Anal. Chem. 2007, 79, 710-717
Selective Sample Cleanup by Reusable Sol-Gel Immunoaffinity Columns for Determination of Deoxynivalenol in Food and Feed Samples Zdenka Brenn-Struckhofova,† Margit Cichna-Markl,*,† Christina Bo 1 hm,‡ and Ebrahim Razzazi-Fazeli‡
Department of Analytical and Food Chemistry, University of Vienna, Wa¨hringer Strasse 38, A-1090 Vienna, Austria, and Department of Veterinary Public Health and Food Science, Institute of Nutrition, University of Veterinary Medicine, Veterina¨rplatz 1, A-1210 Vienna, Austria
The paper describes the development of a simple and highly selective method for the determination of deoxynivalenol (DON) in food and feed samples. It combines sample cleanup with sol-gel immunoaffinity columns containing monoclonal anti-DON antibodies and quantification of DON by high-performance liquid chromatography with ultraviolet detection. The sol-gel immunoaffinity columns are as selective as commercial DON immunoaffinity columns but superior with regard to production costs, storage stability, and reusability. In applying the method for the analysis of maize, wheat, and spaghetti samples, it offers detection limits (LOD, S/N ) 3) of 240, 200, and 207 ng/g, and recoveries of 83, 99, and 97%, respectively. Mycotoxins are toxic fungal secondary metabolites produced either in the field or during production, transportation, processing, or storage of food. Each year ∼25% of all food crops are affected by mycotoxin contamination, causing considerable financial damage worldwide. Trichothecenes, a large group of mycotoxins with a sesquiterpenoid structure, are produced by various Fusarium species, which are growing on agricultural products in northern temperate regions including Europe and North America. The most common trichothecene in cereals is deoxynivalenol (DON), predominantly produced by Fusarium graminearum and Fusarium culmorum. DON causes a variety of toxic effects by inhibiting the synthesis of DNA, RNA, and proteins.1 Consumption of feed contaminated with DON has been reported to lead to vomiting, feed refusal, weight loss, and diarrhea in both experimental animals and livestock.2 Recently published papers describe the influence of harvest and storing conditions on DON concentrations in food,3 while others focus on the possibilities to prevent DON contamination4 or to lower DON concentrations by processing or cooking foods.5,6 * To whom correspondence should be addressed. E-mail: margit.cichna@ univie.ac.at. Tel.:+43-1-4277-52374. Fax: +43-1-4277-9523. † University of Vienna. ‡ University of Veterinary Medicine. (1) Schlatter, J. Toxicol. Lett. 2004, 153, 83-89. (2) Rotter, B. A.; Prelusky, D. B.; Pestka, J. J. J. Toxicol. Environ. Health 1996, 48, 34. (3) Schro ¨dter, R. Toxicol. Lett. 2004, 153, 47-49. (4) Aldred, D.; Magan, N. Toxicol. Lett. 2004, 153, 165-171. (5) Hazel, C. M.; Patel, S. Toxicol. Lett. 2004, 153, 51-59.
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In order to protect the health of humans and animals, most industrialized countries have established regulations for mycotoxins, e.g., maximum tolerated limits for DON in food and feed and tolerable daily intake values for humans. Highly selective analytical methods have to be applied to ensure that DON concentrations in food and feed comply with the legal regulations. In the last years, some review articles covered the topic of trichothecene analysis in food and feed.7,8 Different methods for the determination of DON have been reported, including enzyme immunoassays,9 thin-layer chromatography,10,11 gas chromatography with either electron capture,12,13 or mass spectrometric detection14,15 as well as high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection,16,17 fluorescence detection after postcolumn derivatization,18-20 or MS detection.21-24 In spite of the use of selective separation and sensitive detection methods, low DON levels cannot be determined in (6) Visconti, A.; Haidukowski, E. M.; Pascale, M.; Silvestri, M. Toxicol. Lett. 2004, 153, 181-189. (7) Krska, R.; Baumgartner, S.; Josephs, R. Fresenius J. Anal. Chem. 2001, 371, 285-299. (8) Koch, P. Toxicol. Lett. 2004, 153, 109-112. (9) Papadopoulou-Bouraoui, A.; Vrabcheva, T.; Valzacchi, Stroka, J.; Anklam, E. Food Addit. Contam. 2004, 21, 607-617. (10) Trucksess, M. W.; Nesheim, S. J. Assoc. Off. Anal. Chem. 1984, 67, 40-43. (11) Trucksess, M. W.; Flood, M. T.; Mossoba, M. M.; Page, M. W. J. Agric. Food Chem. 1987, 35, 445-448. (12) Ware, G. M.; Francis, O. J.; Carman, A. S.; Kuan, S. S. J. Assoc. Off. Anal. Chem. 1986, 69, 899-901. (13) Kotal, F.; Holadova, K.; Hajslova, J.; Poustka, J.; Radova, Z. J. Chromatogr., A 1999, 830, 219-225. (14) Onji, Y.; Aoki, Y.; Tani, N.; Umebayashi, K.; Kitada, Y.; Dohi, Y. J. Chromatogr., A 1998, 815, 59-65. (15) Tanaka, T.; Yoneda, A.; Inoue, S.; Sugiura, Y.; Ueno, Y. J. Chromatogr., A 2000, 882, 23-28. (16) Cahill, L. M.; Kruger, S. C.; McAlice, B. T.; Ramsey, C. S.; Prioli, R.; Kohn, B. J. Chromatogr., A 1999, 859, 23-28. (17) Mateo, J. J.; Mateo, R.; Hinojo, M. J.; Llorens, A.; Jime´nez, M. J. Chromatogr., A 2002, 955, 245-256. (18) Sano, A.; Matsutani, S.; Suzuki, M.; Takitani, S. J. Chromatogr. 1987, 410, 427-436. (19) Dall’Asta, C.; Galaverna, G.; Biancardi, A.; Gasparini, M.; Sforza, S.; Dossena, A.; Marchelli, R. J. Chromatogr., A 2004, 1047, 241-247. (20) Klo ¨tzel, M.; Schmidt, S.; Lauber, U.; Thielert, G.; Humpf, H.-U. Chromatographia 2005, 62, 41-48. (21) Razzazi-Fazeli, E.; Bo¨hm, J.; Luf, W. J. Chromatogr., A 1999, 854, 45-55. (22) Razzazi-Fazeli, E.; Bo ¨hm, J.; Jarukamjorn, K.; Zentek, J. J. Chromatogr., B 2003, 796, 21-33. (23) Biancardi, A.; Gasparini, M.; Dall’Asta, C.; Marchelli, R. Food Addit. Contam. 2005, 22, 251-258. (24) Biselli, S.; Hummert, C. Food Addit. Contam. 2005, 22, 752-760. 10.1021/ac061672w CCC: $37.00
© 2007 American Chemical Society Published on Web 12/07/2006
complex food and feed matrixes without carrying out selective sample pretreatment steps. Solid-phase extraction with charcoal/ alumina columns20-22 and immunoaffinity chromatography16,20,25 are the most frequently applied cleanup procedures in DON analysis. Up to now, immunoaffinity columns have been prepared by covalently binding anti-DON antibodies to a solid support material. However, this immobilization technique suffers from several disadvantages. The chemical reaction frequently causes denaturation of the antibodies, thus lowering the affinity for the antigen. In addition, the immobilization procedure usually involves several washing and blocking steps, making the method rather labor- and time-consuming. In order to prevent bacterial degradation of the antibodies, bacteriostatic agents have to be added for storing the columns. These disadvantages can be circumvented by entrapping the antibodies in the pores of a sol-gel glass.26-38 Preparation and application of sol-gel immunoaffinity columns as well as their main characteristics have been reviewed recently.39 Since the entrapment of ligands in the pores of the sol-gel glass can be carried out under mild conditions, their affinity is largely maintained. Since the pore size controls the access of sample components into the pores, sol-gel affinity columns offer an increased selectivity beyond the selectivity of the bioligand. Solgel affinity columns have a high storage stability and can in generally be reused for the cleanup of a number of samples. In addition, alkoxysilanes used for entrapping biomolecules are significantly less costly than support materials commonly used for covalent attachment. In spite of their potential, to our knowledge, sol-gel immunoaffinity columns have never been applied for sample cleanup in DON analysis. The present paper reports the results of our investigations of the applicability of sol-gel immunoaffinity columns containing monoclonal anti-DON antibodies to clean up food and feed samples, focusing on important characteristics such as selectivity, column-to column reproducibility, storage stability, and reusability. EXPERIMENTAL SECTION Reagents and Materials. Purified monoclonal anti-DON antibodies (1 mg/mL phosphate-buffered saline (PBS)) were (25) Kotal, F.; Radova, Z. Czech. J. Food Sci. 2001, 20, 63-68. (26) Zu ¨ hlke, J.; Knopp, D.; Niessner, R. Fresenius, J. Anal. Chem. 1995, 352, 654-659. (27) Cichna, M.; Knopp, D.; Niessner, R. Anal. Chim. Acta 1997, 339, 241250. (28) Scharnweber, T.; Knopp, D.; Niessner, R. Field Anal. Chem. 2000, 4, 4352. (29) Bronshtein, A.; Aharonson, N.; Turniansky, A.; Altstein, M. Chem. Mater. 2000, 12, 2050-2058. (30) Schedl, M.; Wilharm, G.; Achatz, S.; Niessner, K. A. R.; Knopp, D. Anal. Chem. 2001, 73, 5669-5676. (31) Cichna, M.; Markl, P.; Knopp, D.; Niessner, R. J. Chromatogr., A 2001, 919, 51-58. (32) Altstein, M.; Bronshtein, A.; Glattstein, B.; Zeichner, A.; Tamiri, T. J. Almog, Anal. Chem. 2001, 73, 2461-2467. (33) Stalikas, C.; Knopp, D.; Niessner, R. Environ. Sci. Technol. 2002, 36, 33723377. (34) Vazquez-Lira, J. C.; Camacho-Frias, E.; Pena-Alvarez, A.; Vera-Avila, L. E. Chem. Mater. 2003, 15, 154-161. (35) Braunrath, R.; Cichna, M. J. Chromatogr., A 2005, 1062, 189-198. (36) Zhang, X.; Martens, D.; Kra¨mer, P. M.; Kettrup, A. A.; Liang, X. J. Chromatogr., A 2006, 1102, 84-90. (37) Degelmann, P.; Egger, S.; Ju ¨ rling, H.; Mu ¨ ller, J.; Niessner, R.; Knopp, D. J. Agric. Food Chem. 2006, 54, 2003-2011. (38) Podlipna, D.; Cichna-Markl, M. Eur. Food Res. Technol. In press. (39) Cichna-Markl, M. J. Chromatogr., A 2006, 1124, 167-180.
provided by Zheng Zhong Ming (Singapore). Human immunoglobulin G, DON, Tween 20, and Triton X-100 were obtained from Sigma (St. Louis, MO). A certified DON standard solution (100.8 µg/mL in acetonitrile), deepoxy-DON, 3-acetyl-DON, 15-acetylDON, and a maize reference material were obtained from Biopure (Tulln, Austria). Acetonitrile (ACN) and methanol (MeOH), both gradient grade for HPLC, were purchased from Fisher Scientific (Leicestershire, UK). Tetramethoxysilane was from Fluka (Buchs, Switzerland), Roti-Block was from Karl Roth (Karlsruhe, Germany). Immunoaffinity columns DONPREP were received from R-Biopharm (Darmstadt, Germany). Maize and wheat samples were obtained from the Austrian Agency for Health and Food Safety; durum wheat spaghetti was bought in a local supermarket. Ground and sealed samples were stored at 4 °C. Instrumentation. Centrifugation was carried out with a Sigma centrifuge (model 4K 10, Vienna, Austria). Samples were ground in a mechanical mortar (type MM 2000, Retsch, Haan, Germany). Two HPLC systems were used. HPLC system 1 was used to develop the analytical method and to determine DON concentrations in food and feed samples, whereas HPLC system 2 was used to verify the identity of DON. HPLC system 1 consisted of a highpressure gradient pump (model L-7100, Merck), a column thermostat (model bfo-04 dt, W.O. Electronics, Langenzersdorf, Austria), and a six-port injection valve (model 7161, Rheodyne) equipped with a 100-µL stainless steel injection loop. DON was detected with a UV detector (model L-4200, Merck) at 220 nm. Peaks were integrated using the McDacq software (Bischoff, Leonberg, Germany). HPLC system 2 consisted of a Hewlett-Packard Series 1100 gradient pump, an HP Series 1100 autosampler (Agilent, Vienna, Austria) and a UV-detector (Agilent) set at 220 nm. An HCT plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray interface was used for detection in the negative mode. The selected temperature for the heated capillary was 300 °C. The dry-gas flow was set at 10 L/min. The [M-H]of DON, 295 m/z as parent ion, was used in MS/MS studies. The mass range in scan mode was 100-300 m/z; the scan speed was 8100 m/z per second. Pure nitrogen as nebulizing and carrier gas was produced in a Nitrox Dominick hunter N2 generator, UHPLCMS 18 (Alltech, Deerfield, Ireland). Standard Solutions and Buffers. A stock solution of DON was prepared by dissolving 1.0 mg of DON in 10.0 mL of ACN. Working solutions of DON were prepared by diluting the stock solution with bidistilled water. The DON stock solution was stored at -20 °C, and working solutions were stored at 4 °C. Phosphatebuffered saline, pH 7.6, was prepared by dissolving 12.46 g of Na2HPO4·2H2O, 1.56 g of NaH2PO4·2H2O, and 8.5 g of NaCl in 1 L of bidistilled water. Preparation of Sol-Gel Immunoaffinity Columns. Solgel immunoaffinity columns were prepared by entrapping either 0.5, 1, or 2 mg of the purified monoclonal anti-DON antibody in the pores of 1 g of silicate glass according to a previously described protocol.35 The silicate glass was manually ground in an achate mortar andswithout sievingspacked into empty 3-mL plastic columns equipped with polytetrafluorethylene frits (Bartelt, Vienna, Austria). The columns were washed with 20 mL of ACNwater (40:60, v/v), followed by 20 mL of PBS, and stored in PBS at 4 °C. Columns containing 0.5 or 1 mg of antibodies were used Analytical Chemistry, Vol. 79, No. 2, January 15, 2007
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for carrying out experiments with standard solutions, whereas columns containing 2 mg of antibodies were used for cleaning up food or feed samples. HPLC Phase Systems. In HPLC system 1, a Phenomenex, Synegri 4u Polar-RP, 80 Å, 250 mm × 4.6 mm i.d., 5 µm (Phenomenex, Aschaffenburg, Germany) was used as the analytical column. The mobile phase consisted of bidistilled waterACN-MeOH (80:10:10, v/v/v). All separations were carried out at 25 °C applying a flow rate of 1 mL/min. In HPLC system 2, an ACE 3-C18 RP column, 100 Å, 150 mm × 2.1 i.d., 3 µm (ACE, Aberdeen, Scotland) was used. Mobile phase A was bidistilled water and mobile phase B ACN. The gradient program was as follows: 0 min 95% A/5% B, 5 min 95% A/5% B, and 15 min 85% A/15% B. The flow rate of the mobile phase was 0.2 mL/min. A 30-µL sample volume was injected into the system. External Calibration. HPLC system 1 was calibrated by injecting six standard solutions in the concentration range from 25 to 1000 ng/mL in bidistilled water. The analysis function was obtained by linear regression of peak areas on standard concentrations. The system was also calibrated for all cross-reactive substances in the concentration range from 50 to 300 ng/mL in PBS. Sample Preparation with Sol-Gel Columns. Samples were ground in the mechanical mortar for 20 min, and 5 g of the ground samples were weighed in and mixed with 40 mL of bidistilled water. After stirring the suspension for 10 min, an 11-mL aliquot was centrifuged at 2800g for 10 min. After filtering the supernatant through a borosilicate frit (porosity 5), a 5-mL aliquot was applied to an immunoaffinity column containing 2 mg of anti-DON antibodies. After washing the column with 10 mL of MeOH-water (1:99, v/v) and 20 mL of bidistilled water, elution was carried out with 4 mL of ACN-water (40:60, v/v). The eluate was collected in a 5-mL measuring flask. After evaporating ACN under a slight nitrogen stream, the measuring flask was filled up to the ring mark with water. The column was regenerated with 20 mL of PBS. Sample Preparation with Commercially Available Immunoaffinity Columns. According to the instruction of the manufacturer, 40 mL of bidistilled water was added to 5 g of ground sample. After the suspension was stirred for 10 min, an 11-mL aliquot was centrifuged at 2800g for 10 min. The supernatant was filtered through the borosilicate frit. Before the DONPREP column was used, it was allowed to get to room temperature. A 2-mL sample of the extract was passed through the column under gravity. The column was washed with 5 mL of bidistilled water at the same flow rate. Using a syringe pump unit, the column was dried by flushing with air to remove the last few drops of water. DON was eluted from the column by back flushing with 1.5 mL of MeOH. Finally, air was passed through the column while the last drops of MeOH were collected. MeOH was removed from the collected eluate under a nitrogen atmosphere, and the measuring flask was filled up to the ring mark (1 mL) with water. RESULTS AND DISCUSSION Retention Mechanism. In order to investigate whether DON is retained in the immunoaffinity columns due to specific interactions with the anti-DON antibodies, we compared the breakthrough curves obtained with three different sol-gel columns. One column contained 1 mg of anti-DON antibodies, the second one the same amount of “nonspecific” human immunoglobulins, 712 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007
Figure 1. Breakthrough curves obtained with a column packed with 1 g of sol-gel glass containing 0.5 mg of anti-DON antibodies. Loading solution: 100 ng of DON/mL in (×) PBS, (4) ACN-water (5:95, v/v), or (O) ACN-water (10:90, v/v). co, DON concentration in the loading solution; c, DON concentration in the eluate.
and the last one only pure sol-gel matrix. A DON standard solution (15 mL of a solution containing 100 ng of DON/mL in PBS) was applied to each column while the eluates were collected in 1-mL fractions. The DON concentration in the eluate fractions was determined by injecting aliquots into HPLC system 1. The breakthrough curves obtained with the columns that did not contain anti-DON antibodies were almost identical. In both cases, DON was already detected in the second eluate fraction. In contrast, DON was strongly retained in the column having entrapped anti-DON antibodies. Breakthrough of DON was not observed before having applied 1000 ng of DON. These data demonstrate that retention of DON in the immunoaffinity columns occurs due to specific interactions with the anti-DON antibodies. Loading Conditions. In the case of solid sample matrixes, the determination of analyte(s) usually starts with extracting the analyte(s) from the matrix by either aqueous or organic solvents, depending on the solubility of the analyte(s). Due to its polarity, DON is frequently extracted with water or mixtures of water with ACN or MeOH. In order to investigate whether low concentrations of organic solvents influence the interactions between DON and the monoclonal anti-DON antibodies used in the present study, immunoaffinity columns containing 500 µg of anti-DON antibodies were overloaded with a DON standard solution (100 ng of DON/ mL) in either water, PBS, ACN-water (5:95, v/v), or ACN-water (10:90, v/v) while collecting 1-mL eluate fractions. The breakthrough curves obtained with PBS, ACN-water (5:95, v/v), and ACN-water (10:90, v/v) are shown in Figure 1; the breakthrough curve obtained with water was identical with that obtained with PBS. When DON was loaded in ACN-water (10:90, v/v, or 5:95, v/v), DON could already be detected in the second or third eluate fraction, respectively. In contrast, when either water or PBS was used as loading medium, first traces of DON were detected only in the sixth eluate fraction. These results demonstrate that strong retention of DON in the column can only be achieved when pure aqueous solutions are loaded onto the immunoaffinity columns. In order to determine the pH optimum of the loading medium, immunoaffinity columns were loaded with 5 mL of a DON standard solution (100 ng of DON/mL) in PBS at different pH (5, 6, 7, or 8). After the columns were washed with 5 mL of water, DON was eluted with 4 mL of ACN-water (40:60, v/v). Mean
Figure 2. Chromatograms obtained by injecting aliquots of purified wheat samples into HPLC system 1. Wheat samples were spiked with (A) 800 ng of DON/g of sample, (B, C) 400 ng of DON/g of sample. Different washing conditions were used before DON was eluted from the immunoaffinity column: (A) 15 mL of water, (B) 5 mL of MeOH-water (1:99, v/v) plus 5 mL of water, and (C) 10 mL of MeOH-water (1:99, v/v) plus 20 mL of water. Table 1. Influence of the Washing Condition on DON Recovery loading solution
washing medium
standard solution (5 mL, 100 ng/mL in water)
15 mL of Roti-Block/water (5:95, v/v) 15 mL of Triton X-100/water (1:99, v/v) 15 mL of Tween 20/water (1:99, v/v) 15 mL of MeOH/water (1:99, v/v)
extract of a blank maizesample, spiked with 800 ng/g DON
10 mL of Roti-Block/water (5:95, v/v), 5 mL water 10 mL of Triton X-100/water (0.1:99.9, v/v), 5 mL water 10 mL of Tween 20/water (0.1:99.9, v/v), 5 mL water 10 mL of MeOH/water (1:99, v/v), 5 mL water 10 mL of MeOH/water (1:99, v/v), 20 mL water
recoveries (n ) 3) were found to be 97, 95, 94, and 96%, indicating that within the pH range of 5-8 the pH value of the loading solution does not show an influence on DON recovery. Since slow reaction kinetics of antigen-antibody reactions can cause low analyte recoveries at high sample loading flow rates, experiments had to be carried out to investigate the influence of loading flow rate on analyte recovery. A 5-mL aliquot of a DON standard solution (100 ng/mL) in water was loaded onto a column applying flow rates of about 0.5, 1, 1.5, or 2 mL/min. After the column was washed with 5 mL of water, DON was eluted with 4 mL of ACN-water (40:60, v/v). Determination of the DON concentrations in the eluate indicated that DON standard solutions could be loaded up to a flow rate of 2 mL/min without affecting DON recovery. However, when blank wheat extracts spiked with 800 ng of DON/g were loaded onto the immunoaffinity columns, the loading flow rate did show an influence on the recovery of DON. Loading the spiked wheat extract with a flow rate of 1 mL/ min resulted in a recovery of only 57%. However, recoveries of 96% were obtained by decreasing the sample loading flow rate to 0.5 mL/min. Due to these results, all sample extracts were loaded onto immunoaffinity columns applying a flow rate of 0.5 mL/min. Washing Conditions. Preliminary experiments with both maize and wheat samples showed that matrix components nonspecifically bound to the sol-gel glass material could not be completely removed by washing the immunoaffinity columns with pure water. Although the HPLC-UV chromatograms did not contain peaks overlapping with the DON peak, matrix peaks were obtained at the beginning of the chromatograms. In order to increase the selectivity of the immunoaffinity columns, experiments were carried out to improve the efficiency in removing nonspecifically bound matrix components. The following washing media were tested: MeOH-water (1:99, v/v), Triton X-100-water (0.1:99.9, v/v), Tween 20-water (0.1:99.9, v/v), and Roti-Block-
recovery (%) 225 35 69 98 90 26 59 96 95
water (5:95, v/v). Roti-Block is the trade name for a mixture of a high molecular weight poly(vinylpyrrolidone) with a nonionic surfactant dissolved in PBS. In one of our previous studies, the extent of nonspecific binding of polycyclic aromatic hydrocarbons could significantly be reduced by washing the sol-gel immunoaffinity column with 5% (v/v) Roti-Block in ACN-water (10:90, v/v).40 In order to investigate whether washing the immunoaffinity column with low concentrations of organic solvents or surfactants interfered with specific retention of DON in the column, first experiments were carried out with DON standard solutions. Five milliliters of a 100 ng/mL solution were loaded, followed by washing the column with 15 mL of the washing medium under test and eluting DON with ACN-water (40:60, v/v). The data given in Table 1 show that an acceptable DON recovery was only obtained after washing the column with MeOH-water (1:99, v/v). Washing with either Triton X-100-water (1:99, v/v) or Tween 20water (1:99, v/v) significantly decreased the recovery of DON. In contrast, after washing with Roti-Block, traces of the surfactant in the eluate seemed to interfere with UV detection of DON, feigning a significantly too high recovery of DON. Additional experiments were carried out with blank maize samples spiked with 800 ng of DON/g of sample. In contrast to the experiments described above, the extracts were loaded to immunoaffinity columns containing 2 mg instead of 1 mg of antiDON antibodies. The columns were washed with only 10 mL of the washing media and subsequently flushed with 5 mL of water in order to avoid any interferences with the following UV detection of DON in the eluates. In addition, an increase in the recovery of DON, by lowering the concentrations of Triton X-100 and Tween 20 from 1 to 0.1%, was attempted. As can be seen in Table 1, the (40) Cichna, M.; Markl, P.; Knopp, D.; Niessner, R. Chem. Mater. 1997, 9, 26402646.
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Figure 4. HPLC chromatogram of a standard solution containing 300 ng of (1) DON, (2) deepoxy-DON, (3) 3-acetyl-DON, and (4) 15acetyl-DON/mL of water. Figure 3. Distribution of DON in the eluate fractions of the immunoaffinity column, depending on the ACN concentration in the elution medium. Table 2. Influence of Loading Concentration and Loading Volume on DON Recovery loading concn (ng/mL)
loading vol (mL)
total amt of DON loaded (ng)
recovery (%)
25 50 75 100
12 6 4 3
300 300 300 300
95 91 99 90
Table 3. DON Recoveries Obtained with Immunoaffinity Columns from Different Production Batches (Sol-Gel Glasses Prepared on Different Days) production batch A B C D E F G H I mean recovery (%) standard deviation (%)
recovery (%) 100 97 96 99 99 98 98 97 96 97.8 1.4
highest recovery was obtained after washing the column with MeOH-water (1:99, v/v). In the case of washing with Roti-Block, the subsequent flushing of the column with water significantly reduced the interference with UV detection of DON, resulting in an acceptable recovery of 90%. However, lowering the concentration of Triton X-100 and Tween 20 to 0.1% did not improve DON recovery. Figure 2 shows chromatograms obtained by injecting immunoaffinity-purified wheat extracts into HPLC system 1. Different washing conditions were used to remove nonspecifically bound impurities. It can be seen that increasing both the MeOH concentration and the washing volume resulted in an increase in the washing efficiency. However, when MeOH-containing solutions were injected, a slightly negative baseline disturbance was observed at a retention time of 6 min (Figure 2B). By flushing 714 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007
the immunoaffinity column with 20 mL of water before eluting DON, the MeOH concentration in the eluate was decreased and thus the negative baseline drift removed almost completely (Figure 2C). A sample cleanup procedure including a time-consuming washing step is, however, not very attractive even if it is very selective. In the present study, the whole washing procedure (washing the immunoaffinity column with 10 mL of MeOH-water (1:99, v/v) and 20 mL of water) could be carried out within 1 min by using a syringe to press the solutions through the column. In contrast to the loading step, the high flow rate did not affect the recovery of DON. Due to the high washing efficiency achievable in a short time, these washing conditions were then selected for purifying food and feed extracts. Elution Conditions. A number of previous studies showed that mixtures of ACN and water are suitable for eluting various analytes from sol-gel immunoaffinity columns, the actual ACN concentration and the elution volume being dependent on the affinity constants of the antibodies entrapped in the sol-gel glasses.26-28,30,31,34-38,40 In the present study, a series of experiments was carried out to find elution conditions that allow to yield a high DON recovery in a small elution volume without irreversibly denaturating the antibodies. After loading 10 mL of DON standard solutions (100 ng of DON/mL of water), the columns were washed with 5 mL of water and DON eluted with 5 mL of ACN-water (10:90, 20:80, 30:70, or 40:60, v/v) by collecting 0.5-mL eluate fractions. Figure 3 shows the distribution of DON in the eluate fractions depending on the ACN concentration in the elution medium. ACN-water (40:60, v/v) turned out to be the most suitable elution medium since it resulted in quantitative elution of DON. On the other hand, elution with 10, 20, or 30% ACN resulted in recoveries of only 48, 65, and 81%, respectively. In addition, these recoveries were only obtained with relatively large elution volumes resulting in greater DON dilutions with their implications for the detection limit of the method. Since elution flow rates up to 1 mL/min did not show an influence on the recovery, elution of DON was always carried out by applying a flow rate of 1 mL/min. Binding Capacity. The binding capacity of sol-gel immunoaffinity columns was determined by overloading columns containing 1 mg of anti-DON antibody with 20 mL of a DON standard solution (100 ng/mL) in water, washing the columns
Figure 5. Chromatograms obtained by injecting aliquots of purified food and feed samples into HPLC system 1. Sample cleanup (A-C) with sol-gel immunoaffinity columns, (D) with a DONPREP column. (A) Unspiked maize, (B) spaghetti spiked with 400 ng of DON/g of sample, (C) wheat spiked with 400 ng of DON/g of sample, and (D) wheat spiked with 800 ng of DON/g of sample.
with 5 mL of water, and eluting DON with 4 mL of ACN-water (40:60, v/v). Columns containing 1 mg of anti-DON antibody showed a binding capacity of 1000 ng of DON. Influence of Loading Concentration and Loading Volume on DON Recovery. In order to investigate whether the recovery of DON is influenced by the volume or the concentration of the solution loaded onto the column, an immunoaffinity column was repeatedly loaded with a total amount of 300 ng of DON by varying loading volume and loading concentration. The data summarized in Table 2 indicate that similar DON recoveries were obtained within the concentration range of 25-100 ng/mL and a loading volume up to at least 12 mL. Column-to-Column Reproducibility. The column-to-column reproducibility was determined by preparing nine sol-gel immunoaffinity columns on nine different days and subjecting them to recovery tests with DON standard solutions (see Table 3). The mean recovery was found to be 97.8% with a standard deviation of 1.4%. These data indicate that sol-gel immunoaffinity columns could be produced and operated highly reproducibly. Cross-Reactivity. The selectivity of immunoaffinity columns can be limited by the retention of substances showing crossreactivity with the antibodies immobilized. Anti-DON antibodies frequently show cross-reactivity with deepoxy-DON, 3-acetyl-DON, and 15-acetyl-DON. In order to determine if there is any retention of these substances in the sol-gel immunoaffinity columns produced in the present study, a standard solution containing the test substance (5 mL of a 100 ng/mL solution in PBS) was loaded onto a column. After washing the column with 5 mL of water,
elution was carried out with 5 mL of ACN-water (40:60, v/v). Cross-reactivities for deepoxy-DON and 3-acetyl-DON were found to be 42 and 56%, respectively. 15-Acetyl-DON could not be detected in the eluate, indicating that the anti-DON antibody did not show cross-reactivity with 15-acetyl-DON. In the present study, cross-reactivity with deepoxy-DON and 3-acetyl-DON did, however, not pose a problem, since DON was separated from both substances by the chromatographic conditions applied (Figure 4). Preparation of Food and Feed Samples. The analytical method, including the initial extraction of DON with water, sample cleanup by sol-gel immunoaffinity chromatography, and quantification of DON by HPLC with UV detection, was applied to determine DON in wheat, maize, and spaghetti samples. The pH value of aqueous extracts of wheat and maize samples was found to be 6.1 and that of a spaghetti extract 5.9. Since experiments with DON standard solutions have shown that in the pH range 5-8 the DON recovery is not affected by the pH value, the aqueous sample extracts were loaded to immunoaffinity columns without previously having adjusted the pH. Figure 5 shows representative chromatograms of purified food and feed samples, (A) unspiked maize, (B) spaghetti, and (C) wheat, the latter two spiked with 400 ng of DON/g of sample. In all cases, the DON peak was baseline separated from matrix peaks. In order to assess the efficiency of the sol-gel immunoaffinity columns in removing matrix components, a wheat sample (spiked with 800 ng of DON/g of sample) was alternatively purified using a commercially available immunoaffinity column by operating the Analytical Chemistry, Vol. 79, No. 2, January 15, 2007
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Figure 6. MS and MS/MS spectra of the DON peak (retention time 9 min) obtained after injecting an aliquot of a purified maize sample into HPLC system 2.
column according to the instruction of the manufacturer. The chromatogram shown in Figure 5D is similar to that shown in Figure 5C, indicating that there is no significant difference in the selectivity of the sol-gel immunoaffinity columns produced in our laboratory and the commercially available column. Verification of the Identity of DON. In order to confirm the identity of DON, the aliquot (30 µL) of a purified maize sample was analyzed by LC-MS (HPLC system 2). DON was eluted within 9 min (Figure 6). The full scan MS spectrum shows a main signal at m/z 295 ion, which was assigned to the [M-H]- ion. MS/MS analysis showed the most abundant fragment at m/z 264.9, which is in accordance with previously published papers.22 Quantitative Determination of DON. HPLC system 1 was calibrated by injecting six standard solutions in the concentration range from 25 to 1000 ng/mL in water. A linear relationship was obtained between DON concentration and peak area over the whole concentration range, with a correlation coefficient of 0.9999 (n ) 6). The detection limit (signal-to-noise ratio, 3) was 25 ng of DON/mL. The performance of the whole analytical method was assessed by analyzing nonspiked and spiked maize, wheat, and spaghetti samples. As can be seen in Table 4, both high recovery rates and a good reproducibility were obtained for all sample matrixes. In wheat, maize, and spaghetti, limits of detection (S/N ) 3) were found to be 200, 240, and 207 ng/g, respectively. Validation of the Analytical Method by a Certified Reference Material. In order to validate the analytical method developed in the present study, a certified maize reference material was repeatedly analyzed (n ) 4). Sample cleanup was carried out on the same day with one and the same sol-gel immunoaffinity column. Analysis of the reference material resulted in a mean 716 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007
Table 4. Validation Data of the Analytical Method
sample
spiking value (ng/mL)
measured value (ng/mL)
recovery (%)
mean recovery ( standard deviation (%)
0 50 100 150
34.5 74.9 118.5 162.1
80.7a 84.0a 85.0a
83.2 ( 2.3
0 59 117 176
0 56.5 117.5 175.9
96.5 100.3 100.0
99.0 ( 2.1
0 50 100 150
0 49.5 96.6 138.6
99.6 97.2 92.9
96.6 ( 3.4
maize
wheat
spaghetti
a
Recoveries were corrected for the blank value.
DON concentration of 497.8 ng of DON/g with a SD of 12.3 ng/ g. According to the manufacturer, the reference material contained 474 ( 30 ng of DON/g of sample. This result confirms the accuracy of the analytical method developed in the present study. Stability and Reusability of Sol-Gel Immunoaffinity Columns. In contrast to columns prepared by covalently binding the antibodies to a solid support material, in sol-gel columns, the antibodies are entrapped in the pores of a silicate network. Since microorganisms are excluded from the pores, sol-gel immunoaffinity columns are usually stored without adding bacteriostatic agents at 4 °C. In the present study, it was investigated as to whether sol-gel immunoaffinity columns can also be stored at
room temperature without affecting the affinity of the antibodies. After storing two new sol-gel immunoaffinity columns in PBS for 19 weeks at room temperature, the columns were subjected to recovery tests by applying DON standard solutions. With both columns, a DON recovery of 96% was obtained, indicating that the sol-gel columns prepared in the present study can be stored in PBS at room temperature for several weeks, without adding a bacteriostatic agent, which might interfere with subsequent quantification steps. As already mentioned above, in the present study, sol-gel immunoaffinity columns were repeatedly used for the cleanup of a number of samples. After having purified a certain number of samples, the columns were subjected to recovery tests with DON standard solutions to detect any decrease in DON recovery. These experiments showed that the columns could be used for ∼20 sample cleanup cycles without observing any loss in DON recovery. This was found for columns stored at 4 °C as well as at room temperature. After the cleanup of 25 food or feed samples, the recovery decreased to 80%. However, when these columns were stored for ∼20 weeks in PBS without using them for sample cleanup, the recovery returned to 98%. These experiments indicated that repeatedly used sol-gel immunoaffinity columns can be regenerated by long-time storage in PBS. Using the same immunoaffinity column for the cleanup of a number of food and feed samples can lead to a carryover effect causing systematic errors. In the present paper, in none of the numerous recovery tests was carryover observed, neither in experiments with DON standard solutions, nor when purifying food or feed samples. Commercially available DON immunoaffinity columns are provided as single-use columns. The manufacturers usually recommend flushing the column with a few milliliters of ACN or MeOH to dissociate the complex between DON and the anti-DON antibody in order to elute DON from the column. These drastic elution conditions obviously cause irreversible denaturation of the antibodies, preventing the columns from being reused. In the present study, it was investigated as to whether a commercially available DON immunoaffinity column can be reused when being operated under milder elution conditions. After extracting DON from a previously spiked wheat sample, 1 mL of the filtered wheat extract was loaded onto the DONPREP column, which had been
preconditioned with 20 mL of PBS. After the column was washed with 2 mL of water, DON was eluted with 1.5 mL of ACN-water (40:60, v/v). After evaporating part of ACN under a nitrogen stream and filling up the 1-mL measuring flask up to the ring mark with ACN-water (40:60, v/v), an aliquot was injected into HPLC system 1. By flushing the DONPREP column with ACN-water (40:60, v/v)sinstead of pure ACN as recommended by the manufacturers96% of the loaded DON could be recovered. After the elution step, the DONPREP column was flushed with 20 mL of PBS and stored in PBS at 4 °C, analogous to the sol-gel immunoaffinity columns produced in our laboratory. On the next 2 days, cleanup of the spiked wheat sample was repeated with the same column as described above. The second cleanup with the DONPREP column resulted in a DON recovery of 86%, and when the column was used for the third time, the recovery was decreased to 49%. These experiments show that in spite of applying milder elution conditions the DONPREP column should not be used for the cleanup of more than one sample. CONCLUSION Sol-gel immunoaffinity columns containing monoclonal antiDON antibodies were synthesized and their properties characterized. After optimizing their operation parameters, they were used to selectively isolate DON from maize, wheat, and spaghetti samples. Compared to covalently immobilizing bioligands to a solid support material, their entrapment in a porous glass matrix is less time-consuming and less costly and leads to columns with a superior storage stability. Recovery rates obtained with columns from different production batches demonstrated that they can be produced and operated with high reproducibility. When applied for the analysis of real samples, the columns prepared in the present study could be reused for the cleanup of ∼20 food or feed samples without a decline of DON recovery. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Austrian Science Fund (FWF Grant L98-N19). Received for review September 5, 2006. Accepted October 11, 2006. AC061672W
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