Anal. Chem. 2002, 74, 3463-3469
Selective Detection of Biogenic Amines Using Capillary Electrochromatography with an On-Column Derivatization Technique Shigeyuki Oguri,*,† Yukari Yoneya,† Makiko Mizunuma,† Yuhei Fujiki,‡ Koji Otsuka,‡ and Shigeru Terabe‡
Department of Home Economics, Aichi-Gakusen University, 28 Kamikawanari, Hegoshi-cho, Okazaki City, Aichi 444-8520, Japan, and Faculty of Science, Himeji Institute of Technology, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-129, Japan
A selective detection method for biogenic amines present in highly complex matrixes was devised by employing both electrokinetic injection and on-column-derivatization capillary electrochromatographic methods. The on-column derivatization capillary electrochromatography system was evaluated by use of a capillary column (total length of 45 cm, effective length of 25 cm) fabricated using a 100-µm (i.d.) fused-silica capillary tube packed with 5-µm (i.d.) ODS particles that were tolerant of an alkaline environment. The column was filled with a run buffer consisting of a derivatization reagent, o-phthalaldehyde/2-mercaptoethanol, in a mixture of borate buffer (pH 10). After electrokinetic injection of a mixture of five biogenic amines (histamine, serotonin, tyramine, putrescine, cadaverine) as a test sample, the free amines entered into the anodic site of the capillary column and started to travel along the column, during which time the analytes reacted with the derivatization reagent, separated out, and were detected with an absorbance at 340 nm when high voltage was applied to the column. When this system was applied to a mixture containing 5 biogenic amines and 17 amino acids, the 5 biogenic amines plus arginine selectively entered into the capillary with the electrokinetic injection and were observed on the electrochromatogram, but none of the amino acids lacking arginine were detected. The designated method was also tested for its ability to determine the presence of biogenic amines in the crude extracts obtained from two types of aged fish. Mono- or polyamines involved in biological activities and that exist in nature are sometimes called by biogenic amines.1 Some of them are biologically synthesized from amino acids by amino acid decarboxylase degradation and play an important role in living systems.2,3 Examples of monoamines are histamine and serotonin (5-hydroxytryputamine), which are synthesized from histidine and * Corresponding author: (fax) +81-564-1270; (E-mail)
[email protected]; (URL) http://www.gakusen.ac.jp/faculty/s-oguri. † AichiGakusen University. ‡ Himeji Institute of Technology. (1) Slocum, R. D.; Kaur-Sawhney, R.; Galston, A. W. Arch. Biochem. Biophys. 1984, 235, 283-303. (2) Persson, L.; Holm, I.; Stjernborg, L.; Heby, O. Adv. Exp. Med. Biol. 1988, 250, 61-71. 10.1021/ac025592d CCC: $22.00 Published on Web 05/21/2002
© 2002 American Chemical Society
tryptophan, respectively. These are regarded as belonging to the group of chemical mediators that are also known as autacoids or neurotransmitters.4-6 In the case of polyamines, some types of malignant cell proliferation are associated with increased cellular polyamine metabolism, and several investigators have suggested a possible role of polyamine determination in blood and urine samples as markers of the presence of malignancies.7-10 As is well known to researchers in the biological and analytical chemistry fields, the ability to selectively detect biogenic amines, including biogenically derived compounds, leads to deeper understanding of the role of these compounds in biological processes. The assay method for the above purpose has been employed with ELISA based on immunochemistry. Although the ELISA11-13 technique is very simple and sensitive, its ability to target a specific amine is relatively poor in comparison with some chromatographic techniques. Therefore, several chromatographic methods, such as gas chromatography (GC),14 high-performance liquid chromatography (HPLC),15 and capillary electrophoresis (CE),16-18 have been described in the literature for their ability to detect amines. These methods were usually performed by precolumn and postcolumn derivatization techniques in order to (3) Porter, C. W.; Herrera-Ornelas, L.; Pera, P.; Petrelli, N. F. Cancer 1987, 60, 1275-1281. (4) Yamatodani, N.; Inagaki, N.; Panula, P.; Itowi, N.; Watanabe, T.; Wada, H. Histamine and Histamine Antagonists. In Handbook of Experimental Pharmacology; Uvnas, B., Ed.; Springer-Verlag: Berlin, 1991; Vol. 97, Chapter 7, pp 243-283. (5) Cassel, J. C.; Jeltsch, H. Neuroscience 1995, 69, 1-41. (6) Nelson, R. J.; Chiavegatto, S. Trends Neuro Sci. 2001, 24, 713-719. (7) Bachrach, U. Ital. J. Biochem. 1976, 25, 77-93. (8) Russell, D. H. Clin. Chem. 1977, 23, 22-27. (9) Takami, H.; Romsdahl, M. M.; Nishioka, K. Lancet 1979, 2, 912. (10) Uehara, N.; Shirakawa, S.; Uchino, H.; Saeki, Y. Cancer 1980, 45, 108111. (11) Fujiwara, K.; Furukawa, K.; Nakayama, E.; Shiku, H. Histochemistry 1994, 102, 397-404. (12) Catcheside, J. A.; Stead, A. D.; Rider, C. C. Hybridoma 1996, 15, 199-204. (13) Delcros, J. G.; Loeuillet, L.; Chamaillard, L.; Royou, A.; Bouill, N.; Seiler, N.; Moulinoux, J. P. Cytometry 1997, 27, 255-261. (14) Mita, H.; Yasueda, H.; Shida, T. J. Chromatogr. 1980, 221, 1-6. (15) Seiler, N. Methods Biochem. Anal. 1970, 18, 259-265. (16) Oguri, S.; Watanabe, S.; Abe, S. J. Chromatogr., A 1997, 790, 177-183. (17) High Performance Capillary Electrophoresis; Khaledi, M. G., Ed.; John Wiley & Sons Inc.: New York, 1998. (18) Chromatography and Capillaryelectrophoresis in Food Analysis; Sørenson, H., Sørenson, S., Jergegaard, C. B., Michaelsen, S., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 1999.
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enhance detection sensitivity or selectivity. However, these methods sometimes require an extraction procedure to remove excess reagent in the case of the precolumn derivatization method and also sometimes require a special chemical reactor device for labeling of amines on-line in the case of postcolumn derivatization methods, respectively. These extra requirements complicate the operations and, therefore, tend to increase the probability of making human errors. Using CE methodology, new detection platforms have been developed to overcome these problems by several researchers. For example, Kuhr and Yeung19 first demonstrated an indirect detection technique that can be used as a universal CE detection scheme, without the need for timeconsuming precapillary derivatization or experimentally complicated postcapillary derivatization procedures. Using a run buffer containing a background electrolyte (BGE) in which a chromophore or fluorophore is present, the native analytes can be indirectly detected in the capillary with a high degree of sensitivity. This technique was applied to CE20 or a CE chip21 to detect biogenic amines, including amino acids in complex samples. In recent years, the in-capillary derivatization method was developed into a versatile derivatization technique as the alternative method to the pre- and postderivatization method for CE.22 This technique is mainly classified into either “on-site in-capillary derivatization” or “throughout in-capillary derivatization”. Fischman et al.23 first pointed out the possibility and demonstrated this technique by using the inlet of a separation capillary tube as a reaction chamber. The derivatization was performed at the inlet site of the capillary by introducing an analyte into the capillary tube between two plugs of labeling reagent. On-column derivatization was first developed by Saito et al.24 for the determination of biogenic amines in foods with HPLC using a mobile phase containing a derivatization reagent. This idea is based on mobile phase combined with both a separation buffer and a reaction buffer for derivatization. After that, the Oguri group25 applied this idea to the CE method in 1996, and this method is usually called the “throughout in-capillary derivatization method” or “on-line mode in-capillary derivatization method”. The throughout in-capillary derivatization CE performed separation and derivatization of free amines such as amino acids and biogenic amines, simultaneously in a separation capillary tube that was already filled with run buffer at pH 10 in the presence of o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as the derivatization reagent.26,27 Capillary electrochromatography (CEC)28 is currently undergoing rapid growth and is likely to become as important as CE is for microseparation. CEC is a powerful and useful technique because CEC possesses the advantages of both CE and HPLC. Therefore, we decided to use this technique to perform the (19) Kuhr, W. G.; Yeung, F. S. Anal. Chem. 1988, 60, 1832. (20) Arce, L.; Rı´os, A.; Valcarel, M. J. Chromatogr., A 1998, 803, 249-260. (21) Munro, M. J.; Hung, Z.; Finegold, D. N.; Landers, J. P. Anal. Chem. 2000, 72, 2765-2773. (22) Bardelmeijer, H. A.; Lingeman, H.; de Ruiter, C.; Under, W. J. M. J. Chromatogr., A 1998, 807, 3-26. (23) Fischman, H. A.; Shear, J. B.; Colon, L. A.; Zare, R. N. U.S. Patent 5 318 680, 1994. (24) Saito, K.; Horie, M.; Nose, N. J. Chromatogr., A 1992, 595, 163-168. (25) Oguri, S.; Fujiyoshi, T.; Miki, Y. Analyst 1996, 121, 1683-1688. (26) Oguri, S.; Tsukamoto, A.; Yura, A.; Miho, Y. Electrophoresis 1998, 19, 29862990. (27) Oguri, S.; Yokoi, K.; Motohase, Y. J. Chromatogr., A 1997, 787, 253-260. (28) Steiner, F.; Scherer, B. J. Chromatogr., A 2000, 887, 55-83.
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determination of biogenic amines using on-column derivatization CEC instead of throughout in-capillary derivatization CE; in doing so, we observed an interesting property that was useful for further development of a selective biogenic amines assay system. In this paper, the existence of biogenic amines in aged fish meat was also examined using this system. MATERIALS AND METHODS Reagents and Materials. OPA and other reagents were of commercially available biochemical and superfine reagent or HPLC grade, respectively, and were purchased from Wako Pure Chemical Co. (Tokyo, Japan). They were used without further purification. All aqueous solutions were prepared by using water purified with a Milli-Q purification system (Millipore, Milford, MA). Histamine, serotonin, tyramine, putrescine, and cadaverine as the test samples of biogenic amines were individually dissolved in saline to make 10 mM and were stored in a refrigerator before use. When refrigerated, they are thought to have a “shelf life” of approximately one week. A five-biogenic amine standard solution (each concentration of biogenic amine was 1 mM) was also prepared by combining 100 µL of each of the five kinds of 10 mM individual biogenic amine/saline solutions with 500 µL of acetonitrile. The 5-component biogenic amine and 17-component amino acid solution at 1 mM was obtained by mixing 200 µL of a 17component amino acid solution ((Type H; Wako Pure Chemical Co., Osaka, Japan), A 20-µL aliquot of each of the five kinds of 10 mM biogenic amine solution, 20 µL of 1 M sodium hydroxide, and 30 µL of saline were placed in a 1.6-mL polyethylene tube. The run buffers containing the derivatization reagent were prepared as follows. A mixture of acetonitrile and borate buffer (pH adjusted to 10 with 1 M NaOH) was added to a mixture of 1 mL of 50 mM OPA in acetonitrile and 1 mL of 50 mM 2-mercaptoethanol (2ME) in borate buffer to make a 10-mL final volume in a 12-mL glass reservoir. The reservoir was placed in a sonication bath (model UT-52, Sharp Co., Nara, Japan) under reduced pressure for several minutes in order to degas the run buffer. The run buffers were prepared just before use. As a precautionary note, proper care should be taken when handling biogenic amines, OPA/2ME reagent, and organic solvents as they may be sources of hazard to one’s health. Apparatus. The CEC and CE systems used consisted of a Jasco model CE-800 (Tokyo, Japan) equipped with an UV/visible detector, model CE-971-UV (Jasco). All data were printed out with an intelligent data processor, model 807-IT (Jasco). Fusedsilica capillary tubes (Polymicro Technologies LLC) of 100-µm i.d. (25 cm effective length) for the CEC study and 50-µm i.d. (25-cm effective length) for the CE study were used throughout the work. Sample solutions were introduced into the capillary tubes from the anodic side by electrokinetic injection (5 kV, 3 s). The electrochromatograms or electropherograms were produced by monitoring absorbance at 340 nm. The applied voltage in this study was carried out at 15 kV throughout the work. Capillary Column Fabrication. Capillary column fabrication was carried out according to the previously reported procedure29 with a slight modification: (1) A temporary frit was made at an appropriate position, e.g., a few centimeters from one end of the capillary with a sodium silicate solution by heating the capillary (29) Otsuka, K.; Mikami, C.; Terabe, S. J. Chromatogr., A 2000, 887, 457-463.
with an EK 1.2 CE capillary burner (Electro-Kinetic Technologies, West Lothian, U.K.). (2) A slurry of the packing material (20 mg), obtained by taking out the inside of Capcell Pak C18 (5 µm) type UG 120 (Shiseido, Tokyo, Japan) in 2-propanol (1 mL) followed by sonication for several minutes, was packed into the capillary with an LC-5A high-performance liquid chromatographic pump (Shimadzu, Kyoto, Japan) by using acetonitrile as a pressurized solvent at 480 bar. (3) After completing the packing, the capillary was flushed with acetonitrile and then with a 1% (v/v) solution of sodium chloride at 480 bar for 1 h and 20 min, respectively. (4) A retaining frit was made at a point close to the temporary frit, followed by making an end frit at an appropriate position under application of high pressure with the sodium chloride solution. (5) The outside portion of the retaining frit was cut off and then the capillary was flushed with a mobile phase to be used at 60 bar in the reverse direction for flushing out the rest of the packing material and conditioning. (6) A detection window was made just after the end frit by removing a section of the polyimide coating with the capillary burner. Finally, conditioning of the capillary was carried out by flushing with a mobile phase for 1 h with the syringe pump. Precolumn Derivatization CEC. A 50 mM OPA solution was prepared by dissolution of OPA (67.1 mg) in acetonitrile (10 mL). A 200 mM 2ME solution was also prepared by dilution of 2ME (∼20 µL; 20 mg) with 10 mM borate buffer (1.3 mL) at pH 10. A 100-µL aliquot of each of the 10 mM biogenic amine solutions (histamine, serotonin, tyramine, putrescine, cadaverine) was separately transferred into a 1.6-mL polyethylene tube. Then, 400 µL of the OPA solution and 100 µL of the 2ME solution were poured into the tube, combined with the biogenic amine solutions, and stirred well. After keeping the mixture for 5 min at room temperature, the reaction mixture was injected in to a capillary column which had already been filled with 70% acetonitrile/10 mM borate (pH 10) by means of a electrokinetic injection of 5 kV for 3 s. Then, high voltage (15 kV) was applied to the capillary and monitored at 340 nm. On-Column Derivatization. Two reservoirs were attached to the CEC system, one at the anodic site and one at the cathodic site. The anodic and cathodic reservoirs contained 10 mL of a run buffer containing the derivatizing reagent (OPA/2ME) and 10 mL of a run buffer not containing the derivatizing reagent, respectively. The anodic site of the capillary was placed in the run buffer in the reservoir at the anodic site, and the cathodic site of the capillary was connected to a manual syringe pump which was contained the cathodic run buffer. By pushing the manual syringe pump, any remaining residue in the capillary column was swept out and filled with the buffer. The end of the capillary was carefully disconnected from the manual syringe pump and subsequently moved into the cathodic reservoir without any gas bubbles remaining in the capillary column. Then, a capillary column was equilibrated in the same run buffer by applying a high voltage (15 kV, 30 min) to the column. A fivecomponent biogenic amine solution was directly injected into the capillary at the anodic site and analyzed in the same manner as described above. Throughout In-Capillary Derivatization CE. A throughout in-capillary derivatization CE was performed using the same system as the CEC equipped with a fused-silica capillary tube
instead of a capillary column. Operation procedures were presented in detail in a previous paper.24 Extraction of Biogenic Amines from Food Samples. Extractions of biogenic amines from various foods were carried out by means of a very simple method for this work. The food samples used in this study were pieces of raw fish (mackeral, tuna) that had been kept for 1 day at room temperature in order to produce our desired biogenic amines. A 5-g slice of raw fish meat was homogenized with 20 mL of 0.5 M hydrochloric acid, neutralized with 1 M sodium hydroxide, and subsequently diluted to 50 mL with water in a volumetric flask. Before injection of the extract to CEC or CE, an aliquot of the solution was filtered with a 0.45-µm cellulose acetate membrane filter cartridge (Adovantec Dismic 13 cP, Toyo Roshi Kaisha Ltd., Tokyo, Japan). These procedures were performed just prior to use in the experiment. RESULTS AND DISCUSSION Optimization of On-Column Derivatization and Separation. Mixing of the derivatization reagent and the analytes is caused by the differences in the moving velocities between the reagent and analytes in a capillary column during CEC. As the many factors such as the concentrations of the reagent and buffer constituents depend on the efficiency of the on-column derivatization and separation, the following effects on the on-column derivatization performance were evaluated by using a fivecomponent biogenic amine solution containing histamine, serotonin, tyramine, putrescine, and cadaverine as the test sample. The pH of the run buffer is believed to the dominant factor; this applies, as well, to the run buffer for the throughout in-capillary derivatization CE method previously developed.21-24 The reason for this is because the run buffers in both cases serve as both the separation buffer and the derivatization buffer. As an OPA/2ME was used for the derivatization reagent in this study, the pH of the run buffer was fixed at 10, which was the optimum pH of this reagent for labeling biogenic amines. In addition, the molar ratio of OPA and 2ME concentration was always set at equivalence. The polymer-supported ODS particles used in this study had been advertised as being tolerant of an alkaline environment in an instruction manual on the Capcell Pak C18 column. According to the instruction, a run buffer having 50% or more organic solvent is recommended when the run buffer was used at pH 10. Therefore, 50% or greater concentration of acetonitrile in borate buffer (pH 10) was used as the run buffer in this study. The performance of CEC was evaluated by retention time and peak area or theoretical plate number (N/m) of each biogenic amine. Effect of OPA/2ME Concentration. Generally, it is very hard to know the optimal concentration of the reagent for this system because the total concentration of amines present in a highly complex sample is not known before doing the analyses. Actually, the real concentration of each primary amine, for example, that of histamine in a sample of tuna meat that caused allergic food poisoning, was assumed to be less than 2 mmol/100 g. Therefore, the 1 mM amine solution was used for examination of the reagent’s concentration on the on-column derivatization of the amines. After due to consideration of the factors above, the effect of OPA/2ME concentration on peak area and elution time of each amine was investigated over a concentration range of 0.5-7.5 mM while maintaining a constant run buffer concentration of 70% acetonitrile in 10 mM borate (pH 10). The results regarding Analytical Chemistry, Vol. 74, No. 14, July 15, 2002
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Figure 1. Effect of the concentration of OPA/2ME reagent in a 70% acetonitrile in 10 mM borate run buffer (pH 10) on (A) peak area response and (B) retention time. Data were obtained by means of an electrochromatogram after injection of a five-component biogenic amine standard solution.
Figure 2. Effect of the concentration of borate buffer (pH 10) in 5 mM OPA/2ME reagent-70% acetonitrile on (A) peak area response and (B) retention time. Other conditions as described in Figure 1.
retention time and peak area are shown in Figure 1A and B, respectively. Figure 1A shows that the peak area of each amine increases with increasing reagent concentration in the run buffer until 5 mM, and after that, each trace line appears to be almost flat. As for the peak shape, symmetrical peaks were observed above 2.5 mM, while slight leading and tailing shapes were observed below 1 mM. On the other hand, each retention time shows a tendency to decrease slightly with increasing concentrations of the reagent as shown in Figure 1B. However, it was concluded that the reagent’s concentration had little influence on the elution behavior (order and time) of each amine. These results mean that this derivatization system might require more than 5 mM reagent in order to determine amines if the each amine exists at maximum at 1 mM in a sample solution. Effect of Buffer Concentration. The effect of the concentration of borate buffer at pH 10 on these separations was examined over concentration levels of 5, 7.5, 10, and 15 mM while maintaining OPA/2ME and acetonitrile at a constant concentration of 5 mM and 70%, respectively. A 20 mM borate buffer was not included, because precipitation occurred when a borate buffer of greater 3466
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than 20 mM concentration was used. As shown in Figure 2A and B, area and elution time of each amine in both cases slightly increased with increasing buffer concentration. Although the traced lines of histamine, serotonin, and putrescine corresponding to the peak area have positive slopes until 10 mM in Figure 2A, there is no clear improvement in separation with increasing concentration of the borate buffer, because the plots all appear to shift more or less in parallel in Figure 2B. As a result of this study, 10 or 15 mM of borate buffer was chosen as the optimized concentration for this system. Effect of Acetonitrile Concentration. The effect of the percentage of acetonitrile in the borate buffer (pH 10) were also investigated over a range of 50%, 60%, and 70% while keeping OPA/2ME and borate buffer concentration constant at 5 mM and 15 mM, respectively. The retention time of each amine decreased with increasing percentage of acetonitrile in borate in the following order: cadaverine, putrescine, tyramine, serotonin, and histamine, as shown in Figure 3B. Taking these results into consideration with regard to separation time or operation time in the determi-
Figure 3. Effect of the percentage of acetonitrile in 15 mM borate buffer (pH 10) and 5 mM OPA/2ME reagent on (A) peak area response and (B) retention time. Other conditions as described in Figure 1.
nation of biogenic amines, the 70% acetonitrile borate buffer was chosen. Effect of Electrokinetic Injection. The conditions of electrokinetic injection needed to be optimized in order to avoid overcharging the column and, also, to achieve the highest degree of separation. The effects of the injection time at 5 kV on the performance of on-column derivatization CEC were tested by varying the concentration of the five-component biogenic amine solution within a range from 1.0 to 7.5 mM. After considerations of the peak area and N/m data (data not shown) obtained under those conditions, the voltage injection at 5 kV for 3 s was at the optimal condition. Method of Validation. The analytical reproducibility of the present method was tested by five identical injections of 1 mM biogenic amine standard solution into the optimized apparatus, as described above. The reproducibility of each peak area response and retention time were less than 1.95% and 0.78% of center values (cv), respectively, thus confirming that the CE was operating as expected. Linearity of peak-area response was also evaluated using the amine solution at a concentration between 0.25 and 2.0 mM of each amine and showed correlation factors (r) from 0.991 to 0.997. The detection limits, obtained from the signal-to-noise level (S/N) at 3, ranged from 0.05 mM for cadaverine to 0.1 mM for putrescine. Although the detection sensitivity seemed to be weaker in comparison with other reported methods, these data were thought to be relatively good after taking the UV detection system into consideration. If more detection sensitivity is needed, employment of a fluorescence detection system instead of a UV detection system might improve sensitivity by a factor of 10 times or more than that of the present system. It should be noted that, because it is very important to obtain a high degree of reproducibility of the assay results, the CEC column should be aged by working the column with the running buffer containing OPA/2ME reagent until the baseline of the electrochromatogram flattens out. This usually takes more than 1 h. These data are listed in Table 1. Performance of On-Column Derivatization CEC. The performance of the on-column derivatization CEC method was evaluated by comparing a precolumn derivatization CEC and a throughout in-capillary derivatization CE method using the same dimension of capillary, run buffer and running conditions in both
Table 1. Method of Validation of On-Column Derivatization CEC reproducibility (cv, %)a amine
area
time
detection limitb (mM)
linearityc (r)
histamine serotonin tyramine putrescine cadaverine
1.85 1.66 1.92 1.95 1.33
0.65 0.23 0.33 0.78 0.48
0.05 0.05 0.05 0.10 0.05
0.996 0.997 0.994 0.991 0.997
a Each reproducibility value was from five identical CEC runs of 1 mM five-component biogenic amine solution. b Each detection limit was determined based on S/N ) 3. c Each linearity of peak area as correlation factor (r) corresponding to each amine between 0.25 and 2 mM was calculated by the least-squares regression method.
instances. Traces A-C in Figure 4 present a typical separation profile of the five biogenic amines at the 1 mM level obtained with on-column derivatization CEC, the precolumn derivatization CEC, and the throughout in-capillary derivatization using the same run buffer of 5 mM OPA/2ME-70% acetonitrile-10 mM borate buffer (pH 10) in each case, respectively. Although both pre- and on-column derivatization CEC give complete separations of the amines, the throughout in-capillary derivatization CE cannot separate them as shown in the Figure 4C. A previous paper25 presented the derivatization efficiency and reproducibility obtained with precolumn and in-capillary derivatization with CE. According to this paper, where 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA) was used as the derivatization reagent instead of OPA/ 2ME and alanine was used as the standard sample analyte, the reproducibility of the in-capillary method appeared to be superior to the precolumn method. In contrast, no general conclusion as to which way is superior can be drawn in this case. In fact, the N/m of histamine and putrescine derived by the on-column method showed higher N/m than that of the precolumn derivation, but the opposite was true for the other three amines as shown in Table 2. Selective Detection of Biogenic Amines. OPA/2ME is useful as the derivatization reagent for determination of biogenic amines because OPA/2ME reacts selectively to only the amino Analytical Chemistry, Vol. 74, No. 14, July 15, 2002
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Figure 4. Typical electrochromatograms (A) and (B) and electropherogram (C) obtained from on-column, precolumn, and throughout in-capillary derivatization methods, respectively. The conditions of voltage injection (5 kV, 3 s) of the five-component biogenic amine standard solution, separation voltage (15 kV), and run buffer were the same; capillary size was varied as described in the Experimental Section. Table 2. Comparison of the Performance between Precolumn and On-Column Derivatizationa precolumn amine
time (min)
histamine serotonin tyramine putrescine cadaverine
7.27 8.04 9.25 10.99 12.08
on-column
area
N/m (× 104)
time (min)
area
N/m (× 104)
26 221 33 769 41 639 24 737 60 978
12.1 14.9 8.7 6.7 8.2
7.68 8.48 9.58 11.47 11.94
34 586 33 199 24 425 51 673 58 965
17.9 6.0 8.2 11.6 6.3
a Each value was obtained by calculating the peak corresponding to each amine in Figure 4A and B.
group within a variety of mixed compounds, such as would naturally be found in a biological sample or food sample. However, when a sample obtained from biological materials or food extractions was determined with the chromatographic method using the OPA/2ME labeling method, amino acids were also detected together with biogenic amines, and amino acids sometimes disturbed the separation of biogenic amines. When a solution containing 5 biogenic amines plus 17 amino acids was analyzed using the throughout in-capillary derivatization CE method, numerous peaks were observed, as shown in Figure 5C. It seems to be very difficult to identify those peaks corresponding to biogenic amines. By contrast, the CEC system with on-column derivatization method provided a simple chromatogram, as shown in Figure 5A and B when two mixtures of 17 kinds of amino acids, one with and one without an additional mixture of 5 kinds of biogenic amines, were separately analyzed. In the former case (one where five biogenic amines were present), only six peaks were identified as shown in Figure 5A. On the other hand, only one peak corresponding to arginine was observed in the later case (one where five biogenic amines were absent) as shown in Figure 5B. 3468
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Figure 5. Typical electrochromatograms (A) and (B) obtained from the mixture of 17 amino acids plus 5 biogenic amines and the mixture of 17 amino acids, respectively. Conditions A and B were the same as describe in Figure 4A. The electropherogram of (C) was obtained using a run buffer (15.4 mM β-cyclodextrin-2 mM OPA/NAC-100 mM borate-phosphate buffer of pH 10) the same as was employed from the optimized throughout in-capillary derivatization CE conditions reported previously,27 except for the injection and detection methods. Other conditions were as described in Figure 4.
The mechanism of this selectivity can be understood as follows: when high voltage is applied to a capillary containing a biogenic amine in an alkaline environment, the amine is subject to two types of kinetic force, one due to electroosmotic flow (EOF ) and the other due to electrophoretic flow. The moving velocity (νs) of the analyte through the capillary can be calculated by means of the following equation:
νs ) KRνeo + νep where KR, νeo, and νep are the relative delay coefficient, velocity of EOF, and velocity of electrophoretic flow, respectively. In this study, the velocity is defined as “+” when the flow direction of the analyte is toward the cathodic site from the anodic site of the capillary; when the flow is in the reverse direction, it is defined as “-“. When flow does not occur, it is defined as or “0”. In the case of the five biogenic amines, νs of each amine showed + because the νeo was + and νep was 0 in a solution of pH 10. Therefore, all five biogenic amines were able to enter into the capillary column and were subsequently derivatized, separated, and detected. In contrast with biologic amines, amino acid is a “zwitterion” possessing both amino and carboxyl groups in the molecule. Therefore, the νep of an amino acid must be expressed as follows:
νep ) νep‚amino + νep‚caro where νep‚amino and νep‚caro are the velocities of electrophoretic flow due to the amino groups and to the carboxyl groups, respectively. If an amino acid is placed in a solution the pH of which is less than the value of the isoelectric point (pI) of the amino acid, the amino group is protonated to form a cationic ion but the carboxyl
Figure 7. Electrochromatograms obtained from (A) mackerel and (B) tuna, respectively, after keeping them at room temperature for 1 day. Other conditions as described in Figure 4A. Figure 6. Electrokinetic injection mechanism of the analyte into the capillary column. (+) and (-) express the direction of motion of the analyte toward the cathodic site from anodic site and toward the anodic site from cathodic site, respectively. When flow does not occur, it is defined as (0). Other details are described in Results and Discussion.
group does not change. In this case, νep‚amino and νep‚caro show + and 0, respectively. In the case where the pH is the same as the pI, νep‚amino and νep‚caro are both 0, and there is no net flow of νep. The pI of arginine is 10.8, which is almost the same pH as the buffer used in this study. Therefore, the direction of kinetic force acting on arginine is the same as that of the five biogenic amines. On the other hand, if an amino acid is placed in a solution the pH of which is more than the pI, the carboxyl group dissociates into anionic ions and the amino group is not protonated. The pI’s of all amino acids used in this study are less than 10, except for arginine. Therefore, νep‚amino and νep‚caro were 0 and -, respectively, in solution pH 10. Whether an amino acid can enter the capillary column or not depends on which value is greater, νeo or νep. If νeo is greater than νep, the amino acid will enter the column. In this study, the νep of all amino acids except for arginine was greater than νeo, because νeo of this on-column derivatization CEC was about one-fifth to one-tenth in comparison with that of CE (data not shown). Therefore, none of the amino acids, except for arginine, could enter the column or be detected. In addition, when a NAC molecule having one carboxyl group was used instead of a 2ME, no amino compounds at all, including the five biogenic amines, were detected with this system, because the νep of each of the amine-OPA/NAC derivatives was -. This mechanism is illustrated in Figure 6. Tests of Food Samples. In general, the determination of mono- and polyamines in food samples sometimes needs relatively complicated “batchwise” operations for the extraction of amines. For example, a standard method of analysis of His and other amines in food in Japan30 adopts the following method: (1) Sample was extracted by homogenizing with hydrochloric acid. (2) The extract was filtered by means of an ion-exchange column (Amberlite CG-50) in order to remove unwanted compounds. (3) The solution eluted from the column with hydrochloric acid was treated with a derivatization reagent. (4) The derivatives were analyzed using optimized high-performance liquid chromatography. Al(30) Standard Method of Analysis for Hygiene Chemists; Pharamceutical Society of Japan with commentary, 2000; pp 1296-1301.
though this method is an authorized method and has high reliability, it is technically difficult to perform, which increases the probability of human error. By the present method, a crude extract sample obtained from hydrochloric acid extraction can be used and does not need any further purification such as the ion chromatographic purification mentioned above. After neutralization of the hydrochloric acid extraction, aliquots of the extracts were directly injected into the on-column derivatization CEC apparatus. Traces A and B of Figure 7 show the chromatograms obtained from samples of raw mackerel and tuna meat that were allowed to age for 1 day at room temperature, respectively. Histamine, tyramine, putrescine, and cadaverine were identified in the former case, but no amines of any kind were identified in the latter case. CONCLUSIONS In a previous paper,25 we developed a throughout in-capillary derivatization method using CE with OPA/NAC as a derivatization reagent in order to detect amines such as amino acids and biogenic amines.16,26,27 Here, we tried to detect some biogenic amines using CEC with an on-column derivatization method using OPA/2ME as a reagent. The present method provides the following: (1) An excellent separation is given of five biogenic amines as a sample in comparison with that of throughout incapillary derivatization CE. The CE method cannot separate these amines from each other. (2) Unlike with the throughout in capillary derivatization CE method, the on-column derivatization CEC method offers the advantage of selective detection of amines. In the case of determination of biogenic amines in a sample having many kinds of amino acids, such as in a food sample, the present method allows use of a very simple extraction method, such as hydrochloric acid extraction, which requires no further purification to remove unwanted compounds. (3) Neither the present method nor the throughout in-capillary derivatization CE requires specific handling of the derivatization step in order to enhance the detection sensitivity, as is the case with the pre- or postcapillary derivatization methods. The on-column CEC method appears to have large promise as a next-generation technique for rapid, specific, and sensitive biogenic amine assay. Received for review February 19, 2002. Accepted April 20, 2002. AC025592D Analytical Chemistry, Vol. 74, No. 14, July 15, 2002
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