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An Isocratic Assay for Norepinephrine, Dopamine, and 5-Hydroxytryptamine Using Their Native Fluorescence by High-Performance Liquid Chromatography with Fluorescence Detection in Discrete Brain Areas of Rat
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
246, 166–170 (1997)
AB969997
An Isocratic Assay for Norepinephrine, Dopamine, and 5-Hydroxytryptamine Using Their Native Fluorescence by High-Performance Liquid Chromatography with Fluorescence Detection in Discrete Brain Areas of Rat Madepalli K. Lakshmana and Trichur R. Raju1 Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore 560 029, India
Received July 15, 1996
A rapid and simple isocratic chromatographic procedure for simultaneous determination of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) using their native fluorescence by high-performance liquid chromatography coupled to fluorescence detection (HPLC–FD) is described. Since the present procedure does not involve sample prepurification, the recovery of monoamines was more than 97% (n Å 12) and within a given run, coefficient of variation was less than 3.1% (n Å 12). Accordingly, use of an internal standard is not mandatory. In a single chromatographic run, levels of NE, DA, and 5-HT can be determined in less than 30 min. The minimum concentration of monoamines which could be detected by this method was found to be 250 pg for NE and DA and 100 pg for 5-HT. The validity of the method was confirmed by the estimation of levels of monoamines in the hypothalamus and striatum of rat brain following treatment with clorgyline, a monoamine oxidase inhibitor. q 1997 Academic Press
Several methods have been used to determine simultaneously monoamines and their related metabolites in discrete regions of rat brain. In recent years, highperformance liquid chromatography coupled to electrochemical detection (HPLC–ECD)2 (1–5) has been the 1 To whom correspondence should be addressed. Fax: 91 (80) 6631830. 2 Abbreviations used: HPLC–ECD, high-performance liquid chromatography coupled to electrochemical detection; HPLC–FD, HPLC with fluorimetric detection; NE, norepinephrine; DA, dopamine; 5HT, 5-hydroxytryptamine; IP, isoproterenol; PCA, perchloric acid; SF, solvent front; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HTP, 5-hydroxytryptophan; 5-HIAA, 5-hydroxyindole acetic acid; TRP, tryptophan; HSA, heptane sulfonic acid.
most frequently used method. Although electrochemical detection is sensitive enough to estimate monoamines in tissues, problems of maintenance, limited life time of the electrode (6), and variability of sensitivity (7, 8) result in frequent repacking/repolishing of the electrodes (9, 10). Accordingly, fluorimetry is considered as an alternative or additional detection system. HPLC with fluorimetric detection (HPLC–FD) has been used to determine monoamines after derivatization with fluorescamine (11), o-pthalaldehyde (12), or trihydroxy indole (13, 14). Derivatization methods enhance the detectability; however, they involve additional procedures for the analysis of samples. The interpretation of the resulting chromatogram becomes complicated because of the nonspecificity of derivatization agents. Detection of native fluorescence of monoamines without derivatization has also been reported. However, these methods which use native fluorescence employ either alumina for adsorption of monoamines (15) or gradient elution (16) which results in an increase in the number of steps involved in sample prepurification and more time is required for elution of samples. To date, no isocratic method, which is simple and rapid for the determination of monoamines in discrete regions of rat brain by HPLC–FD, has been reported. Here we describe a simplified procedure for simultaneous determination of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) in a single chromatographic run using their native fluorescence in discrete regions of rat brain which does not involve any sample prepurification. The utility of the method has been tested by analyzing the effect of monoamine oxidase A inhibitor clorgyline on the levels of NE, DA, and 5-HT in hypothalamus and striatum of rat brain.
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CHROMATOGRAPHIC ASSAY MEASURING MONOAMINE LEVELS
MATERIALS AND METHODS
Materials. Clorgyline and standards such as norepinephrine bitartrate salt, dopamine hydrochloride, 5-hydroxytryptamine hydrochloride, isoproterenol hydrochloride, and heptane sulfonic acid were all obtained from Sigma Chemical Co. (St. Louis, MO). HPLC-grade methanol was supplied by Spectrochemicals (Bombay, India). Analytical-grade sodium acetate, EDTA, o-phosphoric acid, perchloric acid, and dibutylamine were all obtained from Glaxo Laboratories (Bombay, India). Apparatus. The HPLC system consisted of a delivery pump (Model ERC-8710, Erma Optical Works, Tokyo, Japan), a reversed-phase analytical column (Ultracarb ODS, 3 mm, 150 1 4.6-mm i.d.; Phenomenex, Torrance, CA), protected by a guard column (Ultracarb ODS, 3 mm, 30 1 4.6-mm i.d.; Phenomenex), a degasser (ERC 3310, ERMA (Tokyo, Japan), a recorder (Sekonic SS-100F, Tokyo, Japan), electrochemical detector (BioRad, Model 1640, U.S.A.), and a fluorescence spectrophotometer (Hitachi, Model 650-40, Tokyo, Japan) for detection. Chromatographic conditions. The mobile phase consisted of sodium acetate (0.02 M), methanol (16%), heptane sulfonic acid (0.055%), EDTA (0.2 mM), and dibutylamine (0.01%, v/v). The solution was adjusted to pH 3.92 with o-phosphoric acid and filtered through a 0.45-mm membrane (Sartorius) and degassed. The flow rate was set to 0.9 ml/min, which yielded a pressure of 125–130 kg/cm2. Animals. Adult male Wistar rats weighing 150– 180 g received clorgyline injections intraperitoneally (4 mg/kg body wt) for 3 days. Control group of rats received equimolar quantity of saline. Sample preparation. Rats were sacrificed by decapitation on the third day, 2 h postinjection of clorgyline and heads were dropped into ice-cold 0.1 M PCA. Immediately the brains were removed; hypothalamus and striatum were separated on an ice-chilled petri dish according to a slightly modified method of Glowinsky and Iversen (17). The tissues were weighed and homogenized in 2 ml of 0.1 M PCA containing isoproterenol at the concentration of 30 ng/ml. After centrifugation at 14,000g for 15 min at 47C, the supernatant was filtered through a 0.45-mm membrane (Sartorius) and 100 ml of the filtrate was injected onto an HPLC column. After separation, NE, DA, isoproterenol (IP), and 5-HT were detected at the excitation wavelength of 280 nm and an emission wavelength of 315 nm. The slit width was kept at 10/10 for excitation/emission, respectively. The slit width is expressed as the length and width of the sample-plane zone being quantitated. Standards and calculations. Stock solutions of standards, 1 mg/ml, were prepared in 0.1 M hydrochlo-
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ric acid and were stored at 0207C. If they were not used within a month of preparation, they were discarded. The working standard solutions were prepared freshly in 0.1 M PCA for each experiment. The amounts of standards per injection volume of 100 ml were 3 ng each of NE, DA, IP, and 5-HT. Monoamine peaks were identified by comparing their retention time in the sample (tissue extracts) solution with that of standard solution and also by superimposing the chromatograms of the samples spiked with and without known amounts of the standards. Each monoamine in the tissue was quantified by comparing peak heights in elution profiles of samples with known standards. RESULTS
Chromatograms of diluted monoamines and isoproterenol (500 and 1000 pg/100 ml) using fluorimetric and electrochemical detectors are compared in Fig. 1. With the same chromatographic conditions, the peak heights of monoamines were double under electrochemical detector compared to fluorimetric detector, suggesting that the electrochemical detector is more sensitive. The linearity between 500 and 1000 pg of monoamines was observed under both the detectors. A chromatogram of the blank (0.1 M PCA) is shown in Fig. 2A. Figure 2B shows typical chromatograms of 3 ng/100 ml of each monoamine and the internal standard, isoproterenol. Chromatograms of tissue extracts of hypothalamus and striatum are shown in Figs. 2C and 2D. In both standard and tissue chromatograms, monoamines and isoproterenol were completely separated from the solvent front (SF). Monoamine metabolites and precursors such as 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxytryptophan (5-HTP) and 5-hydroxyindole acetic acid (5-HIAA) and tryptophan (TRP) coeluted. The recovery for monoamines ranged from 97.1 to 98.3%, and the coefficient of variation was in the range of 1.4 to 3.1%; the detection limit determined on the basis of injected amount of standards giving a signalto-noise ratio higher than 3 was 250 pg for NE and DA and 100 pg for 5-HT (Table 1). Levels of monoamine neurotransmitters in hypothalamus and striatum of rat brains following treatment with clorgyline and values from the brains of control animals which did not receive any clorgyline are compared in Table 2. In clorgyline-treated rats, significantly increased levels of NE and 5-HT were observed in striatum and hypothalamus, while DA levels were increased only in striatum. DISCUSSION
The isocratic HPLC–FD method described here makes it possible to determine simultaneously the levels of NE, DA, and 5-HT in less than 30 min in rat
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Vander Hoorn et al. (20), after comparison of five types of buffers, reported higher fluorescence intensity for monoamines with lower molarity of sodium acetate. Accordingly, in the present method low molar sodium acetate buffer was used. The pH and composition of the mobile phase, particularly the concentration of heptane sulfonic acid (HSA), are the main determinants of solute separation and retention time. A relatively high amount of HSA was used in the present experiment to shift monoamine substrates to ‘‘chromatographically clean’’ areas where they can be resolved without interference from
FIG. 1. Chromatograms of diluted standards, norepinephrine (NE), dopamine (DA), isoproterenol (IP), and 5-hydroxytryptamine (5-HT) using fluorimetric and electrochemical detectors. (A and B) Chromatograms of 500 and 1000 pg of monoamines using electrochemical detector. (C and D) Chromatograms of 500 and 1000 pg using fluorimetric detector. Note that the height of each monoamine peak is twice that under electrochemical detector compared to fluorimetric detector. Chromatographic conditions: analytical column; 3 mm, Ultracarb ODS (150 1 4.6 mm, i.d.); mobile phase, 0.02 M sodium acetate (pH 3.92) with 16% methanol, 0.055% heptane sulfonic acid, 0.2 mM EDTA, and 0.01% dibutylamine; temperature, ambient.
brain regions. Previously reported methods for the determination of monoamines using HPLC–FD require either sample prepurification (15, 18) or derivatization of monoamines (11, 12, 13, 14, 19). Vander Hoorn et al. (20) have reported a method involving both a purification and a derivatization procedure to assay monoamines in plasma. This method has also been successfully used to assay monoamines in the brain tissue (21). However, such methods using purification and derivatization steps are time-consuming and require careful control by the use of internal standards. A major advantage of the present procedure is the ease of sample preparation which reduces assay time and chances for technical errors. In addition, it does not require the use of an internal standard as can be judged from high recovery (97.1–98.3%) and good coefficients of variation (1.4–3.1%).
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FIG. 2. Chromatograms of standards and tissue samples using fluorimetric detector. (A) Blank, 0.1 M PCA (solvent front, SF). (B) Typical chromatograms of 3 ng each of NE, DA, IP, and 5-HT in 100 ml. (C) Chromatogram of hypothalamus extract (43 mg). The absolute values of NE, DA, and 5-HT are 1860.1 { 167.8, 364.7 { 35.7, and 1020.3 { 101.2 ng/g wet wt tissue, respectively, (D) Chromatogram of striatal extract (35 mg). The absolute values of NE, DA, and 5HT are 321.6 { 31.7, 7560.2 { 457.8, and 553.8 { 47.9 ng/g wet wt tissue. The values in parentheses represent retention time (minutes). For chromatographic conditions, see legend for Fig. 1.
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CHROMATOGRAPHIC ASSAY MEASURING MONOAMINE LEVELS TABLE 1
Recovery, Precision, and Detection LImits for the HPLC-FD Determination of Three Monoamine Neurotransmitters Monoamines
Recovery (%)a (n Å 12)
Within runb CV (%)
Detectionc limits (pg/100 ml)
Norepinephrine Dopamine 5-Hydroxytryptamine
97.4 { 1.7 97.1 { 1.5 98.3 { 1.4
{2.2 {1.4 {3.1
250 250 100
Note. Values are means { SD. Chromatographic conditions are as in the legend to Fig. 1. a Recoveries were measured by repeated injections of tissue homogenate samples spiked with or without known amounts of standards (3 ng). b Coefficients of variation (CV) were obtained by repeated injections of 3 ng of the standards, from the same working standard solution. c Injected amount of standards giving a signal-to-noise ratio higher than 3.
monoamine metabolites. This concentration of HSA was also sufficient to separate NE from the solvent front. These separations can be achieved at room temperature (25 { 17C) and control of column temperature is not required. An analytical column can be used for more than a thousand sample injections, probably due to the much higher percentage of organic modifier in the mobile phase. To confirm the validity of this method, the effect of monoamine oxidase A inhibitor, clorgyline, on levels of monoamines in hypothalamus and striatum was determined. Our results for normal control rats are in agreement with the previously published data (22, 23). The observed increase in NE and DA levels as shown in Table 2 after clorgyline treatment is in agreement with the previous report (23). Although HPLC–FD has been shown to be more selective and sensitive enough to measure catecholamines compared with HPLC–ECD (20), in the present method DOPAC and 5-HTP on one hand and 5-HIAA and TRP on the other hand coeluted. However, the concentrations of NE and DA were shown to be higher and interassay coefficient of variation was lower in HPLC– FD than in HPLC–ECD (20). In HPLC–ECD, there is
gradual decay of sensitivity of the electrode, since crude homogenate supernatants pollute the electrode surface. This results in gradual broadening of the solvent front which in turn interferes with the separation of other substrates (3). The fluorimetric response remains constant for many months or even years without any attention. With the chromatographic conditions used in this study, DOPAC and 5-HTP coeluted after NE elution. Similarly, 5-HIAA and TRP coeluted after isoproterenol. The only limitation of the present method is the coelution of monoamine metabolites (DOPAC and 5-HTP, 5-HIAA and TRP). Thus we could not determine the levels of monoamine metabolites. However, such coelutions do not interfere with the estimation of monoamines. In the present method we have demonstrated for the first time that HPLC–FD can be used for the rapid assay of brain monoamine levels without involving prepurification steps. Whether HPLC–FD is superior to HPLC–ECD for estimating monoamines is still a debatable issue. On the other hand, a procedure which is simple and rapid and does not involve elaborate purification steps should be exploited at least as an alternative method to HPLC–ECD.
TABLE 2
Levelsa of Norepinephrine (NE), Dopamine (DA), and 5-Hydroxytryptamine (5-HT) in Striatum and Hypothalamus of Rat Brains Treated with Clorgyline (4 mg/kg body wt, ip) Brain region Striatum
Hypothalamus
Monoamines
Control
Clorgyline
Control
Clorgyline
NE DA 5-HT
321.6 { 31.7 7560.2 { 457.8 553.8 { 47.9
390.5 { 37.1** 8216.7 { 462.8* 671.8 { 66.8**
1860.1 { 167.8 364.7 { 35.7 1020.3 { 101.2
2241.8 { 192.3** 367.8 { 35.3 1281.7 { 127.8***
Note. Values are means { SD of 8 rats in each group. Chromatographic conditions are as in the legend to Fig. 1. a ng/g wet wt tissue. * P õ 0.05. ** P õ 0.01. *** P õ 0.001 versus control (Student’s t test).
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REFERENCES 1. Mefford, I. N. (1981) J. Neurosci. Methods 3, 207.
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2. Wagner, J., Vitali, P., Paltreyman, M. G., Zraika, M., and Hont, S. (1982) J. Neurochem. 38, 1241.
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3. Lashley, S. M., Michaelson, I. A., Greenland, R. O., and McGinnis (1984) J. Chromatogr. 305, 27.
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4. Leung, P. Y., and Isao, C. S. (1992) J. Chromatogr. 576, 245.
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5. Cheng, F. C., and Kuo, J-S. (1993) J. Chromatogr. 615, 225. 6. Hallman, H., Farnebo, L. O., Hanberger, B., and Johnsson, G. (1978) Life Sci. 23, 1049.
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7. Hjemdahl, P., Daleskog, M., and Kahan, T. (1979) Life Sci. 25, 131.
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8. Nagao, T., and Tanimura, T. (1989) J. Chromatogr. 496, 39. 9. Adams, R. N., and Marsden, C. A. (1982) in Handbook of Psychopharmacology (Iversen, L. L., Iversen, S. D., and Snyder, S. H., Eds.), Vol. 15, p. 1, Plenum, New York. 10. Anton, A. H. (1984) Life Sci. 35, 79.
20. 21. 22.
11. Imai, K. (1975) J. Chromatogr. 105, 135. 12. Davis, T. P., Gehrke, C. W., Gehrke, C. W., Jr., Cunningham,
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T. D., Cuo, K. C., Gerhardt, K. O., Johnson, H. D., and Williams, C. H. (1978) Clin. Chem. 24, 1317. Okamoto, K. L., Ishida, Y., and Asai, K. (1978) J. Chromatogr. 167, 205. Yu, Y., Itokawa, Y., and Kawai, C. W. (1980) Anal. Biochem. 108, 11. Jackman, G. P., Carlson, V. J., Bobik, A., and Skews, H. (1980) J. Chromatogr. 182, 277. Krstulovic, A. M., and Powell, A. M. (1979) J. Chromatogr. 171, 345. Glowinski, J., and Iversen, L. L. (1966) J. Neurochem. 13, 655. Nohta, H., Yamaguchi, E., Ohkura, Y., and Watanabe, H. (1989) J. Chromatogr. 493, 15. Mitsui, A., Nohta, H., and Ohkura, Y. (1985) J. Chromatogr. 344, 61. Vander Hoorn, F. A. J., Boomsma, F., Manin, A. J., Veld, T., and Schalekamp, M. A. D. H. (1989) J. Chromatogr. 487, 17. Verhage, M., Ghilsen, W. E. J., Boomsma, F., and Lopesda Silva, F. H. (1992) J. Neurochem. 59, 881. Felice, L. J., Felice, J. D., and Kissinger, P. T. (1978) J. Neurochem. 31, 1461. Buu, N. T., and Angers, M. (1987) Biochem. Pharmacol. 36, 1731.
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AB969997
An Isocratic Assay for Norepinephrine, Dopamine, and 5-Hydroxytryptamine Using Their Native Fluorescence by High-Performance Liquid Chromatography with Fluorescence Detection in Discrete Brain Areas of Rat Madepalli K. Lakshmana and Trichur R. Raju1 Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore 560 029, India
Received July 15, 1996
A rapid and simple isocratic chromatographic procedure for simultaneous determination of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) using their native fluorescence by high-performance liquid chromatography coupled to fluorescence detection (HPLC–FD) is described. Since the present procedure does not involve sample prepurification, the recovery of monoamines was more than 97% (n Å 12) and within a given run, coefficient of variation was less than 3.1% (n Å 12). Accordingly, use of an internal standard is not mandatory. In a single chromatographic run, levels of NE, DA, and 5-HT can be determined in less than 30 min. The minimum concentration of monoamines which could be detected by this method was found to be 250 pg for NE and DA and 100 pg for 5-HT. The validity of the method was confirmed by the estimation of levels of monoamines in the hypothalamus and striatum of rat brain following treatment with clorgyline, a monoamine oxidase inhibitor. q 1997 Academic Press
Several methods have been used to determine simultaneously monoamines and their related metabolites in discrete regions of rat brain. In recent years, highperformance liquid chromatography coupled to electrochemical detection (HPLC–ECD)2 (1–5) has been the 1 To whom correspondence should be addressed. Fax: 91 (80) 6631830. 2 Abbreviations used: HPLC–ECD, high-performance liquid chromatography coupled to electrochemical detection; HPLC–FD, HPLC with fluorimetric detection; NE, norepinephrine; DA, dopamine; 5HT, 5-hydroxytryptamine; IP, isoproterenol; PCA, perchloric acid; SF, solvent front; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HTP, 5-hydroxytryptophan; 5-HIAA, 5-hydroxyindole acetic acid; TRP, tryptophan; HSA, heptane sulfonic acid.
most frequently used method. Although electrochemical detection is sensitive enough to estimate monoamines in tissues, problems of maintenance, limited life time of the electrode (6), and variability of sensitivity (7, 8) result in frequent repacking/repolishing of the electrodes (9, 10). Accordingly, fluorimetry is considered as an alternative or additional detection system. HPLC with fluorimetric detection (HPLC–FD) has been used to determine monoamines after derivatization with fluorescamine (11), o-pthalaldehyde (12), or trihydroxy indole (13, 14). Derivatization methods enhance the detectability; however, they involve additional procedures for the analysis of samples. The interpretation of the resulting chromatogram becomes complicated because of the nonspecificity of derivatization agents. Detection of native fluorescence of monoamines without derivatization has also been reported. However, these methods which use native fluorescence employ either alumina for adsorption of monoamines (15) or gradient elution (16) which results in an increase in the number of steps involved in sample prepurification and more time is required for elution of samples. To date, no isocratic method, which is simple and rapid for the determination of monoamines in discrete regions of rat brain by HPLC–FD, has been reported. Here we describe a simplified procedure for simultaneous determination of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) in a single chromatographic run using their native fluorescence in discrete regions of rat brain which does not involve any sample prepurification. The utility of the method has been tested by analyzing the effect of monoamine oxidase A inhibitor clorgyline on the levels of NE, DA, and 5-HT in hypothalamus and striatum of rat brain.
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0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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CHROMATOGRAPHIC ASSAY MEASURING MONOAMINE LEVELS
MATERIALS AND METHODS
Materials. Clorgyline and standards such as norepinephrine bitartrate salt, dopamine hydrochloride, 5-hydroxytryptamine hydrochloride, isoproterenol hydrochloride, and heptane sulfonic acid were all obtained from Sigma Chemical Co. (St. Louis, MO). HPLC-grade methanol was supplied by Spectrochemicals (Bombay, India). Analytical-grade sodium acetate, EDTA, o-phosphoric acid, perchloric acid, and dibutylamine were all obtained from Glaxo Laboratories (Bombay, India). Apparatus. The HPLC system consisted of a delivery pump (Model ERC-8710, Erma Optical Works, Tokyo, Japan), a reversed-phase analytical column (Ultracarb ODS, 3 mm, 150 1 4.6-mm i.d.; Phenomenex, Torrance, CA), protected by a guard column (Ultracarb ODS, 3 mm, 30 1 4.6-mm i.d.; Phenomenex), a degasser (ERC 3310, ERMA (Tokyo, Japan), a recorder (Sekonic SS-100F, Tokyo, Japan), electrochemical detector (BioRad, Model 1640, U.S.A.), and a fluorescence spectrophotometer (Hitachi, Model 650-40, Tokyo, Japan) for detection. Chromatographic conditions. The mobile phase consisted of sodium acetate (0.02 M), methanol (16%), heptane sulfonic acid (0.055%), EDTA (0.2 mM), and dibutylamine (0.01%, v/v). The solution was adjusted to pH 3.92 with o-phosphoric acid and filtered through a 0.45-mm membrane (Sartorius) and degassed. The flow rate was set to 0.9 ml/min, which yielded a pressure of 125–130 kg/cm2. Animals. Adult male Wistar rats weighing 150– 180 g received clorgyline injections intraperitoneally (4 mg/kg body wt) for 3 days. Control group of rats received equimolar quantity of saline. Sample preparation. Rats were sacrificed by decapitation on the third day, 2 h postinjection of clorgyline and heads were dropped into ice-cold 0.1 M PCA. Immediately the brains were removed; hypothalamus and striatum were separated on an ice-chilled petri dish according to a slightly modified method of Glowinsky and Iversen (17). The tissues were weighed and homogenized in 2 ml of 0.1 M PCA containing isoproterenol at the concentration of 30 ng/ml. After centrifugation at 14,000g for 15 min at 47C, the supernatant was filtered through a 0.45-mm membrane (Sartorius) and 100 ml of the filtrate was injected onto an HPLC column. After separation, NE, DA, isoproterenol (IP), and 5-HT were detected at the excitation wavelength of 280 nm and an emission wavelength of 315 nm. The slit width was kept at 10/10 for excitation/emission, respectively. The slit width is expressed as the length and width of the sample-plane zone being quantitated. Standards and calculations. Stock solutions of standards, 1 mg/ml, were prepared in 0.1 M hydrochlo-
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ric acid and were stored at 0207C. If they were not used within a month of preparation, they were discarded. The working standard solutions were prepared freshly in 0.1 M PCA for each experiment. The amounts of standards per injection volume of 100 ml were 3 ng each of NE, DA, IP, and 5-HT. Monoamine peaks were identified by comparing their retention time in the sample (tissue extracts) solution with that of standard solution and also by superimposing the chromatograms of the samples spiked with and without known amounts of the standards. Each monoamine in the tissue was quantified by comparing peak heights in elution profiles of samples with known standards. RESULTS
Chromatograms of diluted monoamines and isoproterenol (500 and 1000 pg/100 ml) using fluorimetric and electrochemical detectors are compared in Fig. 1. With the same chromatographic conditions, the peak heights of monoamines were double under electrochemical detector compared to fluorimetric detector, suggesting that the electrochemical detector is more sensitive. The linearity between 500 and 1000 pg of monoamines was observed under both the detectors. A chromatogram of the blank (0.1 M PCA) is shown in Fig. 2A. Figure 2B shows typical chromatograms of 3 ng/100 ml of each monoamine and the internal standard, isoproterenol. Chromatograms of tissue extracts of hypothalamus and striatum are shown in Figs. 2C and 2D. In both standard and tissue chromatograms, monoamines and isoproterenol were completely separated from the solvent front (SF). Monoamine metabolites and precursors such as 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxytryptophan (5-HTP) and 5-hydroxyindole acetic acid (5-HIAA) and tryptophan (TRP) coeluted. The recovery for monoamines ranged from 97.1 to 98.3%, and the coefficient of variation was in the range of 1.4 to 3.1%; the detection limit determined on the basis of injected amount of standards giving a signalto-noise ratio higher than 3 was 250 pg for NE and DA and 100 pg for 5-HT (Table 1). Levels of monoamine neurotransmitters in hypothalamus and striatum of rat brains following treatment with clorgyline and values from the brains of control animals which did not receive any clorgyline are compared in Table 2. In clorgyline-treated rats, significantly increased levels of NE and 5-HT were observed in striatum and hypothalamus, while DA levels were increased only in striatum. DISCUSSION
The isocratic HPLC–FD method described here makes it possible to determine simultaneously the levels of NE, DA, and 5-HT in less than 30 min in rat
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Vander Hoorn et al. (20), after comparison of five types of buffers, reported higher fluorescence intensity for monoamines with lower molarity of sodium acetate. Accordingly, in the present method low molar sodium acetate buffer was used. The pH and composition of the mobile phase, particularly the concentration of heptane sulfonic acid (HSA), are the main determinants of solute separation and retention time. A relatively high amount of HSA was used in the present experiment to shift monoamine substrates to ‘‘chromatographically clean’’ areas where they can be resolved without interference from
FIG. 1. Chromatograms of diluted standards, norepinephrine (NE), dopamine (DA), isoproterenol (IP), and 5-hydroxytryptamine (5-HT) using fluorimetric and electrochemical detectors. (A and B) Chromatograms of 500 and 1000 pg of monoamines using electrochemical detector. (C and D) Chromatograms of 500 and 1000 pg using fluorimetric detector. Note that the height of each monoamine peak is twice that under electrochemical detector compared to fluorimetric detector. Chromatographic conditions: analytical column; 3 mm, Ultracarb ODS (150 1 4.6 mm, i.d.); mobile phase, 0.02 M sodium acetate (pH 3.92) with 16% methanol, 0.055% heptane sulfonic acid, 0.2 mM EDTA, and 0.01% dibutylamine; temperature, ambient.
brain regions. Previously reported methods for the determination of monoamines using HPLC–FD require either sample prepurification (15, 18) or derivatization of monoamines (11, 12, 13, 14, 19). Vander Hoorn et al. (20) have reported a method involving both a purification and a derivatization procedure to assay monoamines in plasma. This method has also been successfully used to assay monoamines in the brain tissue (21). However, such methods using purification and derivatization steps are time-consuming and require careful control by the use of internal standards. A major advantage of the present procedure is the ease of sample preparation which reduces assay time and chances for technical errors. In addition, it does not require the use of an internal standard as can be judged from high recovery (97.1–98.3%) and good coefficients of variation (1.4–3.1%).
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FIG. 2. Chromatograms of standards and tissue samples using fluorimetric detector. (A) Blank, 0.1 M PCA (solvent front, SF). (B) Typical chromatograms of 3 ng each of NE, DA, IP, and 5-HT in 100 ml. (C) Chromatogram of hypothalamus extract (43 mg). The absolute values of NE, DA, and 5-HT are 1860.1 { 167.8, 364.7 { 35.7, and 1020.3 { 101.2 ng/g wet wt tissue, respectively, (D) Chromatogram of striatal extract (35 mg). The absolute values of NE, DA, and 5HT are 321.6 { 31.7, 7560.2 { 457.8, and 553.8 { 47.9 ng/g wet wt tissue. The values in parentheses represent retention time (minutes). For chromatographic conditions, see legend for Fig. 1.
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CHROMATOGRAPHIC ASSAY MEASURING MONOAMINE LEVELS TABLE 1
Recovery, Precision, and Detection LImits for the HPLC-FD Determination of Three Monoamine Neurotransmitters Monoamines
Recovery (%)a (n Å 12)
Within runb CV (%)
Detectionc limits (pg/100 ml)
Norepinephrine Dopamine 5-Hydroxytryptamine
97.4 { 1.7 97.1 { 1.5 98.3 { 1.4
{2.2 {1.4 {3.1
250 250 100
Note. Values are means { SD. Chromatographic conditions are as in the legend to Fig. 1. a Recoveries were measured by repeated injections of tissue homogenate samples spiked with or without known amounts of standards (3 ng). b Coefficients of variation (CV) were obtained by repeated injections of 3 ng of the standards, from the same working standard solution. c Injected amount of standards giving a signal-to-noise ratio higher than 3.
monoamine metabolites. This concentration of HSA was also sufficient to separate NE from the solvent front. These separations can be achieved at room temperature (25 { 17C) and control of column temperature is not required. An analytical column can be used for more than a thousand sample injections, probably due to the much higher percentage of organic modifier in the mobile phase. To confirm the validity of this method, the effect of monoamine oxidase A inhibitor, clorgyline, on levels of monoamines in hypothalamus and striatum was determined. Our results for normal control rats are in agreement with the previously published data (22, 23). The observed increase in NE and DA levels as shown in Table 2 after clorgyline treatment is in agreement with the previous report (23). Although HPLC–FD has been shown to be more selective and sensitive enough to measure catecholamines compared with HPLC–ECD (20), in the present method DOPAC and 5-HTP on one hand and 5-HIAA and TRP on the other hand coeluted. However, the concentrations of NE and DA were shown to be higher and interassay coefficient of variation was lower in HPLC– FD than in HPLC–ECD (20). In HPLC–ECD, there is
gradual decay of sensitivity of the electrode, since crude homogenate supernatants pollute the electrode surface. This results in gradual broadening of the solvent front which in turn interferes with the separation of other substrates (3). The fluorimetric response remains constant for many months or even years without any attention. With the chromatographic conditions used in this study, DOPAC and 5-HTP coeluted after NE elution. Similarly, 5-HIAA and TRP coeluted after isoproterenol. The only limitation of the present method is the coelution of monoamine metabolites (DOPAC and 5-HTP, 5-HIAA and TRP). Thus we could not determine the levels of monoamine metabolites. However, such coelutions do not interfere with the estimation of monoamines. In the present method we have demonstrated for the first time that HPLC–FD can be used for the rapid assay of brain monoamine levels without involving prepurification steps. Whether HPLC–FD is superior to HPLC–ECD for estimating monoamines is still a debatable issue. On the other hand, a procedure which is simple and rapid and does not involve elaborate purification steps should be exploited at least as an alternative method to HPLC–ECD.
TABLE 2
Levelsa of Norepinephrine (NE), Dopamine (DA), and 5-Hydroxytryptamine (5-HT) in Striatum and Hypothalamus of Rat Brains Treated with Clorgyline (4 mg/kg body wt, ip) Brain region Striatum
Hypothalamus
Monoamines
Control
Clorgyline
Control
Clorgyline
NE DA 5-HT
321.6 { 31.7 7560.2 { 457.8 553.8 { 47.9
390.5 { 37.1** 8216.7 { 462.8* 671.8 { 66.8**
1860.1 { 167.8 364.7 { 35.7 1020.3 { 101.2
2241.8 { 192.3** 367.8 { 35.3 1281.7 { 127.8***
Note. Values are means { SD of 8 rats in each group. Chromatographic conditions are as in the legend to Fig. 1. a ng/g wet wt tissue. * P õ 0.05. ** P õ 0.01. *** P õ 0.001 versus control (Student’s t test).
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