1500

Journal of Food Protection, Vol. 71, No. 7, 2008, Pages 1500–1504 Copyright ᮊ, International Association for Food Protection

Research Note

Identification of B2, HT-2 , , and in Dried Figs by Liquid Chromatography– Time-of-Flight Mass Spectrometry and Liquid Chromatography– Mass Spectrometry

H. Z. S¸ENYUVA1* AND J. GILBERT2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/7/1500/1682434/0362-028x-71_7_1500.pdf by guest on 25 September 2021

1Ankara Test and Analysis Laboratory, Scientific and Technical Research Council of Turkey, Ankara 06330, Turkey; and 2Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK

MS 07-582: Received 31 October 2007/Accepted 20 February 2008

ABSTRACT

Dried figs from Turkey that were visibly moldy (or fluorescent under UV light) and thus rejected as unsuitable for human food were screened for the presence of fungal metabolites. Crude solvent extracts from individual figs were directly analyzed by liquid chromatography combined with time-of-flight mass spectrometry to generate accurate mass data for all detectable components. A comparison of these data with a metabolite database indicated the presence of B2 and B4, patulin, HT-2 toxin, and zearalenone among various other metabolites. Portions of the same figs were reextracted and then analyzed by conventional liquid chromatography–mass spectrometry. On the basis of coincident retention times and by matching selected ion monitoring for coincident ions with that of authentic standards, the identification of fumonisin B2, HT-2 toxin, patulin, and zearalenone was confirmed.

Dried figs are economically important in Turkey. Turk- but with a high reporting limit of 10 ng/g OTA. An exten- ish figs represent about 60% of the world’s dried fig pro- sive survey of the 2003 and 2004 harvest of dried figs in duction (14) and are predominantly exported to the Euro- Turkey revealed a 14 to 15% incidence of OTA with levels pean Union (EU). Dried figs must comply with a regulatory as high as 26 ng/g (18). Others have found patulin and limit of 2 ng/g aflatoxin B1 (4 ng/g total aflatoxins) for ergosterol in culled dried figs (6) with 40 to 150 ng/g pat- export to the EU (3, 4), and to achieve this demanding ulin and ergosterol levels in the milligram per kilogram standard, individual figs are hand screened. Figs showing range, indicating substantial fungal contamination. bright greenish yellow (BGY) fluorescence (22) or other The presence of both OTA and patulin in dried figs visible defects under UV light are rejected. This screening provides strong evidence that in addition to the well-estab- focuses on detecting aflatoxins, which have received wide lished Aspergillus flavus and Aspergillus paraciticus, figs attention from researchers reporting both the incidence and also can become contaminated with other fungal species levels of contamination of aflatoxins in dried figs (2, 6, 11, such as Penicillium ochraceus and Penicillium expansum. 15, 20). Levels of aflatoxins are extremely variable among Beauvericin, fusaproliferin, fumonisins, and fusaric acid individual figs; some dried figs are highly contaminated but also have been reported as being produced by Fusarium the majority are free of contamination (19). fungi isolated from Italian figs (8, 9), which indicates that Attention also has focused on the presence of ochra- figs can provide a suitable medium to support a range of toxin A (OTA) in dried figs. Ozay and Alperden (11) an- fungal species and subsequent toxin production (17). alyzed 103 fig samples from the 1988 Turkish crop and In recent years, liquid chromatography–time-of-flight found OTA in 3% of these samples. Although in subsequent mass spectrometry (LC–TOF-MS) has become accessible work (12) no OTA was detected in 32 samples of dried for use as a routine analytical tool. It uniquely offers the figs, the reporting limit of the method was not given. How- possibility of providing accurate molecular mass data with ever, in dried figs available in Morocco (25) there was a a high level of sensitivity and can function across a wide 60% incidence of OTA in 20 samples surveyed. Bayman mass range without a loss in sensitivity. The possibilities et al. (1) carried out mycological analysis of 50,000 indi- of exploiting applications of LC–TOF-MS as a screening vidual fig samples grown in California, and those that con- tool are only just becoming apparent. However, TOF-MS tained toxigenic fungi were analyzed for OTA. The inci- has been used to confirm the identification of fungal me- dence of contamination in individual figs was 0.1 to 0.6% tabolites from fungi grown in culture media (10) and has * Author for correspondence. Tel: ϩ90 312 2124620; Fax: ϩ90 312 been used in a targeted mode for simultaneous determina- 2123749; E-mail: [email protected]. tion of , zearalenone, and aflatoxins in food- J. Food Prot., Vol. 71, No. 7 LC–TOF-MS ANALYSIS OF IN FIGS 1501

TABLE 1. Identification of mycotoxins in samples of individual dried figs by comparison with a LC–TOF-MS database, with identifi- cation confirmed by LC-MS against matching standardsa

b Fig no. OTA AFB1 AFB2 AFG1 AFG2 ZON FB2 Patulin HT-2

1 ߛ 2 ߛ 4 ߛߛ 5 ߛߛ 6 ߛߛ 11 ߛߛ 12 ߛߛ 16 ߛ 17 ߛ

18 ߛ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/7/1500/1682434/0362-028x-71_7_1500.pdf by guest on 25 September 2021 22 ߛߛߛߛߛ 23 ߛ 24 ߛ 25 ߛߛߛߛߛ ߛߛ 27 ߛ 29 ߛߛ 32 ߛߛ 33 ߛߛ 34 ߛ 35 ߛߛ 36 ߛߛߛ 39 ߛߛߛߛߛߛߛ ߛ 42 ߛߛߛߛߛߛ 45 ߛߛ 49 ߛߛߛ a OTA, A; AF, aflatoxin; ZON, zearalenone; FB2, fumonisin B2; HT-2, HT-2 toxin. b A total of 52 individual figs were analyzed, but mycotoxins were found by both methods in only 25 figs.

stuffs (23). As part of a wider program to assess the use of B2, HT-2 toxin, and zearalenone were obtained from Biopure LC–TOF-MS for screening fungal metabolites, we have (Tulln, Austria), and the internal standard (benzophenone) was been analyzing crude extracts from fungal cultures and from Merck. The stock solutions were prepared in methanol and Ϫ Њ foodstuffs. The methodology has been validated for its abil- stored at 20 C. ity to accurately detect known mycotoxins in crude extracts Extraction. Homogenized 2-g samples from individual figs using a specially constructed metabolite database (16). We were placed into 10-ml disposable screw-cap bottles. Extraction report here for the first time the presence of fumonisins B2 conditions were modified from a published method (21). Sample and B4, HT-2 toxin, patulin, and zearalenone in dried figs, portions were extracted twice with a mixture of 3 ml of acetoni- as determined with this novel technique. The preliminary trile and 2 ml of 1% formic acid in ethyl acetate. After vortexing identification based on accurate mass measurements with for 2 min, the sample was placed for 10 min in an ultrasonic bath LC–TOF-MS was subsequently confirmed by conventional to complete the extraction. The combined extracts were filtered through Whatman no. 4 filter paper (Maidstone, UK) and evapo- targeted LC-MS analysis for fumonisin B , HT-2 toxin, pat- 2 rated gently under a nitrogen stream. The extracts were spiked ulin, and zearalenone. with benzophenone as an internal standard. The residue was dis- MATERIALS AND METHODS solved in 1 ml of methanol, ultrasonicated for 10 min, and passed through a 0.2-␮m-pore-size disposable filter (FM 0599-1, Agilent, Samples. Samples of dried figs (10 kg total) of the Sarilop Santa Clara, Calif.) before either LC–TOF-MS or LC-MS analy- cultivar were obtained from a commercial processing facility from sis. the 2006 harvest (Aydin, Izmir, Turkey). These figs had been iden- tified as unsuitable for food either because of the presence of Apparatus. An Agilent 6210 TOF-MS coupled to an Agilent visible defects or because of BGY fluorescence when screened 1200 series HPLC was used for LC–TOF-MS analyses. The sep- under UV light. Fifty-two individual figs were randomly selected aration of mycotoxins and other fungal metabolites was carried for analysis. out using an HPLC system (a vacuum degasser, an autosampler with thermostat, and binary pump) equipped with a reversed- Reagents. Extraction solvents were ethyl acetate (J. T. Baker, phase C18 column (ZORBAX Eclipse XDB, 100 mm by 2.1 mm Mallinckrodt Baker, Inc., Deventer, The Netherlands), 1% formic by 1.8 ␮m; Agilent) and precolumn. The TOF-MS was equipped acid (Merck, KGaA, Darmstadt, Germany), and acetonitrile with a dual nebulizer electrospray source, allowing continuous (Merck). Gradient grade acetonitrile and ammonium acetate in an introduction of reference mass compounds. All data recorded were aqueous solution of 1% formic acid (Merck) was used as the high- processed with Analyst-QS software. performance LC (HPLC) mobile phase. Zearalenone, fumonisin LC-MS was performed in electrospray and atmospheric pres- 1502 S¸ENYUVA AND GILBERT J. Food Prot., Vol. 71, No. 7

sure chemical ionization modes with analyses performed using an Agilent 1100 HPLC system (Waldbronn, Germany) consisting of a binary pump, an autosampler, and a temperature-controlled col- umn oven coupled to an Agilent 1100 MS detector equipped with an electrospray interface with a reversed-phase C18 column (ZORBAX Eclipse XDB, 4.6 mm by 100 mm by 3.5 ␮m) and precolumn. LC–TOF-MS scanning was from m/z 100 to m/z 1,000 for all samples at a scan rate of 1 cycle per s and 9,429 transients per scan. To perform the real-time lock mass correction, two reference mass compounds, a lock mass solution including purine (C5H4N4 at m/z 121.050873, 10 ␮mol literϪ1) and hexakis (1H,1H,3H-te- trafluoropentoxy)-phosphazene (C18H18O6N3P3F24 at m/z ␮ Ϫ1 922.009798, 2 mol liter ) were used. The injected sample vol- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/7/1500/1682434/0362-028x-71_7_1500.pdf by guest on 25 September 2021 ume was 5 ␮l. The LC analysis used a mobile phase of acetonitrile and 2 mM ammonium acetate in an aqueous solution of 1% for- mic acid at a flow rate of 0.3 ml/min. The gradient elution started with 15% acetonitrile and reached 100% acetonitrile in 20 min. The ZORBAX Eclipse XDB column (100 mm by 2.1 mm by 1.8 ␮m) and precolumn were washed with 100% acetonitrile for 5 min and equilibrated for 5 min between chromatographic runs. The optimum TOF-MS conditions were 3,000 V capillary voltage, 40 psig nebulizer pressure, 10 liters/min drying gas, 300ЊC gas temperature, 150 V fragmentor voltage, 60 V skimmer voltage, 250 V OCT RF, 37.5 V OCT DC, mass range from m/z 100 to m/z 1,000, and reference masses of m/z 121.050873 and m/z 922.009798.

Construction of the database of accurate masses of fungal FIGURE 1. Analysis of an extract from dried fig by LC–TOF- metabolites. An Excel spreadsheet (Microsoft, Redmond, Wash.) MS. (a) Expanded region of the TIC for aflatoxin B1; (b) expanded was constructed containing the exact mass data for each of 465 region of the TIC for ochratoxin A and spectrum; (c) expanded mycotoxins and fungal metabolites and their empirical formulae region of the TIC for fumonisin B2 and spectrum; (d) expanded (16). Theoretical monoisotopic exact masses of the compounds region of the TIC for and spectrum. were calculated based on the molecular formula using an Excel spreadsheet (Formula DB Generator, provided with the Agilent TOF) and put into the csv (comma-separated values) format for considered confirmed when one or more selected ions coeluted at use by the Agilent TOF automated data analysis software. The retention times coincident (Ϯ2%) with mycotoxin standards. csv file was searched automatically by the LC–TOF-MS instru- Analytical quality assurance. Analytical quality assurance ment at the completion of the sample run, and a report was gen- measures were employed to ensure that no cross-contamination of erated on compounds that were found in the database. The crea- samples occurred (blanks were run at regular intervals). The LC- tion of the data analysis method was carried out using a data MS procedure also was verified by the analysis of sample extracts analysis editor. The editor allowed selection of adducts (e.g., in ϩ from Food Analysis Performance Assessment Scheme (FAPAS) positive ion H ,NH4, Na, etc.) and neutral losses to be searched test materials naturally contaminated with fumonisins and zeara- automatically and mass accuracy and retention time tolerances, lenone, where both the correct identification and satisfactory Z- report options, and other search and detection criteria. score were obtained for quantification. Each batch of samples included a blank sample, i.e., con- LC-MS confirmation of fumonisins, HT-2 toxin, patulin, taining Ͻ0.2 ng gϪ1 for individual and the same blank and zearalenone. Optimal conditions were obtained by using flow sample spiked with each mycotoxin. One sample in each batch injection analysis. The instrument was operated in positive elec- was analyzed in duplicate. Recoveries of Ͼ80% were deemed trospray mode (except patulin) with a fragmentor voltage of 100 acceptable; recoveries typically ranged from 83 to 92%. Each eV, a source temperature of 350ЊC, a capillary voltage of 3,500 sample was analyzed singly. The limits of detection for each my- V, and a nebulizer gas (N2) flow of 12 liters/h and a nebulizer cotoxin spiked into figs based on a signal corresponding to three pressure of 50 psig. LC was performed throughout using a ZOR- times the background noise were 0.5 to 5.3 ng gϪ1 for trichothe- ␮ BAX Eclipse XDB C18 (4.6 mm by 100 mm by 3.5 m) column cenes and 0.1 to 0.3 ng gϪ1 for aflatoxins. connected to an Agilent 1100 binary pump, autosampler, and ther- mostatted column compartment. The mobile phase A was 10 mM RESULTS AND DISCUSSION ammonium acetate in 0.1% formic acid, and the mobile phase B Identification of mycotoxins in figs. We used LC– was acetonitrile. The gradient elution started with 10% acetonitrile and reached 95% acetonitrile in 16 min. Initially, scanning from TOF-MS to screen 52 individual dried figs that had been m/z 50 to m/z 1,000 provided preliminary full scan spectral evi- sorted under UV light and rejected for food use because of dence of identification of the target mycotoxins. This scan was possible fungal contamination. We identified the anticipated followed by selected ion monitoring mode for m/z 706 and m/z aflatoxins and OTA (15). However, in several figs there also 707 for fumonisin B2, m/z 442 and m/z 447 for HT-2 toxin, m/z was evidence of other mycotoxins, some of which have 153 for patulin, and m/z 319 for zearalenone. Identification was been previously reported in figs, such as fumonisins (8, 9) J. Food Prot., Vol. 71, No. 7 LC–TOF-MS ANALYSIS OF MYCOTOXINS IN FIGS 1503

2b), the zearalenone standard (Fig. 2c), and zearalenone detected in a sample of dried fig (Fig. 2d). The identification of fumonisins in foods largely has been confined worldwide to preharvest contaminant in corn, although there have been several isolated reports of fumonisins in other commodities such as peanuts (13), sor- ghum syrup (24), beer (5), and asparagus (7). Various Fu- sarium species belonging to the section Liseola have been previously isolated from figs that have been infected with so-called pink or soft rot (9), and in vitro cultures produced a range of toxins, including fumonisins (8). However, we report here for the first time the natural occurrence of fu-

monisins in moldy figs and the presence of zearalenone and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/7/1500/1682434/0362-028x-71_7_1500.pdf by guest on 25 September 2021 HT-2 toxin. These results provide further evidence that figs are a suitable medium for growth of a wide range of fungi, which can lead to subsequent production of a diverse range of mycotoxins.

ACKNOWLEDGMENTS The authors gratefully acknowledge the TUBITAK-ATAL Director- ate for financial support, S¸ebnem O¨ ztu¨rkog˘lu and Su¨reyya O¨ zcan for tre- mendous help, and Agilent Technologies for the loan of an LC–TOF-MS instrument and provision of consumables.

REFERENCES 1. Bayman, P., J. L. Baker, M. A. Doster, T. J. Michailides, and N. E. Mahoney. 2002. Ochratoxin production by the Aspergillus ochraceus ¤ FIGURE 2. LC-MS selected ion monitoring for m/z 706 ( ) group and Aspergillus alliaceus. Appl. Environ. Microbiol. 68: showing (a) the fumonisin B2 standard and (b) fumonisin B2 de- 2326–2329. tected in a dried fig sample, and for m/z 319 (*) showing (c) the 2. Doster, M. A., T. J. Michailides, and D. P. Morgan. 1996. Aspergillus zearalenone standard and (d) zearalenone detected in a dried fig species and mycotoxins in figs from California orchards. Plant Dis. sample. 80:484–489. 3. European Commission. 1998. Commission directive 98/53/EC of 16 July 1998 laying down the sampling methods and the methods for and patulin (6); others, such as HT-2 toxin and zearalenone, analysis for the official control of the levels for certain contaminants had not been previously reported in figs. Table 1 shows the in foodstuffs. Off. J. Eur. Communities L201:93. individual figs and the secondary metabolites initially de- 4. European Commission. 2002. Commission regulation no. 472/2002 tected by LC–TOF-MS and subsequently confirmed by LC- of 12 March 2002 amending regulation (EC) no. 466/2001 setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. MS by selected ion monitoring with authentic standards. Of Communities L75:18. the 52 individual figs analyzed by LC–TOF-MS, mycotox- 5. Hlywka, J. J., and L. B. Bullerman. 1999. Occurrence of fumonisin ins were found by both methods in only 25 individual figs B1 and B2 in beer. Food Addit. Contam. 16:319–324. (Table 1). To give a complete picture of the extent of mul- 6. Karaca, H., and S. Nas. 2006. Aflatoxins, patulin and ergosterol con- timycotoxin contamination of figs, Table 1 also indicates in tents of dried figs in Turkey. Food Addit. Contam. 23:502–508. 7. Liu, C., F. Liu, W. Xu, A. Kofoet, H. U. Humpf, and S. Jiang. 2005. which individual figs OTA and aflatoxins B1,B2,G1, and Occurrence of fumonisins B1 and B2 in asparagus from Shandong G2 were previously reported to occur (17). Figure 1 pro- province, P.R. China. Food Addit. Contam. 22:673–676. vides an illustration of typical accurate mass data (by LC– 8. Moretti, A., R. Ferracane, A. Ritieni, S. Frisullo, A. Lops, and A. Logrieco. 2005. Fusarium species from fig in Apulia: biological and TOF-MS) for aflatoxin B1, OTA, fumonisin B2, and fu- monisin B found in crude extracts from figs matched toxicological characterization, p. 72. In Proceedings of the Third 4 International ISHS Symposium on Fig. International Society for Hor- against the database accurate masses and spectra. In both ticultural Science, Leuven, Belgium. cases, there was a match in accurate masses to less than 5 9. Moretti, A., A. Ritieni, and A. Logrieco. 2005. Toxin profile and ppm and no indications of other structural formulae that morphological identification of Fusarium species isolated from fig could make a reasonable match. To unequivocally confirm in Apulia, p. 71. In Proceedings of the Third International ISHS these findings, samples of the same figs were reextracted Symposium on Fig. International Society for Horticultural Science, Leuven, Belgium. and subjected to direct LC-MS analysis (without derivati- 10. Nielsen, K. F., and J. Smedsgaard. 2003. Fungal metabolite screen- zation) for fumonisins, HT-2 toxin, patulin, and zearale- ing: database of 474 mycotoxins and fungal metabolites for derepli- none. Analysis of these extracts by LC-MS revealed fumo- cation by standardised liquid chromatography–UV–mass spectrom- etry methodology. J. Chromatogr. A 1002:111–136. nisin B2, HT-2 toxin, patulin, and zearalenone with reten- tion times in close agreement with those of standards and 11. Ozay, G., and I. Alperden. 1991. Aflatoxin and ochratoxin—a con- tamination of dried figs from the 1988 crop. Mycotox. Res. 7:85–91. matching electrospray ionization spectra. Figure 2 shows 12. Ozay, G., N. Aran, and M. Pala. 1995. Influence of harvesting and selected ion monitoring for the fumonisin B2 standard (Fig. drying techniques on microflora and mycotoxin contamination of 2a), fumonisin B2 detected in a sample of dried fig (Fig. figs. Die Nahrung 2:156–165. 1504 S¸ENYUVA AND GILBERT J. Food Prot., Vol. 71, No. 7

13. Sangare-Tigori, B., S. Moukha, H. J. Kouadio, A.-M. Betbeder, D. 20. Sharman, M., A. L. Patey, D. A. Bloomfield, and J. Gilbert. 1991.

S. Dano, and E. E. Creppy. 2006. Co-occurrence of aflatoxin B1, Surveillance and control of aflatoxin contamination of dried figs and , ochratoxin A and zearalenone in cereals and peanuts fig paste imported into the United Kingdom. Food Addit. Contam. from Coˆte d’Ivoire. Food Addit. Contam. 23:1000–1007. 8:299–304. 14. Sektor: Kuru Incir, GTIP no. 08 04 20 90. 21. Smedsgaard, J. 1997. Micro-scale extraction procedure for standard- 15. S¸enyuva, H. Z., J. Gilbert, S. O¨ zcan, and U. Ulken. 2005. Survey ized screening of fungal metabolite production in cultures. J. Chro- matogr. A 760:264–270. for the co-occurrence of ochratoxin A and aflatoxin B1 in dried figs in Turkey by using a single laboratory-validated alkaline extraction 22. Steiner, W. E., R. H. Rieker, and R. Battaglia. 1988. Aflatoxin fluo- method for ochratoxin A. J. Food Prot. 68:1512–1515. rescence in dried figs: distribution and association with fluorescence. 16. S¸enyuva, H. Z., J. Gilbert, and S. O¨ ztu¨rkoglu. 2008. Rapid analysis J. Agric. Food Chem. 36:88–91. of fungal cultures and dried figs for secondary metabolites by LC/ 23. Tanaka, H., M. Takino, Y. Sugita-Konishi, and T. Tanaka. 2006. De- velopment of a liquid chromatography/time-of-flight mass spectro- TOF-MS. Anal. Chim. Acta. DOI: 10.1016/j.aca.2008.01.019. metric method for the simultaneous determination of trichothecenes, 17. S¸enyuva, H. Z., J. Gilbert, R. A. Samson, S. O¨ zcan, S. O¨ ztu¨rkog˘lu, zearalenone and aflatoxins in foodstuffs. Rapid Commun. Mass Spec- and D. O¨ nal. 2008. Occurrence of fungi and their mycotoxins in trom. 20:1422–1428. individual Turkish dried figs. World Mycotox. J. 1:79–86. 24. Trucksess, M. W., T. E. Cho, and D. E. Ready. 2000. Liquid chro- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/7/1500/1682434/0362-028x-71_7_1500.pdf by guest on 25 September 2021 18. S¸enyuva, H. Z., J. Gilbert, and U. Ulken. 2007. Aflatoxins in Turkish matographic method for fumonisin B1 in sorghum syrup and corn- dried figs intended for export to the European Union. J. Food Prot. based breakfast cereals Food Addit. Contam. 17:161–166. 70:1029–1032. 25. Zinedine, A., J. M. Soriano, C. Juan, B. Mojemmi, J. C. Molto, A. 19. Sharman, M., S. Macdonald, A. J. Sharkey, and J. Gilbert. 1994. Bouklouze, Y. Cherrah, L. Idrissi, R. El Aouad, and J. Manes. 2007. Sampling bulk consignments of dried figs for aflatoxin analysis. Incidence of ochratoxin A in rice and dried fruits from Rabat and Food Addit. Contam. 11:17–23. Sale area, Morocco. Food Addit. Contam. 24:285–291.