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Serotonin monitoring in microdialysate from rat brain by microbore-liquid chromatography with fluorescence detection
Analytica Chimica Acta 365 (1998) 227±232
Serotonin monitoring in microdialysate from rat brain by microbore-liquid chromatography with ¯uorescence detection Junichi Ishidaa, Takashi Yoshitakeb, Kaoru Fujinob, Ken Kawanoa, Jan Kehrc, Masatoshi Yamaguchia,* a Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan-ku, Fukuoka 814-80, Japan Chemical Biotesting Center, Chemical Inspection and Testing Institute, 3-822 Ishii Machi, Hita City, Oita 877, Japan c Division Cellar and Molecular Neurochemistry, Department of Neuroscience, Karolinska Institute S-171 77 Stockholm, Sweden b
Received 16 June 1997; received in revised form 26 September 1997; accepted 22 October 1997
Abstract A sensitive ¯uorimetric microbore-liquid chromatographic method for the determination of serotonin in microdialysate from rat brain was developed. The method is based on the ¯uorescence derivatization of serotonin by reaction with benzylamine in the presence of potassium hexacyanoferrate(III). A microdialysis probe was implanted in the rat brain, and continuously perfused at 2.0 ml minÿ1 with Ringer solution. The microdialysate, collected every 5 min, was added with a reagent solution composed of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v). The derivatization reaction was completed by standing at room temperature for 2 min. The benzylamine derivative of serotonin could be separated within 10 min on a reversed-phase microbore column (1001.0 mm i.d., 5 mm TSK gel ODS-80TM) with isocratic elution. The detection limit (signal-to-noise ratio3) of serotonin is 80 amol for a 5 ml injection. The effects of increased potassium ion concentration in the Ringer solution and a single intraperitoneal injection of methamphetamine on the serotonin level in microdialysate were examined. # 1998 Elsevier Science B.V. Keywords: Fluorescence derivatization; Microbore HPLC; Serotonin; Microdialysis; Methamphetamine; Rat brain
1. Introduction Serotonin is well known as a neurotransmitter in the control and regulation of many brain functions, and has been strongly implicated in several pathological conditions such as aggressive and predatory behavior [1], migraine [2], depression [3] and carcinoid syndrome [4]. Moreover, various drugs such as antidepressants act on the central serotonergic system. It is very important to measure time-dependent changes of *Corresponding author. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00616-8
serotonin levels for the investigations of the relationships between serotonin levels and various pharmaceutical activities. The brain microdialysis sampling technique has been successfully introduced into in vivo studies of the neurotransmitters [5±7]. Microdialysis can be applied to the continuous measurement of the neurotransmitters of a freely moving animal. Liquid chromatography (LC) with electrochemical (EC) [8±10] or ¯uorimetric [11,12] detection is usually used for the determination of serotonin in a microdialysate. Neither EC detection based on the oxidation reaction of a phenolic group nor ¯uores-
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J. Ishida et al. / Analytica Chimica Acta 365 (1998) 227±232
cence detection based on native ¯uorescence are sensitive for serotonin and related compounds, and microdialysates collected for 20±30 min were needed for the measurement of serotonin [8±12]. Furthermore, those methods are not selective for these compounds. A sensitive and selective detection method which can monitor serotonin level changes in a shorttime is required for microdialysis. In previous work [13,14], we have found that aromatic methylamines such as benzylamine react highly selectively and sensitively with 5-hydroxyindoles and catecholamines in weakly alkaline media in the presence of potassium hexacyanoferrate(III) to produce highly ¯uorescent benzoxazole derivatives. We have reported a sensitive and selective ¯uorimetric LC method with pre- [14] and post-column [15] derivatization using benzylamine for the determination of serotonin and related compounds in plasma and urine with simple pre-treatment. In this study, we have developed a microbore LC method based on precolumn ¯uorescence derivatization with benzylamine for the determination of serotonin in dialysates collected in a short time (2±5 min). The method was applied to serotonin monitoring using high potassium Ringer solution and after intraperitoneal injection of methamphetamine. 2. Experimental 2.1. Chemicals and solutions De-ionized and distilled water, puri®ed with a Milli-Q II (Millipore, Milford, MA) system, was used for all aqueous solutions. Serotonin and its related compounds [5-hydroxytryptophan (5HTP) and 5hydroxyindole-3-acetic acid (5HIAA)] were purchased from Sigma (St. Louis, MO). Benzylamine hydrochloride was purchased from Tokyo Kasei Kogyo (Tokyo) and was used after puri®cation by recrystallization from absolute ethanol. Methamphetamine was obtained from Dainippon Seiyaku (Tokyo). N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS) was purchased from Wako Pure Chemical (Osaka, Japan). Potassium hexacyanoferrate(III) was purchased from Kisida Chemical (Tokyo). Other chemicals were of the highest purity available and were used as received. The derivatization reagent solution
was a mixture of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v). The reagent solution was stable for at least 2 weeks at room temperature. Standard solutions of 5-hydroxyindoles were prepared in water and kept frozen (ÿ208C) in amber coloured test tubes. 2.2. Derivatization procedure To a 10 ml (corresponding to 5 min microdialysis) portion of a standard solution (or microdialysate) placed in a micro test tube (100 mm15 mm i.d.) was added 10 ml of the derivatization reagent solution. The mixture was allowed to stand at room temperature for 2 min. A 5 ml portion of the ®nal reaction mixture was injected into the chromatograph. For the reagent blank, water in place of the sample solution was subjected to the same procedure. 2.3. Chromatography Chromatography was performed with a EP-300 (EICOM, Kyoto, Japan) high-performance liquid chromatograph with CMA/200 refrigerated microsampler (BAS, Tokyo) and L-7480 ¯uorescence spectromonitor (2 ml ¯ow-cell, Hitachi, Japan). The latter was operated at an excitation wavelength of 345 nm and an emission wavelength of 481 nm. The column was of TSK gel ODS-80 TM (100 mm1.0 mm I.D.; particle size 5 mm; Tosoh, Japan). The separation of the benzylamine derivative of serotonin was achieved by using a mixture of acetonitrile and 40 mM phosphate buffer (pH7.5) (53:47, v/v) containing 1 mM disodium EDTA (Mobile phase A) for frontal cortex and hippocampus or a mixture of acetonitrile and 40 mM phosphate buffer (pH7.5) (40:60, v/v) containing 1 mM disodium EDTA and 50 mM 1-octanesulfonic acid sodium salt (Mobile phase B) for striatum. The ¯ow rate was 50 ml minÿ1. The column temperature was ambient (20±238C). 2.4. Animals Male Sprague-Dawley rats (Charles River, Japan) (250±350 g) were used in these experiments. Rats were maintained on a 12 h light±dark cycle (light at 7.00 a.m.). Food and water were freely available.
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2.5. Surgery and brain microdialysis Rats were anaesthetized with pentobarbital sodium (40 mg kgÿ1). Rats were implanted stereotaxically with a guide cannula in the hippocampus (rostral± caudal, ÿ5.8 mm; lateral, ÿ5.0 mm; ventral, 3.5 mm, from the bregma and the dural surface), frontal cortex (rostral±caudal, 3.3 mm; lateral, ÿ2.8 mm; ventral, 0.5 mm) and striatum (rostral±caudal, 0.2 mm; lateral, 3.0 mm; ventral, 3.5 mm). After the implantation, the guide cannula was ®xed ®rmly to the skull with anchor screws and dental cement. 3±7 days after surgery, the straight-type dialysis probe [3.0 mm (hippocampus and striatum), 4.0 mm (frontal cortex) in length, 0.22 mm internal diameter, molecular weight cut off 5000, EICOM] was inserted. The dialysis probe was perfused with a Ringer solution (NaCl, 147 mM; KCl, 4 mM; CaCl2, 3.4 mM) at a rate of 2.0 ml minÿ1 in a freely moving rat. The dialysates were collected every 5 min. 3. Results and discussion 3.1. LC conditions The concentrations of neurotransmitters such as serotonin in the brain change in a short period by the effect of various stimulae and pharmaceuticals. Therefore, it is important to shorten the separation time in LC for measurement of serotonin in dialysates. Fig. 1(a) and (b) show typical chromatograms obtained with a standard solution of serotonin using mobile phases A and B, respectively. Other 5-hydroxyindoles (5HIAA and 5HTP) and catecholamines (dopamine, epinephrine and norepinephrine) also react with benzylamine to give the corresponding ¯uorescent peaks under the present conditions. However, the compounds were co-eluted at retention times between 2 and 4 min for both mobile phases, and did not affect the determination of serotonin. Thus, Mobile phase A was used for the serotonin determination in frontal cortex and hippocampus. Since dopamine and its metabolites exist at high concentrations in striatum, the peaks due to their amines partially overlapped to that of serotonin. Therefore, Mobile phase B was employed for the serotonin assay in striatum. The serotonin derivative gave a single peak at a retention
Fig. 1. Chromatograms of benzylamine derivative of serotonin (250 fmol per injection) with mobile phases A and B. Peak: 1 serotonin. Mobile phase A; acetonitrile: 40 mM phosphate buffer (pH7.5) (53:47, v/v) containing 1 mM disodium EDTA for frontal cortex and hippocampus, Mobile phase B; acetonitrile: 40 mM phosphate buffer (pH7.5) (40:60, v/v) containing 1 mM disodium EDTA and 50 mM 1-octanesulfonic acid sodium salt for striatum.
time of 4.95 min for Mobile phase A and 9.95 min for Mobile phase B. The peaks for benzylamine derivatives of serotonin and their related compounds were sharpened by adding disodium EDTA in the mobile phase. The peak for serotonin with disodium EDTA in the mobile phase was ca. 1.5 times higher than that obtained without it. 3.2. Derivatization conditions of serotonin with benzylamine In our previous method, the derivatization was carried out by adding the derivatization reagents (benzylamine, potassium hexacyanoferrate(III) and methanol) to the sample separately [14]. We have found in this study that a mixture of the reagents can be used for derivatization. The resulting optimal conditions for the derivatization are as follows; the reagent solution, consisting of a mixture of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v), was added to the dialysate sample with the same volume as that of the sample. The reagent solution was stable for at least 2 weeks, even
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Fig. 2. Chromatograms obtained with dialysate samples from intact rats. Peaks: 1 serotonin; 2 unknown.
at room temperature. The ¯uorescent derivative of serotonin in the ®nal solution was fairly stable and gave a constant peak height for at least 5 h in daylight at room temperature. 3.3. Measurement of serotonin in intact rats Fig. 2(a), (b) and (c) show typical chromatograms obtained with dialysate samples from frontal cortex, hippocampus and striatum, respectively. The component of peak 1 (Fig. 2) was identi®ed as the ¯uorescent derivative of serotonin on the basis of its retention time in comparison with that of the standard compound and by co-chromatography of the standard and the sample with 35±60% acetonitrile solutions as mobile phase. The ¯uorescence excitation (maximum, 345 nm) and emission (maximum, 481 nm) spectra of the eluate of peak 1 were in good agreement with those for pure serotonin. In addition, peaks 1 and 2 were absent when benzylamine and/or potassium hexacyanoferrate(III) were not present in the derivatization reagent. These observations support the conclusion that peak 1 in Fig. 2 has a single component, the benzylamine derivative of serotonin. Peak 2 in the chromatogram (Fig. 2) was observed only when the reaction was
performed with benzylamine and potassium hexacyanoferrate(III). Its exact identity, however, is unknown. The amounts of serotonin in the microdialysates (10 ml) from frontal cortex, hippocampus and striatum were 1.0, 1.5 and 0.5 fmol, respectively. 3.4. Calibration graph, precision and detection limit The relationship between peak height and amount of serotonin was linear up to at least 500 fmol per 5 ml injection volume; the linear correlation coef®cient was 0.999 (n5). The precision was established by repeated determination (n7) of a standard solution of serotonin (100 fmol per 5 ml injection). The relative standard deviation was 2.1%. The detection limit for serotonin (signal-to-noise ratio3) was 80 amol in an injection volume of 5 ml. 3.5. Measurement of rat brain serotonin with high K and methamphetamine stimulation The method was applied to monitor the time-dependent changes of serotonin levels in rat brain. The potassium ion concentration in Ringer solution was
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Fig. 3. Effect of high potassium concentration on serotonin levels in the hippocampus. High potassium Ringer solution was perfused for 15 min.
Fig. 4. Effect of methamphetamine on serotonin level in the hippocampus. Methamphetamine (3 mg kgÿ1) was injected intraperitoneally.
changed to 100 mM for 15 min. The perfusion of high potassium Ringer solution [16,17] provoked an increase in extracellular serotonin level in the intact rat hippocampus, which was approximately three times as high as the basal levels (Fig. 3) Fig. 4
The effects of methamphetamine on serotonergic neurons has been characterized extensively in in vivo studies [18,19]. They include release of serotonin and inhibition of monoamine oxidase. As expected, intraperitoneal injection of methamphetamine
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(3.0 mg kgÿ1) induced a rapid and drastic increase in the extracellular level of serotonin in the intact hippocampus. The concentration of serotonin reached a maximum at ca. 40 min after injection and then decreased rapidly to the basal levels. In this study, serotonin in dialysates was measured every 5 min. However, the present method can be scaled down, possibly to 40% (i.e 4 ml). Thus, the sensitivity of the method (detection limit: 80 amol/injection volume) permits the determination of serotonin in only 4 ml of the microdialysates from rat brain (corresponds to 2 min dialysis). 4. Conclusions The proposed microbore LC method coupled with pre-column ¯uorescence derivatization with benzylamine permits the highly sensitive, selective and quick determination of serotonin and can be applied to the measurement of serotonin in microdialysates without prior sample puri®cation. The method requires an extremely small portion of microdialysate (4±10 ml), and, therefore, should be useful for biological investigation of the brain. Acknowledgements The authors are grateful to Dr. M. Nakamura (Faculty of Pharmaceutical Sciences, Fukuoka University) for helpful suggestions. The ®nancial support of the Grant-in-Aid for Scienti®c Research (No. 08672493) from the Ministry of Education, Science and Culture of Japan is greatly acknowledged.
References [1] P. Nebinger, M. Koel, J. Chromatogr. 427 (1988) 326. [2] E. Traiffort, P. Hubert, N. Tayeb, N. Aymard, J. Chromatogr. 571 (1991) 231. [3] B. Takkenderg, E. Endert, H.E. Vaningen, M. Ackermans, J. Chromatogr. 565 (1991) 430. [4] S. Wright-Honari, E.F. Marshall, C.H. Ashton, F. Hassanyeh, Biomed. Chromatogr. 4(5) (1990) 201. [5] U. Ungerstedt, IBRO Handbook Series: Methods in Neuroscience, Wiley, New York, 6, 1984, p. 816. [6] B.H.C. Westerink, G. Damsma, H. Rollema, J.B. Veries, A.S. Horn, Life Sci. 41 (1987) 1763. [7] C. Humpel, T. Ebendal, L. Olson, J. Mol. Med. 74(9) (1996) 523. [8] D. Men, A. Matsui, Y. Matsui, Neurochem. Res. 21(12) (1996) 1515. [9] M. Yoshioka, M. Matsumoto, H. Togashi, C.B. Smith, H. Saito, Brain Res. 613(1) (1993) 74. [10] S. Sarre, Y. Michotte, C.A. Marvin, G. Ebinger, J. Chromatogr. 582 (1992) 29. [11] W.J. Drijfhout, C.J. Grol, B.H. Westerink-BH, Eur. J. Pharmacol. 308(2) (1996) 117. [12] P. Kalen, R.E. Strecker, E. Rosengren, A. Bjorklund, J. Neurochem. 51(5) (1988) 1422. [13] J. Ishida, M. Yamaguchi, M. Nakamura, Analyst 116 (1991) 301. [14] J. Ishida, M. Takada, M. Yamaguchi, J. Chromatogr. 692 (1997) 31. [15] J. Ishida, R. Iizuka, M. Yamaguchi, Analyst 118 (1993) 165. [16] Q.S. Yan, M.E. Reith, P.C. Jobe, J.W. Dailey, Eur. J. Pharmacol. 301(1±3) (1996) 49. [17] M. Yoshioka, M. Matsumoto, R. Numazawa, H. Togashi, C.B. Smith, H. Saoto, Eur. J. Pharmacol. 294(2-3) (1995) 565. [18] A. Shimada, K. Yamaguchi, T. Yanagita, Ann. New York Acad. Sci. 801 (1996) 361. [19] T.H. Tsai, C.F. Chen, 166(2) (1994) 175.
Serotonin monitoring in microdialysate from rat brain by microbore-liquid chromatography with ¯uorescence detection Junichi Ishidaa, Takashi Yoshitakeb, Kaoru Fujinob, Ken Kawanoa, Jan Kehrc, Masatoshi Yamaguchia,* a Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan-ku, Fukuoka 814-80, Japan Chemical Biotesting Center, Chemical Inspection and Testing Institute, 3-822 Ishii Machi, Hita City, Oita 877, Japan c Division Cellar and Molecular Neurochemistry, Department of Neuroscience, Karolinska Institute S-171 77 Stockholm, Sweden b
Received 16 June 1997; received in revised form 26 September 1997; accepted 22 October 1997
Abstract A sensitive ¯uorimetric microbore-liquid chromatographic method for the determination of serotonin in microdialysate from rat brain was developed. The method is based on the ¯uorescence derivatization of serotonin by reaction with benzylamine in the presence of potassium hexacyanoferrate(III). A microdialysis probe was implanted in the rat brain, and continuously perfused at 2.0 ml minÿ1 with Ringer solution. The microdialysate, collected every 5 min, was added with a reagent solution composed of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v). The derivatization reaction was completed by standing at room temperature for 2 min. The benzylamine derivative of serotonin could be separated within 10 min on a reversed-phase microbore column (1001.0 mm i.d., 5 mm TSK gel ODS-80TM) with isocratic elution. The detection limit (signal-to-noise ratio3) of serotonin is 80 amol for a 5 ml injection. The effects of increased potassium ion concentration in the Ringer solution and a single intraperitoneal injection of methamphetamine on the serotonin level in microdialysate were examined. # 1998 Elsevier Science B.V. Keywords: Fluorescence derivatization; Microbore HPLC; Serotonin; Microdialysis; Methamphetamine; Rat brain
1. Introduction Serotonin is well known as a neurotransmitter in the control and regulation of many brain functions, and has been strongly implicated in several pathological conditions such as aggressive and predatory behavior [1], migraine [2], depression [3] and carcinoid syndrome [4]. Moreover, various drugs such as antidepressants act on the central serotonergic system. It is very important to measure time-dependent changes of *Corresponding author. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00616-8
serotonin levels for the investigations of the relationships between serotonin levels and various pharmaceutical activities. The brain microdialysis sampling technique has been successfully introduced into in vivo studies of the neurotransmitters [5±7]. Microdialysis can be applied to the continuous measurement of the neurotransmitters of a freely moving animal. Liquid chromatography (LC) with electrochemical (EC) [8±10] or ¯uorimetric [11,12] detection is usually used for the determination of serotonin in a microdialysate. Neither EC detection based on the oxidation reaction of a phenolic group nor ¯uores-
228
J. Ishida et al. / Analytica Chimica Acta 365 (1998) 227±232
cence detection based on native ¯uorescence are sensitive for serotonin and related compounds, and microdialysates collected for 20±30 min were needed for the measurement of serotonin [8±12]. Furthermore, those methods are not selective for these compounds. A sensitive and selective detection method which can monitor serotonin level changes in a shorttime is required for microdialysis. In previous work [13,14], we have found that aromatic methylamines such as benzylamine react highly selectively and sensitively with 5-hydroxyindoles and catecholamines in weakly alkaline media in the presence of potassium hexacyanoferrate(III) to produce highly ¯uorescent benzoxazole derivatives. We have reported a sensitive and selective ¯uorimetric LC method with pre- [14] and post-column [15] derivatization using benzylamine for the determination of serotonin and related compounds in plasma and urine with simple pre-treatment. In this study, we have developed a microbore LC method based on precolumn ¯uorescence derivatization with benzylamine for the determination of serotonin in dialysates collected in a short time (2±5 min). The method was applied to serotonin monitoring using high potassium Ringer solution and after intraperitoneal injection of methamphetamine. 2. Experimental 2.1. Chemicals and solutions De-ionized and distilled water, puri®ed with a Milli-Q II (Millipore, Milford, MA) system, was used for all aqueous solutions. Serotonin and its related compounds [5-hydroxytryptophan (5HTP) and 5hydroxyindole-3-acetic acid (5HIAA)] were purchased from Sigma (St. Louis, MO). Benzylamine hydrochloride was purchased from Tokyo Kasei Kogyo (Tokyo) and was used after puri®cation by recrystallization from absolute ethanol. Methamphetamine was obtained from Dainippon Seiyaku (Tokyo). N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS) was purchased from Wako Pure Chemical (Osaka, Japan). Potassium hexacyanoferrate(III) was purchased from Kisida Chemical (Tokyo). Other chemicals were of the highest purity available and were used as received. The derivatization reagent solution
was a mixture of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v). The reagent solution was stable for at least 2 weeks at room temperature. Standard solutions of 5-hydroxyindoles were prepared in water and kept frozen (ÿ208C) in amber coloured test tubes. 2.2. Derivatization procedure To a 10 ml (corresponding to 5 min microdialysis) portion of a standard solution (or microdialysate) placed in a micro test tube (100 mm15 mm i.d.) was added 10 ml of the derivatization reagent solution. The mixture was allowed to stand at room temperature for 2 min. A 5 ml portion of the ®nal reaction mixture was injected into the chromatograph. For the reagent blank, water in place of the sample solution was subjected to the same procedure. 2.3. Chromatography Chromatography was performed with a EP-300 (EICOM, Kyoto, Japan) high-performance liquid chromatograph with CMA/200 refrigerated microsampler (BAS, Tokyo) and L-7480 ¯uorescence spectromonitor (2 ml ¯ow-cell, Hitachi, Japan). The latter was operated at an excitation wavelength of 345 nm and an emission wavelength of 481 nm. The column was of TSK gel ODS-80 TM (100 mm1.0 mm I.D.; particle size 5 mm; Tosoh, Japan). The separation of the benzylamine derivative of serotonin was achieved by using a mixture of acetonitrile and 40 mM phosphate buffer (pH7.5) (53:47, v/v) containing 1 mM disodium EDTA (Mobile phase A) for frontal cortex and hippocampus or a mixture of acetonitrile and 40 mM phosphate buffer (pH7.5) (40:60, v/v) containing 1 mM disodium EDTA and 50 mM 1-octanesulfonic acid sodium salt (Mobile phase B) for striatum. The ¯ow rate was 50 ml minÿ1. The column temperature was ambient (20±238C). 2.4. Animals Male Sprague-Dawley rats (Charles River, Japan) (250±350 g) were used in these experiments. Rats were maintained on a 12 h light±dark cycle (light at 7.00 a.m.). Food and water were freely available.
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2.5. Surgery and brain microdialysis Rats were anaesthetized with pentobarbital sodium (40 mg kgÿ1). Rats were implanted stereotaxically with a guide cannula in the hippocampus (rostral± caudal, ÿ5.8 mm; lateral, ÿ5.0 mm; ventral, 3.5 mm, from the bregma and the dural surface), frontal cortex (rostral±caudal, 3.3 mm; lateral, ÿ2.8 mm; ventral, 0.5 mm) and striatum (rostral±caudal, 0.2 mm; lateral, 3.0 mm; ventral, 3.5 mm). After the implantation, the guide cannula was ®xed ®rmly to the skull with anchor screws and dental cement. 3±7 days after surgery, the straight-type dialysis probe [3.0 mm (hippocampus and striatum), 4.0 mm (frontal cortex) in length, 0.22 mm internal diameter, molecular weight cut off 5000, EICOM] was inserted. The dialysis probe was perfused with a Ringer solution (NaCl, 147 mM; KCl, 4 mM; CaCl2, 3.4 mM) at a rate of 2.0 ml minÿ1 in a freely moving rat. The dialysates were collected every 5 min. 3. Results and discussion 3.1. LC conditions The concentrations of neurotransmitters such as serotonin in the brain change in a short period by the effect of various stimulae and pharmaceuticals. Therefore, it is important to shorten the separation time in LC for measurement of serotonin in dialysates. Fig. 1(a) and (b) show typical chromatograms obtained with a standard solution of serotonin using mobile phases A and B, respectively. Other 5-hydroxyindoles (5HIAA and 5HTP) and catecholamines (dopamine, epinephrine and norepinephrine) also react with benzylamine to give the corresponding ¯uorescent peaks under the present conditions. However, the compounds were co-eluted at retention times between 2 and 4 min for both mobile phases, and did not affect the determination of serotonin. Thus, Mobile phase A was used for the serotonin determination in frontal cortex and hippocampus. Since dopamine and its metabolites exist at high concentrations in striatum, the peaks due to their amines partially overlapped to that of serotonin. Therefore, Mobile phase B was employed for the serotonin assay in striatum. The serotonin derivative gave a single peak at a retention
Fig. 1. Chromatograms of benzylamine derivative of serotonin (250 fmol per injection) with mobile phases A and B. Peak: 1 serotonin. Mobile phase A; acetonitrile: 40 mM phosphate buffer (pH7.5) (53:47, v/v) containing 1 mM disodium EDTA for frontal cortex and hippocampus, Mobile phase B; acetonitrile: 40 mM phosphate buffer (pH7.5) (40:60, v/v) containing 1 mM disodium EDTA and 50 mM 1-octanesulfonic acid sodium salt for striatum.
time of 4.95 min for Mobile phase A and 9.95 min for Mobile phase B. The peaks for benzylamine derivatives of serotonin and their related compounds were sharpened by adding disodium EDTA in the mobile phase. The peak for serotonin with disodium EDTA in the mobile phase was ca. 1.5 times higher than that obtained without it. 3.2. Derivatization conditions of serotonin with benzylamine In our previous method, the derivatization was carried out by adding the derivatization reagents (benzylamine, potassium hexacyanoferrate(III) and methanol) to the sample separately [14]. We have found in this study that a mixture of the reagents can be used for derivatization. The resulting optimal conditions for the derivatization are as follows; the reagent solution, consisting of a mixture of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v), was added to the dialysate sample with the same volume as that of the sample. The reagent solution was stable for at least 2 weeks, even
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Fig. 2. Chromatograms obtained with dialysate samples from intact rats. Peaks: 1 serotonin; 2 unknown.
at room temperature. The ¯uorescent derivative of serotonin in the ®nal solution was fairly stable and gave a constant peak height for at least 5 h in daylight at room temperature. 3.3. Measurement of serotonin in intact rats Fig. 2(a), (b) and (c) show typical chromatograms obtained with dialysate samples from frontal cortex, hippocampus and striatum, respectively. The component of peak 1 (Fig. 2) was identi®ed as the ¯uorescent derivative of serotonin on the basis of its retention time in comparison with that of the standard compound and by co-chromatography of the standard and the sample with 35±60% acetonitrile solutions as mobile phase. The ¯uorescence excitation (maximum, 345 nm) and emission (maximum, 481 nm) spectra of the eluate of peak 1 were in good agreement with those for pure serotonin. In addition, peaks 1 and 2 were absent when benzylamine and/or potassium hexacyanoferrate(III) were not present in the derivatization reagent. These observations support the conclusion that peak 1 in Fig. 2 has a single component, the benzylamine derivative of serotonin. Peak 2 in the chromatogram (Fig. 2) was observed only when the reaction was
performed with benzylamine and potassium hexacyanoferrate(III). Its exact identity, however, is unknown. The amounts of serotonin in the microdialysates (10 ml) from frontal cortex, hippocampus and striatum were 1.0, 1.5 and 0.5 fmol, respectively. 3.4. Calibration graph, precision and detection limit The relationship between peak height and amount of serotonin was linear up to at least 500 fmol per 5 ml injection volume; the linear correlation coef®cient was 0.999 (n5). The precision was established by repeated determination (n7) of a standard solution of serotonin (100 fmol per 5 ml injection). The relative standard deviation was 2.1%. The detection limit for serotonin (signal-to-noise ratio3) was 80 amol in an injection volume of 5 ml. 3.5. Measurement of rat brain serotonin with high K and methamphetamine stimulation The method was applied to monitor the time-dependent changes of serotonin levels in rat brain. The potassium ion concentration in Ringer solution was
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Fig. 3. Effect of high potassium concentration on serotonin levels in the hippocampus. High potassium Ringer solution was perfused for 15 min.
Fig. 4. Effect of methamphetamine on serotonin level in the hippocampus. Methamphetamine (3 mg kgÿ1) was injected intraperitoneally.
changed to 100 mM for 15 min. The perfusion of high potassium Ringer solution [16,17] provoked an increase in extracellular serotonin level in the intact rat hippocampus, which was approximately three times as high as the basal levels (Fig. 3) Fig. 4
The effects of methamphetamine on serotonergic neurons has been characterized extensively in in vivo studies [18,19]. They include release of serotonin and inhibition of monoamine oxidase. As expected, intraperitoneal injection of methamphetamine
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(3.0 mg kgÿ1) induced a rapid and drastic increase in the extracellular level of serotonin in the intact hippocampus. The concentration of serotonin reached a maximum at ca. 40 min after injection and then decreased rapidly to the basal levels. In this study, serotonin in dialysates was measured every 5 min. However, the present method can be scaled down, possibly to 40% (i.e 4 ml). Thus, the sensitivity of the method (detection limit: 80 amol/injection volume) permits the determination of serotonin in only 4 ml of the microdialysates from rat brain (corresponds to 2 min dialysis). 4. Conclusions The proposed microbore LC method coupled with pre-column ¯uorescence derivatization with benzylamine permits the highly sensitive, selective and quick determination of serotonin and can be applied to the measurement of serotonin in microdialysates without prior sample puri®cation. The method requires an extremely small portion of microdialysate (4±10 ml), and, therefore, should be useful for biological investigation of the brain. Acknowledgements The authors are grateful to Dr. M. Nakamura (Faculty of Pharmaceutical Sciences, Fukuoka University) for helpful suggestions. The ®nancial support of the Grant-in-Aid for Scienti®c Research (No. 08672493) from the Ministry of Education, Science and Culture of Japan is greatly acknowledged.
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