MS method for the simultaneous quantification of free and

Wageningen Academic P u b l i s h e r s

World Mycotoxin Journal, August 2008; 1(3): 237-246

A LC/MS/MS method for the simultaneous quantification of free and masked

http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2008.x040 - Thursday, August 20, 2015 1:03:13 PM - IP Address:117.169.6.88

fumonisins in maize and maize-based products C. Dall’Asta1, G. Galaverna1, G. Aureli2, A. Dossena1 and R. Marchelli1 1Dipartimento di Chimica Organica e Industriale, Università degli Studi di Parma, Viale G.P. Usberti 17/A, 43100 Parma,

Italy; 2C.R.A.-Istituto Sperimentale per la Cerealicoltura, Sezione Merceologia dei Prodotti, Via Cassia 176, 00191 Roma, Italy; [email protected] Received: 28 June 2007 / Accepted: 17 December 2007 © 2008 Wageningen Academic Publishers

Abstract An LC-ESI-MS/MS method for the simultaneous detection of the main fumonisins and their hydrolysed derivatives is described, allowing for a simplified sample preparation without previous clean up. The method has a very low quantification limit (10 µg/kg for FB1, 12 µg/kg for FB2 and FB3, 70 µg/kg for HFB1, HFB2 and HFB3 in maize flour) and a very good recovery for all the analytes. The method has been applied to check several maize-based foods for the presence of free and bound forms of fumonisins, the latter being determined after alkaline hydrolysis as hydrolysed derivatives. Bound fumonisins were found to be present not only in thermally treated maize-based products but also in mild processed or even raw products (pasta, bread, cakes, crisps, flour) and they were always present in almost similar or even higher amounts than the free forms. Osborne fractions of maize proteins showed that fumonisins were particularly bound to prolamins and glutelins. Model systems and extracts of these protein fractions gave positive response to ELISA tests, thus confirming the cross reactivity of these masked forms. Keywords: free fumonisins; bound fumonisins; masked mycotoxins; ELISA; mass spectrometry

1. Introduction O

OH

O

O

Fumonisins are a group of structurally related mycotoxins, produced mainly by Fusarium verticillioides and F. proliferatum, which are the most important seed-borne fungi associated with maize (Sewram et al., 2005). These mycotoxins, often found as natural contaminants of maize and maize-products for animal feed as well as for human consumption, are structurally characterised by a 20 carbon aminopolyhydroxy-alkyl chain diesterified with propane-1,2,3-tricarboxylic acid (tricarballylic acid, TCA) (Figure 1).

Figure 1. Chemical structures of the main fumonisins.

Fumonisin B1 (FB1) is known to cause a range of speciesspecific toxic responses (Bezuidenhout et al., 1988; Gelderblom et al., 1988), such as leucoencephalomalacia in horses, pulmonary oedema in swine as well as hepatocarcinogenic, hepatotoxic, nephrotoxic and

cytotoxic effects in rodents. Moreover, consumption of maize highly contaminated with fumonisins has been associated with increased risk of human oesophageal cancer and clear evidence for the carcinogenic activity of

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2008.x040

R1

O

HO

CH3

HO O

O

OH

O

O

CH3

OH

R2

NH2

FB1: R1 = OH, R2 = OH FB2: R1 = OH, R2 = H FB3: R1 = H, R2 = OH

237


C. Dall’Asta et al.

fumonisin B1 has been found in rodents (Voss et al., 2005). For these reasons, fumonisin B1 has been declared by the International Agency for Research on Cancer (IARC) as a class 2B carcinogen. The European Union has recently enforced the new legislation for fumonisins in food: according to this regulation the limits for total fumonisins in maize (4,000 µg/kg), maize-based product for human consumption (1,000 µg/kg), maize-based breakfast cereals and maize-based snacks (800 µg/kg) and in baby-food (200 µg/kg) have been established (EC No 1126/2007). Fumonisins are relatively heat stable and are appreciably degraded only at higher temperatures such as those connected to baking, roasting or extrusion or, alternatively, by alkali processing. In particular, alkali treatment (nixtamalization) leads to the formation of hydrolysed fumonisins (loss of TCA side chains) in processed foods (Scott and Lawrence, 1996; Humpf and Voss, 2004). Degradation was thought to be a method for detoxification: however, in a short-term liver cancer initiation/promotion model, hydrolysed fumonisin B1 (HFB1) revealed greater cytotoxicity than FB1 (Hartl and Humpf, 2000; Seefelder et al., 2003a). Indeed, experimental evidence showed that, despite the low absorption and bioavailability of FB1 after oral administration, toxic effects were recorded also after ingestion of low contaminated feed: this resulted in the formulation of the hypothesis that FB1-derivatives may be present and preferentially adsorbed, then reconverted to the active forms in the body. One of the hypotheses is that FB1 may be bound to macromolecular components such as carbohydrates or proteins, in particular in thermally treated or extruded products (cornflakes, etc.). Several reports on the fate of fumonisins during maize processing have shown that analytical methods often underestimate the levels of fumonisins in heat- or alkali plus heat-processed maize foods because of low recoveries (Scott and Lawrence, 1994; De Girolamo et al., 2001). This might be due to the binding of fumonisins to the food matrix or to the modification of fumonisins structure which lead to compounds not easily detectable by the normal methods of analysis. In an experiment using radio-labelled fumonisin B1 (FB1) added to maize meal dough, Shier et al. (1997) found that only 37% of the radioactivity was detected by the conventional analysis after roasting, while an additional 46% was extracted by a solution of sodium dodecyl sulphate (SDS), a detergent used to dissolve proteins. Shier et al. (1997) partially characterised the covalent binding of radiolabelled FB1 to maize proteins and starch. Seefelder et al. (2003b) demonstrated that upon thermal treatment fumonisin B 1 reacts with methyl-α-Dglucopyranoside and protected amino acids, as model compounds for starch and proteins, respectively. Adducts are formed via the tricarballylic acid moieties. Moreover, recently large amounts of N-fatty acylated derivatives of 238

fumonisins have been detected in tortilla chips by using radioactive studies (Shier et al., 2003). Therefore, the presence of bound (hidden) fumonisins conjugated to proteins and other food components in heatprocessed foods are of concern with respect to food safety, as they may be expected to release FB1 and HFB1 in the gastrointestinal tract (Shier, 2000). Recently, several authors (Kim et al., 2003; Park et al., 2004) demonstrated the presence of bound fumonisins in maize-based foods by using HPLC-FLD and HPLC-MS. Protein-bound fumonisins are extractable with SDS, which is removed by liquid-liquid partition and HFB1 released by hydrolysis is cleaned up by OASIS HLB extraction columns. Total-bound fumonisin is also purified by using the same clean-up column after alkali-hydrolysis of the food sample. Compared with FB1 detected by traditional analysis, about 1.3 and 0.9 times more FB1 in bound form is detected in alkali-processed maize-based food. In this study a method for the simultaneous determination of FB1, FB2, FB3 and their hydrolysed derivatives HFB1, HFB2 and HFB3 is described. Moreover, the optimisation of the alkaline hydrolysis conditions for the indirect quantification of bound fumonisins is reported and applied to both thermally treated and untreated maizebased foods. Finally, the interaction of several fumonisin derivatives with an antibody against FB1 are studied using a competitive ELISA assay.

2. Materials and methods Chemicals Fumonisin FB 1, FB 2 and FB 3 standards (1 mg) were purchased from Biopure (Tulln, Austria). All solvents used (LC grade) were obtained from Carlo Erba (Milan, Italy); bidistilled water was produced in our laboratory utilising an Alpha-Q system (Millipore, Marlborough, MA, USA). All salts were from Baker (Mallinckrodt Baker, Phillipsburg, NJ, USA), orthophosphoric acid and formic acid were from Fluka (Basel, Switzerland). The certified reference material T2204 (maize flour containing fumonisins FB1, FB2 and FB3: 1,600±300 µg/kg, 700±100 µg/kg, 200±100 µg/kg, respectively), tested under the supervision of the Food Analysis Performance Assessment Scheme (FAPAS) (DEFRA, Central Science Laboratories, York, UK), was used. N-α-acetyl-L-lysine methyl ester, glucose and starch were from Sigma-Aldrich (Steinheim, Germany).

Hydrolysed fumonisins preparation A standard solution of the three main fumonisins (50 µg/ ml of each, 5 ml) was prepared in acetonitrile:water 1:1 and evaporated to dryness. The residue was redissolved World Mycotoxin Journal 1 (3)


A LC/MS/MS method for the simultaneous quantification of free and masked fumonisins in maize and maize-based products

in 2 M NaOH, then allowed to react overnight. After hydrolysis, the mixture was extracted twice by liquid-liquid partition using ethyl acetate. The organic phase was pooled, evaporated under N 2 stream and redissolved in 1 ml acetonitrile:water. The reaction yield was checked by LCMS and found to be higher than 99%. Calibration curves were prepared by proper dilution of the standard solution, assuming the total conversion of the native compounds to the hydrolysed forms.

Sample preparation for the analysis of free fumonisins Aliquots (25 g) of finely ground maize or maize-based products were blended in a high speed blender (Ultraturrax T25, IKA, Stauffen, Germany) with 100 ml of water: acetonitrile:methanol (50:25:25, v/v) for 5 min at 6000 rpm. The extract was then filtered through Whatman No. 4 filter papers. An aliquot of the filtrate (4 ml) was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 1 ml of water:acetonitrile 1:1 v/v, filtered through a 0.45 µm nylon filter and analysed by LC-MS/MS.

Sample preparation for the analysis of bound fumonisins After extraction of the free forms, the residue was washed with 50 ml of water:acetonitrile:methanol (50:25:25, v/v) and dried under a vacuum bell overnight. Then, 1 g of solid residue was subjected to alkaline hydrolysis (15 ml 2 M NaOH, 25 °C, 60 min). Afterwards, the aqueous phase was extracted twice with 15 ml ethyl acetate. The two phases were collected separately and checked for hydrolysed FB1, which was proven to be only in the organic layer. Thus the organic phases were pooled and evaporated by rotavapor. The residue was dissolved in 2 ml water:acetonitrile 1:1 v/v, filtered through a 0.45 µm nylon filter and analysed by LC-MS/MS.

LC-MS/MS analysis The LC-MS/MS system consisted of a 2695 Alliance (Waters Co., Milford, MA, USA) equipped with a QuattroTM triple quadrupole mass spectrometer with an electrospray source (Micromass, Waters, Manchester, UK). Chromatographic conditions were the following: column, C18 XTerra Waters narrow bore (250×2.1 mm, 5 µm) equipped with a C18 precolumn cartridge; flow rate, 0.2 ml/min; column temperature, 30 °C; injection volume, 10 µl. Gradient elution was performed using water (eluent A) and methanol (eluent B), both acidified with 0.1% formic acid: 0-3 min, isocratic step 100% A, switched to the waste in order to wash out the salts and to focus the analytes on the C18 precolumn cartridge; 3-5 min to 45% B; 510 min isocratic 45% B; 10-25 min to 85% B, 25-35 min isocratic 85% B; finally, a re-equilibration step at 100% A (initial conditions) for 10 minutes was performed (total analysis time, 45 minutes). MS parameters: ESI+ (positive ion mode); capillary voltage, 3.2 kV; cone voltage, 30 V; extractor voltage, 3 V; source block temperature, 120 °C; desolvation temperature, 160 °C; desolvation and cone gas (nitrogen) 650 l/h and 70 l/h, respectively. Detection was performed using a multiple reaction monitoring (MRM) mode, by monitoring three transitions for each analyte, as reported in Table 1. The main transition was used for quantification, while two more transitions were chosen as qualifiers. Linearity and calibration experiments were based on the analysis of spiked fumonisin-free maize samples at six concentration levels in the range 250-4,000 µg/kg (three determinations at each level were performed). Recovery experiments were performed by spiking a blank maize sample at two concentration levels close to the proposed EU legal limits for final products and raw materials (500 µg/kg and 2,000 µg/kg, respectively). The detection limits (LOD) and the quantification limits (LOQ) were determined by

Table 1. MRM conditions for LC-ESI-MS/MS analysis of fumonisins (relative abundance of mass ions in brackets). Compound-specific ions (m/z)

FB1 FB2 FB3 HFB1 HFB2 HFB3

Precursor ion [M+H]+

Main transition

Identification

CE (eV)

First qualifier

CE (eV)

Second qualifier

CE (eV)

722.4 706.4 706.4 406.5 390.5 390.5

352.5 (68) 336.5 (80) 336.5 (80) 334.5 (57) 318.5 (58) 318.5 (58)

[M+H-2TCA-H2O]+ [M+H-2TCA-H2O]+ [M+H-2TCA-H2O]+ [M+H-4H2O]+ [M+H-4H2O]+ [M+H-4H2O]+

35 35 35 21 21 20

334.6 (20) 354.2 (15) 354.2 (15) 370.5 (21) 354.5 (26) 354.5 (26)

35 35 35 13 13 15

704.7 (13) 688.6 (5) 688.6 (5) 388.5 (22) 372.5 (16) 372.5 (16)

27 27 35 11 11 15

Figures in italics refer to relative abundance of mass ions. TCA = Tricarballylic acid moiety; CE = Collisional Energy.

World Mycotoxin Journal 1 (3)

239


spiking blank samples with proper amounts of each analyte (FB1, FB2, FB3, HFB1, HFB2 and HFB3) and were calculated at a signal to noise ratio of 3:1 and 10:1, respectively.


Proteins isolation from maize (Osborne fractions) The main maize protein groups (albumins, globulins, glutelins and prolamins) have been isolated from raw maize using the Osborne fractions method. Briefly, the protein groups have been isolated from maize according to their solubility, using the following scheme. First, raw maize or maize-based product was extracted with water to obtain the albumin fraction. Then, globulins were dissolved by 0.4 M NaCl solution. The residue was then extracted with ethanol:water 70:30 (v/v) to obtain the prolamins. Finally, a last extraction step was performed with a solution of 1-propanol:water 1:1 (v/v) containing 1% dithiothreithol to obtain the glutelin fraction. Each fraction has been collected and purified on a Waters Oasis C18 cartridge: after a conditioning step with methanol (2 ml) and water (2 ml), the fraction (4 ml) was applied on the cartridge. A washing step with bidistilled water was performed to separate starch and sugars from the sample, then the proteins were eluted with acetonitrile (2 ml). Before and after purification, the co-occurrence of carbohydrates were tested using the Fehling test for reducing sugars and the iodine test for starch. Both tests were negative after purification on SPE cartridges.

Model reactions of fumonisin B1 with lysine, glucose and starch Model reactions using fumonisin B1 were performed in vitro according to Seefelder et al. (2003b). In particular, FB1 was reacted with N-α-acetyl-L-lysine methyl ester, glucose and starch at 100 °C for 60 minutes, respectively. Blank samples were also obtained by heating FB1 alone under the same conditions. The reaction with glucose was also performed for HFB1. The reaction mixtures were analysed using LC-ESI-MS.

Immunoenzymatical analysis of FB1 derivatives using ELISA assay The FB1 derivatives and the maize fractions containing bound fumonisins were analysed using a competitive ELISA assay, according to the manufacturer’s instructions (R-Biopharm Ridascreen® Fumonisin assay).

µg/ml, water:acetonitrile 1:1 v/v). The same experiments were also performed for the hydrolysed fumonisins, obtaining the best ionisation for both groups in the positive ion mode. For each analyte, the most abundant transition was chosen for MRM quantification, while two other transitions were established as qualifiers, as reported in Table 1. The LC-MS/MS analysis was performed using a C18 column and a water-acetonitrile mobile phase acidified with 0.1% formic acid, using a gradient elution. Recovery experiments were performed for FB1, FB2, FB3 and their hydrolysed forms HFB1, HFB2 and HFB3, by spiking a maize flour sample at two concentration levels (500 µg/kg and 2,000 µg/kg). The results are reported in Table 2. The recovery is higher than 90% in all cases. Moreover, our procedure precludes the need for the purification step, which is the most important source of error in the determination of hydrolysed forms, giving higher recoveries for these derivatives in comparison to those reported by many authors (Maragos et al., 1996; Smith and Takhur, 1996; Churchwell et al., 1997). The sensitivity was also good for all the considered analytes (see Table 2), being the LOD and LOQ values comparable with those from other recently published LC-MS/MS methods, although those methods required a sample purification and pre-concentration step (Cavaliere et al., 2005; Faberi et al., 2005; Paepens et al., 2005). Detection limits were slightly higher for HFBs, probably on account of a lower ionisation efficiency in comparison to their precursors due to the loss of the TCA moieties and to the consequent lower polarity of the hydrolysed forms. In order to estimate the matrix effect, which can greatly affect the analyte response in biological matrices, the peak areas of each matrix-matched standard calibration curves obtained for maize flour samples were compared to normal Table 2. Limits of detection, limits of quantification and accuracy of the method for FB1, FB2 and FB3, HFB1, HFB2 and HFB3 in maize flour. Accuracy is expressed as recovery (n=3) of spiked samples. Compound LOD (μg/kg)

LOQ (μg/kg)

3. Results and discussion LC-MS/MS method development and optimisation The optimisation of the fumonisin ionisation and fragmentation in the electrospray source was performed by direct infusion of FB1, FB2 and FB3 standard solutions (2.5 240


1 1 8 20 25 20

5 5 12 70 70 70

Recovery (%) 500 μg/kg

2,000 μg/kg

95 96 95 93 94 95

97 98 96 93 92 98




standard calibration curves using two-tailed paired t-test at 95% confidence limit, as reported in Table 3. The |t|-values of these curves which were all lower than a critical value (P=0.05) of 2.18 (n=12) imply that the data of these two sets are not significantly different. No matrix effect was indeed observed for maize flour during this study. On the contrary, an ion enhancement effect was observed when a great number of samples (n>100) was run in the same batch, thus the source was routinely cleaned after 80 samples. The repeatability of the method was tested by analysing the maize flour blank samples spiked at two contamination levels (500 µg/kg and 2,000 µg/kg) and both intra-assay (six extractions at each level on the same day) and intermediate precision (six extractions at each level on two days) were calculated. The results were satisfactory at all the concentration levels, the RSDs being lower than the critical

value obtained by the Horwitz equation for the considered concentration levels according to the Commission Decision 2002/657/CE, as reported in Table 4. The method has been applied to different maize-based food products. As an example, the natural occurrence of native and hydrolysed fumonisins in contaminated maize flakes is reported in Figure 2. The accuracy of the method was also checked with a Certified Reference Material (CRM, maize flour), contaminated with FB1 (declared value: 1,600±300 µg/kg), FB2 (declared value: 700±100 µg/kg) and FB3 (declared value: 200±100 µg/kg). The following concentrations were obtained: 1,622±30 µg/ kg for FB1, 640±10 µg/kg for FB2, 220±12 µg/kg for FB3. The results showed a good accuracy, with a mean recovery for the simplified procedure of 95±2%. A comparison with the CRM declared concentrations showed a very good z-score value lower than 0.6 for all three analytes.

Table 3. Matrix effect evaluation (maize flour): comparison between standard and matrix calibration curves for FB 1, FB2, FB3, HFB1, HFB2 and HFB3. Compound


Standard calibration curves

Matrix calibration curves

t-value

Slope

Y intercept

r2

Slope

Y intercept

r2

36.8 29.5 15.9 4.0 3.8 4.0

-11.48 -13.12 10.95 -18.30 57.5 -16.3

0.9998 0.9997 0.9978 0.9998 0.9999 0.9992

43.2 29.6 19.8 3.4 3.3 4.2

-14.30 -9.85 7.17 -17.93 52.2 -18.0

0.9997 0.9999 0.9999 0.9997 0.9998 0.9993

0.119 0.922 0.146 0.225 0.249 0.015

Linear regression parameters from standard and matrix calibration curves: range from 50-5,000 µg/kg (6 points, duplicate analyses) and from 2002,000 µg/kg (6 points, duplicate analyses) for FBs and HFBs, respectively. |t|-values of two-tailed paired t-test at 95% confidence limit: |tcrit| = 2.18.

Table 4. The method precision expressed as RSD of spiked maize samples. RSD% at 2,000 μg/kg


RSD% at 500 μg/kg

Day 1a

Day 2a

Overallb

Acceptable valuec

Day 1a

Day 2a

Overallb

Acceptable valuec

7.6 9.8 8.5 4.5 4.0 5.1

4.3 10.4 6.8 4.4 4.7 4.5

5.1 0.3 0.7 0.3 0.6 1.1

14.1

13.9 18.2 15.7 12.2 11.4 13.2

9.8 10.1 18.6 10.5 7.6 11.1

3.8 9.3 6.9 1.4 1.9 2.2

18.9

a

Intra-assay precision of data analysed within the same day (n=6). Intermediate precision of data analysed on different day (n=2). c RSD values obtained from Horwitz equation (RSDr = 0.67×2(1-0.5 logC)). b


241


2: MRM of 3 Channels ES+ TIC 1.61e4

16.39

100

%

HFB1

0 5.00

10.00

15.00

20.00 18.85 20.20

25.00

30.00

35.00

40.00

45.00 1: MRM of 3 Channels ES+ TIC 1.76e4

25.00

30.00

35.00

40.00


25.00

30.00

35.00

40.00


25.00

30.00

35.00

40.00

HFB2

%

HFB3

0

5.00

10.00

15.00

20.00 21.74

100

%

FB3

0

5.00

10.00

15.00

20.29

20.00

FB2

18.24

100

FB1

%


100

0

5.00

10.00

15.00

20.00

45.00

Time

Figure 2. MRM chromatogram of a corn flakes naturally contaminated with FB1, FB2, FB3 and the hydrolysed forms HFB1, HFB2 and HFB3.

Bound fumonisins Several commercial maize-based products have been analysed for total and bound fumonisins. The amount of bound derivatives was found to be very close or even higher than the free toxins in all the analysed samples. As an example, results obtained with a contaminated maize flakes sample are reported in Figure 3. In this case, the amount of HFB1 released after hydrolysis (bound fumonisin) is 4 times higher than the sum of FB1 and HFB1. Many other samples showed a similar situation (Figure 4). Interestingly, also in those samples in which free fumonisin contamination was found to be lower than the proposed legal limits (800 µg/kg), the sum of free and bound forms led to this value being exceeded, thus posing a new problem as regards the determination of total fumonisins in foods. Moreover, bound forms were found to occur not only in thermally extruded products (i.e. cornflakes), as already suggested by several authors (Kim et al., 2003; Park et al., 2004; Seefelder et al., 2003b), but also in mild processed foods (i.e. maize-based bread, cakes) and even maize flour. These data suggested that the transformation of native fumonisins to bound derivatives may be also ascribed to natural phenomena due to the plant metabolism, as already proven for other Fusarium mycotoxins such

242

as deoxynivalenol (Berthiller et al., 2005, 2006) and zearalenone (Schneweis et al., 2002). In order to study which macromolecular components preferentially bind fumonisins, we separated carbohydrates and proteins by selective extraction and chromatographic separation. In order to investigate if fumonisins are bound to proteins, as expected on the basis of the literature reports, Osborne fractions of maize proteins were obtained using sequential extraction with solutions of different polarity (starting from water up to isopropanol:water mixtures). Each fraction was successively purified on C18 SPE columns in order to separate carbohydrates (in particular, from the most water soluble fractions) and hydrolysed to be analysed by LC-MS/MS. After hydrolysis, significant amounts of HFB 1 were found among globulins and prolamins (Figure 5). Since the iodine test for starch was positive before SPE purification and negative after clean up for the more polar fractions (albumins and globulins), the washing solution was collected and found to contain starch. Thus, this fraction was also hydrolysed under alkaline conditions in order to check whether hidden fumonisins occurred. The LC-MS/MS analyses showed that no HFB1 was released from starch under alkaline conditions. Since this approach was essentially qualitative, we were not able to quantify the amount of hydrolysed fumonisins World Mycotoxin Journal 1 (3)


Mercanti_Post Sm (Mn, 2x3) 100

1: MRM of 5 Channels ES+ TIC 3.98e5 Area

13.58 434385

%

HFB1 from bound FB1 (820 µg/kg)

7.50

10.00

12.50

15.00

17.50

20.00

22.50

27.50

30.00

32.50

35.00

37.50

40.00

Free FB1 (120 µg/kg)

18.12 116951

0 2.50 5.00 Mercanti Sm (Mn, 2x3) 100

25.00


%

2.50 5.00 Mercanti Sm (Mn, 2x3) 100

7.50

10.00

12.50

15.00

17.50

20.00

22.50

25.00

27.50

30.00

32.50

35.00

37.50

40.00


%

Free HFB1 (120 µg/kg)

13.36 46585 0

2.50

5.00

7.50

10.00

12.50

15.00

17.50

20.00

22.50

25.00

27.50

30.00

32.50

35.00

37.50

Time

40.00

Figure 3. Free (FB1 and HFB1) and bound fumonisins in cornflakes sample.

500 450 400 Concentration (µg/kg)


0

350 300 250 200 150 100 50 0

pasta

cornflakes 1 cornflakes 2 cornflakes 3 cornflakes 4

free FB1

flour 1

flour 2

cakes

crisp

bread

bound FB1

Figure 4. Free and bound fumonisins in different food samples.

associated with protein fraction. We can’t therefore speculate as to whether the sum of the HFB1 protein fraction equal the HFB1 amount released in the sample after hydrolysis. Further experiments should be performed to check the occurrence of bound fumonisins also associated with other food macroconstituents.


ELISA analysis of fumonisin B1 derivatives In order to check the cross-reactivity of an anti-fumonisin antibody with FB1 derivatives, several reactions have been performed ‘in vitro’ using both FB1 and HFB1, according to Seefelder et al. (2003). The reaction mixtures were analysed by LC-MS/MS, proving the formation of N-deoxyfructosyl243


H_glut_M Sm (SG, 3x1) 100

MRM of 4 Channels ES+ TIC 7.31e4

%

Glutelins

0 6.00

8.00

10.00

7.72 754

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00 MRM of 4 Channels ES+ TIC 7.31e4

24.00

26.00


24.00

26.00


24.00

26.00

HFB1

%

Prolamins

0

2.00 4.00 H_glob_M Sm (SG, 3x1) 100

6.00

8.00

%

7.81 764

0

2.00 4.00 6.00 H_alb_M Sm (SG, 3x1); Sm (SG, 3x1) 100

8.00

10.00

12.00

14.00

16.00

18.00

HFB1

10.00

20.00

22.00

Globulins

12.00

14.00

16.00

18.00

20.00

22.00

Albumins %


2.00 4.00 H_prol_M Sm (SG, 3x1) 100

0

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

28.00

Time

Figure 5. Distribution of bound fumonisins in the protein fractions of maize (cornflakes sample).

FB1 (NDF-FB1) and N-deoxy-fructosyl-HFB1 (NDF-HFB1) from the reaction of FB1 and HFB1 with glucose and the formation of both the mono- and di-lysil-fumonisin B1 derivatives from FB1 and N-α-acetyl-lysine methyl ester. The synthesised derivatives were purified using RPHPLC (same conditions reported for FB1 analysis in the experimental part) and used for a competitive ELISA assay, in order to evaluate whether the anti-fumonisin antibody is also able to bind masked forms of the mycotoxin. After chromatographic purification, all the derivatives were checked by LC-MS/MS for the absence of free FB1, which could give a false positive result to the ELISA. The reaction of FB1 with starch also led to the formation of different derivatives, which have not yet been characterised. First, a preliminary ELISA test was performed using fumonisin B1 and its hydrolysed form HFB1: the results confirmed the reactivity of the antibody towards fumonisin B1, whereas no signal was reported in the presence of the hydrolysed form HFB1. These data is in agreement with several authors reporting that the tricarballylic side chains are required for the antibody recognition of fumonisins (Barna-Vetró et al., 2000).

244

The ELISA assay was then repeated using the synthesised derivatives NDF-FB1, NDF-HFB1, mono- and di-lysyl-FB1, as well as the adduct formed by fumonisin B1 and starch. Moreover, the ELISA experiment was also performed using a protein fraction extracted from contaminated cornflakes and found to release HFB1 after alkaline hydrolysis, in order to check whether the antibody was able to give crossreaction with bound fumonisins. The results are reported in Table 5, where a negative response is depicted as ‘–’, a positive response is reported as ‘+’, a good signal is reported as ‘++’ and a very high interaction is reported as ‘+++’. Table 5. ELISA response to different free and bound fumonisin forms. Target Standard solution

Response

FB1 HFB1 Model system NDF-FB1 NDF-HFB1 Lysil-FB1 FB1-starch adduct Sample naturally containing Protein extract (prolamin bound fumonisins from cornflakes)

++ – + – +++ + +++




These preliminary data showed that the antibody against FB1 is able to cross-react with NDF-FB1 and with the starch-FB1 adduct. Moreover, the response recorded for lysyl-fumonisin derivatives was very strong, suggesting a good interaction between the antibody and the amino acidic derivatives of the mycotoxins. Also, the protein extract containing bound fumonisins gave a very intense response to ELISA, suggesting that the binding naturally occurring among FB1 and the prolamin fraction may be comparable to what was obtained for the lysyl-FB1 model system. It is well known that the ELISA test may often overestimate the FB1 level in naturally contaminated samples. This phenomenon is usually ascribed to the cross-reactivity of the antibody towards several interfering compounds occurring in the matrix, probably formed by fumonisins’ reaction with reducing sugars (Meister, 2001). The preliminary experiments performed in this study suggested that this over-estimation may also be due to the occurrence of protein bound-fumonisin, which can also be recognised by the antibody.

4. Conclusions This LC/MS/MS method without sample clean up allows for the determination of free and bound fumonisins (after alkaline hydrolysis) in maize-based foods. A preliminary survey of several maize-based products showed that bound-fumonisins occurrence is not restricted to thermally treated products, but may also be found in raw products. Thus, besides thermal effects, as reported in the literature, other masking mechanisms should be taken into account for the evaluation of the occurrence of bound fumonisins in food. In particular, plant metabolism may be responsible for the transformation of native fumonisins into bound conjugates due to the effect of chemical compartimentation exerted by the plant towards xenobiotics produced by inhabiting moulds. From the data reported in the present study, fumonisins seem to be particularly bound to maize prolamins and globulins. Moreover, antibodies against FB1 seem to be able to recognise protein-bound fumonisins both in model systems and in naturally contaminated samples. These results may partially explain the systematic over-estimation of FB1 levels obtained by ELISA tests, which may be due to the occurrence of protein-bound fumonisins.

Acknowledgements The present work has been performed within a project (SIQUAL) partially financed by the Emilia-Romagna Region (Italy). The authors are grateful to Dr. Maria Grazia D’Egidio (Istituto Sperimentale per la Cerealicoltura, Roma) for the helpful discussions.


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