, 8.6 67.1 8.5 6.7Greece 5.5, 6.0 77.3 ND ND 5.8 4.6More than one country 3.6 77.3 ND ND 3.6 NDMore than one country 0.9 77.3 ND ND 0.9 NDUSA 1.7 77.3 ND ND 1.7 NDUSA 0.9 73.1 ND ND 0.9 NDUSA 0.4 77.3 ND ND 0.4 NDMore than one country > 19.5 77.3 20.1, 19.9 73.1 20.0 9.9USA 0.5 77.3 ND ND 0.5 NDUSA 0.4 77.3 ND ND 0.4 NDUSA 0.3 77.3 ND ND 0.3 NDUSA < 0.2 77.3 ND ND < 0.2 NDChile < 0.2 77.3 ND ND < 0.2 NDAustralia 0.6 77.3 ND ND 0.6 NDForeign produce 8.8 77.3 10.1, 9.7 67.1 9.5 6.6More than one country 0.7 77.3 ND ND 0.7 NDUSA 0.4 77.3 ND ND 0.4 NDUSA 0.5 77.3 ND ND 0.5 NDUSA 1.2 77.3 ND ND 1.2 NDChile < 0.2 73.1 ND ND < 0.2 NDaAs stated on product packaging.bND, not done.cMS, mass spectrometry.

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Con® rmation was additionally performed via liquidchromatography± mass spectrometry (LC± MS). Ali-quots of eluents from immunoa� nity clean-up repre-senting blank samples spiked at 5 m g/kg withochratoxin A were found to contain 4.0± 6.5 m g/kgtoxin, representing recoveries of 80± 130%. LC± MSgenerally provides only one ion for an analyte, but asochratoxin A contains a chlorine atom it is possible toobtain additional con® rmation by monitoring the twoisotopic forms. However, because the detection limitis set by the m/ z 404 ion, it is not always possible tomonitor the m/ z 406 ion with su� cient precision. Theimmunoa� nity column residues were found to con-tain large amounts of co-extractives by LC± MS and itis possible that ion suppression occurred because ofthis, which would explain the bias observed betweenthe HPLC and LC± MS results for samples, and theobservation that standards were not a� ected. Thus, arange of 20% was accepted for samples, which hada mean ratio of 2.99, compared with 2.76 and 2.87 forstandards and extracts of spiked samples respectively,and the expected value for 35Cl/ 37Cl of 3.13.

Regulations for ochratoxin A in food have not yetbeen proposed by the European Commission, but apossible limit of 4 or 5 m g/kg has been discussed.Ochratoxin A at concentrations exceeding 4 m g/kgwas found only in dried vine fruit from Mediterra-nean countries, which however were the source ofmost of the fruit analysed. Dried vine fruits on retailsale may be derived from more than one year’sharvest, so it is not clear whether the generally highconcentrations of ochratoxin A found arose becauseof unusually poor weather conditions. Further detailsof this work are available elsewhere (MAFF 1997).

Conclusions

The method developed for ochratoxin A in dried vinefruits is e� ective and reliable. Of the currant, raisinand sultana samples analysed, 88% were found tocontain ochratoxin A in the range 0.2± 53.6 m g/kg.

258 S. MacDonald et al.

Table 2c. Dried vine fruit ochratoxin A concentrations (sultanas), corrected for recovery.

Country of origina

Ochratoxin Ainitial analysis

( m g/kg)Recovery

%

Ochratoxin Areplicateanalysisb

( m g/ kg)Recoveryb

%

Ochratoxin Amean value

( m g/ kg)

Ochratoxin AMSc valueb

( m g/kg)Greece 1.6 80.5 ND ND 1.6 NDMore than one country 8.8 80.5 9.2, 9.4 69.3 9.1 7.5Greece < 0.2 78.4 ND ND < 0.2 NDGreece 1.1 80.5 ND ND 1.1 NDTurkey 1.5 80.5 ND ND 1.5 NDGreece 12.3 80.5 13.0, 12.7 69.3 12.7 9.5Turkey 7.2 80.5 8.6, 7.1 69.3 7.6 6.1Greece 0.9 80.5 ND ND 0.9 NDGreece > 20.0 80.5 17.9, 18.3 78.4 18.1 15.2Greece 1.0 80.5 ND ND 1.0 NDGreece 2.4 80.5 ND ND 2.4 NDGreece 12.3 80.5 10.0, 11.3 69.3 11.2 8.9More than one country 7.4 80.5 8.8, 8.3 69.3 8.2 5.5Greece 1.5 80.5 ND ND 1.5 NDImported < 0.2, < 0.2 80.5 ND ND < 0.2 NDTurkey < 0.2 80.5 ND ND < 0.2 NDTurkey 4.4 80.5 4.7, 4.9 69.3 4.7 3.7More than one country 3.9 80.5 ND ND 3.9 NDTurkey 10.7 78.4 11.6, 11.2 69.3 11.2 7.6Greece 0.5 78.4 ND ND 0.5 NDa As stated on product packaging.b ND, not done.c MS, mass spectrometry.

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259Ochratoxin A in dried vine fruit

Figure 1. Chromatograms: OA in dried vine fruit. (a) Currant < 0.2¹g/kg OA; (b) currant 13.5 ¹g/kg OA, AA570; (c)raisin < 0.02 ¹g/kg OA; (d) raisin 8.8¹g/kg OA, AA615; (e) sultana < 0.2¹g/kg OA; (f) sultana 10.7 ¹g/kg OA,AA604.

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However, no a¯ atoxins were found at > 0.2 m g/kg inany UK retail dried vine fruit sample analysed.

Acknowledgements

This work was funded by the Joint Food Safety andStandards Group of the UK Ministry of Agriculture,Fisheries and Food, and the Department of Health.

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