Anal Bioanal Chem (2010) 397:8792 DOI 10.

1007/s00216-010-3530-1

ORIGINAL PAPER

Determination of aflatoxin B1 in alcoholic beverages: comparison of one- and two-photon-induced fluorescence

Claudia Rasch & Maike Bttcher & Michael Kumke

Received: 15 October 2009 / Revised: 25 January 2010 / Accepted: 30 January 2010 / Published online: 4 March 2010 # Springer-Verlag 2010

Abstract The qualitative and quantitative analysis of aflatoxin B1 in a model system (water/ethanol), in different wines and in beer using one- and two-photon-induced fluorescence is discussed. The absorption and fluorescence properties of aflatoxin B1 depend on the solvent and pH. The two-photon-absorption cross-section was calculated for aflatoxin B1 in beer and wine (2 25 GM) for excitation at 720 nm. A comparison of the one- and two-photoninduced fluorescence results showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photonexcitation conditions. The limit of detection for the oneand two-photon-induced fluorescence was determined. Keywords Aflatoxin B1 . Fluorescence . Two-photon absorption . Two-photon-absorption cross-section . Beer . Wine

Introduction The aflatoxins are a group of mycotoxins produced as secondary metabolites by certain strains of the fungi Aspergillus flavus and A. parasiticus. Among 18 different types of aflatoxins identified, major members are aflatoxins B1 (AFB1), B2, G1, G2, M1 and M2 [13]. These types of mycotoxins structurally refer to the group of difuranocoumarins and are classified in two broad groups according to
C. Rasch : M. Bttcher : M. Kumke (*) Department of Chemistry (Physical Chemistry), University of Potsdam, Karl-Liebknecht-Str. 2425, 14476 Potsdam-Golm, Germany e-mail: kumke@uni-potsdam.de

the chemical structure: the difurocoumarocyclopentenone (e.g. aflatoxin M1) and the difurocoumarolactone (e.g. aflatoxin G1) series, respectively. Aflatoxin-producing members of Aspergillus are common and widespread in nature. They can colonize and contaminate grain before harvest or during storage if water is allowed to exceed critical values for mould growth. Aflatoxins are detected occasionally in cheese, corn, peanuts, cottonseed, nuts, almond, figs, fruits and spices and a variety of other foods and feeds. Milk, eggs and meat products are sometimes contaminated because of animal consumption of aflatoxin-contaminated feed (carry-over) [4, 5]. AFB1 (Fig. 1) is frequently found in cultures as well as in food products and is the most toxic aflatoxin [68]. This toxin can also be found in wine and beer, produced by Aspergillus carbonarius (black aspergilli). Pure AFB1 is a pale-white to yellow crystalline, odourless solid. AFB1 is soluble in methanol, chloroform, acetone, acetonitrile and water. Studies with different mycotoxins added at various stages of the brewing process show that aflatoxins may be transmitted from contaminated grains into beer. They originate from the malted grain or from food additives [9]. Reducing the fungal contamination of malt and barley is the most promising strategy for reducing mycotoxins in beer. At present, the European Union has not set a maximum allowable limit for AFB1 in beer, although there is a limit in barley and malt [10]. With regard to wine, a gradient of concentration is usually recognized; AFB1 levels decreases in the order red, rose and white wine, but also with increasing latitude of the producing countries [11]. The presence of AFB1 in wines is caused by fungi which grow on grapes in the vineyards. At grape crushing, the juice can become contaminated with AFB1 and this contamination is subsequently carried over into the wine [12, 13]. A major concern is that the mycotoxins are very

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O O OCH3

Fig. 1 Chemical structure of aflatoxin B1 (AFB1)

heat stable, so cooking (and even less freezing) only destroys them upon harsh treatment. They remain on the food indefinitely during the production process. Pre- and postharvest treatments are being investigated to diminish contamination of wines as much as possible. Aflatoxins are potentially toxic, carcinogenic, mutagenic and immunosuppressive agents. The ability of aflatoxins to cause cancer and related diseases in humans given their seemingly unavoidable occurrence in food and feed makes the prevention and detoxification of these mycotoxins one of the most challenging toxicology issues of today [14, 15]. Absorption and fluorescence spectroscopy are highly attractive for a rapid and reliable online and in-line monitoring because of their non-invasive and nondestructive approach. Here, results with special emphasis on two-photon-induced fluorescence (TPIF) with 1ex = 720 nm for the identification of AFB1 in alcoholic beverages are presented [16]. First, the basic photophysical parameters such as selected excitation and emission wavelengths of AFB1 in a model system were determined. In a further step, the fluorescence of AFB1 excited by two-photon absorption (TPA) in the model system and in selected alcoholic beverages was measured and compared with the results of regular fluorescence (induced by the absorption of one photon). TPIF is of interest in the analysis of complex samples because of the limitations when using regular fluorescence techniques due to background luminescence signals. TPIF has an improved spatial resolution owing to its quadratic dependence on the excitation irradiance and negligible linear absorption at the pumping wavelength resulting in an increased penetration depth.

solutions (3.2 10-5 M) were prepared in ethanol and Millipore water. The model solvent consisted of water and 12 vol% ethanol, pH 4. For the pH adjustment of the solutions 0.1 M HCl or 0.1 M NaOH was added. The different wines and beers were bought from a supermarket and originated from southern Germany. The pH of the commercial beverage was determined by a pH electrode and ranged between 3.5 (white wine) and 4.3 (beer). The absorption measurements were carried out using a Lambda 750 UV/vis spectrometer (PerkinElmer) with 10-mm quartz cells. The spectra obtained were corrected by the absorption spectra of the solvents or alcoholic beverages. The standard steady-state fluorescence measurements were performed with a FluoroMax3 spectrometer (Jobin Yvon). The fluorescence quantum efficiencies were determined using an integrating sphere set-up (Hamamatsu C9920-02 photoluminescence quantum yield measurement system). The TPIF measurements were performed using an FLS920 spectrofluorometer (Edingburgh Instruments). For the excitation, a titaniumsapphire laser (Tsunami 3960, Spectra Physics) was used operating at 720 nm and emission in the range 350 nm< 1 <650 nm was detected. Fluorescein (1.510-5 M in 0.1 M NaOH, pH 11) and water were used as reference samples; fluorescein as a positive standard for TPIF and water as a negative control [17]. The laser beam was approximately collimated over the path length of the cuvette (1 cm) and the fluorescence was collected at a right angle with respect to the incident beam by a lens and directed to a photomultiplier tube. Data analysis The theory of the TPA was established by Maria GppertMayer [18] in the 1930s. The first experimental evidence of this phenomenon came 30 years later when Kaiser and Garret [19] demonstrated two-photon excitation in a CaF2: Eu2+ crystal. TPA is the simultaneous absorption of two photons of identical or different frequencies to excite a molecule from one state (usually the ground state) to a higher-energy electronic state. In the first step, the molecule is excited to a virtual state, which does not need to correspond to any electronic or vibrational energy eigenstate. The energy difference between the lower and upper states of the molecule involved is equal to the sum of the energies of the two photons. TPA is many orders of magnitude weaker than linear absorption and is therefore not an everyday phenomenon. It differs from linear absorption in that the strength of absorption depends on the square of the light intensity; thus, it is a nonlinear optical process [20]. In particular, the imaginary part of the third-order nonlinear susceptibility is related to the extent of TPA in a given molecule.

Experimental Sample preparation and spectroscopic methods The pure solid AFB1 (98% or better purity, produced by A. flavus) was purchased from Sigma-Aldrich. The sample

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Aflatoxin B1 in model solvent

The selection rules for TPA are therefore different from those for one-photon absorption (OPA), which is dependent on the first-order susceptibility. For example, in a centrosymmetric molecule, one- and two-photon allowed transitions are mutually exclusive. In quantum mechanical terms, this difference results from the need to conserve angular momentum. Since photons have spin of 1, OPA requires excitation to involve an electron changing its molecular orbital to one with an angular momentum different by 1. TPA requires a change of +2, 0 or 2 [21]. Measurements of the relation between laser power and fluorescence intensity of AFB1 were carried out to prove there was TPIF. In addition, fluorescein and water were used as positive and negative standards for TPIF. In the experiments, the power of the titaniumsapphire laser was successively decreased with a grey-wheel filter and monitored with a power meter. For every laser power chosen, a TPIF emission spectrum of the substances was recorded and the integral of the emission spectrum was calculated. Equations 1 and 2 describe the relation between the laser power (P) and the fluorescence emission intensity (I): I / P2 ; log I / 2 log P: 1 2

1.4 1.2 1.0

pH = 4 pH = 7 pH = 11

Absorbance

0.8 0.6 0.4 0.2 0.0 250 300 350 400 450

Wavelength / nm

Fig. 3 Absorption spectra of AFB1 in the model solution with different pH values

efficiency is known. Although the fluorescence method provides much better sensitivity, accurate determination of absolute TPA cross-sections is still hampered by the strong dependence of absorption rates on the temporal and spatial coherence of the excitation light: R S S S R F dn C f s2 s2 : 3 F S dn C f For the calculation of the TPA cross-section using Eq. 3, the quantum yields of the substances in the different solutions are needed. In Eq. 3, the index S refers to the reference fluorescein, 2 is the TPA cross-section, F is the integral of the fluorescence spectrum, C is the concentration and f is the fluorescence quantum yield [22]. The spectra were corrected by a correction factor to account for the wavelength dependence of the response functions of the photomultiplier tube.

In the data evaluation plotting the fluorescence intensity versus the laser power on a loglog scale, a slope of (nearly) 2 indicates that a two-photon transition is observed. Direct measurement of TPA cross-sections is usually difficult because only a small fraction of photons are absorbed in a two-photon process. TPIF is an alternative approach for determining TPA cross-sections, provided that the material is fluorescent and that its fluorescence quantum

1.4

Rel. fluorescence intensity / a.u.

1.2 1.0

Fluorescence excitation AFB1 in water AFB1 in ethanol

Fluorescence emission AFB1 in water AFB1 in ethanol

2.5

1.4

Rel. fluorescence intensity (ex = 360 nm)

Absorbance AFB1 in white wine AFB1 in model solvent

Fluorescence emission AFB1 in white wine AFB1 in model solvent

1.2 1.0 0.8

2.0

Absorbance

0.8 0.6 0.4 0.2 0.0 250 300 350 400 450 500 550 600 650

1.5 0.6 1.0 0.4 0.5 0.2 0.0 600

0.0 200

250

300

350

400

450

500

550

Wavelength / nm

Wavelength / nm

Fig. 2 Fluorescence excitation as well as fluorescence emission spectra of aflatoxin B1 in water and ethanolic solution

Fig. 4 Absorption and fluorescence emission spectra of AFB1 in wine and the model solvent at pH 4

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Table 1 Absorption maxima (1abs,max), absorption coefficient (), fluorescence emission maxima (1em,max) and fluorescence quantum efficiency (f) of aflatoxin B1 in different solvents Solvent Water Ethanol Model solvent, pH 4 1abs,max (nm) (, lmol1 cm1) 224 (16,800) 224 (22,800) 224 265 (11,400) 265 (12,200) 265 365 (18,200) 360 (21,500) 365 (18,800) 1em,max (nm) 440 430 440 f(%) (1ex =360nm) 1.3 4.2 3.8

Results and discussion Absorption and steady-state fluorescence In Fig. 2 the fluorescence excitation and emission spectra of AFB1 in water and in ethanol at pH 7 are shown. A red shift of the fluorescence emission maxima is symmetric to a spectral shift found in the corresponding excitation maxima. In ethanol the fluorescence emission maximum is observed at 430 nm and in water at 440 nm. Generally, an increase of solvent polarity causes a red shift of the absorption/excitation maximum, e.g. it indicates the * character of the 365-nm band. The emission spectra in both solvents show a distinct mirror symmetry in relation to the long-wavelength maximum of the fluorescence excitation spectra [23]. Figure 3 shows the absorption spectra of AFB1 in the model solvent at different pH values. The spectrum of AFB1 in neutral solution shows three typical bands in the UV/vis range. AFB1 has two intense absorption bands around 224 and 360 nm as well as a lower band at 265 nm. With increase of pH to 11, a broader band and a small, but distinct bathochromic shift to 365 nm can be seen. At pH around 4, there is a hypsochromic shift to 340 nm and a decrease of intensity of the other spectral bands. The shape of the spectra and the formation of a shoulder suggest the
350

formation of a second species with decreasing pH, which may refer to formation of alfatoxin B2A, from photochemically induced hydroxylation of AFB1 with the addition of water [24]. The absorption and fluorescence spectra of AFB1 in wine, e.g. white wine, and the model system (pH 4) were found to be almost identical (Fig. 4). In Table 1, the basic spectroscopic parameters of AFB1 are summarized. It can be seen that the solvents tested influence the absorption and fluorescence emission spectra as well as the fluorescence quantum efficiencies of AFB1 only moderately. Two-photon-induced fluorescence and two-photonabsorption cross-section Because of the strong intrinsic absorption of beer and wine, a direct selective excitation of AFB1 is very difficult in such matrices. As an alternative approach, TPIF was tested. Therefore, the dependence of the observed fluorescence induced at 1ex =720 nm on the laser power as well as the TPA cross-section were evaluated. In the TPIF experiment, a grey-wheel filter was used to adjust the laser power (at 1ex =720 nm). The solution of the mycotoxin in red wine was diluted 1:10 because of strong scattering effects. The TPIF emission was measured in the wavelength range 350 nm< 1 <650 nm. The experimental

Fluorescence intensity / counts ex = 720 nm

300 250 200 150 100

white wine rose wine beer

10 10

Intensity

10 10 10

Fluoresceine Aflatoxin B1 in: model solvent beer rose wine white wine

50

10
0 350 400 450 500 550 600 650

60

80

100

120

140 160 180 200

Laser power / mW

Wavelength / nm

Fig. 5 Two-photon-induced fluorescence emission spectra of beer as well as of rose and white wine (1ex =720 nm)

Fig. 6 Double-logarithmic plot of the intensity versus laser power of fluorescein as a reference and AFB1 in the model solvent and alcoholic beverages

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emission spectra of AFB1 in alcoholic beverages were corrected for the intrinsic fluorescence contribution (due to ingredients) of the beverages investigated. Figure 5 shows the TPIF emission spectra of the alcoholic beverages with 1ex =720 nm. The spectra of the intrinsic background fluorescence are very similar for the beverages under investigation. The results of the laser power emission intensity measurements of AFB1 in the model system as well as in the alcoholic beverages are shown in Fig. 6. For all systems investigated, the slopes of the linear fits obtained were around 2 and confirm the presence of TPIF. For the determination of the TPA cross-section, the fluorescence quantum yield of AFB1 in the different beverages is needed. In the present approach, the quantum yields determined under regular (one-photon) excitation conditions (1 =360 nm) were used, which seems to be justified since the spectral distribution of the fluorescence spectra obtained under one-photon and two-photon excitation are identical (see Fig. 7); this has also been reported for other systems [25, 26]. From comparison of the fluorescence spectra (see Fig. 7), it can be concluded that the same electronically excited state (independent of OPA or TPA) is the origin of the apparent fluorescence and, consequently, the fluorescence quantum yields obtained from one-photon excitation experiments may be used in the TPA cross-section calculations. An average value of 2.3% for the fluorescence quantum yield of AFB1 in the different beverages was determined, which is smaller than for the model system. This could be related to an additional fluorescence quenching effect of ingredients present in the beverages [27]. The calculation of the TPA cross-section of AFB1 in different solvents using Eq. 3 yields comparable values for all beverages investigated (Table 2).Within the experimental accuracy, in the model solution (27 GM) the TPA cross-

Table 2 Two-photon-absorption cross-section (2) of aflatoxin B1 in different solvents Solvent Model solvent Beer White wine Rose wine Red wine (1:10 dilution)a
a

2 (GM) of aflatoxin B1 273 265 273 245 238

Dilution was made by adding model solvent

section is as high as in the alcoholic beverages (approximately 25 GM). Limit of detection To test the potential applicability of TPIF for the determination of AFB 1 in alcoholic beverages, test solutions were spiked with known amounts of AFB1. The limit of detection (LOD) was determined for the onephoton-induced fluorescence (OPIF) and TPIF with excitation at 360 and 720 nm, respectively. The TPIF emission intensity of each spectrum was evaluated in the wavelength range 435 nm < 1 <445 nm and corrected for contributions from the intrinsic background emission of the beverage investigated. In this work, the LOD was determined using calibration data (different concentration of AFB1) and regression statistics [28]. The results obtained are summarized in Table 3. The LOD of AFB1 determined in the model solvent is excellent, less than 1 ppb (equivalent to micrograms per litre) for the tests of both excitation conditions. A major factor here is that there are no disturbing background signals from the solvent. In wine and beer, a LOD of 31 ppb<LOD<43 ppb in the different wines and 62 ppb in beer in the case of OPIF was determined. The LOD calculated from the TPIF measurements is improved, which can mainly be attributed to a decreased contribution from matrix-related background signals.

1.4

1.4

Norm. fluorescence intensity / a.u. ex = 360 nm

Aflatoxin B1 in white wine

1.2 1.0 0.8 0.6 0.4 0.2 0.0 350

Norm. fluorescence intensity / a.u. ex = 720 nm

after one-Photon-induced fluorescence after two-Photon-induced fluorescence

1.2 1.0 0.8 0.6 0.4 0.2 0.0 650

Table 3 Limit of detection (LOD) of aflatoxin B1 in the model solution and alcoholic beverages: comparison of one-photon-induced fluorescence (OPIF; 1ex =360 nm) and two-photon-induced fluorescence (TPIF; 1ex =720 nm) Model solution White wine Rose wine Beer LOD (ppb) OPIF <1 TPIF <1 31 6 43 20 62 46

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450

500

550

600

Wavelength / nm

Fig. 7 Fluorescence emission spectra of AFB1 in white wine after one-photon-induced and two-photon-induced fluorescence

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Summary The absorption and fluorescence properties of AFB1 were analysed in different solvents. The positions of the absorption maxima and the fluorescence excitation as well as the emission maxima depend on the solvent and the pH. The formation of alfatoxin B2A with increasing pH (adding NaOH) is observed as the generation of a shoulder in the absorption spectra. In the spectra a mirror symmetry between absorption and emission was observed. The results of direct optical detection of AFB1 obtained for the model solvent were applied in real-world samples, wine and beer. In these matrices, the mycotoxin showed spectroscopic properties similar to those in ethanol and water. The determination of the TPA cross-section was carried out for AFB1 at 720 nm. A comparison of the OPIF and TPIF results showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon-excitation conditions. No differences were found for the alcoholic beverages investigated. The spectroscopic results presented are promising for the development of an improved in situ analysis and as an online-method for non-invasive and non-destructive determination of AFB1 in the brewery and in wine production. Other mycotoxins, such as ochratoxin A, which occur in wine and beer too may also be monitored using TPIF. Here, work is in progress to determine the corresponding photophysical properties in real-world samples, such as alcoholic beverages.

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Acknowledgements The authors express their thanks to the German Federal Ministry of Education and Research (BMBF) and the project executing organisation Jlich (PtJ) for financial support within the joint research project ProSenso.net2 (PSn2).