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Speciation analysis of selenium enriched green onions (Allium fistulosum) by HPLC-ICP-MS
Microchemical Journal 84 (2006) 56 – 62 www.elsevier.com/locate/microc
Speciation analysis of selenium enriched green onions (Allium fistulosum) by HPLC-ICP-MS Emese Kápolna ⁎, Péter Fodor Department of Applied Chemistry, Corvinus University of Budapest, Villányi út 29-33, 1118 Budapest, Hungary Received 3 February 2006; received in revised form 11 April 2006; accepted 11 April 2006 Available online 21 June 2006
Abstract Green onions (Allium fistulosum) enriched with 10 or 100 μg mL− 1 Se(IV) or SeMet were analyzed for total selenium and species distribution. Anion and cation exchange chromatographies were applied for the separation of selenium species with mass spectrometric detection. Two different sample preparation methods (NaOH and enzymatic) were compared from the Se extraction efficiency point of view. Total selenium concentration accumulated by the onions reached the 200 μg g− 1 level expressed for dry weight when applying SeMet at a concentration of 100 μg mL− 1 as the source of Se. Speciation studies revealed that both in onion bulbs and leaves the predominant form of organic selenium is Se-methylselenocysteine (MeSeCys). When Se(IV) was applied for Se-enrichment at a concentration level of 100 μg mL− 1 both onion leaf and bulb contained a significant amount of inorganic selenium. An unknown compound was also detected. © 2006 Elsevier B.V. All rights reserved. Keywords: Green onion; HPLC-ICP-MS; Selenium supplementation; Se-methyl-selenocysteine; Speciation
1. Introduction Selenium (Se) plays an important role in human and animal nutrition being an essential component of a number of enzymes like glutathione peroxidase (GPX) [1,2]. Insufficient selenium intake tends to cause several health problems including cardiac failure. For example in a region of China (Keshan) where the selenium deficiency accompanying with a virus infection resulted in cardiomyopathy affected young women and children [3]. Furthermore, the amount of cancer patients is also increasing in consequence with the selenium deficiency in everyday diet [4]. Although selenium is required for health in a certain concentration, high doses can be toxic [5]. The intoxication depends on selenium concentration and the chemical forms of this element. In humans the gap between the estimated average requirement and the upper limit of safe intake is relatively narrow [6]. Recommended daily dietary
⁎ Corresponding author. Tel.: +36 1 482 6164; fax: +36 1 466 4272. E-mail address: [email protected] (E. Kápolna). 0026-265X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2006.04.014
allowance (RDA) for selenium in European countries is set from 30 to 70 μg [7]. Selenium content of food varies widely between regions throughout the world. In several European countries including Hungary, the soil is selenium depleted. This deficiency should be made up by introducing natural selenium sources into the human diet. For selenium supplementation purposes, several commercial dietary supplements are available in the market. Knowing only the total selenium content of a proposed Se-supplement is not unambiguous that the given product could be appropriate for supplementation. The bioavailability of selenium depends on several factors like selenium species distribution in the food product and the special physiological conditions of the consumer/e.g. age, the state of health etc./ [8]. Several animal studies have proved that selenium supplementation significantly reduces tumor incidence [9]. Most common Se supplements are selenized yeasts whose major selenocompound is selenomethionine (SeMet) [10]. A paper by Ip. C. et al. [11] compared the anticarcinogenic attribute of Se-enriched yeast and garlic on rats with both chemically and virally induced tumors. This study revealed that the ingestion of selenized yeast caused an undesirable accumulation of selenium in tissues while this
E. Kápolna, P. Fodor / Microchemical Journal 84 (2006) 56–62
phenomenon did not appear in the case of garlic. The explanation for the different behaviour is due to the diverse chemical forms of selenium present in the sample. Plants acquire selenium from the soil and therefore vary in content depending upon the region they are grown and tend to incorporate it non-specifically into compounds usually containing sulfur [12,13]. Selenium enriched garlic, onion, ramp, cabbage, sprout and broccoli have been extensively studied. Major Se species found in these selenized vegetables are the methylated forms of selenium, like Se-methyl-seleno-cysteine and γ-glutamyl-Se-methyl-seleno-cysteine. These species seem to be more effective inhibitors of tumor formation than other organic or inorganic ones [9,14–19]. Allium family vegetables, like onion and garlic are consumed world-wide due to their flavor compounds. Allium species have the capability to accumulate selenium up to high μg g − 1 levels meanwhile converting inorganic selenium being fortified with into organic compounds [17,20]. As Allium family vegetables are kindly consumed everywhere, the aim of this work was to produce a Se-supplement focusing on green onions (Allium fistulosum), which can be easily built into the everyday diet. Within the confines of this study the determination of not only the total selenium concentration of the enriched onion samples but also the species distribution was performed. The goal was to compare the supposedly various selenium distribution of samples enriched in different ways. 2. Materials and methods 2.1. Instrumentation An ICP-MS Agilent 7500ce (Agilent Technologies, Tokyo, Japan) was applied for total selenium and speciation analysis as an element specific detector. The ICP-MS is equipped with an octopole reaction cell and an ASX-510 autosampler. The reaction cell conditions were optimized using H2 as collision gas with a flow rate between 0–5 mL min− 1 monitoring 77Se, 78Se, 80Se and 82Se isotopes. A conventional Babington nebulizer was used for the sample introduction under standard plasma conditions. For chromatographic separations the ICP-MS was coupled to a Merck Model L-7100 4-channel HPLC pump with a six port Rheodyne (Cotati, CA, USA) sample injection valve (Merck, Darmstadt, Germany) used for eluent delivery and sample introduction. The coupling between the corresponding column outlet and the sample introduction system of ICP-MS was obtained through a 500 mm long 0.5 mm id. PEEK tubing. Chromatographic columns used were a silica-based strong cation exchange column (Ionospher 5C, Varian BV, Middelburg, The Netherlands) and a polystyrene-divinylbenzenebased anion exchange column (Hamilton PRP X-100, Reno, NV, USA) equipped with a guard column with the same stationary phase material. The analytical peaks obtained were evaluated in terms of peak area mode by external standard calibration and standard addition methods. The instrumental operating conditions are given in Table 1.
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Table 1 Instrumental operating conditions for the HPLC-ICP-MS set-up ICP-MS parameters Forward power Plasma gas flow rate Carrier gas flow rate Dwell time Isotopes monitored
1500 W 14.5 L min− 1 0.94 L min− 1 0.1 s per isotope 77 Se, 78Se, 80Se, 82Se
SCX-HPLC parameters Column Mobile phase Flow rate Injection volume
Chrompack IonoSpher 5C (4.6 mm × 150 mm × 5 μm) 2 mM pyridinium formate, pH 2.8, isocratic elution 0.8 mL min− 1 100 μL
SAX-HPLC Column Mobile phase
Flow rate Injection volume
Hamilton PRP X-100 (4.1 mm × 250 mm × 10 μm) Start buffer (A): 10 mM NH4H2PO4, 1% (v/v) methanol, pH 5.0 Elution buffer (B): 50 mM NH4H2PO4, 1% (v/v) methanol, pH 5.0 0–7 min: 100% A 7.1–21 min: 100% B 21.1–26 min: 100% A 1.5 mL min− 1 100 μL
2.2. Reagents All reagents were of analytical reagent grade. 100 mg L− 1 (for Se) stock solutions of Se-(methyl) selenocysteine hydrochloride (MeSeCys; 95%; Sigma Chemicals, St. Louis, MO, USA), seleno-DL-methionine (SeMet; 99%+, Acros Organics, Geel, Belgium), seleno-L-cysteine (SeCys2; 98%, Acros Organics) and Se(VI) (prepared from Na2SeO4; 98%; Aldrich, Milwaukee, WI, USA) were obtained by dissolving the appropriate amount of the corresponding compound in deionized water. Se(IV) 1000 mg L− 1 stock solution was obtained from Merck (Darmstadt, Germany). For chromatographic purposes, NH4H2PO4, NH4OH, H3PO4, conc. HCOOH, NaOH and pyridine were obtained from Reanal (Budapest, Hungary). For the sample preparation procedures, conc. HNO3, H2O2 and Pronase E (10191.4 U mg− 1 solid) were purchased from Merck. Elgacan Ultra-Pure water (R N 10 MΩ; Vivendi Water Systems Ltd., High Wycombe Bucks, England) was used throughout the whole experiment. Chromatographic standard and other working solutions were prepared daily as required. 2.3. Cultivation and preparation of Se-enriched green onion For this study, commercially available green onion, Allium fistulosum was selected. Onion sets purchased from a local food market were planted into pots (5 pickles in a pot) and were watered daily with deionized water. After 3 weeks when onions had attained full growth, selenium supplementation was started. Plants were treated either with the solution of Se(IV) or SeMet at the concentration of 10 or 100 μg Se mL− 1 every other day for
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2 weeks. Each pot containing 5 full-grown onions were selenized altogether with 200 mL of the corresponding Se species solution. The total cultivation period (including the time of growing and the enrichment) took 5 weeks. Treatments were carried out in eight replicates. To determine the average selenium content of non-selenized onions, control samples cultivated without Se supplementation were also analyzed in this study. Five replicates were applied. After harvesting, green onions were washed first with tap water then deionized water to exclude contamination from the surface. Samples were then separated into bulbs and leaves. These two parts were handled separately during this study. Bulbs and leaves were cut into small pieces to increase the specific surface of the samples thus improving the heat transfer during freeze-drying. After eliminating the moisture content, which found to be 90% of the sample weight, samples were ground in a household coffee-grinder (Bomann CB425, Germany) for complete homogenisation of the sample.
et al. [17] 0.050 g of freeze-dried onion sample was weighed into a plastic centrifuge tube and 3 mL of sodium hydroxide solution (0.1 mol L− 1) was added. This mixture was shaken on a Vortex device for 15 min. In order to extract the protein bound selenium species, enzymatic extraction was also performed with the help of a proteolytic enzyme. Deionized water (5 mL) was added to 0.05 g of the sample and Pronase E (0.005 g) was applied. This mixture was shaken at room temperature for 24 h. After both extraction procedures, samples were centrifuged at 5000 rpm for 10 min. The supernatant was decanted from the residue and filtered through a 0.45 μm cellulose nitrate syringe filter (Millipore, Tullagreen, Ireland). Samples kept frozen till speciation analysis started. Three repetitions were applied in the case of both extractions. 3. Results and discussion 3.1. Total selenium determination
2.4. Procedure for total Se-determination For the determination of total Se-content of onions, 100 mg sample was placed into PTFE vials and dissolved in a mixture of 2.0 mL of conc. HNO3 and 2.0 mL of 30% H2O2. After 12 h of contact time, total digestion was carried out at 110 °C for 20 min in a laboratory pressure cooker to get totally digested sample. Then final solutions were made up to 10.0 mL in a volumetric flask with deionized water. From each sample three digestions were carried out. Digestion blank was also applied so that the possible Se-contamination during sample preparation could be controlled. The total selenium concentration was determined by the method of standard addition and external standard calibration. The detection limit in terms of amount of selenium was calculated as three times the standard deviation of the background signal and was established to be 2 ng g− 1 as Se under the operating conditions given in Table 1. 2.5. Sample preparation for speciation analysis The importance of sample preparation is of paramount interest as selenium compounds can exist both in free form or covalently bound to proteins in the sample. Several extraction alternatives have already been reported such as shaking or stirring the sample with either slightly concentrated NaOH [17] or HCl [21,22] solutions but hot water extraction is also often used [9,14]. By applying any of these procedures, mostly the free forms of selenium can only be extracted. Thus, for liberating the selenium incorporated into proteins the peptide bonds should be broken down inside the molecules while keeping the Se-containing amino acids or their derivatives intact. The usual analytical practice applies proteolytic enzymes as the tool of this process [23–28]. Two types of sample preparation methods based on the above mentioned studies were compared from the point of view of Se extraction efficiency in the Se-enriched onions. The first extraction was carried out on the basis of the paper by M. Shah
Se-uptake rates were studied by determining the total selenium content of selenized onion bulbs and leaves separately with an ICP-MS by monitoring 77Se, 78Se, 80Se and 82Se isotopes. All concentration values presented in this paper are calculated for dry weight of the sample. The total selenium concentration of control samples (without selenium treatment) was approximately 1 μg g− 1 (1.24 ± 0.07 μg Se g− 1 in the leaves and 0.7 ± 0.23 μg Se g− 1 in the bulbs). Since the control sample does not contain adequate amount of selenium and taking into consideration the extraction efficiency during sample preparation, these samples were not Se-speciated. Results derived from the total selenium measurements are presented in Table 2. As this table shows, treating the onions with 10 μg mL− 1 solution results in approximately 10 μg g− 1 total Se for dry weight in either part of the sample independently the Se species applied. When onion was enriched with Se(IV) at a concentration of 100 μg mL − 1 , the total Se content increased significantly. Bulbs contained more than two times higher amount of Se than leaves, 61.8 and 24.5 μg g− 1 respectively. Onions supplemented with 100 μg mL− 1 SeMet showed to have accumulated up to 200 μg Se g− 1. No significant difference was found between the Se content of the bulbs and the leaves. Table 2 Total selenium content of selenium enriched onions. Data represent the mean value of the samples measured in 5 replicates Se conc. used for Se form used for Onion part Total Se in freeze-dried enrichment (μg g− 1) enrichment analyzed samples ± SD (μg g− 1) 10
Se(IV) SeMet
100
Se(IV) SeMet
Leaf Bulb Leaf Bulb Leaf Bulb Leaf Bulb
11.8 ± 0.4 14.4 ± 0.8 11.2 ± 0.6 10.9 ± 0.5 24.5 ± 1.1 61.8 ± 1.5 224 ± 1.1 213 ± 1.2
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3.2. Speciation studies To study the effect of the source of selenium enrichment (Se (IV) or SeMet) on the Se species distribution, speciation studies were accomplished. For identification, standard solutions containing inorganic Se(IV) and Se(VI), organic selenomethionine (SeMet), selenocysteine (SeCys2) and Se-(methyl) selenocysteine (SeMeCys) were used. The calibration curves for each of the species were linear over three orders of magnitude of concentration.
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Fig. 1(b) depicts the cation-exchange separation of onion bulbs enriched with SeMet at the concentration of 100 μg Se mL− 1 and extracted with proteolytic enzyme. MeSeCys (No. 2) and SeMet (No. 3) were successfully identified from this sample. An unknown compound (marked with ‘?’) was detected eluting after the inorganic compounds. Inorganic species eluted with the void volume having no retention on this column. Therefore the separation of selenite and selenate was performed with anion exchange chromatography. 3.4. Anion exchange chromatography
3.3. Cation exchange chromatography Fig. 1(a) shows a typical chromatogram of a mixture of selenium standards with the following retention times: Se(IV) (tret = 1.23 ± 0.02 min), MeSeCys (tret = 3.27 ± 0.03 min), SeMet (t ret = 5.07 ± 0.07 min) and SeCys 2 (t ret = 8.76 ± 0.03 min). These retentions are only valid for standard solutions when species of interest are mostly in water medium. Analyzing the different extracts, like enzymatic or alkaline, the method of standard addition is necessary to apply for identification purposes. As the figure presents baseline separation was obtained for all the organic Se species investigated. Using both chromatographies extracts were appropriately diluted for the better separation before being injected onto the column.
Anion exchange chromatography was also employed for the investigation of selenized onions. Co-elution was observed between SeCys2 and MeSeCys under the conditions applied (Table 1) thus this column was addressed to the investigation of inorganic compounds. Fig. 1(c) shows a typical chromatogram of a mixture of selenium standards with the following retention times obtained with anion exchange separation: SeCys2 (tret = 0.89 ± 0.05 min), SeMet (tret = 1.75 ± 0.02 min), Se(IV) (tret = 5.46 ± 0.05 min) and Se(VI) (tret = 17.52 ± 0.07 min). Gradient elution was applied to make Se(VI) elute from the column. This buffer-change caused a baseline shift around 9 min. Analyzing the enzymatic extract of onion bulbs enriched with SeMet at the concentration of 100 μg Se mL− 1, SeMet was unambiguously identified, see Fig.
Fig. 1. Separation of a mixture of selenium standards containing 100 ng Se mL− 1 separately with (a) cation exchange (c) anion exchange chromatography. Peak: 1 = Se (IV); 2 = MeSeCys; 3 = SeMet; 4 = SeCys2; 5 = Se(VI); 100 μg mL− 1 SeMet enriched onion bulb analyzed with (b) cation exchange; (d) anion exchange chromatography after enzymatic digestion. Isotope 80Se was monitored.
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1(d). As mentioned above, SeCys2 co-elutes with MeSeCys on this column therefore the identification of the first peak (No. 2) based on the cation exchange analysis where the method of standard addition proved the presence of MeSeCys. Se(VI) was present in both extracts (enzymatic and NaOH) of onion bulb enriched with 100 μg mL− 1 SeMet representing the total inorganic content of the sample, while analyzing samples supplemented with Se(IV) at higher concentration Se(IV) was identified as the main inorganic species. An unknown compound was also detected with this separation, which could not be identified with the available standards. Further studies are in progress for the identification of the unknown Se species found. Our assumption is that this peak could be γglutamyl-Se-methyl-selenocysteine, identified by several times from Allium vegetables [9,15].
3.5. Species distribution in the Se-enriched onions Fig. 2 compares the extractability of Se species from the point of view of applying different extractants. Data presented in Fig. 2 are based on the cation exchange studies. 100% (sum of the species) indicated is concerned to the total Se content measured in both extracts. All data bars depict the percentage distribution of Se species extracted with a corresponding extractant as a function of the supplementation carried out either with Se(IV) and SeMet at a given concentration level (10 or 100 μg mL− 1). The reported distribution numbers are average values of three replicate measurements expressed as relative percentage concentrations. Results derived from the analysis of the onions enriched with 10 μg Se mL− 1 in the form of SeMet or Se(IV) and extracted
Fig. 2. Selenium species distribution expressed in the percentage of total selenium content of (a) NaOH extract-samples enriched with 100 μg Se mL− 1 solution; (b) enzymatic extract-samples enriched with 10 μg Se mL− 1 solution; (c) enzymatic extract-samples enriched with 100 μg Se mL− 1 solution. Isotope 80Se was monitored.
E. Kápolna, P. Fodor / Microchemical Journal 84 (2006) 56–62
with NaOH (not presented in Fig. 2) showed the presence of MeSeCys as the only Se species could be identified with the available standards. Both enrichments resulted in a less intensive unknown peak, corresponding approximately 10% of the total Se content of the samples. When analyzing onion leaves and bulbs supplemented with 100 μg Se(IV) mL− 1 and extracted with NaOH, only a slight difference was observed in the species distribution of the two onion parts (Fig. 1a). Approximately 80% of inorganic selenium was detected while the organic species did not exceed the 20% value. An unknown compound is responsible for 3% and 1% of the total Se content of the extract of the leaves and bulbs, respectively. When enrichment of the onions were carried out with the same concentration of Se solution but Se was in the form of SeMet, leaves showed to contain about 85% of inorganic and 10% of MeSeCys, and some traces of SeMet was also detected. On the contrary, bulbs comprised 50% of inorganic Se and the MeSeCys content of these bulbs increased to 40%. SeMet and the unknown compound are responsible for the rest 10% of the total Se in the extract. On the grounds of the above results, the higher inorganic Se concentration indicates a lower inorganic to organic conversion when supplementation was carried out with the solution containing either of the Se species at 100 μg mL− 1 concentration. To release the covalently bound selenoamino acids enzymatic or acidic extraction is necessary. Using enzymatic extraction for releasing the Se from the sample, extraction efficiency increased to 78% compared to the NaOH extract (55%). Analyzing onions enriched with 10 μg mL− 1 SeMet or Se (IV) more organic compounds were detected due to the function of enzymes releasing the species located in the intracellular space (Fig. 2b). Main species identified are the MeSeCys and SeMet. Unknown species was also detected here, contributing up to 8% of the total Se content of the extracts. The highest amount of MeSeCys (85%) was observed in the case of onion bulbs enriched with 10 μg mL− 1 Se(IV). Selenium species distribution found in this study is in agreement with other papers reported in this topic being MeSeCys is the main species of selenized Allium plants [14,29]. When samples were supplemented with 100 μg mL− 1 SeMet or Se(IV), high amount of inorganic Se was detected just like in the case of NaOH extraction but the proteolytic enzyme released the SeMet content from the sample as well (Fig. 2c). SeMet enriched onions contained higher amount of organic Se compared to Se(IV)-enriched. 4. Conclusions Green onions were enriched with SeMet or Se(IV) at two concentrations (10 and 100 μg Se mL− 1) to produce a possible Se supplement. Results derived from total selenium determination showed that onion enriched with 100 μg Se mL− 1 in the form of SeMet accumulated Se up to 220 μg g− 1. Using proteolytic enzyme 80% of the total Se could be extracted from the sample, while NaOH extraction yielded 55% as the extraction efficiency. However much higher total Se content was attained either with Se(IV) or SeMet at
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100 μg Se mL− 1 concentration, speciation studies revealed that this type of enrichment resulted in 80% or more inorganic Se in the plant. This Se concentration is too high for the onion plants to metabolize either inorganic or organic Se into anticarcinogenic species, like MeSeCys and therefore could not be used for producing Se supplements. The highest amount of MeSeCys (85%) was observed in the case of onion bulbs enriched with 10 μg mL− 1 Se(IV). Using this type of lyophilized onion for dietary purposes, 2–6 g should be consumed to cover the adequate RDA range (30 to 70 μg). When these Se-enriched onions are in the natural form (water content is not removed from the plant) the above range is ten times higher.
Acknowledgements Special thanks to Éva Bányai for her help with the cultivation of the onions. References [1] J.T. Rotruck, H.E. Ganther, A.B. Swanson, D.G. Hafeman, W.G. Hoekstra, Science 179 (1973) 588. [2] J.R. Arthur, F. Nicol, G.J. Beckett, Biochem. J. 272 (1990) 537. [3] L.H. Foster, S. Sumar, Crit. Rev. Food Sci. Nutr. 37 (1997) 211. [4] P.A. van den Brandt, R.A. Goldbohm, P. van't Veer, P. Bode, E. Dorant, R.J. Hermus, F. Sturmans, J. Natl. Cancer Inst. 85 (1993) 224. [5] D.H. Holben, A.M. Smith, J. Am. Diet. Assoc. 99 (1999) 836. [6] W.N. Choy, P.R. Henika, C.C. Willhite, A.F. Tarantal, Environ. Mol. Mutagen. 21 (1993) 73. [7] M.P. Rayman, Lancet 356 (2000) 233. [8] C.D. Thomson, Br. J. Nutr. 91 (2004) 661. [9] A.M. Roussel, Selenium in cancer prevention, 10th ed. Plenum Press, New York, 2000. [10] M. Kotrebai, M. Birringer, J.F. Tyson, E. Block, P.C. Uden, Anal. Commun. 36 (1999) 249. [11] C. Ip, M. Birringer, E. Block, M. Kotrebai, J.F. Tyson, P.C. Uden, D.J. Lisk, J. Agric. Food Chem. 48 (2000) 2062. [12] J.L. Groff, S.S. Gropper, S.M. Hunt, Advanced nutrition and human metabolism, West Publishing Company, Minneapolis, 1995. [13] M. Shils, J.A. Olson, M. Shike, A.C. Ross, Nutrition in health and disease, 9th ed.Williams & Wilkins, Baltimore, 1999. [14] P.D. Whanger, C. Ip, C.E. Polan, P.C. Uden, G. Welbaum, J. Agric. Food Chem. 48 (2000) 5723. [15] M. Kotrebai, M. Birringer, J.F. Tyson, E. Block, P.C. Uden, Analyst 125 (2000) 71. [16] J.W. Hamilton, J. Agric. Food Chem. 23 (1975) 1150. [17] M. Shah, S.S. Kannamkumarath, J.C.A. Wuilloud, R.G. Wuilloud, J.A. Caruso, J. Anal. At. Spectrom. 19 (2004) 381. [18] M.T. Roberge, A.J. Borgerding, J.W. Finley, J. Agric. Food Chem. 51 (2003) 4191. [19] J. Lintschinger, N. Fuchs, J. Moser, D. Kuehnelt, W. Goessler, J. Agric. Food Chem. 48 (2000) 5362. [20] D.A. Kopsell, W.M. Randle, Euphytica 96 (1997) 385. [21] T.D. Grant, M. Montes-Bayon, D. LeDuc, M.W. Fricke, N. Terry, J.A. Caruso, J. Chromatogr., A 1026 (2004) 159. [22] M. Montes-Bayon, T.D. Grant, J. Meija, J.A. Caruso, J. Anal. At. Spectrom. 17 (2002) 1015. [23] S.S. Kannamkumarath, K. Wrobel, K. Wrobel, A. Vonderheide, J.A. Caruso, Anal. Bioanal. Chem. 373 (2002) 454. [24] E.T. Bodó, Zs. Stefánka, I. Ipolyi, Cs. Sörös, M. Dernovics, P. Fodor, Anal. Bioanal. Chem. 377 (2003) 32.
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Speciation analysis of selenium enriched green onions (Allium fistulosum) by HPLC-ICP-MS Emese Kápolna ⁎, Péter Fodor Department of Applied Chemistry, Corvinus University of Budapest, Villányi út 29-33, 1118 Budapest, Hungary Received 3 February 2006; received in revised form 11 April 2006; accepted 11 April 2006 Available online 21 June 2006
Abstract Green onions (Allium fistulosum) enriched with 10 or 100 μg mL− 1 Se(IV) or SeMet were analyzed for total selenium and species distribution. Anion and cation exchange chromatographies were applied for the separation of selenium species with mass spectrometric detection. Two different sample preparation methods (NaOH and enzymatic) were compared from the Se extraction efficiency point of view. Total selenium concentration accumulated by the onions reached the 200 μg g− 1 level expressed for dry weight when applying SeMet at a concentration of 100 μg mL− 1 as the source of Se. Speciation studies revealed that both in onion bulbs and leaves the predominant form of organic selenium is Se-methylselenocysteine (MeSeCys). When Se(IV) was applied for Se-enrichment at a concentration level of 100 μg mL− 1 both onion leaf and bulb contained a significant amount of inorganic selenium. An unknown compound was also detected. © 2006 Elsevier B.V. All rights reserved. Keywords: Green onion; HPLC-ICP-MS; Selenium supplementation; Se-methyl-selenocysteine; Speciation
1. Introduction Selenium (Se) plays an important role in human and animal nutrition being an essential component of a number of enzymes like glutathione peroxidase (GPX) [1,2]. Insufficient selenium intake tends to cause several health problems including cardiac failure. For example in a region of China (Keshan) where the selenium deficiency accompanying with a virus infection resulted in cardiomyopathy affected young women and children [3]. Furthermore, the amount of cancer patients is also increasing in consequence with the selenium deficiency in everyday diet [4]. Although selenium is required for health in a certain concentration, high doses can be toxic [5]. The intoxication depends on selenium concentration and the chemical forms of this element. In humans the gap between the estimated average requirement and the upper limit of safe intake is relatively narrow [6]. Recommended daily dietary
⁎ Corresponding author. Tel.: +36 1 482 6164; fax: +36 1 466 4272. E-mail address: [email protected] (E. Kápolna). 0026-265X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2006.04.014
allowance (RDA) for selenium in European countries is set from 30 to 70 μg [7]. Selenium content of food varies widely between regions throughout the world. In several European countries including Hungary, the soil is selenium depleted. This deficiency should be made up by introducing natural selenium sources into the human diet. For selenium supplementation purposes, several commercial dietary supplements are available in the market. Knowing only the total selenium content of a proposed Se-supplement is not unambiguous that the given product could be appropriate for supplementation. The bioavailability of selenium depends on several factors like selenium species distribution in the food product and the special physiological conditions of the consumer/e.g. age, the state of health etc./ [8]. Several animal studies have proved that selenium supplementation significantly reduces tumor incidence [9]. Most common Se supplements are selenized yeasts whose major selenocompound is selenomethionine (SeMet) [10]. A paper by Ip. C. et al. [11] compared the anticarcinogenic attribute of Se-enriched yeast and garlic on rats with both chemically and virally induced tumors. This study revealed that the ingestion of selenized yeast caused an undesirable accumulation of selenium in tissues while this
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phenomenon did not appear in the case of garlic. The explanation for the different behaviour is due to the diverse chemical forms of selenium present in the sample. Plants acquire selenium from the soil and therefore vary in content depending upon the region they are grown and tend to incorporate it non-specifically into compounds usually containing sulfur [12,13]. Selenium enriched garlic, onion, ramp, cabbage, sprout and broccoli have been extensively studied. Major Se species found in these selenized vegetables are the methylated forms of selenium, like Se-methyl-seleno-cysteine and γ-glutamyl-Se-methyl-seleno-cysteine. These species seem to be more effective inhibitors of tumor formation than other organic or inorganic ones [9,14–19]. Allium family vegetables, like onion and garlic are consumed world-wide due to their flavor compounds. Allium species have the capability to accumulate selenium up to high μg g − 1 levels meanwhile converting inorganic selenium being fortified with into organic compounds [17,20]. As Allium family vegetables are kindly consumed everywhere, the aim of this work was to produce a Se-supplement focusing on green onions (Allium fistulosum), which can be easily built into the everyday diet. Within the confines of this study the determination of not only the total selenium concentration of the enriched onion samples but also the species distribution was performed. The goal was to compare the supposedly various selenium distribution of samples enriched in different ways. 2. Materials and methods 2.1. Instrumentation An ICP-MS Agilent 7500ce (Agilent Technologies, Tokyo, Japan) was applied for total selenium and speciation analysis as an element specific detector. The ICP-MS is equipped with an octopole reaction cell and an ASX-510 autosampler. The reaction cell conditions were optimized using H2 as collision gas with a flow rate between 0–5 mL min− 1 monitoring 77Se, 78Se, 80Se and 82Se isotopes. A conventional Babington nebulizer was used for the sample introduction under standard plasma conditions. For chromatographic separations the ICP-MS was coupled to a Merck Model L-7100 4-channel HPLC pump with a six port Rheodyne (Cotati, CA, USA) sample injection valve (Merck, Darmstadt, Germany) used for eluent delivery and sample introduction. The coupling between the corresponding column outlet and the sample introduction system of ICP-MS was obtained through a 500 mm long 0.5 mm id. PEEK tubing. Chromatographic columns used were a silica-based strong cation exchange column (Ionospher 5C, Varian BV, Middelburg, The Netherlands) and a polystyrene-divinylbenzenebased anion exchange column (Hamilton PRP X-100, Reno, NV, USA) equipped with a guard column with the same stationary phase material. The analytical peaks obtained were evaluated in terms of peak area mode by external standard calibration and standard addition methods. The instrumental operating conditions are given in Table 1.
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Table 1 Instrumental operating conditions for the HPLC-ICP-MS set-up ICP-MS parameters Forward power Plasma gas flow rate Carrier gas flow rate Dwell time Isotopes monitored
1500 W 14.5 L min− 1 0.94 L min− 1 0.1 s per isotope 77 Se, 78Se, 80Se, 82Se
SCX-HPLC parameters Column Mobile phase Flow rate Injection volume
Chrompack IonoSpher 5C (4.6 mm × 150 mm × 5 μm) 2 mM pyridinium formate, pH 2.8, isocratic elution 0.8 mL min− 1 100 μL
SAX-HPLC Column Mobile phase
Flow rate Injection volume
Hamilton PRP X-100 (4.1 mm × 250 mm × 10 μm) Start buffer (A): 10 mM NH4H2PO4, 1% (v/v) methanol, pH 5.0 Elution buffer (B): 50 mM NH4H2PO4, 1% (v/v) methanol, pH 5.0 0–7 min: 100% A 7.1–21 min: 100% B 21.1–26 min: 100% A 1.5 mL min− 1 100 μL
2.2. Reagents All reagents were of analytical reagent grade. 100 mg L− 1 (for Se) stock solutions of Se-(methyl) selenocysteine hydrochloride (MeSeCys; 95%; Sigma Chemicals, St. Louis, MO, USA), seleno-DL-methionine (SeMet; 99%+, Acros Organics, Geel, Belgium), seleno-L-cysteine (SeCys2; 98%, Acros Organics) and Se(VI) (prepared from Na2SeO4; 98%; Aldrich, Milwaukee, WI, USA) were obtained by dissolving the appropriate amount of the corresponding compound in deionized water. Se(IV) 1000 mg L− 1 stock solution was obtained from Merck (Darmstadt, Germany). For chromatographic purposes, NH4H2PO4, NH4OH, H3PO4, conc. HCOOH, NaOH and pyridine were obtained from Reanal (Budapest, Hungary). For the sample preparation procedures, conc. HNO3, H2O2 and Pronase E (10191.4 U mg− 1 solid) were purchased from Merck. Elgacan Ultra-Pure water (R N 10 MΩ; Vivendi Water Systems Ltd., High Wycombe Bucks, England) was used throughout the whole experiment. Chromatographic standard and other working solutions were prepared daily as required. 2.3. Cultivation and preparation of Se-enriched green onion For this study, commercially available green onion, Allium fistulosum was selected. Onion sets purchased from a local food market were planted into pots (5 pickles in a pot) and were watered daily with deionized water. After 3 weeks when onions had attained full growth, selenium supplementation was started. Plants were treated either with the solution of Se(IV) or SeMet at the concentration of 10 or 100 μg Se mL− 1 every other day for
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2 weeks. Each pot containing 5 full-grown onions were selenized altogether with 200 mL of the corresponding Se species solution. The total cultivation period (including the time of growing and the enrichment) took 5 weeks. Treatments were carried out in eight replicates. To determine the average selenium content of non-selenized onions, control samples cultivated without Se supplementation were also analyzed in this study. Five replicates were applied. After harvesting, green onions were washed first with tap water then deionized water to exclude contamination from the surface. Samples were then separated into bulbs and leaves. These two parts were handled separately during this study. Bulbs and leaves were cut into small pieces to increase the specific surface of the samples thus improving the heat transfer during freeze-drying. After eliminating the moisture content, which found to be 90% of the sample weight, samples were ground in a household coffee-grinder (Bomann CB425, Germany) for complete homogenisation of the sample.
et al. [17] 0.050 g of freeze-dried onion sample was weighed into a plastic centrifuge tube and 3 mL of sodium hydroxide solution (0.1 mol L− 1) was added. This mixture was shaken on a Vortex device for 15 min. In order to extract the protein bound selenium species, enzymatic extraction was also performed with the help of a proteolytic enzyme. Deionized water (5 mL) was added to 0.05 g of the sample and Pronase E (0.005 g) was applied. This mixture was shaken at room temperature for 24 h. After both extraction procedures, samples were centrifuged at 5000 rpm for 10 min. The supernatant was decanted from the residue and filtered through a 0.45 μm cellulose nitrate syringe filter (Millipore, Tullagreen, Ireland). Samples kept frozen till speciation analysis started. Three repetitions were applied in the case of both extractions. 3. Results and discussion 3.1. Total selenium determination
2.4. Procedure for total Se-determination For the determination of total Se-content of onions, 100 mg sample was placed into PTFE vials and dissolved in a mixture of 2.0 mL of conc. HNO3 and 2.0 mL of 30% H2O2. After 12 h of contact time, total digestion was carried out at 110 °C for 20 min in a laboratory pressure cooker to get totally digested sample. Then final solutions were made up to 10.0 mL in a volumetric flask with deionized water. From each sample three digestions were carried out. Digestion blank was also applied so that the possible Se-contamination during sample preparation could be controlled. The total selenium concentration was determined by the method of standard addition and external standard calibration. The detection limit in terms of amount of selenium was calculated as three times the standard deviation of the background signal and was established to be 2 ng g− 1 as Se under the operating conditions given in Table 1. 2.5. Sample preparation for speciation analysis The importance of sample preparation is of paramount interest as selenium compounds can exist both in free form or covalently bound to proteins in the sample. Several extraction alternatives have already been reported such as shaking or stirring the sample with either slightly concentrated NaOH [17] or HCl [21,22] solutions but hot water extraction is also often used [9,14]. By applying any of these procedures, mostly the free forms of selenium can only be extracted. Thus, for liberating the selenium incorporated into proteins the peptide bonds should be broken down inside the molecules while keeping the Se-containing amino acids or their derivatives intact. The usual analytical practice applies proteolytic enzymes as the tool of this process [23–28]. Two types of sample preparation methods based on the above mentioned studies were compared from the point of view of Se extraction efficiency in the Se-enriched onions. The first extraction was carried out on the basis of the paper by M. Shah
Se-uptake rates were studied by determining the total selenium content of selenized onion bulbs and leaves separately with an ICP-MS by monitoring 77Se, 78Se, 80Se and 82Se isotopes. All concentration values presented in this paper are calculated for dry weight of the sample. The total selenium concentration of control samples (without selenium treatment) was approximately 1 μg g− 1 (1.24 ± 0.07 μg Se g− 1 in the leaves and 0.7 ± 0.23 μg Se g− 1 in the bulbs). Since the control sample does not contain adequate amount of selenium and taking into consideration the extraction efficiency during sample preparation, these samples were not Se-speciated. Results derived from the total selenium measurements are presented in Table 2. As this table shows, treating the onions with 10 μg mL− 1 solution results in approximately 10 μg g− 1 total Se for dry weight in either part of the sample independently the Se species applied. When onion was enriched with Se(IV) at a concentration of 100 μg mL − 1 , the total Se content increased significantly. Bulbs contained more than two times higher amount of Se than leaves, 61.8 and 24.5 μg g− 1 respectively. Onions supplemented with 100 μg mL− 1 SeMet showed to have accumulated up to 200 μg Se g− 1. No significant difference was found between the Se content of the bulbs and the leaves. Table 2 Total selenium content of selenium enriched onions. Data represent the mean value of the samples measured in 5 replicates Se conc. used for Se form used for Onion part Total Se in freeze-dried enrichment (μg g− 1) enrichment analyzed samples ± SD (μg g− 1) 10
Se(IV) SeMet
100
Se(IV) SeMet
Leaf Bulb Leaf Bulb Leaf Bulb Leaf Bulb
11.8 ± 0.4 14.4 ± 0.8 11.2 ± 0.6 10.9 ± 0.5 24.5 ± 1.1 61.8 ± 1.5 224 ± 1.1 213 ± 1.2
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3.2. Speciation studies To study the effect of the source of selenium enrichment (Se (IV) or SeMet) on the Se species distribution, speciation studies were accomplished. For identification, standard solutions containing inorganic Se(IV) and Se(VI), organic selenomethionine (SeMet), selenocysteine (SeCys2) and Se-(methyl) selenocysteine (SeMeCys) were used. The calibration curves for each of the species were linear over three orders of magnitude of concentration.
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Fig. 1(b) depicts the cation-exchange separation of onion bulbs enriched with SeMet at the concentration of 100 μg Se mL− 1 and extracted with proteolytic enzyme. MeSeCys (No. 2) and SeMet (No. 3) were successfully identified from this sample. An unknown compound (marked with ‘?’) was detected eluting after the inorganic compounds. Inorganic species eluted with the void volume having no retention on this column. Therefore the separation of selenite and selenate was performed with anion exchange chromatography. 3.4. Anion exchange chromatography
3.3. Cation exchange chromatography Fig. 1(a) shows a typical chromatogram of a mixture of selenium standards with the following retention times: Se(IV) (tret = 1.23 ± 0.02 min), MeSeCys (tret = 3.27 ± 0.03 min), SeMet (t ret = 5.07 ± 0.07 min) and SeCys 2 (t ret = 8.76 ± 0.03 min). These retentions are only valid for standard solutions when species of interest are mostly in water medium. Analyzing the different extracts, like enzymatic or alkaline, the method of standard addition is necessary to apply for identification purposes. As the figure presents baseline separation was obtained for all the organic Se species investigated. Using both chromatographies extracts were appropriately diluted for the better separation before being injected onto the column.
Anion exchange chromatography was also employed for the investigation of selenized onions. Co-elution was observed between SeCys2 and MeSeCys under the conditions applied (Table 1) thus this column was addressed to the investigation of inorganic compounds. Fig. 1(c) shows a typical chromatogram of a mixture of selenium standards with the following retention times obtained with anion exchange separation: SeCys2 (tret = 0.89 ± 0.05 min), SeMet (tret = 1.75 ± 0.02 min), Se(IV) (tret = 5.46 ± 0.05 min) and Se(VI) (tret = 17.52 ± 0.07 min). Gradient elution was applied to make Se(VI) elute from the column. This buffer-change caused a baseline shift around 9 min. Analyzing the enzymatic extract of onion bulbs enriched with SeMet at the concentration of 100 μg Se mL− 1, SeMet was unambiguously identified, see Fig.
Fig. 1. Separation of a mixture of selenium standards containing 100 ng Se mL− 1 separately with (a) cation exchange (c) anion exchange chromatography. Peak: 1 = Se (IV); 2 = MeSeCys; 3 = SeMet; 4 = SeCys2; 5 = Se(VI); 100 μg mL− 1 SeMet enriched onion bulb analyzed with (b) cation exchange; (d) anion exchange chromatography after enzymatic digestion. Isotope 80Se was monitored.
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1(d). As mentioned above, SeCys2 co-elutes with MeSeCys on this column therefore the identification of the first peak (No. 2) based on the cation exchange analysis where the method of standard addition proved the presence of MeSeCys. Se(VI) was present in both extracts (enzymatic and NaOH) of onion bulb enriched with 100 μg mL− 1 SeMet representing the total inorganic content of the sample, while analyzing samples supplemented with Se(IV) at higher concentration Se(IV) was identified as the main inorganic species. An unknown compound was also detected with this separation, which could not be identified with the available standards. Further studies are in progress for the identification of the unknown Se species found. Our assumption is that this peak could be γglutamyl-Se-methyl-selenocysteine, identified by several times from Allium vegetables [9,15].
3.5. Species distribution in the Se-enriched onions Fig. 2 compares the extractability of Se species from the point of view of applying different extractants. Data presented in Fig. 2 are based on the cation exchange studies. 100% (sum of the species) indicated is concerned to the total Se content measured in both extracts. All data bars depict the percentage distribution of Se species extracted with a corresponding extractant as a function of the supplementation carried out either with Se(IV) and SeMet at a given concentration level (10 or 100 μg mL− 1). The reported distribution numbers are average values of three replicate measurements expressed as relative percentage concentrations. Results derived from the analysis of the onions enriched with 10 μg Se mL− 1 in the form of SeMet or Se(IV) and extracted
Fig. 2. Selenium species distribution expressed in the percentage of total selenium content of (a) NaOH extract-samples enriched with 100 μg Se mL− 1 solution; (b) enzymatic extract-samples enriched with 10 μg Se mL− 1 solution; (c) enzymatic extract-samples enriched with 100 μg Se mL− 1 solution. Isotope 80Se was monitored.
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with NaOH (not presented in Fig. 2) showed the presence of MeSeCys as the only Se species could be identified with the available standards. Both enrichments resulted in a less intensive unknown peak, corresponding approximately 10% of the total Se content of the samples. When analyzing onion leaves and bulbs supplemented with 100 μg Se(IV) mL− 1 and extracted with NaOH, only a slight difference was observed in the species distribution of the two onion parts (Fig. 1a). Approximately 80% of inorganic selenium was detected while the organic species did not exceed the 20% value. An unknown compound is responsible for 3% and 1% of the total Se content of the extract of the leaves and bulbs, respectively. When enrichment of the onions were carried out with the same concentration of Se solution but Se was in the form of SeMet, leaves showed to contain about 85% of inorganic and 10% of MeSeCys, and some traces of SeMet was also detected. On the contrary, bulbs comprised 50% of inorganic Se and the MeSeCys content of these bulbs increased to 40%. SeMet and the unknown compound are responsible for the rest 10% of the total Se in the extract. On the grounds of the above results, the higher inorganic Se concentration indicates a lower inorganic to organic conversion when supplementation was carried out with the solution containing either of the Se species at 100 μg mL− 1 concentration. To release the covalently bound selenoamino acids enzymatic or acidic extraction is necessary. Using enzymatic extraction for releasing the Se from the sample, extraction efficiency increased to 78% compared to the NaOH extract (55%). Analyzing onions enriched with 10 μg mL− 1 SeMet or Se (IV) more organic compounds were detected due to the function of enzymes releasing the species located in the intracellular space (Fig. 2b). Main species identified are the MeSeCys and SeMet. Unknown species was also detected here, contributing up to 8% of the total Se content of the extracts. The highest amount of MeSeCys (85%) was observed in the case of onion bulbs enriched with 10 μg mL− 1 Se(IV). Selenium species distribution found in this study is in agreement with other papers reported in this topic being MeSeCys is the main species of selenized Allium plants [14,29]. When samples were supplemented with 100 μg mL− 1 SeMet or Se(IV), high amount of inorganic Se was detected just like in the case of NaOH extraction but the proteolytic enzyme released the SeMet content from the sample as well (Fig. 2c). SeMet enriched onions contained higher amount of organic Se compared to Se(IV)-enriched. 4. Conclusions Green onions were enriched with SeMet or Se(IV) at two concentrations (10 and 100 μg Se mL− 1) to produce a possible Se supplement. Results derived from total selenium determination showed that onion enriched with 100 μg Se mL− 1 in the form of SeMet accumulated Se up to 220 μg g− 1. Using proteolytic enzyme 80% of the total Se could be extracted from the sample, while NaOH extraction yielded 55% as the extraction efficiency. However much higher total Se content was attained either with Se(IV) or SeMet at
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100 μg Se mL− 1 concentration, speciation studies revealed that this type of enrichment resulted in 80% or more inorganic Se in the plant. This Se concentration is too high for the onion plants to metabolize either inorganic or organic Se into anticarcinogenic species, like MeSeCys and therefore could not be used for producing Se supplements. The highest amount of MeSeCys (85%) was observed in the case of onion bulbs enriched with 10 μg mL− 1 Se(IV). Using this type of lyophilized onion for dietary purposes, 2–6 g should be consumed to cover the adequate RDA range (30 to 70 μg). When these Se-enriched onions are in the natural form (water content is not removed from the plant) the above range is ten times higher.
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