Anal. Chem. 2000, 72, 4009-4014
An Automatic Analyzer for Catecholamines and Their 3-O-Methyl Metabolites Using a Micro Coulometric Flow Cell as a Postcolumn Reactor for Fluorogenic Reaction Kazuko Takezawa,† Makoto Tsunoda,† Noriyuki Watanabe,‡ and Kazuhiro Imai*,†
Graduate School of Pharmaceutical Sciences and Graduate School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
A coulometric flow cell for a miniaturized LC system was developed. The cell was examined, as 3-O-methyl catecholamines were converted to their relative o-quinones for subsequent fluorometric and chemiluminescence detection. Its performance was evaluated in comparison with commercially available amperometric and coulometric detectors in terms of specification of the low dead volume and high conversion efficiency. The fully automated smallbore LC analyzer for simultaneous determination of catecholamines and their 3-O-methyl metabolites included precolumn pretreatment, column switching, column separation, postcolumn oxidative conversion, fluorometric derivatization, and chemiluminescence detection. The detection limits were 0.3-2.0 fmol for catecholamines and their 3-O-methyl metabolites. Because of the high sensitivity, the required volume of rat plasma sample was only 15 µL. Recent advances in the development of small-bore liquid chromatographic columns have given a tremendous impetus to the miniaturization of liquid chromatography (LC).1-3 On those small-bore columns, the flow rate is low and the substances are less diluted so that they have the advantages of increased sensitivity and reduced mobile-phase consumption. Because of their small elution peak volume obtained from those columns, detectors with small dead volume are a prerequisite. Electrochemical detection in combination with LC has been most commonly used for its sensitivity, selectivity, and simplicity.4-11 * To whom correspondence should be addressed: (tel) +81-3-5841-4760; (fax) +81-3-5841-4885; (e-mail)
[email protected]. † Graduate School of Pharmaceutical Sciences. ‡ Graduate School of Engineering. (1) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 185, 27-41. (2) Novotny, M. Anal. Chem. 1988, 60, 500A-510A. (3) Shirota, O.; Suzuki, A.; Kanda, T.; Ohtsu, Y.; Yamaguchi, M. J. Microcolumn Sep. 1995, 7, 29-35. (4) Hollenbach, E.; Schultz, C.; Lehnert, H. Life Sci. 1998, 63, 737-750. (5) Raggi, M. A.; Sabbioni, C.; Casamenti, G.; Gerra, G.; Calonghi, N.; Masotti, L. J. Chromatogr., B 1999, 730, 201-211 (6) Chi, J. D.; Odontiadis, J.; Franklin, M. J. Chromatogr., B 1999, 731, 361367. (7) Pagliari, R.; Cottet-Emard, J. M.; Peyrin, L. J. Chromatogr. 1991, 563, 2326. (8) Lenders, J. W. M.; Eisenhofer, G.; Armando, I.; Keiser, H. R.; Goldstein, D. S.; Kopin, I. J. Clin. Chem. 1993, 39, 97-103. 10.1021/ac0003697 CCC: $19.00 Published on Web 08/03/2000
© 2000 American Chemical Society
Moreover, amperometric detection lends itself to miniaturization. Detectors with a submicroliter cell volume have been described by several workers.12-16 But as far as we know, there has been no report on a coulometric flow cell for the miniaturized system. This is because it demands a large electrode surface area and thus results in considerable dead volume. This disadvantage also obstructed the application of the coulometric flow cell as on-line oxidation on a small-bore column LC. In this study, we report a new coulometric flow cell. The cell suppresses the occurrence of an additional band broadening, even at a low flow rate. Two pieces of stainless tubing (0.2 mm i.d. × 5.0 cm), one of which was packed with glassy carbon powder, were connected with three polyetheretherketone (PEEK) unions. Since the dead volume of the flow cell is extremely small, when it is used in conjunction with a proper detector, it is useful to an on-line coulometric oxidation. Here we also report an application of the flow cell to the miniaturization of a fully automated analyzer for catecholamines and their 3-O-methyl metabolites. Catecholamines and their 3-O-methyl metabolites were coulometrically oxidized into their related o-quinones without additional peak broadening. It enabled a previous method17 to be miniaturized for greater sensitivity. A sample volume of 15 µL of rat plasma was found sufficient. EXPERIMENTAL SECTION Reagents. Catecholamines (CAs; norepinephrine, NE; epinephrine, E; dopamine, DA), their 3-O-methyl metabolites (MNs; normetanephrine, NMN; metanephrine, MN; 3-methoxytyramine, 3-MT), and 4-methoxytyramine (4-MT, internal standard) were (9) Mashige, F.; Matsushima, Y.; Miyata, C.; Yamada, R.; Kanazawa, H.; Sakuma, I.; Takai, N.; Shinozuka, N.; Ohkubo, A.; Nakahara, K. Biomed. Chromatogr. 1995, 9, 221-225. (10) Hay, M.; Mormede, P. J. Chromatogr., B 1997, 703, 15-23. (11) Watanabe, N.; Toyo’oka, T.; Imai, K. Biomed. Chromatogr. 1987, 2, 99103. (12) Cheng, F. C.; Kuo, J. S. J. Chromatogr. 1995, 665, 1-13. (13) Carlsson, A.; Lundstroˆm, K. J. Chromatogr. 1985, 350, 169-178. (14) Sagar, K. A.; Kelly, M. T.; Smyth, M. R. J. Chromatogr. 1992, 577, 109116. (15) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A537A. (16) Mao, L.; Shi, G.; Tian, Y.; Liu, H.; Jin, L.; Yamamoto, K.; Tao, S.; Jin, J. Talanta 1998, 46, 1547-1556. (17) Tsunoda, M.; Takezawa, K.; Santa, T.; Imai, K. Anal. Biochem. 1999, 269, 386-392.
Analytical Chemistry, Vol. 72, No. 17, September 1, 2000 4009
Figure 1. Diagram of a homemade coulometric flow cell for on-line electrochemical oxidation of CAs and MNs. Two pieces of stainless steel tubing (50 mm × 0.2 mm i.d. × 1.6 mm o.d.) and three pieces of union were arranged.
all purchased from Sigma (St. Louis, MO). Trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL). Acetonitrile, ethanol, 1,4-dioxane, and ethyl acetate, all of HPLC grade, were purchased from Wako Pure chemicals (Osaka, Japan). Hydrogen peroxide and bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl] oxalate (TDPO) were also from Wako. Sodium 1-hexanesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate, and imidazole (zone-refined) were obtained from Tokyo Kasei (Tokyo, Japan). The purified ethylenediamine (ED) for washing for semiconductors was a gift from Wako. All other reagents were of analytical grade. The water used was purified on a Milli RO-Milli Q system (Nihon Millipore, Tokyo, Japan). Optimization of the Coulometric Flow Cell. A coulometric flow cell was employed for the postcolumn oxidative conversion of NE and NMN. Three kinds of flow cells were examined: a radial flow cell (BAS, Tokyo, Japan), a coulometric flow cell (Coulochem 5100A, ESA), and a homemade flow cell. The homemade flow cell consists of three PEEK unions and two pieces of stainless steel tubing (0.2-mm i.d., 1.6-mm o.d., 50-mm length) as shown in Figure 1. One of the two tubings was a working electrode packed with powdered glassy carbon (∼50 µm diameter) (Tokai Carbon, Tokyo, Japan) with stainless steel frits of 0.5-µm pore size (c-409, Upchrch Scientific, WA) at both ends. The other was used as a counter electrode. The potential on the cell was applied using LC-3E (BAS, West Lafayette, IN). The counter electrode was connected to both counter and reference electrode terminals of LC-3E. Fifty picomoles of NE and NMN were injected, separated on a separation column, oxidized in a coulometric flow 4010
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cell, and then derivatized with fluorogenic reagent in a reaction coil. The fluorometric detection was carried out at an excitation wavelength of 410 nm and an emission wavelength of 500 nm with a fluorescence detector connected after the reaction coil. The HPLC conditions were as follows: the composition of the mobile phase was 75 mM potassium acetate buffer (pH 3.2)/100 mM potassium phosphate buffer (pH 3.2)/acetonitrile (93.1:4.9:2, v/v/v) containing 6 mM sodium 1-hexanesulfonate at a flow rate of 100 µL/min; the composition of the fluorogenic reagent was 105 mM ED and 175 mM imidazole in acetonitrile/ethanol/water (85:10:5, v/v/v) at a flow rate of 96 µL/min. The length of the reaction coil and its temperature were fixed at 6.5 m and 80 °C, respectively. The effect of the applied potential on the oxidation by the coulometric cell was also investigated under the same HPLC conditions. Optimization of the HPLC Conditions. The separation of CAs, MNs, and an internal standard was investigated under the following conditions: the composition of the mobile phase was 75 mM potassium acetate buffer (pH 3.2)/50 or 100 mM potassium phosphate buffer (pH 3.2)/acetonitrile containing sodium alkanesulfonate (sodium 1-hexanesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate) at a flow rate of 100 µL/min. The conditions of fluorescence derivatization were examined. The length (2-8.5 m) of the reaction coil (Teflon tubing, 0.25 mm i.d. × 1.6 mm o.d.) winding in a knitted type was investigated. The temperature of the reaction coil, which was kept in a thermostatically controlled bath (ASB 200D, Jasco, Tokyo, Japan) at 40-95 °C, was also optimized.
Figure 2. Block diagram of the column-switching semi-microcolumn LC system for the automated analyzer of CAs and their 3-O-methyl metabolites: (Pump-1) pretreatment buffer, 10 mM potassium phosphate buffer (pH 7.5), 100 µL/min; (Pump-2) mobile phase, 75 mM potassium acetate buffer (pH 3.2)/100 mM potassium phosphate buffer (pH 3.2)/acetonitrile (93.1:4.9:2, v/v/v) containing 6 mM sodium 1-hexanesulfonate, 100 µL/min; (Pump-3) fluorogenic reagent; 105 mM ED and 175 mM imidazole in acetonitrile/ethanol/water (85:10:5, v/v/v), 96 µL/min; (Pump4) chemiluminogenic reagent, 0.25 mM TDPO, 150 mM H2O2, and 110 mM TFA in 1,4-dioxane/ethyl acetate (50:50, v/v), 490 µL/min. Precolumn, Capcell Pak MF-SCX (10 mm × 2.0 mm i.d., Shiseido); separation column, Capcell Pak C18 UG120 (250 mm × 1.5 mm i.d., Shiseido); column oven, 35 °C; reaction coil, 0.25 mm i.d. × 6.5 m, 80 °C. The applied potential on the coulometric flow cell was set at +1.2 V.
The duration of adsorption and desorption on and from the precolumn was optimized. The adsorption time (0.5-3.5 min) in the precolumn was investigated with the desorption time fixed at 5 min, and the desorption time (2.5-5.0 min) was investigated with the adsorption time fixed at 2.5 min. A 45-µL portion of the diluted rat plasma sample was injected into the HPLC. HPLC-PO-CL Detection System. The automatic system, shown in Figure 2, consisted of the Nanospace series (Shiseido, Tokyo, Japan) without the thermostatically controlled bath, pump 4 (PU-980, Jasco), CL detector (FP-920 (CL option), Jasco), and integrator (807-IT, Jasco). Three 2001 pumps, a 2003 autosampler, a 2004 column oven, and a 2011 six-way valve, and other instruments were arranged as shown in Figure 2. PEEK tubing (0.25-mm i.d.) was used for all of the connections among all of these HPLC components. A separation column, Capcell Pak C18 UG120 (250 mm × 1.5 mm i.d., Shiseido), and a precolumn, Capcell Pak MF-SCX (10 mm × 2.0 mm i.d., Shiseido), were used. The flow cell of the CL detector used was the one optimized for the semi-micro LC system as described previously.18 Rat Plasma Collection and Preparation. Male SpragueDawley (SD) rats (8 weeks old, 340-370 g) purchased from Charles River Japan Inc. (Kanagawa, Japan) were anesthetized with diethyl ether. Blood was taken from the abdominal aorta, transferred to a heparinized polyethylene tube, and centrifuged at 3000g for 20 min at 4 °C. The plasma fraction was collected and diluted with twice the volume of the sample dilution buffer (10 mM glutathione, 10 mM citric acid, 0.1% Triton X-100, 100 mg/L EDTA‚2Na and 33 nM 4-MT (internal standard) (pH 4.5)). A 45-µL aliquot of the diluted mixture was injected into the HPLC. Validation of the HPLC System. A 5-µL aliquot of sample containing 5, 25, 50, 250, or 500 fmol of NE, E, DA, NMN, MN, or 3-MT, respectively, was injected into the HPLC. The calibration curves for relative peak area to internal standard (4-MT) versus the concentrations of these compounds were obtained. Least(18) Takezawa, K.; Tsunoda, M.; Murayama, K.; Santa, T.; Imai, K. Analyst 2000, 125, 293-296.
squares regression was used for the calibration of the slope, intercept, and correlation coefficient. In an accuracy experiment, to 15 µL of rat plasma, 30 µL of dilution buffer containing 100, 200, 400, or 800 fmol of NE, E, DA, NMN, MN, or 3-MT, respectively, was added. A 45-µL aliquot of the mixture was injected into the HPLC. The slopes of the curve of the standards were compared with the calibration curves obtained from the same mixtures as described above. The precision of this system was evaluated by analyzing the same rat plasma samples five consecutive times or five successive days. The mixture of 15 µL of rat plasma and 30 µL dilution buffer was injected into the HPLC. Values of all the data in this paper are presented as the mean ( SEM. RESULTS AND DISCUSSION Optimization of the Coulometric Flow Cell. There are two types of electrochemical detector cells, amperometric and coulometric. An amperometric flow cell is designed to convert 1-5% of the electroactive species so that it does not have enough surface area. On the other hand, a coulometric flow cell is designed to achieve 100% conversion. By reason of gaining better electrochemical oxidation efficiency, a coulometric cell demands a large electrode surface area, which results in considerable dead volume.12 The commercially available coulometric cells are for conventional HPLC and have large dead volumes. Generally, most amperometric flow cells are not anticipated to be followed by another detector, that is, no consideration is taken to keep the dead volume to a minimum. The radial flow cell, which is expected to work coulometrically at a low flow rate, also suffered a similar disadvantage. In fact, the chromatogram obtained with this radial flow cell (Figure 3a) was significantly broadened. While the coulometric flow cell provided by ESA was designed to be followed by another detector, it is intended for conventional LC. Thus, it also caused peak broadening and diminution of peak response (Figure 3b). The homemade flow cell was composed of two pieces of stainless steel tubing (0.2-mm i.d., 1.6-mm o.d., 50-mm length) Analytical Chemistry, Vol. 72, No. 17, September 1, 2000
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Figure 3. Chromatograms of a standard mixture of NE (peak 1) and NMN (peak 2). Fifty picomoles of each was injected and electrochemically oxidized by the radial flow cell (BAS) (a), coulometric flow cell (ESA) (b), or the newly developed coulometric flow cell (c) and derivatized at the optimum fluorogenic derivatization condition. The applied potential was set at +1.0 V (a), +0.6 V (vs H2/H+ reference electrode described in the manual of ESA Coulochem 5100A) (b), and +1.2 V (c). The fluorescence intensity was monitored at an excitation wavelength of 410 nm and an emission wavelength of 500 nm.
and three PEEK unions connected as shown in Figure 1. The two stainless steel tubes serve as a working electrode and a counter electrode. Since the working electrode was packed with powdered glassy carbon (∼50 µm of diameter), the active electrode surface area was large enough to convert CAs and MNs into their respective o-quinone compounds. As the glassy carbon was packed with high pressure in the stainless steel tubing, the void volume of the flow cell was much smaller than usual. The surface area and void volume of the cell were roughly estimated to be 1.2 cm2 and 0.5 µL, respectively. As shown in Figure 3c, the on-line coulometric oxidation of catechol and 4-hydroxy-3-methoxyphenyl compounds into the respective o-quinone compounds was achieved without band broadening using the homemade flow cell. The effects of the applied potential on the coulometric detector on the conversion reaction were evaluated by measurement of the fluorescence intensities of the fluorescent products of CAs and MNs with ethylenediamine. As shown in Figure 4, the fluorescence intensities for CAs were almost constant over the range of +1.0 to +1.4 V, which were the same as those obtained without application of the potential. MNs, however, were not oxidized at voltages of
