Mycotoxin Res DOI 10.1007/s12550-014-0218-y
ORIGINAL PAPER
Structure elucidation and in vitro cytotoxicity of ochratoxin α amide, a new degradation product of ochratoxin A Andrea Bittner & Benedikt Cramer & Henning Harrer & Hans-Ulrich Humpf
Received: 29 October 2014 / Revised: 17 December 2014 / Accepted: 18 December 2014 # Society for Mycotoxin Research and Springer-Verlag Berlin Heidelberg 2015
Abstract The mycotoxin ochratoxin A is a secondary metabolite occurring in a wide range of commodities. During the exposure of ochratoxin A to white and blue light, a cleavage between the carbon atom C-14 and the nitrogen atom was described. As a reaction product, the new compound ochratoxin α amide has been proposed based on mass spectrometry (MS) experiments. In the following study, we observed that this compound is also formed at high temperatures such as used for example during coffee roasting and therefore represents a further thermal ochratoxin A degradation product. To confirm the structure of ochratoxin α amide, the compound was prepared in large scale and complete structure elucidation via nuclear magnetic resonance (NMR) and MS was performed. Additionally, first studies on the toxicity of ochratoxin α amide were performed using immortalized human kidney epithelial (IHKE) cells, a cell line known to be sensitive against ochratoxin A with an IC50 value of 0.5 μM. Using this system, ochratoxin α amide revealed no cytotoxicity up to concentrations of 50 μM. Thus, these results propose that the thermal degradation of ochratoxin A to ochratoxin α amide might be a detoxification process. Finally, we present a sample preparation and a HPLC-tandem mass spectrometry (HPLCMS/MS) method for the analysis of ochratoxin α amide in extrudates and checked its formation during the extrusion of artificially contaminated wheat grits at 150 and 180 °C, whereas no ochratoxin α amide was detectable under these conditions. Keywords Ochratoxin A . Degradation . Detoxification . Roasting . Extrusion . Ochratoxinα amide . Thermalstability . Light . Coffee A. Bittner : B. Cramer : H. Harrer : H.98 % (NMR) (Cramer et al. 2008). 14R-ochratoxin A, 14-decarboxy-ochratoxin A, and ochratoxin α were available from a previous study (Cramer et al. 2008). Fourier transformation mass spectrometry The exact mass measurement and the higher energy collision dissociation (HCD) fragmentation were carried out on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a direct flow infusion of 5 μL/min. Data acquisition was performed with Xcalibur 2.07 SP I (Thermo Fisher Scientific). The mass spectrometer was operated in the positive mode, and the ionization was performed with heated electrospray ionization. Further conditions were as follows: capillary temperature, 225 °C; vaporizer temperature, 50 °C; sheath gas flow, 10 units; auxiliary gas flow, 5 units; source voltage, 3.5 kV; tube lens, 55 V; and capillary voltage, 14 V. The HCD cell was used at the optimal relative energy of 65 % determined in a range from 10 to 70 %. The mass resolution was set to 30,000, and the maximum ion injection time was set to 100 ms. NMR spectroscopy 1
H-, 13C-, and 2D-NMR data were acquired on a 600-MHz DD2 NMR spectrometer (Agilent Technologies, Waldbronn, Germany). Signals are reported in parts per million relative to tetramethylsilane (TMS). For structural elucidation and NMR signal assignment, 2D NMR experiments, such as gradientselected correlated spectroscopy (gs-COSY), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear multiple-bond correlation (HMBC), were carried out. Pulse programs for these experiments were taken from the Bruker software library. Heating experiment
A 445-μL stock solution with an ochratoxin A concentration of 226 μg/mL in acetonitrile, prepared by dissolving 45.2 mg of ochratoxin A in 200 mL of acetonitrile using a volumetric flask, was evaporated in a 1.5-mL vial under a stream of nitrogen at 40 °C to achieve an ochratoxin A amount of 100 μg per vial. In this way, three vials were prepared, capped, and heated for 9 min at a temperature of 200, 220, and 240 °C, respectively. After cooling, the reaction mixture was dissolved in 1 mL of a mixture of acetonitrile/water/formic acid (20:80:0.1, v/v/v) and afterwards analyzed by HPLC with fluorescence detection (HPLC-FLD) and Fourier transform mass spectrometry (HPLC-FTMS). Each heating experiment was performed in triplicate.
HPLC-FLD The reaction mixture was analyzed by a HPLC system consisting of two LC-10ATVP pumps coupled with a SIL10AF autosampler and a RF-10A fluorescence detector (Shimadzu, Kyoto, Japan). Data acquisition was performed with LC solution software 1.25 (Shimdazu). The chromatographic separation of the thermal degradation products of ochratoxin A was carried out on a 150×4.6 mm inner diameter, 5 μm, Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) with a 4×3 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany) using a binary gradient. The column oven was set to 40 °C, and an injection volume of 10 μL was used. Solvent A was water and solvent B was acetonitrile both containing 0.1 % formic acid. The flow rate was set to 1 mL/min and the HPLC programmed isocratic for the first 3 min at 20 % solvent B followed by a linear gradient to 70 % solvent B at 20 min, which was held for 5 min. After each run, the column was equilibrated at the starting conditions for 5 min. The fluorescence detector was set to a wavelength of 330 nm for excitation and 460 nm for emission. HPLC-FTMS For HPLC-FTMS analysis, an Accela LC 60057-60010 system (Thermo Fisher Scientific, Bremen, Germany) was connected to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) which was used in the scan mode. Data acquisition was performed with Xcalibur 2.07 SP1 (Thermo Scientific). The thermal degradation products were separated on a 150×2.1 mm inner diameter, 5 μm, Agilent Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) with a 4×2 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany) using a binary gradient as described above but with a flow rate of 300 μL/min. The column temperature was 40 °C, and the injection volume was 20 μL. The mass spectrometer was operated in the positive mode, and the ionization was performed with heated electrospray ionization. The reaction products were measured in a scan mode in a mass range of m/z 100 to 1000 with a resolution of 30,000. Further conditions were as follows: capillary temperature, 225 °C; vaporizer temperature, 350 °C; sheath gas flow, 40 units; auxiliary gas flow, 20 units; source voltage, 3.5 kV; tube lens, 120 V; and capillary voltage, 49 V. Preparation of ochratoxin α amide Pure ochratoxin A (214.4 mg) was heated in a 4-mL screwcapped brown glass vial at 240 °C for 20 min. The reaction mixture was dissolved in 3 mL of acetonitrile/1 % formic acid (20:80, v/v) and sonicated for 10 min. Insoluble components
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were separated by membrane filtration using a Phenomenex®RC membrane syringe filter (0.45-μm pore size; 15-mm diameter) (Phenomenex, Aschaffenburg, Germany) followed by separation on a semi-preparative HPLC. For the isolation of ochratoxin α amide, the filtrate was purified in two steps on a 250×9.4 mm inner diameter, 5 μm, Agilent Eclipse XDBC18 column (Agilent Technologies, Böblingen, Germany) with a 4×3 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany). In the first step, ochratoxin α amide was separated from compounds with a higher retention on the C18 material by fractionation. The first fraction was achieved by using an isocratic mixture of acetonitrile/1 % formic acid (35:65, v/v) delivered by two Varian ProStar 210 HPLC solvent delivery modules (Varian, Darmstadt, Germany). The flow rate was set at 4 mL/min. The ochratoxin α amide containing fraction 1 was collected for the first 9 min after peak detection by a Varian ProStar 325 UV/vis detector (Varian, Darmstadt, Germany) set at 330 nm. The fraction 2, containing predominantly ochratoxin A and 14R-ochratoxin A, was obtained by a followed linear gradient to 100 % solvent B in 6 min, which was held for 5 min. The two fractions were separately collected over three runs and combined, and the solvent was removed under reduced pressure. Afterwards, fraction 1 was further purified to remove the ochratoxin α impurities from ochratoxin α amide. Therefore, the evaporated residue of fraction 1 was dissolved in 2 mL of acetonitrile/1 % formic acid (20:80, v/v) and separated on the column and HPLC system, as described above using an isocratic mixture of acetonitrile/1 % formic acid (20:80, v/v). After detection of the ochratoxin α amide peak, it was collected over four runs and combined. The solvent was removed under reduced pressure, and ochratoxin α amide was obtained as a white solid (1.60 mg, 6.26 μmol, 1.2 % yield). For the determination of the purity of the isolated ochratoxin α amide, a LC-20AT system (Shimadzu, Kyoto, Japan) was coupled to an Evaporative Light Scattering Detector (ELSD) (Shimadzu). Data acquisition was performed with LC solution 1.25 (Shimadzu). Separation was carried out on a 150× 4.6 mm inner diameter, 5 μm, Agilent Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) with a 4 × 3 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany) using a binary gradient. Solvent A was water and solvent B was acetonitrile both containing 0.1 % formic acid. The injection volume was 10 μL and the flow rate was set to 1 mL/min. The HPLC was programmed to run isocratic for the first 5 min at 20 % solvent B followed by a linear gradient to 100 % solvent B at 25 min. Afterwards, the column was washed with 100 % solvent B for 2 min and then equilibrated to starting conditions. Ochratoxin α amide ESI-MS (positive mode): HRMS m/z: 256.0371 (calculated formula [C 11 H 10 ClNO 4 + H] + (0.038 ppm). MS/MS (HCD, 65 %; [M + H]+) m/z (%):
239.0105 (100), 193.0050 (71), 221.0000 (44), 211.0156 (32), 165.0099 (25), 137.0150 (12), 257.0211 (7). 1H NMR (600 MHz, CDCl3) δ: 1.61 (3H, d, J=6.3 Hz, H-11), 2.87 (1H, dd, J=17.4, 11.6 Hz, H-4A), 3.31 (1H, dd, J=17.4, 3.4 Hz, H-4B), 4.82–4.74 (1H, m, H-3), 5.85 (1H, s, H-13A), 7.88 (1H, s, H-13B), 8.48 (1H, s, H-6), 12.82 (1H, s, C-8OH). 13C NMR (151 MHz, CDCl3) δ: 20.7 (C-11), 32.3 (C-4), 75.9 (C-3), 110.1 (C-9), 120.6 (C-7), 123.2 (C-5), 139.4 (C-6), 141.1 (C-10), 159.1 (C-8), 164.3 (C-12), 169.9 (C-1). Cytotoxicity assay (CCK-8) IHKE cells, which were kindly provided by Prof. Dr. M. Gekle (Institut für Physiologie, Martin-Luther-Universität Halle Wittenberg, Germany), were used for the cytotoxicity experiments. These cells were cultivated, as described by Tveito et al., and as previously applied by Cramer et al. (Cramer et al. 2010; Ishiyama et al. 1996; Tveito et al. 1989). The determination of the cytotoxicity was performed by a colorimetric assay using the Cell Counting Kit-8 (CCK8) (Dojindo, Laboratories, Tokyo, Japan) according to the manufacturer’s instructions the metabolic activity of the cells was measured. Therefore, the cells were seeded in 96-well plates. After a growth of 48 h, the cells were cultivated in a serum-free medium for 24 h in order to exclude any binding of the tested compounds to serum proteins. Then solutions of ochratoxin α amide were added to the cells in a concentration range from 0.01 nM to 50 μM (stock solution: 10 mM in methanol) and incubated for 24 h. Cells with an equal solvent concentration were incubated as control. Afterwards, the viability of the cells was measured as previously described (Cramer et al. 2010). The incubation time with the dye solution WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-(2.4-disulfophenyl)-2H-tetrazolium, monosodium salt] was changed to 45 min. This experimental setup was simultaneously done with ochratoxin α, ochratoxin A, and 14Rochratoxin A for comparison. Sampling For the extrusion experiments, commercial durum wheat grits were bought from a local supermarket (Münster, Germany). The natural moisture content of the durum wheat grits was determined by the weight loss after oven drying at a temperature of 130 °C to a constant weight and was measured to be approximately 12.6 %. For the preparation of a stock solution, 6.0 mg of ochratoxin A was dissolved in 25 mL of acetonitrile. The concentration of the stock solution was determined as described in the AOAC 2000.03 method by photometric analysis to be 232 μg/mL ochratoxin A (Entwisle et al. 2000). One kilogram of wheat grits containing no detectable amount of ochratoxin A was spiked with 86.2 μL of the ochratoxin A stock solution (232 μg/mL) and with 197-mL
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distillated water. The stock solution contained no detectable ochratoxin α amide (LOD 0.06 ng/mL). The mixture was homogenized by blending for 90 min with a Bosch Profi 67 mixer (Bosch, Stuttgart, Germany) to achieve an ochratoxin A concentration of 20 μg/kg and a moisture content of 27 % [dry weight basis (dw)]. A blank sample was produced without an addition of ochratoxin A for the preparation of the matrix-matched calibration and the determination of the recovery rate. The samples were stored in polyethylene bags in a freezer at −18 °C. Extrusion processing The extrusion cooking was performed on the basis of the experimental setup as previously described by Wu et al. (Wu et al. 2011). A model KE19/25D 150 Nm/150 rpm laboratoryscale conical single-screw extruder (Brabender, Duisburg, Germany) was used with compression ratio screw 1/1 and a 5-mm diameter cylindrical die. The screw speed was set to 35 rpm and the feed speed to 10 rpm. The residence time was determined to be 2.0 min. By using these conditions, the ochratoxin A-spiked wheat grits and the blank material were extruded at 150 °C. The ochratoxin A-spiked wheat grits were also extruded at 180 °C. For each extruded sample, a 100-g material was collected, ground with a Fritsch Pulverisette 14 mill (Fritsch, Idar-Oberstein, Germany), and stored at −18 °C until the analysis. Sample preparation A total of 5 g of the ground extruded sample was weighted into a 100-mL Erlenmeyer flask. Afterwards, 50 mL of extraction solvent containing acetonitrile/water/formic acid (60:40:0.1v/v/v) was added. The suspension was homogenized for 3 min by using an Ultra-Turrax T25 mixer (Janke & Kunkel IKA®, Staufen, Germany) at a rotation of 8000 min−1. Subsequently, the sample was filtered over a fluted filter and then concentrated by the evaporation of 3-mL filtrate under a stream of nitrogen at 50 °C. The residue was dissolved in a 150-μL acetonitrile/water/formic acid (20:80:0.1, v/v/v) and analyzed by HPLC-MS/MS. The sample preparation was done in duplicate and each sample injected twice into the HPLC-MS/MS System. Matrix calibration and determination of the limit of detection, limit of quantification, and recovery rate A matrix-matched calibration of ochratoxin α amide was prepared and used for the determination of the limit of detection (LOD), limit of quantification (LOQ), and the recovery rate. First, a stock solution of ochratoxin α amide with a concentration of 80 μg/mL was prepared by dissolving 1.6 mg of the prepared ochratoxin α amide in 20 mL of
methanol using a volumetric flask. Afterwards, the stock solution was diluted to an ochratoxin α amide concentration of 80 ng/mL by diluting 25 μL of the stock solution with methanol in a 25-mL volumetric flask (standard solution). Out of this solution, a matrix-matched calibration was prepared. Therefore, the standard solution was diluted with the extract of the blank matrix to ochratoxin α amide concentrations of 0.06, 0.10, 0.14, 0.18, and 0.22 ng/mL. The blank matrix extract was achieved by the extrusion of blank durum wheat grits at 150 °C following sample preparation, as described above. Each concentration of the matrix-matched calibration was injected twice. The LOD was determined at a signal-tonoise ratio (S/N) higher than 3:1 and the LOQ with S/N higher than 10:1. For the determination of the recovery rate, the extruded blank grits (5 g) were spiked with 87.5 μL of the ochratoxin α amide standard solution (80 ng/mL) to achieve an ochratoxin α amide concentration of 1.4 μg/kg. This preparation was done in duplicate. After the ochratoxin α amide allowed to soak for 10 min, the samples were prepared, as described above, and analyzed by HPLC-MS/MS. The samples were injected twice.
HPLC-MS/MS A QTRAP 5500 MS system (AB SCIEX, Darmstadt, Germany) coupled to a LaChrom Ultra HPLC system (VWR Hitachi, Darmstadt, Germany) was used for the detection of ochratoxin α amide in multiple reaction monitoring mode (MRM). Data acquisition was performed with Analyst 1.5.2 software. The chromatography was carried out on a 150× 2.1 mm inner diameter, 5 μm, Agilent Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) using a linear binary gradient at a column temperature of 40 °C. The injection volume was 40 μL, and the flow rate was set at 350 μL/min. Solvent A was acetonitrile and solvent B was water, both containing 0.1 % formic acid. The linear gradient was used as follows: 0 min, 20 % A; 3 min, 20 % A; 23 min, 70 % A; 26 min, 70 % A followed by equilibration of the column at starting conditions for 4 min. The mass spectrometer was operated in the positive mode. For the electrospray ionization, the voltage was set to +5500 V and nitrogen was used as the curtain gas (35 psi). Zero-grade air was used as nebulizer gas (35 psi) and as a drying gas (45 psi) heated to 350 °C. The transition reactions were monitored with a dwell time of 40 ms each. The protonated ochratoxin α amide was measured with an entrance potential (EP) of 10 V and a declustering potential (DP) of 61 V. The used collision energy (CE) and collision cell exit energy (CXP) are as follows: ochratoxin α amide, [M + H]+ 255.8→220.8 (CE, 31 V; CXP, 12 V); [M + H]+ 255.8→102.0 (CE, 57 V; CXP, 8 V). The first MRM transition listed was used as a quantifier, and the second was used as a qualifier.
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Results and discussion Heating of ochratoxin A at roasting temperatures of 200 °C and above resulted in a partial decomposition of ochratoxin A and the formation of degradation products. In Fig. 2, HPLC-FLD chromatograms of the reaction residue of ochratoxin A heated at 200 °C (A), 220 °C (B), and 240 °C (C) for 9 min are shown. Besides ochratoxin A, three additional peaks appeared after the heating experiment with the highest intensities for the experiment performed at 240 °C. By comparison with reference material, the peak eluting at 19.1 min could be assigned as 14R-ochratoxin A, the thermal isomerization product of ochratoxin A previously found in roasted coffee (Cramer et al. 2008). 14-decarboxy-ochratoxin A, also detected as a degradation product in roasted coffee could also be assigned based on a reference, to a small peak with a retention time of
21.0 min. As in coffee, 14-decarboxy-ochratoxin A is formed only in traces. Two further new compounds with retention times of 9.8 and 11.7 min appear in concentrations increasing with raising temperature. Using HPLC-FTMS, the compound eluting at 9.8 min could be assigned to ochratoxin α. The compound eluting with a retention time of 11.7 min revealed a m/z of 256.0371 in the HPLC-FTMS analysis in the positive mode and could not be assigned to a known thermal degradation product of ochratoxin A. Fragmentation in the collision cell resulted in the product ion spectrum shown in Fig. 3. The typical product ions m/z 239.0105, and 221.0000, characteristic for the complete dihydroisocoumarin moiety of ochratoxin A as well as the smaller fragments of 211.0156, 193.0050, and 165.0099 are detected with high signal intensity. Comparison of the mass spectra with literature data revealed that the new thermal degradation product is identical to a photolytic reaction product previously proposed (Schmidt-Heydt et al. 2012a, b). Measurements of a sample from the respective study confirmed the compounds identity. However, until now, only mass spectrometric data of ochratoxin α amide have been recorded and the structure assignment is based only on these data. Thus, we optimized and repeated the heating experiments of ochratoxin A in a larger scale in order to isolate sufficient amounts of this thermal degradation product to perform comprehensive NMR studies. In detail, 214-mg ochratoxin A were heated at 240 °C for 20 min without light and the residue purified by repeated semipreparative HPLC-UV using a C18-column. After isolation, a 1.6-mg ochratoxin α amide with a purity of ≥96 % (HPLCELSD) was obtained. With this substance in hand, it was possible to perform a complete structure elucidation by means of NMR and MS experiments (see “Materials and methods” for spectroscopic data) in order to confirm the structure of ochratoxin α amide, a photolytic and thermal degradation product of ochratoxin A. Structure elucidation of ochratoxin α amide In agreement with the mass spectrometric data previously published, the detected m/z 256.0371 can be assigned to the sum formula [C11H10ClNO4 + H]+ of ochratoxin α amide (5, Fig. 1). The calculated formula is supported by the
Fig. 2 HPLC-FLD chromatograms of ochratoxin A after heating at 200 °C (a), 220 °C (b), and 240 °C (c) for 9 min (see Fig. 1 for chemical structures)
Fig. 3 HCD spectrum of ochratoxin α amide (m/z 256.0371) in the positive mode
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characteristic isotopic pattern with relative signal intensity of 29 % for m/z 258.0343, the 37Cl-isotopologue. The obtained 13 C-NMR spectrum of the isolated compound shows signals for all 11 C-atoms, of the ochratoxin α backbone. Only for C-12, C-7, and C-6 minimal shifts of 1.0 to 164.3 ppm for C-12 and −0.4 ppm for C-7 and C-6 could be observed. The 1H-NMR spectrum of the ochratoxin α amide showed also signals for the protons of the ochratoxin α backbone of ochratoxin A. However, two further broad resonance signals at 5.85 and 7.88 ppm could be observed at the 1H-spectrum, which are characteristics for the protons of an amide group. The different shifts of the proton signals arise from the nonchemical equivalence of the protons resulting from the double-bound character of the linkage between the nitrogen atom and the C-12 atom based on the mesomeric effect. These results confirm in addition to the 13C-NMR data the presence of an amide group. Thus, the achieved NMR data prove the structure of ochratoxin α amide (5) shown in Fig. 1. Cytotoxicity of ochratoxin α amide The formation of ochratoxin α amide as a thermal degradation product as well as the formation under irradiation with light gives rise to the question if this new described compound is still toxic or if it is a detoxification product. In order to get first informations concerning the cytotoxicity, we performed cell culture experiments using ochratoxin A-sensitive IHKE cells, as the kidney is known as the primary target for ochratoxin A (Cramer et al. 2008). Besides ochratoxin α amide (5), also ochratoxin A (1), 14R-ochratoxin A (2), and ochratoxin α, (6) were tested for comparison (see Fig. 1 for chemical structures). Figure 4 shows the results of the viability tests of the IHKE cells after treatment with the tested substances in a concentration range from 0.01 nM to 50 μM for a period of 24 h as determined by the CCK-8 assay. As expected, ochratoxin A (IC50 value, 0.5 μM) and 14R-ochratoxin A (IC50 value, 4.6 μM) have a cytotoxic effect on the IHKE cells with 14R-ochratoxin A being about tenfold less cytotoxicity compared to ochratoxin A. Ochratoxin α and ochratoxin α amide did not affect the viability of the IHKE cells in the tested concentration range up to 50 μM (Fig. 4). This lack of cytotoxicity of both compounds can be attributed to the missing phenylalanine moiety, which seems to be a critical factor for the toxicity of ochratoxin A in general (Cramer et al. 2010). Thus, based on these first results, the degradation of ochratoxin A to ochratoxin α amide might be a promising pathway for ochratoxin A detoxification. HPLC-MS/MS analysis of ochratoxin α amide As two different processes can lead to the degradation of ochratoxin A to ochratoxin α amide, it is important do detect this compound in processed food. Thus, we tested different techniques for the extraction and purification of ochratoxin α
Fig. 4 Viability of IHKE cells after 24-h exposure with the following compounds: ochratoxin A (IC50 value, 0.5 μM), 14R-ochratoxin A (IC50 value, 4.6 μM), ochratoxin α (no cytotoxic effect in the tested concentration range), and ochratoxin α amide (no cytotoxic effect in the tested concentration range) dependent on the tested concentrations determined by using a CCK-8 assay; values are means with n=12 for the concentration range 0.01 nM–10 μM, n=3 for the concentration of 50 μM. The asterisks indicate means which differ significantly from the solvent-treated negative control (100 %). Statistical analysis was performed using ANOVA with the Scheffe post-hoc test (p≤0.05)
amide from food samples and developed a HPLC-MS/MS method for ochratoxin α amide. In a first step, the suitability of two different immunoaffinity cleanup columns for the isolation of ochratoxin α amide was studied. The columns OchraTest (VICAM, Watertown, MA) and OtaCLEAN (LCTech GmbH, Dorfen, Germany) contain ochratoxin A-specific antibodies and were tested for a cross reactivity against ochratoxin α amide. Unfortunately, antibodies of the columns from both manufactures showed no cross reactivity with ochratoxin α amide, making the cleanup and detection of ochratoxin α amide in complex processed food samples very difficult. Thus, the matrix was changed to cereal samples, where “dilute and shoot” approaches for analyte detection have been shown to be very successful. As sample material for method development and testing, we used artificially contaminated extruded wheat grits. For method development, the extruded grits were spiked to an ochratoxin α amide concentration of 1.4 μg/kg and extracted with a solution of acetonitrile/water/formic acid (60:40:0.1 v/v/v). With this approach and matrix-matched calibration, a recovery rate of 97.8 % was obtained. The achieved LOD and LOQ for the used analytical HPLC-MS/MS method for the ochratoxin α amide analysis in extruded wheat grits were 0.3 and 1.0 μg/kg, respectively. The calculation of these parameters was based on an external matrix-matched calibration curve of ochratoxin α amide in a concentration range of 0.06, 0.10, 0.14, 0.18, and 0.22 ng/mL. With this method, we analyzed wheat grits, artificially contaminated with ochratoxin A to a level of 20 μg/kg, and extruded at temperatures of 150 and 180 °C, respectively. However, despite the high temperatures and pressure applied
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in the system, no formation of ochratoxin α amide up to the LOD could be observed during extrusion cooking. As 180 °C is the maximum temperature for the used laboratory extruder further experiments at higher temperatures could not be performed. From the obtained results, we conclude that ochratoxin A seems to be stable with respect to the degradation to ochratoxin α amide during extrusion cooking up to 180 °C but might be degraded to ochratoxin α amide at higher temperatures as was shown in the model heating experiments at 240 °C described above. Also it might be possible that ochratoxin α amide is just an intermediate product that undergoes further reactions such as binding to food constituents. Nevertheless with these results, a first method for the analysis of ochratoxin α amide in processed wheat samples was developed, which can be used in subsequent studies on the impact of light or temperature on the degradation of ochratoxin A to ochratoxin α amide. Although ochratoxin α amide was not detectable in the preliminary model experiments using extruded corn grits, further thermally processed samples should be analyzed in the future. In summary, we could show that ochratoxin α amide can also be formed during thermal treatment. Using a fast heating procedure and isolation by HPLC, ochratoxin α amide could be isolated for complete structure elucidation. First toxicity studies for ochratoxin α amide indicate that this compound is less cytotoxic compared to ochratoxin A. In addition, we successfully developed a HPLC-MS/MS method for the analysis of ochratoxin α amide in extruded wheat grits. However, so far, no ochratoxin α amide could be detected during model experiments with artificial contaminated wheat grits in combination with extrusion cooking at temperatures up to 180 °C.
Acknowledgments The authors thank ABSciex for supplying us with a QTRAP 5500 mass spectrometer, Merck VWR for supplying us with a VWR Hitachi LaChrom Ultra HPLC system, Rolf Geisen for providing P. nordicum BFE487, K. Bergander for NMR measurements, and A. Klusmeier-König for the performed cell culture experiments. Source of funding The authors thank the Deutsche Forschungsgemeinschaft (HU 730/10-1) for the financial support. Conflict of interests None.
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Brown MH, Szczech GM, Purmalis BP (1976) Teratogenic and toxic effects of ochratoxin A in rats. Toxicol Appl Pharmacol 37:331–338 Castells M, Pardo E, Ramos AJ, Sanchis V, Marin S (2006) Reduction of ochratoxin A in extruded barley meal. J Food Prot 69:1139–1143 Commission E (2006) Commission regulation No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union L364:5–24 Cramer B, Königs M, Humpf HU (2008) Identification and in vitro cytotoxicity of ochratoxin A degradation products formed during coffee roasting. J Agric Food Chem 56:5673–5681 Cramer B, Harrer H, Nakamura K, Uemura D, Humpf HU (2010) Total synthesis and cytotoxicity evaluation of all ochratoxin A stereoisomers. Bioorg Med Chem 18:343–347 Entwisle AC, Williams AC, Mann PJ, Slack PT, Gilbert J (2000) Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley: collaborative study. J AOAC Int 83:1377–1383 European Commission Scientific Committee for Food (2002), SCOOP task 3.2.7. Reports on tasks for scientific cooperation: assessment of dietary intake of ochratoxin A by the population of EU Member States. Available from: http://ec.europa.eu/food/fs/scoop/3.2.7_en. pdf (Accessed 17 December 2014) European Food Safety Authority (EFSA) (2006) Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. EFSA J 365:1–56 International Agency for Research on Cancer (IARC) (1993) Some naturally occurring substance: some food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monogr Eva Carcinog Risks Hum 56:489 Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno KA (1996) Combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull 19:1518–1520 Jalili M, Jinap S, Son R (2011) The effect of chemical treatment on reduction of aflatoxins and ochratoxin A in black and white pepper during washing. Food Addit Contam Part A 28:485–493 Jaynes WF, Zartman RE (2011) Aflatoxin toxicity reduction in feed by enhanced binding to surface-modified clay additives. Toxins (Basel) 3:551–565 Krogh P (1992) Role of ochratoxin in disease causation. Food Chem Toxicol 30:213–224 Petzinger E, Weidenbach A (2002) Mycotoxins in the food chain: the role of ochratoxins. Livest Prod Sci 76:245–250 Romani S, Pinnavaia GG, Dalla Rosa M (2003) Influence of roasting levels on ochratoxin A content in coffee. J Agric Food Chem 51: 5168–5171 Schmidt-Heydt M, Cramer B, Graf I, Lerch S, Humpf HU, Geisen R (2012a) Wavelength-dependent degradation of ochratoxin and citrinin by light in vitro and in vivo and its implications on Penicillium. Toxins (Basel) 4:1535–1551 Schmidt-Heydt M, Graf E, Stoll D, Geisen R (2012b) The biosynthesis of ochratoxin A by Penicillium as one mechanism for adaptation to NaCl rich foods. Food Microbiol 29:233–241 Scudamore KA, Banks JN, Guy RC (2004) Fate of ochratoxin A in the processing of whole wheat grain during extrusion. Food Addit Contam 21:488–497 Tveito G, Hansteen IL, Dalen H, Haugen A (1989) Immortalization of normal human kidney epithelial cells by nickel (II). Cancer Res 49: 1829–1835 van der Merwe KJ, Steyn PS, Fourie L, Scott DB, Theron JJ (1965) Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus. Wilh Nat 205:1112–1113 Wu Q, Lohrey L, Cramer B, Yuan Z, Humpf HU (2011) Impact of physicochemical parameters on the decomposition of deoxynivalenol during extrusion cooking of wheat grits. J Agric Food Chem 59:12480–12485
ORIGINAL PAPER
Structure elucidation and in vitro cytotoxicity of ochratoxin α amide, a new degradation product of ochratoxin A Andrea Bittner & Benedikt Cramer & Henning Harrer & Hans-Ulrich Humpf
Received: 29 October 2014 / Revised: 17 December 2014 / Accepted: 18 December 2014 # Society for Mycotoxin Research and Springer-Verlag Berlin Heidelberg 2015
Abstract The mycotoxin ochratoxin A is a secondary metabolite occurring in a wide range of commodities. During the exposure of ochratoxin A to white and blue light, a cleavage between the carbon atom C-14 and the nitrogen atom was described. As a reaction product, the new compound ochratoxin α amide has been proposed based on mass spectrometry (MS) experiments. In the following study, we observed that this compound is also formed at high temperatures such as used for example during coffee roasting and therefore represents a further thermal ochratoxin A degradation product. To confirm the structure of ochratoxin α amide, the compound was prepared in large scale and complete structure elucidation via nuclear magnetic resonance (NMR) and MS was performed. Additionally, first studies on the toxicity of ochratoxin α amide were performed using immortalized human kidney epithelial (IHKE) cells, a cell line known to be sensitive against ochratoxin A with an IC50 value of 0.5 μM. Using this system, ochratoxin α amide revealed no cytotoxicity up to concentrations of 50 μM. Thus, these results propose that the thermal degradation of ochratoxin A to ochratoxin α amide might be a detoxification process. Finally, we present a sample preparation and a HPLC-tandem mass spectrometry (HPLCMS/MS) method for the analysis of ochratoxin α amide in extrudates and checked its formation during the extrusion of artificially contaminated wheat grits at 150 and 180 °C, whereas no ochratoxin α amide was detectable under these conditions. Keywords Ochratoxin A . Degradation . Detoxification . Roasting . Extrusion . Ochratoxinα amide . Thermalstability . Light . Coffee A. Bittner : B. Cramer : H. Harrer : H.98 % (NMR) (Cramer et al. 2008). 14R-ochratoxin A, 14-decarboxy-ochratoxin A, and ochratoxin α were available from a previous study (Cramer et al. 2008). Fourier transformation mass spectrometry The exact mass measurement and the higher energy collision dissociation (HCD) fragmentation were carried out on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a direct flow infusion of 5 μL/min. Data acquisition was performed with Xcalibur 2.07 SP I (Thermo Fisher Scientific). The mass spectrometer was operated in the positive mode, and the ionization was performed with heated electrospray ionization. Further conditions were as follows: capillary temperature, 225 °C; vaporizer temperature, 50 °C; sheath gas flow, 10 units; auxiliary gas flow, 5 units; source voltage, 3.5 kV; tube lens, 55 V; and capillary voltage, 14 V. The HCD cell was used at the optimal relative energy of 65 % determined in a range from 10 to 70 %. The mass resolution was set to 30,000, and the maximum ion injection time was set to 100 ms. NMR spectroscopy 1
H-, 13C-, and 2D-NMR data were acquired on a 600-MHz DD2 NMR spectrometer (Agilent Technologies, Waldbronn, Germany). Signals are reported in parts per million relative to tetramethylsilane (TMS). For structural elucidation and NMR signal assignment, 2D NMR experiments, such as gradientselected correlated spectroscopy (gs-COSY), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear multiple-bond correlation (HMBC), were carried out. Pulse programs for these experiments were taken from the Bruker software library. Heating experiment
A 445-μL stock solution with an ochratoxin A concentration of 226 μg/mL in acetonitrile, prepared by dissolving 45.2 mg of ochratoxin A in 200 mL of acetonitrile using a volumetric flask, was evaporated in a 1.5-mL vial under a stream of nitrogen at 40 °C to achieve an ochratoxin A amount of 100 μg per vial. In this way, three vials were prepared, capped, and heated for 9 min at a temperature of 200, 220, and 240 °C, respectively. After cooling, the reaction mixture was dissolved in 1 mL of a mixture of acetonitrile/water/formic acid (20:80:0.1, v/v/v) and afterwards analyzed by HPLC with fluorescence detection (HPLC-FLD) and Fourier transform mass spectrometry (HPLC-FTMS). Each heating experiment was performed in triplicate.
HPLC-FLD The reaction mixture was analyzed by a HPLC system consisting of two LC-10ATVP pumps coupled with a SIL10AF autosampler and a RF-10A fluorescence detector (Shimadzu, Kyoto, Japan). Data acquisition was performed with LC solution software 1.25 (Shimdazu). The chromatographic separation of the thermal degradation products of ochratoxin A was carried out on a 150×4.6 mm inner diameter, 5 μm, Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) with a 4×3 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany) using a binary gradient. The column oven was set to 40 °C, and an injection volume of 10 μL was used. Solvent A was water and solvent B was acetonitrile both containing 0.1 % formic acid. The flow rate was set to 1 mL/min and the HPLC programmed isocratic for the first 3 min at 20 % solvent B followed by a linear gradient to 70 % solvent B at 20 min, which was held for 5 min. After each run, the column was equilibrated at the starting conditions for 5 min. The fluorescence detector was set to a wavelength of 330 nm for excitation and 460 nm for emission. HPLC-FTMS For HPLC-FTMS analysis, an Accela LC 60057-60010 system (Thermo Fisher Scientific, Bremen, Germany) was connected to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) which was used in the scan mode. Data acquisition was performed with Xcalibur 2.07 SP1 (Thermo Scientific). The thermal degradation products were separated on a 150×2.1 mm inner diameter, 5 μm, Agilent Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) with a 4×2 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany) using a binary gradient as described above but with a flow rate of 300 μL/min. The column temperature was 40 °C, and the injection volume was 20 μL. The mass spectrometer was operated in the positive mode, and the ionization was performed with heated electrospray ionization. The reaction products were measured in a scan mode in a mass range of m/z 100 to 1000 with a resolution of 30,000. Further conditions were as follows: capillary temperature, 225 °C; vaporizer temperature, 350 °C; sheath gas flow, 40 units; auxiliary gas flow, 20 units; source voltage, 3.5 kV; tube lens, 120 V; and capillary voltage, 49 V. Preparation of ochratoxin α amide Pure ochratoxin A (214.4 mg) was heated in a 4-mL screwcapped brown glass vial at 240 °C for 20 min. The reaction mixture was dissolved in 3 mL of acetonitrile/1 % formic acid (20:80, v/v) and sonicated for 10 min. Insoluble components
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were separated by membrane filtration using a Phenomenex®RC membrane syringe filter (0.45-μm pore size; 15-mm diameter) (Phenomenex, Aschaffenburg, Germany) followed by separation on a semi-preparative HPLC. For the isolation of ochratoxin α amide, the filtrate was purified in two steps on a 250×9.4 mm inner diameter, 5 μm, Agilent Eclipse XDBC18 column (Agilent Technologies, Böblingen, Germany) with a 4×3 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany). In the first step, ochratoxin α amide was separated from compounds with a higher retention on the C18 material by fractionation. The first fraction was achieved by using an isocratic mixture of acetonitrile/1 % formic acid (35:65, v/v) delivered by two Varian ProStar 210 HPLC solvent delivery modules (Varian, Darmstadt, Germany). The flow rate was set at 4 mL/min. The ochratoxin α amide containing fraction 1 was collected for the first 9 min after peak detection by a Varian ProStar 325 UV/vis detector (Varian, Darmstadt, Germany) set at 330 nm. The fraction 2, containing predominantly ochratoxin A and 14R-ochratoxin A, was obtained by a followed linear gradient to 100 % solvent B in 6 min, which was held for 5 min. The two fractions were separately collected over three runs and combined, and the solvent was removed under reduced pressure. Afterwards, fraction 1 was further purified to remove the ochratoxin α impurities from ochratoxin α amide. Therefore, the evaporated residue of fraction 1 was dissolved in 2 mL of acetonitrile/1 % formic acid (20:80, v/v) and separated on the column and HPLC system, as described above using an isocratic mixture of acetonitrile/1 % formic acid (20:80, v/v). After detection of the ochratoxin α amide peak, it was collected over four runs and combined. The solvent was removed under reduced pressure, and ochratoxin α amide was obtained as a white solid (1.60 mg, 6.26 μmol, 1.2 % yield). For the determination of the purity of the isolated ochratoxin α amide, a LC-20AT system (Shimadzu, Kyoto, Japan) was coupled to an Evaporative Light Scattering Detector (ELSD) (Shimadzu). Data acquisition was performed with LC solution 1.25 (Shimadzu). Separation was carried out on a 150× 4.6 mm inner diameter, 5 μm, Agilent Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) with a 4 × 3 mm inner diameter universal C18 guard column (Phenomenex, Aschaffenburg, Germany) using a binary gradient. Solvent A was water and solvent B was acetonitrile both containing 0.1 % formic acid. The injection volume was 10 μL and the flow rate was set to 1 mL/min. The HPLC was programmed to run isocratic for the first 5 min at 20 % solvent B followed by a linear gradient to 100 % solvent B at 25 min. Afterwards, the column was washed with 100 % solvent B for 2 min and then equilibrated to starting conditions. Ochratoxin α amide ESI-MS (positive mode): HRMS m/z: 256.0371 (calculated formula [C 11 H 10 ClNO 4 + H] + (0.038 ppm). MS/MS (HCD, 65 %; [M + H]+) m/z (%):
239.0105 (100), 193.0050 (71), 221.0000 (44), 211.0156 (32), 165.0099 (25), 137.0150 (12), 257.0211 (7). 1H NMR (600 MHz, CDCl3) δ: 1.61 (3H, d, J=6.3 Hz, H-11), 2.87 (1H, dd, J=17.4, 11.6 Hz, H-4A), 3.31 (1H, dd, J=17.4, 3.4 Hz, H-4B), 4.82–4.74 (1H, m, H-3), 5.85 (1H, s, H-13A), 7.88 (1H, s, H-13B), 8.48 (1H, s, H-6), 12.82 (1H, s, C-8OH). 13C NMR (151 MHz, CDCl3) δ: 20.7 (C-11), 32.3 (C-4), 75.9 (C-3), 110.1 (C-9), 120.6 (C-7), 123.2 (C-5), 139.4 (C-6), 141.1 (C-10), 159.1 (C-8), 164.3 (C-12), 169.9 (C-1). Cytotoxicity assay (CCK-8) IHKE cells, which were kindly provided by Prof. Dr. M. Gekle (Institut für Physiologie, Martin-Luther-Universität Halle Wittenberg, Germany), were used for the cytotoxicity experiments. These cells were cultivated, as described by Tveito et al., and as previously applied by Cramer et al. (Cramer et al. 2010; Ishiyama et al. 1996; Tveito et al. 1989). The determination of the cytotoxicity was performed by a colorimetric assay using the Cell Counting Kit-8 (CCK8) (Dojindo, Laboratories, Tokyo, Japan) according to the manufacturer’s instructions the metabolic activity of the cells was measured. Therefore, the cells were seeded in 96-well plates. After a growth of 48 h, the cells were cultivated in a serum-free medium for 24 h in order to exclude any binding of the tested compounds to serum proteins. Then solutions of ochratoxin α amide were added to the cells in a concentration range from 0.01 nM to 50 μM (stock solution: 10 mM in methanol) and incubated for 24 h. Cells with an equal solvent concentration were incubated as control. Afterwards, the viability of the cells was measured as previously described (Cramer et al. 2010). The incubation time with the dye solution WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-(2.4-disulfophenyl)-2H-tetrazolium, monosodium salt] was changed to 45 min. This experimental setup was simultaneously done with ochratoxin α, ochratoxin A, and 14Rochratoxin A for comparison. Sampling For the extrusion experiments, commercial durum wheat grits were bought from a local supermarket (Münster, Germany). The natural moisture content of the durum wheat grits was determined by the weight loss after oven drying at a temperature of 130 °C to a constant weight and was measured to be approximately 12.6 %. For the preparation of a stock solution, 6.0 mg of ochratoxin A was dissolved in 25 mL of acetonitrile. The concentration of the stock solution was determined as described in the AOAC 2000.03 method by photometric analysis to be 232 μg/mL ochratoxin A (Entwisle et al. 2000). One kilogram of wheat grits containing no detectable amount of ochratoxin A was spiked with 86.2 μL of the ochratoxin A stock solution (232 μg/mL) and with 197-mL
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distillated water. The stock solution contained no detectable ochratoxin α amide (LOD 0.06 ng/mL). The mixture was homogenized by blending for 90 min with a Bosch Profi 67 mixer (Bosch, Stuttgart, Germany) to achieve an ochratoxin A concentration of 20 μg/kg and a moisture content of 27 % [dry weight basis (dw)]. A blank sample was produced without an addition of ochratoxin A for the preparation of the matrix-matched calibration and the determination of the recovery rate. The samples were stored in polyethylene bags in a freezer at −18 °C. Extrusion processing The extrusion cooking was performed on the basis of the experimental setup as previously described by Wu et al. (Wu et al. 2011). A model KE19/25D 150 Nm/150 rpm laboratoryscale conical single-screw extruder (Brabender, Duisburg, Germany) was used with compression ratio screw 1/1 and a 5-mm diameter cylindrical die. The screw speed was set to 35 rpm and the feed speed to 10 rpm. The residence time was determined to be 2.0 min. By using these conditions, the ochratoxin A-spiked wheat grits and the blank material were extruded at 150 °C. The ochratoxin A-spiked wheat grits were also extruded at 180 °C. For each extruded sample, a 100-g material was collected, ground with a Fritsch Pulverisette 14 mill (Fritsch, Idar-Oberstein, Germany), and stored at −18 °C until the analysis. Sample preparation A total of 5 g of the ground extruded sample was weighted into a 100-mL Erlenmeyer flask. Afterwards, 50 mL of extraction solvent containing acetonitrile/water/formic acid (60:40:0.1v/v/v) was added. The suspension was homogenized for 3 min by using an Ultra-Turrax T25 mixer (Janke & Kunkel IKA®, Staufen, Germany) at a rotation of 8000 min−1. Subsequently, the sample was filtered over a fluted filter and then concentrated by the evaporation of 3-mL filtrate under a stream of nitrogen at 50 °C. The residue was dissolved in a 150-μL acetonitrile/water/formic acid (20:80:0.1, v/v/v) and analyzed by HPLC-MS/MS. The sample preparation was done in duplicate and each sample injected twice into the HPLC-MS/MS System. Matrix calibration and determination of the limit of detection, limit of quantification, and recovery rate A matrix-matched calibration of ochratoxin α amide was prepared and used for the determination of the limit of detection (LOD), limit of quantification (LOQ), and the recovery rate. First, a stock solution of ochratoxin α amide with a concentration of 80 μg/mL was prepared by dissolving 1.6 mg of the prepared ochratoxin α amide in 20 mL of
methanol using a volumetric flask. Afterwards, the stock solution was diluted to an ochratoxin α amide concentration of 80 ng/mL by diluting 25 μL of the stock solution with methanol in a 25-mL volumetric flask (standard solution). Out of this solution, a matrix-matched calibration was prepared. Therefore, the standard solution was diluted with the extract of the blank matrix to ochratoxin α amide concentrations of 0.06, 0.10, 0.14, 0.18, and 0.22 ng/mL. The blank matrix extract was achieved by the extrusion of blank durum wheat grits at 150 °C following sample preparation, as described above. Each concentration of the matrix-matched calibration was injected twice. The LOD was determined at a signal-tonoise ratio (S/N) higher than 3:1 and the LOQ with S/N higher than 10:1. For the determination of the recovery rate, the extruded blank grits (5 g) were spiked with 87.5 μL of the ochratoxin α amide standard solution (80 ng/mL) to achieve an ochratoxin α amide concentration of 1.4 μg/kg. This preparation was done in duplicate. After the ochratoxin α amide allowed to soak for 10 min, the samples were prepared, as described above, and analyzed by HPLC-MS/MS. The samples were injected twice.
HPLC-MS/MS A QTRAP 5500 MS system (AB SCIEX, Darmstadt, Germany) coupled to a LaChrom Ultra HPLC system (VWR Hitachi, Darmstadt, Germany) was used for the detection of ochratoxin α amide in multiple reaction monitoring mode (MRM). Data acquisition was performed with Analyst 1.5.2 software. The chromatography was carried out on a 150× 2.1 mm inner diameter, 5 μm, Agilent Eclipse XDB-C18 column (Agilent Technologies, Böblingen, Germany) using a linear binary gradient at a column temperature of 40 °C. The injection volume was 40 μL, and the flow rate was set at 350 μL/min. Solvent A was acetonitrile and solvent B was water, both containing 0.1 % formic acid. The linear gradient was used as follows: 0 min, 20 % A; 3 min, 20 % A; 23 min, 70 % A; 26 min, 70 % A followed by equilibration of the column at starting conditions for 4 min. The mass spectrometer was operated in the positive mode. For the electrospray ionization, the voltage was set to +5500 V and nitrogen was used as the curtain gas (35 psi). Zero-grade air was used as nebulizer gas (35 psi) and as a drying gas (45 psi) heated to 350 °C. The transition reactions were monitored with a dwell time of 40 ms each. The protonated ochratoxin α amide was measured with an entrance potential (EP) of 10 V and a declustering potential (DP) of 61 V. The used collision energy (CE) and collision cell exit energy (CXP) are as follows: ochratoxin α amide, [M + H]+ 255.8→220.8 (CE, 31 V; CXP, 12 V); [M + H]+ 255.8→102.0 (CE, 57 V; CXP, 8 V). The first MRM transition listed was used as a quantifier, and the second was used as a qualifier.
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Results and discussion Heating of ochratoxin A at roasting temperatures of 200 °C and above resulted in a partial decomposition of ochratoxin A and the formation of degradation products. In Fig. 2, HPLC-FLD chromatograms of the reaction residue of ochratoxin A heated at 200 °C (A), 220 °C (B), and 240 °C (C) for 9 min are shown. Besides ochratoxin A, three additional peaks appeared after the heating experiment with the highest intensities for the experiment performed at 240 °C. By comparison with reference material, the peak eluting at 19.1 min could be assigned as 14R-ochratoxin A, the thermal isomerization product of ochratoxin A previously found in roasted coffee (Cramer et al. 2008). 14-decarboxy-ochratoxin A, also detected as a degradation product in roasted coffee could also be assigned based on a reference, to a small peak with a retention time of
21.0 min. As in coffee, 14-decarboxy-ochratoxin A is formed only in traces. Two further new compounds with retention times of 9.8 and 11.7 min appear in concentrations increasing with raising temperature. Using HPLC-FTMS, the compound eluting at 9.8 min could be assigned to ochratoxin α. The compound eluting with a retention time of 11.7 min revealed a m/z of 256.0371 in the HPLC-FTMS analysis in the positive mode and could not be assigned to a known thermal degradation product of ochratoxin A. Fragmentation in the collision cell resulted in the product ion spectrum shown in Fig. 3. The typical product ions m/z 239.0105, and 221.0000, characteristic for the complete dihydroisocoumarin moiety of ochratoxin A as well as the smaller fragments of 211.0156, 193.0050, and 165.0099 are detected with high signal intensity. Comparison of the mass spectra with literature data revealed that the new thermal degradation product is identical to a photolytic reaction product previously proposed (Schmidt-Heydt et al. 2012a, b). Measurements of a sample from the respective study confirmed the compounds identity. However, until now, only mass spectrometric data of ochratoxin α amide have been recorded and the structure assignment is based only on these data. Thus, we optimized and repeated the heating experiments of ochratoxin A in a larger scale in order to isolate sufficient amounts of this thermal degradation product to perform comprehensive NMR studies. In detail, 214-mg ochratoxin A were heated at 240 °C for 20 min without light and the residue purified by repeated semipreparative HPLC-UV using a C18-column. After isolation, a 1.6-mg ochratoxin α amide with a purity of ≥96 % (HPLCELSD) was obtained. With this substance in hand, it was possible to perform a complete structure elucidation by means of NMR and MS experiments (see “Materials and methods” for spectroscopic data) in order to confirm the structure of ochratoxin α amide, a photolytic and thermal degradation product of ochratoxin A. Structure elucidation of ochratoxin α amide In agreement with the mass spectrometric data previously published, the detected m/z 256.0371 can be assigned to the sum formula [C11H10ClNO4 + H]+ of ochratoxin α amide (5, Fig. 1). The calculated formula is supported by the
Fig. 2 HPLC-FLD chromatograms of ochratoxin A after heating at 200 °C (a), 220 °C (b), and 240 °C (c) for 9 min (see Fig. 1 for chemical structures)
Fig. 3 HCD spectrum of ochratoxin α amide (m/z 256.0371) in the positive mode
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characteristic isotopic pattern with relative signal intensity of 29 % for m/z 258.0343, the 37Cl-isotopologue. The obtained 13 C-NMR spectrum of the isolated compound shows signals for all 11 C-atoms, of the ochratoxin α backbone. Only for C-12, C-7, and C-6 minimal shifts of 1.0 to 164.3 ppm for C-12 and −0.4 ppm for C-7 and C-6 could be observed. The 1H-NMR spectrum of the ochratoxin α amide showed also signals for the protons of the ochratoxin α backbone of ochratoxin A. However, two further broad resonance signals at 5.85 and 7.88 ppm could be observed at the 1H-spectrum, which are characteristics for the protons of an amide group. The different shifts of the proton signals arise from the nonchemical equivalence of the protons resulting from the double-bound character of the linkage between the nitrogen atom and the C-12 atom based on the mesomeric effect. These results confirm in addition to the 13C-NMR data the presence of an amide group. Thus, the achieved NMR data prove the structure of ochratoxin α amide (5) shown in Fig. 1. Cytotoxicity of ochratoxin α amide The formation of ochratoxin α amide as a thermal degradation product as well as the formation under irradiation with light gives rise to the question if this new described compound is still toxic or if it is a detoxification product. In order to get first informations concerning the cytotoxicity, we performed cell culture experiments using ochratoxin A-sensitive IHKE cells, as the kidney is known as the primary target for ochratoxin A (Cramer et al. 2008). Besides ochratoxin α amide (5), also ochratoxin A (1), 14R-ochratoxin A (2), and ochratoxin α, (6) were tested for comparison (see Fig. 1 for chemical structures). Figure 4 shows the results of the viability tests of the IHKE cells after treatment with the tested substances in a concentration range from 0.01 nM to 50 μM for a period of 24 h as determined by the CCK-8 assay. As expected, ochratoxin A (IC50 value, 0.5 μM) and 14R-ochratoxin A (IC50 value, 4.6 μM) have a cytotoxic effect on the IHKE cells with 14R-ochratoxin A being about tenfold less cytotoxicity compared to ochratoxin A. Ochratoxin α and ochratoxin α amide did not affect the viability of the IHKE cells in the tested concentration range up to 50 μM (Fig. 4). This lack of cytotoxicity of both compounds can be attributed to the missing phenylalanine moiety, which seems to be a critical factor for the toxicity of ochratoxin A in general (Cramer et al. 2010). Thus, based on these first results, the degradation of ochratoxin A to ochratoxin α amide might be a promising pathway for ochratoxin A detoxification. HPLC-MS/MS analysis of ochratoxin α amide As two different processes can lead to the degradation of ochratoxin A to ochratoxin α amide, it is important do detect this compound in processed food. Thus, we tested different techniques for the extraction and purification of ochratoxin α
Fig. 4 Viability of IHKE cells after 24-h exposure with the following compounds: ochratoxin A (IC50 value, 0.5 μM), 14R-ochratoxin A (IC50 value, 4.6 μM), ochratoxin α (no cytotoxic effect in the tested concentration range), and ochratoxin α amide (no cytotoxic effect in the tested concentration range) dependent on the tested concentrations determined by using a CCK-8 assay; values are means with n=12 for the concentration range 0.01 nM–10 μM, n=3 for the concentration of 50 μM. The asterisks indicate means which differ significantly from the solvent-treated negative control (100 %). Statistical analysis was performed using ANOVA with the Scheffe post-hoc test (p≤0.05)
amide from food samples and developed a HPLC-MS/MS method for ochratoxin α amide. In a first step, the suitability of two different immunoaffinity cleanup columns for the isolation of ochratoxin α amide was studied. The columns OchraTest (VICAM, Watertown, MA) and OtaCLEAN (LCTech GmbH, Dorfen, Germany) contain ochratoxin A-specific antibodies and were tested for a cross reactivity against ochratoxin α amide. Unfortunately, antibodies of the columns from both manufactures showed no cross reactivity with ochratoxin α amide, making the cleanup and detection of ochratoxin α amide in complex processed food samples very difficult. Thus, the matrix was changed to cereal samples, where “dilute and shoot” approaches for analyte detection have been shown to be very successful. As sample material for method development and testing, we used artificially contaminated extruded wheat grits. For method development, the extruded grits were spiked to an ochratoxin α amide concentration of 1.4 μg/kg and extracted with a solution of acetonitrile/water/formic acid (60:40:0.1 v/v/v). With this approach and matrix-matched calibration, a recovery rate of 97.8 % was obtained. The achieved LOD and LOQ for the used analytical HPLC-MS/MS method for the ochratoxin α amide analysis in extruded wheat grits were 0.3 and 1.0 μg/kg, respectively. The calculation of these parameters was based on an external matrix-matched calibration curve of ochratoxin α amide in a concentration range of 0.06, 0.10, 0.14, 0.18, and 0.22 ng/mL. With this method, we analyzed wheat grits, artificially contaminated with ochratoxin A to a level of 20 μg/kg, and extruded at temperatures of 150 and 180 °C, respectively. However, despite the high temperatures and pressure applied
Mycotoxin Res
in the system, no formation of ochratoxin α amide up to the LOD could be observed during extrusion cooking. As 180 °C is the maximum temperature for the used laboratory extruder further experiments at higher temperatures could not be performed. From the obtained results, we conclude that ochratoxin A seems to be stable with respect to the degradation to ochratoxin α amide during extrusion cooking up to 180 °C but might be degraded to ochratoxin α amide at higher temperatures as was shown in the model heating experiments at 240 °C described above. Also it might be possible that ochratoxin α amide is just an intermediate product that undergoes further reactions such as binding to food constituents. Nevertheless with these results, a first method for the analysis of ochratoxin α amide in processed wheat samples was developed, which can be used in subsequent studies on the impact of light or temperature on the degradation of ochratoxin A to ochratoxin α amide. Although ochratoxin α amide was not detectable in the preliminary model experiments using extruded corn grits, further thermally processed samples should be analyzed in the future. In summary, we could show that ochratoxin α amide can also be formed during thermal treatment. Using a fast heating procedure and isolation by HPLC, ochratoxin α amide could be isolated for complete structure elucidation. First toxicity studies for ochratoxin α amide indicate that this compound is less cytotoxic compared to ochratoxin A. In addition, we successfully developed a HPLC-MS/MS method for the analysis of ochratoxin α amide in extruded wheat grits. However, so far, no ochratoxin α amide could be detected during model experiments with artificial contaminated wheat grits in combination with extrusion cooking at temperatures up to 180 °C.
Acknowledgments The authors thank ABSciex for supplying us with a QTRAP 5500 mass spectrometer, Merck VWR for supplying us with a VWR Hitachi LaChrom Ultra HPLC system, Rolf Geisen for providing P. nordicum BFE487, K. Bergander for NMR measurements, and A. Klusmeier-König for the performed cell culture experiments. Source of funding The authors thank the Deutsche Forschungsgemeinschaft (HU 730/10-1) for the financial support. Conflict of interests None.
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