Focusing Review

Chromatography, Vol.26 No.3 (2005)

Focusing Review

Development of an Analytical Method for Catecholamines withHPLC−Chemiluminescence Detection and its Application
Makoto Tsunoda
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo−ku, Tokyo 113−0033, Japan
Received for review November 10, 2005. Accepted November 24, 2005.

Abstract
Catecholamines play important roles as neurotransmitters and hormones, and are metabolized by catechol−O−methyltransferase (COMT) andinactivated. We have developed the highly selective and sensitive determination methods for catecholamines (and their 3−O−methyl metabolites) with high−performance liquid chromatography−peroxyoxalate chemiluminescence reaction detection. The developed methods were applicable to the clarification of the roles of catecholamines and their 3−O−methyl metabolites in blood pressure regulation.
Keywords: peroxyoxalate chemiluminescence; high−performance liquid chromatography (HPLC); catecholamines; catechol−O−methyltransferase (COMT); blood pressure

as an index for several diseases such as hypertension and pheochro-1. Introduction
Catecholamines (norepinephrine (NE), epinephrine (E) andmocytoma. Therefore, many methods have been developed for thedopamine (DA)) play important roles as neurotransmitters or hor-determination of catecholamines and/or their metabolites. Most ofmones, and are known to be metabolized by catechol−O−methyl-them adopted high−performance liquid chromatography (HPLC)−transferase (COMT) and monoamine oxidase. As shown in Figure

electrochemical detection or fluorescence detection. However,

1, catecholamines are metabolized into their respective 3−O−

there are many endogenous substances in bio−samples such as

methyl metabolites (normetanephrine (NMN), metanephrine (MN)

plasma or urine. Therefore, these methods have been used in com-and 3−methoxytyramine (3−MT)) by COMT. Catecholamines and/bination with complicated sample pre−treatment such as solidor their metabolites concentrations in plasma and/or urine are usedphase extraction or liquid−liquid extraction to determine catechola-Figure 1. Metabolic pathway of catecholamines by COMT.
Correspondence: Dr. Makoto Tsunoda, Graduate School of Pharmaceutical Sciences, The University of Tokyo,
7−3−1, Hongo, Bunkyo−ku, Tokyo 113−0033, Japan.
Phone: +81−3−5841−4761, Fax: +81−3−5802−3339
E−mail: makotot@mol.f.u−tokyo.ac.jp―９５―Chromatography, Vol.26 No.3 (2005)

3. Investigation of baroreflex−mediated sympathetic nervous

mines selectively. Furthermore, since the plasma catecholamine

system with the developed methods

concentrations are very low (pmol order per 1 ml of plasma), 1 ml

Blood pressure is regulated by the integrated action of cardio-of plasma is needed for the sensitivity of electrochemical or fluo-vascular, renal, neural and endocrine system, and the sympathetic

rescence detection.
In this article, the development of analytical methods for both

nervous system is important for momentary regulation. Sympa-

catecholamines and catecholamines and their 3−O−methyl metabo-

thetic nerve outflow is controlled by baroreflex sensor systems. In

lites with HPLC−chemiluminescence detection is described. Appli-

response to a sudden fall in arterial pressure, afferent signals arecation of the developed methods to the clarification of the roles ofsent to central nervous system to activate sympathetic nervous sys-

the sympathetic nervous system and COMT in blood pressure regu-tem resulting in increased NE outflow.
We investigated the role of baroreflex−mediated sympathetic

lation is also described.

nervous system in blood pressure regulation in conscious rats [5].
When blood pressure was reduced by the administration of a cal-

2. Development of an analytical method for catecholamines
Considering the background as mentioned above, Higashidate

cium antagonist, blood pressure reduction correlated with the in-et al. applied HPLC−peroxyoxalate chemiluminescence (POCL)

crease of plasma NE and E concentrations. Further studies of ad-reaction detection to the determination of catecholamines [1], since

ministration of a calcium antagonist to spontaneously hypertensive

higher sensitivity should be obtained with POCL detection thanrats (SHR) and Wistar−Kyoto (WKY) rats revealed the reduced re-with fluorescence detection [2]. This method was as follows: (1)sponse of baroreflex−mediated sympathetic nerve system in SHR

pre−treatment of samples with alumina extraction; (2) separation ofcompared with WKY rats.

catecholamines on an ODS column; (3) post−column derivatization
4. Development of an analytical method for catecholamines andof catecholamines with ethylenediamine, which produced fluores-

their 3−O−methyl metabolites in plasma

cence derivatives; (4) on−line POCL reaction detection. As expected, the limits of detection for catecholamines were 1 fmol on

Simultaneous determination of catecholamines and their 3−O−column, which were better than those previously reported. Prados

methyl metabolites was thought to be useful for the examination ofet al. modified the method and reported the column−switching sys-sympathoadrenal activity as well as metabolic activity of catechola-

tem for the determination of catecholamines [3].

mines by COMT. Hence, we attempted to develop the simultane-

We have adopted a semi−micro column (1.5 mm I.D.) to im-

ous determination method for catecholamines and their 3−O−

prove the sensitivity [4]. The block−diagram of the system ismethyl metabolites in rat plasma [6]. However, since the fluores-

shown in Figure 2. Figure 3 shows the chromatograms of a mixture

cence reaction of catecholamines with ethylenediamine is selectiveof standard catecholamines and of a rat plasma sample. As a result,for catechol compounds, catecholamines metabolites were not able

3−10 times higher sensitivity was obtained compared with theto react with ethylenediamine. With the coulometric oxidation of
catecholamines metabolites into o−quinone compounds followed

method with conventional column.

by fluorescence reaction with ethylenediamine, the simultaneous
determination of catecholamines and their 3−O−methyl metabolites

Figure 2. Block diagram of the column−switching HPLC system for catecholamine analyzer.

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Makoto Tsunoda

Figure 3. (a) Chromatogram of a standard mixture of catecholamines (250 fmoleach). (b) Chromatogram obtained from a Sprague−Dawley rat plasma
sample. Peaks: 1, norepinephrine; 2, epinephrine; 3, dopamine; 4, N −methyldopamine (internal standard).

Figure 4. Block diagram of the column−switching HPLC system for the automated analyzer of catecholamines and their 3−O−methyl metabolites.
was achieved. Figure 4 shows the block diagram for the system,compounds. The concentrations in rat plasma were 1.05 ± 0.03,

which included on−line extraction of amines using a precolumn

0.64 ± 0.02, 0.19 ± 0.01, 0.51 ± 0.02, 0.26 ± 0.01 pmol/ml (n=3,packed with cation exchange resin, separation of catecholamines

mean ± SEM) for NE, E, DA, NMN and MN, respectively.

and their 3−O−methyl metabolites on an ODS column, coulometricImproved sensitivity could be obtained with the use of a semioxidation of metabolites, fluorogenic derivatization with ethyle-−micro column [7]. To avoid extra−column peak diffusion, a micro

nediamine, and postcolumn POCL detection.

coulometric flow cell was developed. It enabled to lower the detection limits for catecholamines and their 3−O−methyl metabolites toA typical chromatogram of 250 fmol of NE, E, DA, NMN,

0.3−2.0 fmol.

MN, 3−MT, and 4−MT (internal standard) is shown in Figure 5 (a).
Figure 5 (b) shows a typical chromatogram obtained from a Spra-

The developed method was applicable to the determination ofgue−Dawley rat plasma sample (50 µl). With the exception of 3−

catecholamines in 15 µl of mouse plasma [8]. Figure 6 shows theMT, the peaks for NE, E, DA, NMN, and MN in the rat plasma

chromatograms of a standard mixture of catecholamines and their 3

were clearly shown with no interference from endogenous com-−O−methyl metabolites and obtained from a mouse plasma sample.

pounds. This implies the high selectivity of the proposed method,Previously, due to the lack of the sensitivity, we were not able tosince it includes on−line selective extraction of amines by the pre−determine plasma catecholamines concentrations continuously

column and a selective ethylenediamine condensation for catechol

from a mouse. However, we could obtain some plasma samples―９７―

Chromatography, Vol.26 No.3 (2005)

from a mouse with the developed method, and we have found thatsyl−L−methionine (SAMe) to catechol compounds including

after the administration of minoxidil, which is an antihypertensive

catecholamines. COMT is distributed in most tissues, and there areagent, plasma catecholamines were significantly increased by thetwo forms, one is soluble form (S−COMT) and the other is mem-baroreflex−mediated response in mouse.

brane−bound form (MB−COMT).

5. Development of an analytical method for catechol−O−meth-However, many adopted exogenous substrates, and we have devel-

There are many measurement methods for COMT activity.
oped an analytical method for COMT activity with catecholamines

yltransferase activity

as the endogenous substrate. COMT activities were evaluated by

COMT catalyses the transfer of the methyl group of S−adeno-

the quantitation of catecholamines metabolites produced by the enzymatic reaction. The analytical method for catecholamines metabolites was the same as that for plasma sample. COMT activities
in rat erythrocytes [9], rat kidney and liver [10, 11], rat adrenal
gland [12], rat brain [13] and human erythrocytes [14] were measured. Typical chromatograms obtained from the reaction solutionof rat erythrocyte S−COMT and MB−COMT, with NE as the substrate, are shown in Figure 7.
6. Investigation of catechol−O−methyltransferase in blood pressure regulation
As mentioned above, in blood pressure regulation, catecholamines play important roles, and therefore, COMT is also importantfactor for the regulation of catecholamines. However, so far, relationship between COMT and blood pressure has not been examined. We determined plasma catecholamines and their 3−O−methyl
Figure 5. Chromatograms (a) of a standard sample of catecholamines and their 3−O−methyl metabolites (each 250 fmol)and (b) obtained from rat plasma sample (50 µl). Peaks:
1, norepinephrine; 2, epinephrine; 3, normetanephrine; 4,dopamine; 5, metanephrine; 6, 3−methoxytyramine; 7, 4
−methoxytyramine (internal standard).

metabolites concentrations after the administration of a calcium antagonist with the developed method, and found that methylation ofcatecholamines by COMT in SHR was reduced as compared toWKY rats [15]. Furthermore, we found the reduced amounts andactivity of liver MB−COMT in SHR [16]. These results indicatethat liver MB−COMT plays an important role in blood pressure(a)(b)

Figure 6. Chromatograms (a) of a standard mixture of catecholamines and their
3−O−methyl metabolites (each 250 fmol), and (b) obtained from amouse plasma when 30 µL of blood were collected from veins in bothorbitae. Peaks: 1, norepinephrine; 2, epinephrine; 3, normetanephrine;
4, dopamine; 5, metanephrine; 6, 3−methoxytyramine; 7, 4−methoxytyramine (internal standard).

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Chromatography, Vol.26 No.3 (2005)

Makoto Tsunoda

Figure 7. Chromatograms obtained from reaction solution of rat erythrocytes(a) S−COMT and (b) MB−COMT. Peaks: 1, norepinephrine; 2,normetanephrine; 3, 4−methoxytyramine (internal standard).

K. Analyst 2000, 125, 293−296.

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[5]

Tsunoda, M.; Takezawa, K.; Santa, T.; Ina, Y.; Nagashima,K.; Ohmori, K.; Kobayashi, S.; Imai, K. Jpn. J. Pharmacol.

7. Conclusions

2000, 83, 39−45.

We have developed the simultaneous and sensitive determination methods for catecholamines and their 3−O−methyl metabolites[6]

Tsunoda, M.; Takezawa, K.; Santa, T.; Imai, K. Anal. Bio-

[7]

Takezawa, K.; Tsunoda, M.; Watanabe, N.; Imai, K. Anal.

chem. 1999, 269, 386−392.

with HPLC−POCL detection [17]. These methods enabled us theclarification of the role of COMT in blood pressure regulation. InChem. 2000, 72, 4009−4014.

the near future, further clarification of the physiological role of[8]catecholamines and their metabolism should be expected.

Tsunoda, M.; Takezawa, K.; Yanagisawa, T.; Kato, M.;
Imai, K. Biomed. Chromatogr. 2001, 15, 41−44.

[9]Acknowledgement

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The author acknowledges to Emeritus Professor Kazuhiro[10] Tsunoda, M.; Takezawa, K.; Masuda, M.; Imai, K. Biomed.

Imai, The University of Tokyo, for his kind help and encourage-Chromatogr. 2002, 16, 536−541.

ment in this work. The author also expresses his gratitude to Prof.

[11] Hirano, Y.; Tsunoda, M.; Funatsu, T.; Imai, K. J. Chroma-Takashi Funatsu, The University of Tokyo, Prof. Kazuichi Hayak-togr. B 2005, 819, 41−46.

awa, Kanazawa University, and all the collaborators in this work.

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The present author is very grateful to the Japan Society for Chro-−890.

matographic Sciences who selected him as the recipient of the En-[13] Masuda, M.; Tsunoda, M.; Imai, K. Anal. Bioanal. Chem.

couragement Award in 2005 and gave him an opportunity to pub-2003, 376, 1069−1073.

lish this review paper. The author thanks Dr. Chang Kee Lim,[14] Masuda, M.; Tsunoda, M.; Yusa, Y.; Yamada, S.; Imai, K.

MRC Bioanalytical Science Group, for his valuable discussions inAnn. Clin. Biochem. 2002, 39, 589−594.

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