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Simultaneous determination of 3,4-dihydroxyphenylacetic acid and homovanillic acid using high performance liquid chromatography–fluorescence detection and application to rat kidney microdialysate
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 307 (2002) 153–158 www.academicpress.com
Simultaneous determination of 3,4-dihydroxyphenylacetic acid and homovanillic acid using high performance liquid chromatography–fluorescence detection and application to rat kidney microdialysateq Makoto Tsunoda, Kazuto Mitsuhashi, Mayumi Masuda, and Kazuhiro Imai* Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 28 January 2002
Abstract We established a sensitive and simultaneous determination method of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) using HPLC–fluorescence detection. This method adopted the column-switching system, which included an online extraction of carboxylic acids by a strong anion-exchange column followed by separation on an ODS column, coulometric oxidation, fluorogenic reaction with ethylenediamine, and fluorescence detection. The detection limits were 50 and 100 fmol/injection for DOPAC and HVA, respectively (a signal-to-noise ratio of 3). The method was applicable to 50 ll of rat kidney microdialysate with a sufficient accuracy and precision. The concentrations of DOPAC and HVA in rat kidney microdialysate were 131 29 and 404 44 nM, respectively (n ¼ 5). This is the first report of DOPAC and HVA quantified in rat kidney microdialysate. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Catecholamines; Metabolism; Kidney; Microdialysis
Catecholamines (norepinephrine, epinephrine, and dopamine (DA)1) play important roles as neurotransmitters or hormones. We previously reported a method for the determination of catecholamines in rat plasma using HPLC-peroxyoxalate chemiluminescence reaction detection [1,2]. By the use of this method, we delineated the relationship between the changes of plasma catecholamine concentrations and blood pressure after the infusion of calcium antagonists [3–6]. Furthermore, to clarify the role of the methyl metabolism of catecholamines by catechol-O-methyltrans-
q A part of this work was presented at the 11th Conference of the Society for Chromatographic Sciences in Kyoto (October 2000, Japan). * Corresponding author. Fax: +81-3-5841-4885. E-mail address: [email protected] (K. Imai). 1 Abbreviations used: DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; DA, dopamine; COMT, catechol-O-methyltransferase; MAO, monoamine oxidase.
ferase (COMT) in blood pressure regulation, a method was developed for the simultaneous determination of catecholamines and their 3-O-methyl metabolites [7,8]. This method allowed us to show that catecholamine methylation in spontaneously hypertensive rats is attenuated, compared to normotensive Wistar–Kyoto rats [9]. As we were interested in delineating the role of catecholamines in organ levels, dopamine metabolism in the kidney was investigated. Presumably, DA acted as an intrarenal natriuretic hormone, and this activity depended on DA metabolism by COMT and monoamine oxidase (MAO). As shown in Fig. 1, DA can be metabolized to 3-methoxytyramine (3-MT) by COMT and to 3,4-dihydroxyphenylacetic acid (DOPAC) by MAO. Further, both 3-MT and DOPAC are metabolized to homovanillic acid (HVA). Microdialysis techniques can be used not only to obtain rat renal interstitial fluid (RIF) but also in studies on the role of DA metabolism in kidney where
0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 2 6 9 7 ( 0 2 ) 0 0 0 0 6 - 4
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Optimization of the fluorescence derivatization reaction and the coulometric oxidation
Fig. 1. Metabolic pathways of dopamine by COMT and MAO.
DA in rat kidney microdialysate was analyzed [10–12]. As far as we are aware, DA metabolites can be determined in rat urine, but not in rat kidney microdialysate. As it is doubtful that DA metabolites in urine reflect the intrarenal function, we have decided to develop a method to determine DA metabolites in rat kidney microdialysate so that we may gain insight into the metabolism of DA in kidney. Here we developed a sensitive determination method of DOPAC and HVA in rat kidney microdialysate. Optimization of chromatographic conditions, coulometric oxidation, and fluorescence derivatization reactions were performed. Validation of the method and an application to the determination of DOPAC and HVA in 50 ll of rat kidney microdialysate are described.
The effects of the length of reaction coil and the temperature of the fluorescence derivatization and the effect of the applied potential in the coulometric converter were investigated under the following HPLC conditions. The separation column used was TSK-gel ODS-80 Ts, 150 4:6 mm i.d. (Tosoh, Tokyo, Japan). The composition of the mobile phase was 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (85.5/4.5/10, v/v/v) and the composition of the fluorogenic reagent was 105 mM ethylenediamine and 175 mM imidazole in acetonitrile/ ethanol/water (80/10/10, v/v/v). The flow rates of the mobile phase and the fluorogenic reagent were fixed at 0.5 and 0.32 ml/min, respectively, which are the same as those in the previous reports [1,7,13]. One picomole each of DOPAC and HVA was injected into the HPLC system. The fluorescence detection was made at an emission wavelength of 495 nm with excitation at 417 nm. The temperature of a reaction coil (1:6 mm o:d: 0:5 mm i:d: 10–30 m), which was kept in a thermostatically controlled bath (ASB 200D, JASCO), was controlled at 85–120 °C. The applied potential versus a H2 =Hþ reference electrode was set at 0.05–0.5 V. Optimization of the on-line extraction of DOPAC and HVA A weak anion-exchange column (N(CH3)2-1031-N, 30 4:6 mm i.d., Senshu Scientific, Tokyo, Japan) and a strong anion-exchange column (SAX-1031-N, 30 4:6 mm i.d., Senshu Scientific) were used as the precolumn. The composition of the pretreatment buffer was 10 mM potassium phosphate buffer (pH 3.2) at a flow rate of 0.5 ml/min. In this experiment, the native fluorescence detection was carried out at an excitation wavelength of 280 nm and an emission wavelength of 315 nm connected after the precolumn. Five hundred picomoles each of DOPAC and HVA was injected onto the column. Apparatus and HPLC conditions
Materials and methods Chemicals 3,4-Dihydroxyphenylacetic acid and homovanillic acid were obtained from Sigma Chemical (St. Louis, MO). The purified ethylenediamine for washing the semiconductors was a gift from Wako Pure Chemicals (Osaka, Japan). Imidazole was from Merck (Darmstadt, Germany). Acetonitrile and ethanol, HPLC grade, were from Kanto Chemicals (Tokyo, Japan). All other reagents were of analytical grade.
A block diagram of the automated system is shown in Fig. 2. This system consisted of three pumps (PU-880 and PU-980, Jasco, Tokyo, Japan), an autosampler (851AS, Jasco), a rotatory six-way switching valve (HV-99201, Jasco), a fluorescence detector (820-FP, Jasco), and an integrator (807-IT, Jasco). HPLC conditions were as follows: precolumn, SAX-1031-N, 30 4:6 mm i.d. (Senshu Scientific); pretreatment buffer, 10 mM potassium phosphate buffer (pH 3.2); flow rate, 0.5 ml/min; precolumn cleanup solution, 0.1% phosphoric acid/ acetonitrile (50/50, v/v); flow rate, 0.5 ml/min; separation
M. Tsunoda et al. / Analytical Biochemistry 307 (2002) 153–158
Fig. 2. Block diagram of the automated analyzer of DOPAC and HVA in the present method. (Pump l) pretreatment buffer, 10 mM potassium phosphate buffer (pH 3.2), 0.5 ml/min; (Pump 2) mobile phase, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (90.25/4.75/5, v/v/v), 0.5 ml/min; (Pump 3) fluorogenic reagent, 105 mM ethylenediamine, and 175 mM imidazole in acetonitrile/ethanol/water (80/10/10, v/v/v), 0.32 ml/min. Precolumn, SAX-1031-N (30 4:6 mm i.d., Senshu Scientific); separation column, TSK-gel ODS-80 Ts (150 4:6 mm i.d., TOSOH); reaction coil, 0:5 mm i:d: 30 m, 120 °C. The applied potential on the coulometric converter was set at +0.4 V versus a H2 =Hþ reference electrode.
column, as described above; mobile phase, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (90.25/4.75/5, v/v/v); flow rate, 0.5 ml/min; fluorogenic reagent, as described above; coulometric converter (the Coulochem 5100A, ESA), +0.4 V versus a H2 =Hþ reference electrode; and a reaction coil (1:6 mm o:d: 0:5 mm i:d: 30 m), kept at 120 °C, which was the indication of the thermostatically controlled bath. Animal experiments Male Sprague–Dawley rats (8–9 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and housed in an environmentally controlled room with free access to tap water and diet for at least 1 week before use. All animals received humane care in compliance with the National Institutes of Health guidelines. To obtain rat kidney microdialysate, the left kidney in the anesthetized rats was exposed, and a 5-mm microdialysis probe (Bioanalytical Systems, West Lafayette, IN) was placed into the renal cortex. Ringer solution was infused at a flow rate of 2 ll/min. In vitro recoveries of DOPAC and HVA were evaluated by immersing dialysis membranes of the probes (n ¼ 3) in a beaker containing 50 nM each of DOPAC and HVA. Ringer solution was infused at a flow rate of 2 ll/min.
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the correlation coefficient. To 50-ll aliquots of rat kidney microdialysate, DOPAC and HVA (each 0, 150, 300, and 500 fmol) were spiked and analyzed by the HPLC system noted above. The accuracies were expressed as the reciprocal percentage of the amount of DOPAC or HVA recovered from the spiked samples to the expected values. Both intra- and interday precision was calculated by analyzing the same rat kidney microdialysate samples five consecutive times or five successive days, respectively. The precision was estimated based on the coefficient of variation (CV, %).
Results and discussion Optimization of the fluorescence derivatization reaction Catechol compounds selectively react with ethylenediamine to produce the fluorescent compounds, and the optimum conditions for the fluorescence derivatization of DOPAC with ethylenediamine were first investigated. The temperature was set to vary from 85 to 120 °C, and the reaction time was determined by varying the length of the reaction coil from 10 to 30 m. As shown in Fig. 3, with the increases of both the temperature and the length of reaction coil, the recorder response for DOPAC increased. When the length of the reaction coil was more than 30 m, it resulted in a peak broading. From these data shown in Fig. 3, a condition of 120 °C and 30 m was selected for the optimal fluorescence derivatization reaction. Optimization of coulometric oxidation To determine DOPAC and HVA simultaneously, HVA must be converted into o-quinone compounds prior to fluorescence derivatization. The electrochemical coulometric converter was employed to oxidize 3-O-
Validation of the HPLC system DOPAC or HVA (each 0, 150, 300, 500, 1000, 2000, or 5000 fmol) was injected into the HPLC. The calibration curves for peak height versus the amount of each compound were obtained. Least-squares regression was used for the calibration of the slope, intercept, and
Fig. 3. Effects of the reaction time and temperature on the fluorescence intensity of DOPAC. The temperature was changed from 85 to 120 °C. The reaction coil was investigated using 10 (r), 20 (j), 25 (N), and 30 m ( ) of the reaction coil.
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Fig. 4. Effects of applied potential on the fluorescence intensities for DOPAC ( ) and HVA (j). The experimental conditions are described under Materials and methods.
anion-exchange column was better than a weak anionexchange column for the extraction and separation of DOPAC and HVA from neutral and amino compounds (data not shown). Using a strong anion-exchange column (SAX-1031-N), DOPAC and HVA were retained in the precolumn in a pretreatment buffer with a low ionic concentration (10 mM potassium phosphate buffer (pH 3.2)), and eluted for 8– 12 min. Therefore, we decided to introduce this 4-min portion of the eluate into the ODS column. Under these conditions, catecholamines or 3,4-dihydroxyphenylglycol was eluted within 1 min from the position of the void volume, suggesting that a sufficient separation of DOPAC and HVA from neutral and amino compounds was achieved. Optimization of chromatographic separation conditions
methyl metabolites of catecholamines into the respective o-quinone compounds [7,8,14]. As shown in Fig. 4, while the peak of DOPAC was not influenced by the coulometric oxidation, the peak of HVA increased in the range of 0.1–0.4 V, and maximum fluorescence intensity was obtained at 0.4 V. Therefore, a potential of 0.4 V was selected for the simultaneous determination of DOPAC and HVA. Optimization of the on-line extraction of DOPAC and HVA We have previously reported an on-line extraction of catecholamines using a cation-exchange column [1,2]. To extract carboxylic acids including DOPAC and HVA from the biological samples, an anion-exchange column was used as a precolumn. In a preliminary experiment, we have learned that a strong
In the separation of the authentic of DOPAC and HVA, a mobile phase of 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (85.5/4.5/10, v/v/v) was used. This was because DOPAC and HVA gave good separation, with an analysis time within 25 min. However, in microdialysate samples from rat kidney, the peaks of DOPAC and the unknown endogenous compound overlapped. Therefore, by changing the ratio of the buffer to acetonitrile, the separation of these two peaks was investigated. As shown in Fig. 5c, when the concentration of acetonitrile in the mobile phase was 5%, a sufficient separation was obtained. Fig. 5 shows the typical chromatograms obtained (a) from Ringer solution, (b) from 5 pmol of a standard sample of DOPAC and HVA, and (c) from the microdialysate sample from rat kidney.
Fig. 5. Representative chromatograms obtained (a) from Ringer solution, (b) from 5 pmol of a standard sample of DOPAC and HVA, and (c) from the rat kidney microdialysate (50 ll). Peaks: 1, DOPAC; and 2, HVA. HPLC conditions are described under Materials and methods.
M. Tsunoda et al. / Analytical Biochemistry 307 (2002) 153–158 Table 1 Accuracy of the determination of DOPAC and HVA in rat kidney microdialysate (n ¼ 3) Added amounts (fmol) 0
150
300
DOPAC
Found (fmol) Accuracy (%)
248
393 96.9
563 105
HVA
Found (fmol) Accuracy (%)
666
808 94.4
954 96.0
157
Acknowledgment We thank Dr. Chi-Ho Lee for his generous advice on the preparation of the manuscript.
500 745 99.4 1163 99.4
Validation of the proposed method The calibration curves for DOPAC and HVA showed linearity in the range of 150 fmol (DOPAC) or 300 fmol (HVA) to 5000 fmol. The correlation coefficients were 0.997 and 0.998 for DOPAC and HVA, respectively. The limits of detection (a signal-to-noise ratio of 3) were 50 and 100 fmol for DOPAC and HVA, respectively. These values were similar to or better than those previously reported [15–17]. The accuracies for DOPAC and HVA are summarized in Table 1. The intraday precision of this method was 2.68 and 2.91% for DOPAC and HVA (n ¼ 5), respectively. The interday precision of this method was 5.12 and 3.88% for DOPAC and HVA (n ¼ 5), respectively. Further, the reproducibilities of the retention times were 0.046 and 0.16% for DOPAC and HVA (n ¼ 5), respectively. These suggest that, although the internal standard was not used, the proposed method is appropriate for a routine assay of DOPAC and HVA in rat kidney microdialysate. Determination of DOPAC and HVA in rat kidney microdialysate In vitro recoveries of DOPAC and HVA from the beaker containing 50 nM each of DOPAC and HVA were investigated. The relative recoveries for DOPAC and HVA (percentage of DOPAC or HVA concentration in dialysate/DOPAC or HVA concentration in the beaker) were 3.8 and 3.3%, respectively, with a perfusion rate of 2 ll/min. The concentrations of DOPAC and HVA in rat kidney microdialysate determined by the present method were 131 29 and 404 44 nM (n ¼ 5), respectively, estimated with the respective recoveries from the probe in vitro. In summary, a selective and sensitive determination method of DOPAC and HVA utilizing a HPLC–fluorescence detection system was developed for rat kidney microdialysate samples. DOPAC can be assayed at a concentration as low as 150 fmol and HVA at 300 fmol. The present method is considered to be the first method applied to the determination of DOAPC and HVA in rat kidney microdialysate.
References [1] P. Prados, S. Higashidate, K. Imai, A fully automated HPLC method for the determination of catecholamines in biological samples utilizing ethylenediamine condensation and peroxyoxalate chemiluminescence detection, Biomed. Chromatogr. 8 (1994) 1–8. [2] K. Takezawa, M. Tsunoda, K. Murayama, T. Santa, K. Imai, Automatic semi-microcolumn liquid chromatographic determination of catecholamines in rat plasma utilizing peroxyoxalate chemiluminescence reaction, Analyst 125 (2000) 293–296. [3] S. Higashidate, K. Imai, P. Prados, S. Adachi-Akahane, T. Nagao, Relations between blood pressure and plasma norepinephrine concentrations after administration of diltiazem to rats: HPLC-peroxyoxalate chemiluminescence determination on an individual basis, Biomed. Chromatogr. 8 (1994) 19–21. [4] K. Imai, S. Higashidate, P. Prados, T. Santa, S. Adachi-Akahane, T. Nagao, Relation between blood pressure and plasma catecholamine concentration after administration of calcium antagonists to rats, Biol. Pharm. Bull. 17 (1994) 907–914. [5] P. Prados, T. Santa, H. Homma, H. Doi, H. Narita, B. Del Castillo, M.A. Martin, K. Imai, Comparison of the sympathetic nervous system activity between spontaneously hypertensive and Wistar–Kyoto rats to respond to blood pressure reduction, Biol. Pharm. Bull. 20 (1997) 341–344. [6] M. Tsunoda, K. Takezawa, T. Santa, Y. Ina, K. Nagashima, K. Ohmori, S. Kobayashi, K. Imai, New approach for measurement of sympathetic nervous abnormality in conscious, spontaneously hypertensive rats, Jpn. J. Pharmacol. 83 (2000) 39–45. [7] M. Tsunoda, K. Takezawa, T. Santa, K. Imai, Simultaneous automatic determination of catecholamines and their 3-O-methyl metabolites in rat plasma by high-performance liquid chromatography using peroxyoxalate chemiluminescence reaction, Anal. Biochem. 269 (1999) 386–392. [8] K. Takezawa, M. Tsunoda, N. Watanabe, K. Imai, An automatic analyzer for catecholamines and their 3-O-methyl metabolites using a micro coulometric flow cell as a postcolumn reactor for fluorogenic reaction, Anal. Chem. 72 (2000) 4009– 4014. [9] K. Imai, M. Tsunoda, K. Takezawa, K. Mitsuhashi, T. Santa, K. Nagashima, K. Katayama, K. Ohmori, Attenuation of methylation of catecholamines and treatment with a coenzyme of catechol-O-methyltransferase in spontaneously hypertensive rats (SHR), Proc. Jpn. Acad. 75B (1999) 224–227. [10] Z.-Q. Wang, H.M. Siragy, R.A. Felder, R.M. Carey, Intrarenal dopamine production and distribution in the rat. Physiological control of sodium excretion, Hypertension 29 (1997) 228– 234. [11] Y. Wang, T.J. Berndt, J.M. Gross, M.A. Peterson, M.J. So, F.G. Knox, Effect of inhibition of MAO and COMT on intrarenal dopamine and serotonin and on renal function, Am. J. Physiol. 280 (2001) R248–R254. [12] R.M. Carey, Z.Q. Wang, H.M. Siragy, R.A. Felder, Renal dopamine production and release in the rat: a microdialysis study, Adv. Pharmacol. 42 (1998) 873–876. [13] M. Tsunoda, K. Takezawa, K. Imai, A method for catechol-Omethyltransferase activity using norepinephrine, an endogenous substrate, Analyst 126 (2001) 637–640.
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[14] M. Tsunoda, K. Takezawa, T. Yanagisawa, M. Kato, K. Imai, Determination of catecholamines and their 3-O-methyl metabolites in mouse plasma, Biomed. Chromatogr. 15 (2001) 41–44. [15] H.-K. Jeon, H. Nohta, Y. Ohkura, High-performance liquid chromatographic determination of catecholamines and their precursor and metabolites in human urine and plasma by postcolumn derivatization involving chemical oxidation followed by fluorescence reaction, Anal. Biochem. 200 (1992) 332–338.
[16] G.-M. Cao, T. Hoshino, High-performance liquid chromatographic determination of catecholamines, serotonin and their metabolites with three-potential electrochemical detection, Anal. Sci. 12 (1996) 183–188. [17] M.E. Alburges, N. Narang, J.K. Wamsley, A sensitive and rapid HPLC–ECD method for the simultaneous analysis of norepinephrine, dopamine, serotonin and their primary metabolites in brain tissues, Biomed. Chromatogr. 7 (1993) 306–310.
Simultaneous determination of 3,4-dihydroxyphenylacetic acid and homovanillic acid using high performance liquid chromatography–fluorescence detection and application to rat kidney microdialysateq Makoto Tsunoda, Kazuto Mitsuhashi, Mayumi Masuda, and Kazuhiro Imai* Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 28 January 2002
Abstract We established a sensitive and simultaneous determination method of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) using HPLC–fluorescence detection. This method adopted the column-switching system, which included an online extraction of carboxylic acids by a strong anion-exchange column followed by separation on an ODS column, coulometric oxidation, fluorogenic reaction with ethylenediamine, and fluorescence detection. The detection limits were 50 and 100 fmol/injection for DOPAC and HVA, respectively (a signal-to-noise ratio of 3). The method was applicable to 50 ll of rat kidney microdialysate with a sufficient accuracy and precision. The concentrations of DOPAC and HVA in rat kidney microdialysate were 131 29 and 404 44 nM, respectively (n ¼ 5). This is the first report of DOPAC and HVA quantified in rat kidney microdialysate. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Catecholamines; Metabolism; Kidney; Microdialysis
Catecholamines (norepinephrine, epinephrine, and dopamine (DA)1) play important roles as neurotransmitters or hormones. We previously reported a method for the determination of catecholamines in rat plasma using HPLC-peroxyoxalate chemiluminescence reaction detection [1,2]. By the use of this method, we delineated the relationship between the changes of plasma catecholamine concentrations and blood pressure after the infusion of calcium antagonists [3–6]. Furthermore, to clarify the role of the methyl metabolism of catecholamines by catechol-O-methyltrans-
q A part of this work was presented at the 11th Conference of the Society for Chromatographic Sciences in Kyoto (October 2000, Japan). * Corresponding author. Fax: +81-3-5841-4885. E-mail address: [email protected] (K. Imai). 1 Abbreviations used: DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; DA, dopamine; COMT, catechol-O-methyltransferase; MAO, monoamine oxidase.
ferase (COMT) in blood pressure regulation, a method was developed for the simultaneous determination of catecholamines and their 3-O-methyl metabolites [7,8]. This method allowed us to show that catecholamine methylation in spontaneously hypertensive rats is attenuated, compared to normotensive Wistar–Kyoto rats [9]. As we were interested in delineating the role of catecholamines in organ levels, dopamine metabolism in the kidney was investigated. Presumably, DA acted as an intrarenal natriuretic hormone, and this activity depended on DA metabolism by COMT and monoamine oxidase (MAO). As shown in Fig. 1, DA can be metabolized to 3-methoxytyramine (3-MT) by COMT and to 3,4-dihydroxyphenylacetic acid (DOPAC) by MAO. Further, both 3-MT and DOPAC are metabolized to homovanillic acid (HVA). Microdialysis techniques can be used not only to obtain rat renal interstitial fluid (RIF) but also in studies on the role of DA metabolism in kidney where
0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 2 6 9 7 ( 0 2 ) 0 0 0 0 6 - 4
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Optimization of the fluorescence derivatization reaction and the coulometric oxidation
Fig. 1. Metabolic pathways of dopamine by COMT and MAO.
DA in rat kidney microdialysate was analyzed [10–12]. As far as we are aware, DA metabolites can be determined in rat urine, but not in rat kidney microdialysate. As it is doubtful that DA metabolites in urine reflect the intrarenal function, we have decided to develop a method to determine DA metabolites in rat kidney microdialysate so that we may gain insight into the metabolism of DA in kidney. Here we developed a sensitive determination method of DOPAC and HVA in rat kidney microdialysate. Optimization of chromatographic conditions, coulometric oxidation, and fluorescence derivatization reactions were performed. Validation of the method and an application to the determination of DOPAC and HVA in 50 ll of rat kidney microdialysate are described.
The effects of the length of reaction coil and the temperature of the fluorescence derivatization and the effect of the applied potential in the coulometric converter were investigated under the following HPLC conditions. The separation column used was TSK-gel ODS-80 Ts, 150 4:6 mm i.d. (Tosoh, Tokyo, Japan). The composition of the mobile phase was 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (85.5/4.5/10, v/v/v) and the composition of the fluorogenic reagent was 105 mM ethylenediamine and 175 mM imidazole in acetonitrile/ ethanol/water (80/10/10, v/v/v). The flow rates of the mobile phase and the fluorogenic reagent were fixed at 0.5 and 0.32 ml/min, respectively, which are the same as those in the previous reports [1,7,13]. One picomole each of DOPAC and HVA was injected into the HPLC system. The fluorescence detection was made at an emission wavelength of 495 nm with excitation at 417 nm. The temperature of a reaction coil (1:6 mm o:d: 0:5 mm i:d: 10–30 m), which was kept in a thermostatically controlled bath (ASB 200D, JASCO), was controlled at 85–120 °C. The applied potential versus a H2 =Hþ reference electrode was set at 0.05–0.5 V. Optimization of the on-line extraction of DOPAC and HVA A weak anion-exchange column (N(CH3)2-1031-N, 30 4:6 mm i.d., Senshu Scientific, Tokyo, Japan) and a strong anion-exchange column (SAX-1031-N, 30 4:6 mm i.d., Senshu Scientific) were used as the precolumn. The composition of the pretreatment buffer was 10 mM potassium phosphate buffer (pH 3.2) at a flow rate of 0.5 ml/min. In this experiment, the native fluorescence detection was carried out at an excitation wavelength of 280 nm and an emission wavelength of 315 nm connected after the precolumn. Five hundred picomoles each of DOPAC and HVA was injected onto the column. Apparatus and HPLC conditions
Materials and methods Chemicals 3,4-Dihydroxyphenylacetic acid and homovanillic acid were obtained from Sigma Chemical (St. Louis, MO). The purified ethylenediamine for washing the semiconductors was a gift from Wako Pure Chemicals (Osaka, Japan). Imidazole was from Merck (Darmstadt, Germany). Acetonitrile and ethanol, HPLC grade, were from Kanto Chemicals (Tokyo, Japan). All other reagents were of analytical grade.
A block diagram of the automated system is shown in Fig. 2. This system consisted of three pumps (PU-880 and PU-980, Jasco, Tokyo, Japan), an autosampler (851AS, Jasco), a rotatory six-way switching valve (HV-99201, Jasco), a fluorescence detector (820-FP, Jasco), and an integrator (807-IT, Jasco). HPLC conditions were as follows: precolumn, SAX-1031-N, 30 4:6 mm i.d. (Senshu Scientific); pretreatment buffer, 10 mM potassium phosphate buffer (pH 3.2); flow rate, 0.5 ml/min; precolumn cleanup solution, 0.1% phosphoric acid/ acetonitrile (50/50, v/v); flow rate, 0.5 ml/min; separation
M. Tsunoda et al. / Analytical Biochemistry 307 (2002) 153–158
Fig. 2. Block diagram of the automated analyzer of DOPAC and HVA in the present method. (Pump l) pretreatment buffer, 10 mM potassium phosphate buffer (pH 3.2), 0.5 ml/min; (Pump 2) mobile phase, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (90.25/4.75/5, v/v/v), 0.5 ml/min; (Pump 3) fluorogenic reagent, 105 mM ethylenediamine, and 175 mM imidazole in acetonitrile/ethanol/water (80/10/10, v/v/v), 0.32 ml/min. Precolumn, SAX-1031-N (30 4:6 mm i.d., Senshu Scientific); separation column, TSK-gel ODS-80 Ts (150 4:6 mm i.d., TOSOH); reaction coil, 0:5 mm i:d: 30 m, 120 °C. The applied potential on the coulometric converter was set at +0.4 V versus a H2 =Hþ reference electrode.
column, as described above; mobile phase, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (90.25/4.75/5, v/v/v); flow rate, 0.5 ml/min; fluorogenic reagent, as described above; coulometric converter (the Coulochem 5100A, ESA), +0.4 V versus a H2 =Hþ reference electrode; and a reaction coil (1:6 mm o:d: 0:5 mm i:d: 30 m), kept at 120 °C, which was the indication of the thermostatically controlled bath. Animal experiments Male Sprague–Dawley rats (8–9 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and housed in an environmentally controlled room with free access to tap water and diet for at least 1 week before use. All animals received humane care in compliance with the National Institutes of Health guidelines. To obtain rat kidney microdialysate, the left kidney in the anesthetized rats was exposed, and a 5-mm microdialysis probe (Bioanalytical Systems, West Lafayette, IN) was placed into the renal cortex. Ringer solution was infused at a flow rate of 2 ll/min. In vitro recoveries of DOPAC and HVA were evaluated by immersing dialysis membranes of the probes (n ¼ 3) in a beaker containing 50 nM each of DOPAC and HVA. Ringer solution was infused at a flow rate of 2 ll/min.
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the correlation coefficient. To 50-ll aliquots of rat kidney microdialysate, DOPAC and HVA (each 0, 150, 300, and 500 fmol) were spiked and analyzed by the HPLC system noted above. The accuracies were expressed as the reciprocal percentage of the amount of DOPAC or HVA recovered from the spiked samples to the expected values. Both intra- and interday precision was calculated by analyzing the same rat kidney microdialysate samples five consecutive times or five successive days, respectively. The precision was estimated based on the coefficient of variation (CV, %).
Results and discussion Optimization of the fluorescence derivatization reaction Catechol compounds selectively react with ethylenediamine to produce the fluorescent compounds, and the optimum conditions for the fluorescence derivatization of DOPAC with ethylenediamine were first investigated. The temperature was set to vary from 85 to 120 °C, and the reaction time was determined by varying the length of the reaction coil from 10 to 30 m. As shown in Fig. 3, with the increases of both the temperature and the length of reaction coil, the recorder response for DOPAC increased. When the length of the reaction coil was more than 30 m, it resulted in a peak broading. From these data shown in Fig. 3, a condition of 120 °C and 30 m was selected for the optimal fluorescence derivatization reaction. Optimization of coulometric oxidation To determine DOPAC and HVA simultaneously, HVA must be converted into o-quinone compounds prior to fluorescence derivatization. The electrochemical coulometric converter was employed to oxidize 3-O-
Validation of the HPLC system DOPAC or HVA (each 0, 150, 300, 500, 1000, 2000, or 5000 fmol) was injected into the HPLC. The calibration curves for peak height versus the amount of each compound were obtained. Least-squares regression was used for the calibration of the slope, intercept, and
Fig. 3. Effects of the reaction time and temperature on the fluorescence intensity of DOPAC. The temperature was changed from 85 to 120 °C. The reaction coil was investigated using 10 (r), 20 (j), 25 (N), and 30 m ( ) of the reaction coil.
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Fig. 4. Effects of applied potential on the fluorescence intensities for DOPAC ( ) and HVA (j). The experimental conditions are described under Materials and methods.
anion-exchange column was better than a weak anionexchange column for the extraction and separation of DOPAC and HVA from neutral and amino compounds (data not shown). Using a strong anion-exchange column (SAX-1031-N), DOPAC and HVA were retained in the precolumn in a pretreatment buffer with a low ionic concentration (10 mM potassium phosphate buffer (pH 3.2)), and eluted for 8– 12 min. Therefore, we decided to introduce this 4-min portion of the eluate into the ODS column. Under these conditions, catecholamines or 3,4-dihydroxyphenylglycol was eluted within 1 min from the position of the void volume, suggesting that a sufficient separation of DOPAC and HVA from neutral and amino compounds was achieved. Optimization of chromatographic separation conditions
methyl metabolites of catecholamines into the respective o-quinone compounds [7,8,14]. As shown in Fig. 4, while the peak of DOPAC was not influenced by the coulometric oxidation, the peak of HVA increased in the range of 0.1–0.4 V, and maximum fluorescence intensity was obtained at 0.4 V. Therefore, a potential of 0.4 V was selected for the simultaneous determination of DOPAC and HVA. Optimization of the on-line extraction of DOPAC and HVA We have previously reported an on-line extraction of catecholamines using a cation-exchange column [1,2]. To extract carboxylic acids including DOPAC and HVA from the biological samples, an anion-exchange column was used as a precolumn. In a preliminary experiment, we have learned that a strong
In the separation of the authentic of DOPAC and HVA, a mobile phase of 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (85.5/4.5/10, v/v/v) was used. This was because DOPAC and HVA gave good separation, with an analysis time within 25 min. However, in microdialysate samples from rat kidney, the peaks of DOPAC and the unknown endogenous compound overlapped. Therefore, by changing the ratio of the buffer to acetonitrile, the separation of these two peaks was investigated. As shown in Fig. 5c, when the concentration of acetonitrile in the mobile phase was 5%, a sufficient separation was obtained. Fig. 5 shows the typical chromatograms obtained (a) from Ringer solution, (b) from 5 pmol of a standard sample of DOPAC and HVA, and (c) from the microdialysate sample from rat kidney.
Fig. 5. Representative chromatograms obtained (a) from Ringer solution, (b) from 5 pmol of a standard sample of DOPAC and HVA, and (c) from the rat kidney microdialysate (50 ll). Peaks: 1, DOPAC; and 2, HVA. HPLC conditions are described under Materials and methods.
M. Tsunoda et al. / Analytical Biochemistry 307 (2002) 153–158 Table 1 Accuracy of the determination of DOPAC and HVA in rat kidney microdialysate (n ¼ 3) Added amounts (fmol) 0
150
300
DOPAC
Found (fmol) Accuracy (%)
248
393 96.9
563 105
HVA
Found (fmol) Accuracy (%)
666
808 94.4
954 96.0
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Acknowledgment We thank Dr. Chi-Ho Lee for his generous advice on the preparation of the manuscript.
500 745 99.4 1163 99.4
Validation of the proposed method The calibration curves for DOPAC and HVA showed linearity in the range of 150 fmol (DOPAC) or 300 fmol (HVA) to 5000 fmol. The correlation coefficients were 0.997 and 0.998 for DOPAC and HVA, respectively. The limits of detection (a signal-to-noise ratio of 3) were 50 and 100 fmol for DOPAC and HVA, respectively. These values were similar to or better than those previously reported [15–17]. The accuracies for DOPAC and HVA are summarized in Table 1. The intraday precision of this method was 2.68 and 2.91% for DOPAC and HVA (n ¼ 5), respectively. The interday precision of this method was 5.12 and 3.88% for DOPAC and HVA (n ¼ 5), respectively. Further, the reproducibilities of the retention times were 0.046 and 0.16% for DOPAC and HVA (n ¼ 5), respectively. These suggest that, although the internal standard was not used, the proposed method is appropriate for a routine assay of DOPAC and HVA in rat kidney microdialysate. Determination of DOPAC and HVA in rat kidney microdialysate In vitro recoveries of DOPAC and HVA from the beaker containing 50 nM each of DOPAC and HVA were investigated. The relative recoveries for DOPAC and HVA (percentage of DOPAC or HVA concentration in dialysate/DOPAC or HVA concentration in the beaker) were 3.8 and 3.3%, respectively, with a perfusion rate of 2 ll/min. The concentrations of DOPAC and HVA in rat kidney microdialysate determined by the present method were 131 29 and 404 44 nM (n ¼ 5), respectively, estimated with the respective recoveries from the probe in vitro. In summary, a selective and sensitive determination method of DOPAC and HVA utilizing a HPLC–fluorescence detection system was developed for rat kidney microdialysate samples. DOPAC can be assayed at a concentration as low as 150 fmol and HVA at 300 fmol. The present method is considered to be the first method applied to the determination of DOAPC and HVA in rat kidney microdialysate.
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