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Food Additives & Contaminants: Part A

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Use of cyclodextrins as modifiers of fluorescence in the detection of mycotoxins

C. M. Maragosa; M. Appella; V. Lippolisb; A. Viscontib; L. Catuccic; M. Pascaleb a Mycotoxin Research Unit, USDA-ARS-NCAUR, Peoria, IL 61604, USA b Istituto di Scienze delle Produzioni Alimentari (ISPA), Bari 70126, Italy c Dipartimento di Chimica, Universit di Bari, Via Orabona 4, Bari 70126, Italy

To cite this Article Maragos, C. M. , Appell, M. , Lippolis, V. , Visconti, A. , Catucci, L. and Pascale, M.(2008) 'Use of

cyclodextrins as modifiers of fluorescence in the detection of mycotoxins', Food Additives & Contaminants: Part A, 25: 2, 164 171 To link to this Article: DOI: 10.1080/02652030701564555 URL: http://dx.doi.org/10.1080/02652030701564555

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Food Additives and Contaminants, February 2008; 25(2): 164171

Use of cyclodextrins as modifiers of fluorescence in the detection of mycotoxins

C. M. MARAGOS1, M. APPELL1, V. LIPPOLIS2, A. VISCONTI2, L. CATUCCI3, & M. PASCALE2

Mycotoxin Research Unit, USDA-ARS-NCAUR, 1815 N. University Street, Peoria, IL 61604, USA, Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche, Via G. Amendola, 122/o, Bari 70126, Italy, and 3Dipartimento di Chimica, ` Universita di Bari, Via Orabona 4, Bari 70126, Italy
2 1

(Received 11 May 2007; revised 28 June 2007; accepted 10 July 2007)

Abstract Cyclodextrins, cyclic oligosaccharides composed of amylose subunits, are known to interact with mycotoxins. The interactions may be useful to analytical chemists by altering the properties of the mycotoxin of interest, namely the chromatographic properties, electrophoretic properties, fluorescence, or absorption of these fungal metabolites. Practical applications of these effects have been the incorporation of cyclodextrins into high-performance liquid chromatography and capillary electrophoresis methods for mycotoxin detection. Specific mycotoxins include those with a native fluorescence such as the aflatoxins, ochratoxin A (OTA) and zearalenone (ZEN) as well as those that can be rendered fluorescent through derivatization, such as T-2 toxin. The literature describing the applications of cyclodextrins in mycotoxin analysis is reviewed and an attempt to extend the use of cyclodextrins to the detection of labelled T-2 toxin is presented. Twenty cyclodextrins were evaluated for their ability to enhance the fluorescence emission of T-2 toxin derivatized with pyrene1-carbonyl cyanide (T2-Pyr). This evaluation revealed that heptakis (2,6-di-O-methyl)--cyclodextrin (DIMEB), in particular, enhanced T2-Pyr fluorescence. DIMEB was used as a buffer modifier in a capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for detecting T-2 in maize. Because of the effects that certain cyclodextrins have, especially under aqueous conditions, they may make useful additives for a variety of mycotoxin analytical methods.

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Keywords: Mycotoxin, trichothecene, zearalenone, cyclodextrin, fluorescence, analysis

Introduction Among the mycotoxins known to have a native fluorescence emission are certain of the aflatoxins, ochratoxins, zearalenone, and related congeners. The structures, toxic effects, and fluorescence characteristics of these three groups of toxins differ immensely. The fluorophores are derived from a variety of molecular structures. The aflatoxins contain the coumarin moiety, which is highly fluorescent. Similarly, the ochratoxins contain an isocoumarin moiety linked to the amino acid phenylalanine, while zearalenone and related compounds are resorcyclic acid lactones (Figure 1). Even small changes in a toxins structure can dramatically influence fluorescence. The fluorescence emission of
Correspondence: C. M. Maragos. E-mail: chris.maragos@ars.usda.gov ISSN 0265203X print/ISSN 14645122 online 2008 Taylor & Francis DOI: 10.1080/02652030701564555

certain of the aflatoxins, those containing a double bond in the furan moiety, can be enhanced by a number of techniques, including halogenation, hydrolysis and rearrangement to the more fluorescent phenolate ions (aflatoxins B2a, G2a, M2a), or photochemical reaction. The fluorescence emission of many fluorophores are also known to be sensitive to the local environment, and this is true for the aflatoxins, ochratoxin A (OTA) and zearalenone (ZEN) as well. Fluorescence emission of aflatoxins B1 and G1 (AFB1, AFG1) are substantially greater in solvents such as methanol or chloroform than in water (Vazquez et al. 1991). The emission maximum is also influenced by solvation, with a shift towards shorter wavelengths (blue shift) as the environment

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fluorophore and water in the presence of the CD. Thus the fluorescence emission of fluorophores small enough to form inclusion complexes may be affected (Easton & Lincoln 1999). Certain of the mycotoxins fit these criteria. This manuscript provides a short review of the applications of this phenomenon to mycotoxin analysis and a description of an attempt to extend the usefulness of the effect to a mycotoxin lacking a native fluorescence (T-2 toxin) after labelling it with the fluorophore pyrene-1-carbonyl cyanide (PCC), yielding T2-pyrene (T2-Pyr).

Applications of CDs to the analysis of mycotoxins having a native fluorescence The aflatoxins have a native fluorescence that can be excited with ultraviolet light (360365 nm). The nomenclature of the B or G aflatoxins was derived from the colour of the fluorescence: AFB1 and AFB2 have a blue fluorescence (emission circa 440 nm), while AFG1 and AFG2 have a blue/green fluorescence (emission circa 460 nm). Aflatoxin emission can be affected by solvent composition, temperature, and interactions with solid phases, such as silica gel (Robertson & Pons 1968, Van Duuren et al. 1968, Dirr & Schabort 1987, Vazquez et al. 1991). The effects of cyclodextrins on substituted coumarins, such as the aflatoxins, have also been known for some time (Francis et al. 1988, Vazquez et al. 1991) and have been applied in several different ways. Among these include the use of CDs in fungal culture media as an aid in the identification of toxinproducing isolates (Fente et al. 2001, Abbas et al. 2004, Jaimez et al. 2004, Rojas et al. 2005), and the use of CDs as reagents in chemical analyses for the aflatoxins (Francis et al. 1988, Cepeda et al. 1996, Franco et al. 1998, Vazquez et al. 1999, DallAsta et al. 2003). The latter include pre-column (Chiavaro et al. 2001) and post-column (Francis et al. 1988, Cepeda et al. 1996, Vazquez et al. 1999) HPLC assays. Chiavaro et al. (2001) used 6 mM of various CDs added to the mobile phase pre-column and reported that the greatest fluorescence enhancements for AFB1 and AFM1 were obtained with succinyl--CD, while the best enhancement for AFG1 was obtained with heptakis (2,6-di O-methyl)--cyclodextrin (DIMEB). Vazquez et al. (1999) reported better resolution and lower back pressure with the post-column approach. The earliest report (Francis et al. 1988) used unmodified -CD, while the later reports used DIMEB, which was found to provide greater enhancement (Cepeda et al. 1996). The CDs have also been examined for use as aids in the separation of aflatoxins by

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Figure 1. Representative structures of some of the mycotoxins. Aflatoxin B1(AFB1), ochratoxin A (OTA), and zearalenone (ZEN).

becomes less polar, an effect observed for AFB1 in various solvents (Dirr & Schabort 1987, Vazquez et al. 1991). Cyclodextrins (CDs) have been shown previously to enhance the fluorescence emission from a number of hydrophobic fluorophores. The CDs are cyclic oligosaccharides composed of multiple subunits of glucose in an (14) configuration. They are classified by the number of subunits ( 6,  7, 8) and by the type and degree of substitution. The CDs have a cavity (pore) that may accommodate small molecules as guests, forming inclusion complexes. The size of the pore and the environment within it can be modulated through changes to the subunits, with cavity diameters of 4.7, 6.8, and 7.5 A for the -, -, and -CD, respectively, and annular depths of 7.98.0 A (Easton & Lincoln 1999). A possible mechanism for the fluorescence enhancement is believed to be by providing favourable interactions between the fluorophore and the cyclodextrin. The effect might also be derived from a reduction of the interactions between the

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C. M. Maragos et al. decreased, increasing AFB1 fluorescence (RamrezGalicia et al. 2007). Based on models of the docking of AFB1 with - and -CDs it has been suggested that inclusion of the fluorophore into the CD cavity may reduce the quenching effect of the solvent, thereby enhancing fluorescence (Amadasi et al. 2007). The exact mechanism of interaction of the CDs with aflatoxins has not been reported, although formation of a 1 : 1 inclusion complex has been suggested (DallAsta et al. 2003). Recently modelling studies have also suggested the dihydrofuran portion of AFB1 is inserted into -CD, with possible hydrogen bonding between the carbonyl groups of the toxin and the secondary hydroxyl groups of the CD (Amadasi et al. 2007). It should be noted that significant enhancement of aflatoxin emission has generally required using high ratios of CD: aflatoxin, ranging from more than 103 : 1 to 105 : 1. Therefore, regardless of whether the stoichiometry of the host guest complex may be 1 : 1, 2 : 1, or 1 : 2, the need for high ratios of CDs suggest the interaction of aflatoxins with the CDs may be more complicated than the formation of stable inclusion complexes, perhaps including interactions with the outer surface of the CDs (Vazquez et al. 1991). Ochratoxin A (OTA), is a substituted isocoumarin related to the fungal metabolite mellein, which is also fluorescent (Sachse 1992). The fluorescence emission of OTA is sensitive to the hydrophobicity and pH of the environment (Golinski & Chelkowski 1978, Hunt et al. 1979). The absorption spectrum of OTA changes dramatically with pH, with the band near 320 nm decreasing and the band near 370 nm increasing with increasing pH over the range from 3.5 to 11 (Bohs et al. 1995, Verrone et al. 2007). The fluorescence excitation maxima for OTA on silica gel was reported as 340 nm, with an emission maximum of 475 nm (Chu 1970), and HPLC of OTA with excitation at 330340 nm and emission at 460470 nm is commonly used (Scott 2002). The effect of -CD on the spectroscopic properties of OTA in aqueous solution has been recently investigated by Verrone et al. (2007). A 1 : 1 stoichiometry of OTA/-CD was observed at all tested pHs (range 3.59.5) with an increase in emission intensity of up to about twofold (excitation at 330 nm, emission at 450 nm). It was reported that -CD interacted with both the protonated and deprotonated forms of OTA, with binding constants of 2140 and 9790 M1 at pHs 3.5 and 9.5, respectively. This suggests that the dianion form of OTA interacts more strongly with -CD than the protonated form. Molecular modelling simulations have also suggested that the phenylalanine portion of OTA is involved in the inclusion complexation with -CD (Amadasi et al. 2007).

capillary electrophoresis. The initial report by Holland & Sepaniak (1993) tested -, -, and CDs in combination with sodium dodecyl sulfate (SDS) and acetonitrile as buffer additives in the separation of ten mycotoxin standards. Unfortunately, the study used absorbance, rather than fluorescence for detection, so potential effects on fluorescence were not reported. Additionally, -, -, and -CDs had minimal effects upon retention of aflatoxins B1, B2, G1, and G2 (Holland & Sepaniak 1993). The first report to incorporate the fluorescence enhancement effects of CDs with capillary electrophoresis of aflatoxins was that of Wei et al. (2000). The approach used a titanium: sapphire laser (730770 nm) to excite aflatoxins B1, B2, G1, and G2, with either carboxymethyl--CD or sulfated-CD present at 210 mM in the electrophoretic buffer. The carboxymethyl--CD was useful in helping to resolve the aflatoxins. The result was a rapid (80 s) separation of AFB2, AFG1, and AFB1, and limits of detection of 0.20.4 nM. Several CDs have been screened for their ability to enhance aflatoxin fluorescence, and many of these caused a blue shift in the emission from 440 to 435 nm, suggestive of the formation of an inclusion complex (DallAsta et al. 2003). The greatest relative enhancement was observed with succinyl-CD, but others such as DIMEB and carboxymethyl--CD were also quite effective. Based upon binding constants of aflatoxins B1, B2, G1, G2, and M1, the authors suggested the furan moiety of the aflatoxin was involved in the CD inclusion complex (DallAsta et al. 2003). Interestingly, three hydroxylated aflatoxins (AFQ1, AFP1, AFM1) did not show a shift in excitation or emission maxima in the presence of CDs (Franco et al. 1998). AFM1 differs from AFB1 by a hydroxyl group on the dihdryofuran moiety at the C14 position, while AFQ1 differs from AFB1 by a hydroxyl group on the other end of the molecule: the cyclopentenone moiety. AFM1 exhibited a fivefold enhancement of the emission when tested with DIMEB, while AFQ1 exhibited a much greater enhancement of 39-fold (Franco et al. 1998). Furthermore, aflatoxins B2 and G2, which are analogues of AFB1 and AFG1 with the furan double bond reduced, show little enhancement with CDs (Vazquez et al. 1991). These spectroscopic results support the hypothesis that the DIMEB is interacting with the dihydrofuran portion of the molecule: an interaction which is hindered in the case of AFM1 by the presence of the hydroxyl in this region. Alternatively, a theoretical study has proposed a mechanism whereby vibrational coupling of the carbonyl groups of AFB1 with water allows deexcitation of the AFB1. When -CD is added the interactions of the carbonyls with the water may be

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Cyclodextrins and mycotoxin fluorescence While the effect of -CD on OTA fluorescence intensity is not as large as it is for the aflatoxins, the CDs have still been found to be useful in chromatographic assays. Specifically, -CD was reported to facilitate the separation of OTA from ZEN in reverse phase HPLC (Seidel et al. 1993), and to facilitate the separation of OTA from moniliformin in a capillary electrophoresis assay (Bohs et al. 1995). With HPLC, a slight (15%) increase was observed in the sensitivity of the assay to OTA with the inclusion of -CD, which corresponded with a higher ultraviolet light absorption of OTA over the range 310350 nm (Seidel et al. 1993). The increased emission was slight, perhaps because the mobile phase that was used (methanol/water 45 : 55) may have had sufficient solvent strength to obscure the effect. With capillary electrophoresis, 20 mM hydroxypropyl--CD (HP--CD) added to the electrophoresis buffer enhanced the separation factor for OTA from moniliformin, but did not affect the separation factors for ZEN/OTA or OTA/ochratoxin B pairs (Bohs et al. 1995). The apparent mobility of OTA was increased when -CD, HP--CD, HP- -CD, or -CD were added to the electrophoresis buffer. Conversely, the migration of OTA in a micellar electrokinetic capillary chromatography assay was not affected appreciably by 7 mM of either -, -, or -CD, although effects upon OTA fluorescence were not examined (Holland & Sepaniak 1993). The report of Seidel et al. (1993), as described previously, also suggested that ZEN forms an inclusion complex with -CD. Despite the use of fluorescence detection, the effect upon fluorescence intensity of ZEN caused by formation of the inclusion complex was not reported perhaps, as with the OTA, because of the strength of the mobile phase (45% methanol), or the low concentration of -CD used (0.15 mM). The lower CD concentration was recommended in order to reduce backpressure of the HPLC system. The use of CDs to influence the electrophoretic separation of ZEN has also been investigated (Holland & Sepaniak 1993, Bohs et al. 1995, Maragos & Appell 2007). The early work described a strong interaction between -CD and ZEN as measured by an effect upon electrophoretic migration (Holland & Sepaniak 1993). Conversely, -CD and -CD did not show significant effects upon ZEN migration. In the latter report ultraviolet light (254 nm) rather than fluorescence detection was used, and the potential effect of the inclusion complex formation on ZEN fluorescence was therefore not described. Bohs et al. (1995) examined the effects of several CDs on the separation of mycotoxins, including ZEN. CDs examined included: -CD, 2,3,6-methyl--CD, HP--CD, -CD, and HP- -CD. The apparent mobility of

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ZEN was reduced for the 2,3,6-methyl--CD and increased for the other CDs, relative to the mobility in the absence of a CD. Of the CDs, the methylated -CD also yielded the greatest separation factor between ZEN and OTA. It was suggested that ZEN may be too large to form inclusion complexes with HP--CD (Bohs et al. 1995). Although the authors examined several CDs, the method of detection was by photodiode array, and therefore potential effects of the CDs upon ZEN fluorescence were not reported. Recently a systematic description of the effects of 22 cyclodextrins upon ZEN fluorescence was reported (Maragos & Appell 2007). In that work, several of the CDs were shown to influence both the magnitude of the fluorescence emission and electrophoretic mobility in a CE-LIF format. The -CDs had little effect upon fluorescence, suggesting the cavity of the -CDs may be too small for ZEN to form an inclusion complex. The CDs giving the greatest enhancement of fluorescence (325 nm excitation) were DIMEB, 6-monodeoxy-6-monoamino--CD, carboxyethylated--CD, and -CD. The presence of DIMEB increased the mobility of ZEN, while the presence of carboxyethylated--CD reduced it. DIMEB, which gave the greatest fluorescence enhancement, was used to develop a CE-LIF method for detecting ZEN in maize, with a limit of quantitation of 5 ng ZEN g1 maize (Maragos & Appell 2007).

Application of CDs to analysis of fluorescently labelled T-2 toxin T-2 toxin has been reported to interact with CD-bonded phases in an HPLC format (Armstrong et al. 1985). Given this, the effect of CDs upon the native fluorescence of mycotoxins, and the ability of CDs to influence chromatographic and electrophoretic separations, we endeavoured to determine the effect of commonly available CDs upon the fluorescence of a pyrene derivative of T-2 toxin (T2-Pry). The T2-Pyr was made by reacting T-2 toxin with pyrene-1-carbonyl cyanide (PCC) using reaction chemistry equivalent to that of the 1-anthroylnitrile (1-anthroylcyanide) derivative (Pascale et al. 2003, Lippolis et al., personal communication). T-2 toxin was solubilized with 0.05 ml of 15 mM 4-dimethylaminopyridine (DMAP) in toluene. The solution was mixed and 0.1 ml of PCC (1.8 mM in toluene) was added, vortexed, and held at 50 C for 20 min. The mixture was dried at 50 C under nitrogen. Dried mixtures were dissolved in 0.6 ml of acetonitrile, diluted with 0.4 ml of water, and applied to a C18 Sep-Pak Plus cartridge (Waters

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C. M. Maragos et al. (sodium salt), 6-monodeoxy-6-monoamino -CD, mono-6-N-allylammonium-6-deoxy--CD chloride, -CD, succinyl- -CD, carboxymethylated -CD, and carboxyethylated -CD. The effect of CDs upon the fluorescence emission of the T2-Pyr was greatly influenced by the type of CD used. The relative fluorescence response of T2-Pyr with each CD is listed in Table I. Of the CDs tested, the three with the greatest effect were DIMEB, HP--CD, and hexakis(2,3,6-triO-methyl)--CD. With a 64-fold enhancement, DIMEB gave the greatest effect and was used in the development of a CE-LIF method for T2-Pyr, discussed below. In aqueous solution DIMEB also provided substantial fluorescence enhancement (up to 30-fold) of T-2 toxin labelled with the 1-anthroylnitrile (data not shown). It is worth noting that DIMEB is the same modified CD found to be among the most effective at improving the fluorescence emission of AFB1, AFG1 (DallAsta et al. 2003), and ZEN (Maragos & Appell 2007). The fluorescence vibronic structure of pyrene depends upon the hydrophobicity of the environment (Kalyanasundaram & Thomas 1977) and the influence of CDs upon the fluorescence of pyrene is well documented (Street 1987). From the effects of -CD, -CD, and -CD upon pyrene fluorescence, it has been suggested that pyrene interacts with but does not completely fit into the cavity of -CD

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Corp., Milford, MA, USA) previously conditioned with 7 3 methanol/water (v/v). The cartridge was washed with 6 ml of methanol/water (7 3), and the T2-Pyr was eluted with 1.5 ml acetonitrile into a silane-treated vial. The contents were dried under nitrogen at 50 C. The derivatization of T-2 toxin replaced the C-3 hydroxyl of the toxin with a pyrene ester rendering the product, T2-Pyr, more hydrophobic. One of the aspects of pyrene that makes it useful as a probe for hydrophobicity is the ability to self associate into complexes that have different emission spectra than the monomers. Specifically, pyrene dimers have a fluorescence emission, known as an excimer emission at a wavelength (489 nm) higher than that of the pyrene monomer (389 and 409 nm) (Dyck et al. 2003). Because modifications to CDs can affect the incorporation of guest molecules, we tested 20 CDs for the ability to enhance the fluorescence of T2-Pyr when held in phosphate buffer, pH 7.3, at ambient temperature. The CDs examined included: -CD, methyl--CD, hexakis(2,3,6-triO-methyl)--CD, (2-hydroxy)propyl--CD, carboxymethylated -CD, carboxyethylated -CD, -CD phosphate (sodium salt), -CD, methyl-CD, DIMEB, heptakis(2,3,6-tri-O-methyl)-CD, HP--CD, carboxymethylated -CD, carboxyethylated -CD, -CD phosphate

Table I. Effect of 20 cyclodextrins upon the fluorescence of T2-Pyr.a Relative enhancement of responsec Cyclodextrins No cyclodextrin -Cyclodextrins Modification of cyclodextrinb at 389 nm (1.0) 1.2 2.5 9.6 2.6 1.2 2.4 1.9 3.4 64.3 6.3 12.1 2.0 6.1 1.7 1.0 2.5 4.8 3.4 4.4 1.5 at 489 nm (1.0) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.8 0.8 0.9 1.0 1.0 1.0 1.0 1.0 0.9 1.0 1.0 0.8

-Cyclodextrins

-Cyclodextrins

Unmodified Methyl Hexakis(2,3,6-tri-O-methyl) (2-Hydroxy)propyl Carboxymethylated Carboxyethylated Phosphate Unmodified Heptakis (2,6-di-O-methyl) Heptakis (2,3,6-tri-O-methyl) (2-Hydroxy)propyl Carboxymethylated Carboxyethylated Phosphate Sulfate 6-Monodeoxy-6-monoamino Unmodified Succinyl Carboxymethylated Carboxyethylated

a Determined using 1 mM T2-Pyr and 10 mg ml1 of the indicated CD. Data were collected with a FluoroMax fluorometer (SPEX, Edison, NJ, USA), 355 nm excitation, with excitation and emission monochromators set to allow 1.5 nm band-pass. b Chemical modification of the indicated cyclodextrin (CD) backbone. c Calculated as the response with 10 mg ml1 added CD divided by the response without added CD.

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Figure 2. Molecular representation of interactions between T2Pyr and DIMEB. Shown are a possible configuration of a 1:1 ratio (a) and possible configuration of a 1:2 ratio (b) of T2Pyr:DIMEB. Modelling was done with HyperChem, version 7.52 (Gainesville, FL, USA).

Figure 3. Effect of 2 to 50 mM DIMEB upon the fluorescence emission spectrum of 1 mM T2-Pyr. Solutions were in 10 mM sodium phosphate buffer, pH 7.3 (excitation 355 nm). Emission spectra were collected in 1 nm increments with an integration time of 0.25 s.

(Street 1987). Pyrene is known to complex very rapidly with -CD with stoichiometries of 1 : 1, 1 : 2 and 2 : 2 (pyrene: CD) forming within milliseconds (Dyck et al. 2003). Spectroscopic studies suggest a substantial portion of the pyrene resides within the CD cavity and is aligned parallel to its axis (Easton & Lincoln 1999). The stoichiometry and the binding constant of the T2-Pyr/DIMEB inclusion complex were determined by means of the modified Benesi Hildebrand equation (Indirapriyadharshini et al. 2001). In the range of concentrations studied a 1 : 2 (T2-Pyr: DIMEB) stoichiometry was found, with a binding constant of 7700 M1, suggesting a strong interaction between the T-2 derivative and the cyclodextrin. Molecular representations of T2-Pyr inclusion complexes are shown in Figure 2. The configuration relevant to the interaction between T2-Pyr and the first CD molecule, shows the pyrene moiety partially inserted into DIMEB (Figure 2(a)). The iso-valerate portion of T-2 toxin

interacting with a second DIMEB cavity leads to the 1 : 2 configuration depicted in Figure 2(b). The interaction of the T2-Pyr with the DIMEB was greatly influenced by the DIMEB concentration over the range from 2 to 50 mM in phosphate buffer (Figure 3). The dependence of the emission intensity upon the CD concentration is an effect that has also been reported for AFB1, AFG1 (DallAsta et al. 2003), and OTA (Verrone et al. 2007). We observed a gradual decrease in the emission corresponding to the pyrene excimer (489 nm, Figure 3) and a concomitant increase in the emission corresponding to the pyrene monomer (389 nm, Figure 3) as the concentration of DIMEB increased. We speculate that the T2-Pyr stock solution contained T2-Pyr as dimers that, upon addition to the CD solution, dissociate, with the resulting monomers associating with the DIMEB, increasing the fluorescence at 389 nm 409 nm. The hydrophobicity of T2-Pyr suggested that non-aqueous CE, or approaches using high solvent strength or detergents, such as micellar electrokinetic capillary chromatography (MECC), would be necessary in addition to the CD in order for the T2-Pyr to remain soluble. Control maize containing nondetectable levels of T-2 toxin and HT-2 toxin was spiked with T-2 toxin, extracted with 9 1 methanol/ water (v/v), and isolated by immunoaffinity column (IAC) cleanup as described by Visconti et al. (2005). T-2 toxin from samples, or T-2 standards, were derivatized and the T2-Pyr isolated as described above. Derivatized samples were dissolved in 0.15 ml of acetonitrile and 0.45 ml of SDS-CD buffer, composed of 42.5 mM SDS, 2.5 mM sodium borate, 4.3 mM dibasic sodium phosphate and 10 mM DIMEB in water. The electrophoresis buffer consisted of a mixture of 42.5 mM SDS, 2.5 mM sodium borate, 4.3 mM dibasic sodium phosphate, 10 mM DIMEB and 25% (v/v) acetonitrile, pH 9.2. A variety of solvents were tested in combination with DIMEB to arrive at the electrophoresis buffer described above (data not shown). The buffer was found to enhance the emission at 389 nm by 220-fold relative to T2-Pyr in phosphate buffer alone. This seems to be because in phosphate buffer the T2-Pyr appears to exist almost solely as the excimer, while in the more hydrophobic environment of the electrophoresis buffer it exists predominantly as the monomer. Using this buffer the T2-Pyr was separated from the reactants and non-specific products within 10 min (Figure 4). The CE-LIF method could be used to detect T-2 toxin in maize containing as little as 50 ng g1. This demonstrates that detection of a fluorescently derivatized mycotoxin by CE-LIF using CDs is certainly possible. However, the sensitivity of the CE-LIF method relative to an HPLC-FL method

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C. M. Maragos et al. available for HPLC detectors. Furthermore, with CE-LIF analysis it was necessary to isolate the T2-Pyr after derivatization. This additional step was not required for HPLC analysis of the same derivative. This combination of factors suggests that, while CE-LIF of T2-Pyr is feasible, HPLC detection of this derivative is preferable.

Conclusions Certain of the cyclodextrins, in particular heptakis (2,6-di-O-methyl)--CD (DIMEB), enhance the fluorescence of mycotoxins with a native fluorescence such as the AFB1, AFG1, and ZEN. This effect has proven useful in HPLC and CE-fluorescence-based systems for detecting these toxins. This paper demonstrated that the enhancement of fluorescence produced by CDs can also be observed with fluorescently derivatized T-2 toxin (T2-Pyr). The type of CD used and the concentration were factors that influenced the magnitude of the effect. The most effective of 20 CDs that were tested, DIMEB, was used in the development of a capillary electrophoresis-laser induced fluorescence method for T-2 toxin in maize. The method was capable of detecting as little as 50 ng g1 of T-2 toxin in maize. However, the additional clean-up steps for the CE method, which are not required in the HPLC method, suggest that HPLC is preferred for measuring the T2-Pyr derivative. Despite this result, the ability of CDs to enhance fluorescence of mycotoxins or their derivatives is still a useful effect and suggests examination of aqueous-based systems, where this effect can be used to its full potential, should be further explored.
Figure 4. Electropherograms of T2-Pyr. Standard T-2 equivalent to 50 ng T-2 g1 (a), unspiked control maize (b), and maize spiked with 50 ng T-2 g1 (c). Data were collected with a Beckman Coulter P/ACE MDQ system equipped with a LIF detector (Beckman Coulter, Fullerton, CA, USA). Excitation at 325 nm was provided by a model 100 He/Cd laser (Omnichrome, Chino, CA, USA). Emitted light was filtered through a 405 nm bandpass filter (Andover Corporation, Salem, NH, USA). Before each injection the capillary was rinsed for 2 min at 20 psi with electrophoresis buffer (described in the text). Samples were injected for 5 s at 0.5 psi. Separation was performed with a 75 mm i.d. 50.2 cm fused silica capillary at 30 C by applying 30 KV (approximately 102 mA).

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Acknowledgements The authors express their appreciation to Mr John Bobell for excellent technical assistance. They would also like to thank the National Research Council, Italy, for the short Short-Term Mobility Award, 2005. Mention of trade names or commercial products in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

of the same derivative (Lippolis et al., personal communication) was poorer. In part this was likely due to the excitation wavelength selected. The excitation source, a He/Cd laser, provided coherent light at 325 nm, while the excitation maximum for the T2-Pyr was circa 355 nm. Using a 355 nm light source would likely have further improved sensitivity, and such sources are readily

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