Serotonin monitoring in microdialysate from rat brain bymicrobore-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, Japanb 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

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 (100�1.0 mm i.d., 5 mm TSK

gel ODS-80TM) with isocratic elution. The detection limit (signal-to-noise ratio�3) 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 syn-

drome [4]. Moreover, various drugs such as antide-

pressants act on the central serotonergic system. It is

very important to measure time-dependent changes of

serotonin levels for the investigations of the relation-

ships between serotonin levels and various pharma-

ceutical activities. The brain microdialysis sampling

technique has been successfully introduced into in

vivo studies of the neurotransmitters [5±7]. Micro-

dialysis 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-

Analytica Chimica Acta 365 (1998) 227±232

*Corresponding author.

0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.

P I I S 0 0 0 3 - 2 6 7 0 ( 9 7 ) 0 0 6 1 6 - 8

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]. Further-

more, those methods are not selective for these com-

pounds. A sensitive and selective detection method

which can monitor serotonin level changes in a short-

time is required for microdialysis.

In previous work [13,14], we have found that aro-

matic methylamines such as benzylamine react highly

selectively and sensitively with 5-hydroxyindoles and

catecholamines in weakly alkaline media in the pre-

sence 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] deriva-

tization 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 pre-

column ¯uorescence derivatization with benzylamine

for the determination of serotonin in dialysates col-

lected 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 5-

hydroxyindole-3-acetic acid (5HIAA)] were pur-

chased 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. Methamphe-

tamine 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 che-

micals 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 mm�15 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 micro-

sampler (BAS, Tokyo) and L-7480 ¯uorescence spec-

tromonitor (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 mm�1.0 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 phos-

phate buffer (pH�7.5) (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 (pH�7.5) (40:60, v/v) con-

taining 1 mM disodium EDTA and 50 mM 1-octane-

sulfonic 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.

228 J. Ishida et al. / Analytica Chimica Acta 365 (1998) 227±232

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-hydro-

xyindoles (5HIAA and 5HTP) and catecholamines

(dopamine, epinephrine and norepinephrine) also

react with benzylamine to give the corresponding

¯uorescent peaks under the present conditions. How-

ever, 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 determina-

tion in frontal cortex and hippocampus. Since dopa-

mine and its metabolites exist at high concentrations in

striatum, the peaks due to their amines partially over-

lapped 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

time of 4.95 min for Mobile phase A and 9.95 min for

Mobile phase B. The peaks for benzylamine deriva-

tives 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

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

(pH�7.5) (53:47, v/v) containing 1 mM disodium EDTA for

frontal cortex and hippocampus, Mobile phase B; acetonitrile:

40 mM phosphate buffer (pH�7.5) (40:60, v/v) containing 1 mM

disodium EDTA and 50 mM 1-octanesulfonic acid sodium salt for

striatum.

J. Ishida et al. / Analytica Chimica Acta 365 (1998) 227±232 229

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 compo-

nent 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 deri-

vative of serotonin. Peak 2 in the chromatogram

(Fig. 2) was observed only when the reaction was

performed with benzylamine and potassium hexacya-

noferrate(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 (n�5).

The precision was established by repeated determi-

nation (n�7) 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

ratio�3) 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-depen-

dent changes of serotonin levels in rat brain. The

potassium ion concentration in Ringer solution was

Fig. 2. Chromatograms obtained with dialysate samples from intact rats. Peaks: 1� serotonin; 2� unknown.

230 J. Ishida et al. / Analytica Chimica Acta 365 (1998) 227±232

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

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.

J. Ishida et al. / Analytica Chimica Acta 365 (1998) 227±232 231

(3.0 mg kgÿ1) induced a rapid and drastic increase in

the extracellular level of serotonin in the intact hip-

pocampus. 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 benzyla-

mine 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 inves-

tigation of the brain.

Acknowledgements

The authors are grateful to Dr. M. Nakamura

(Faculty of Pharmaceutical Sciences, Fukuoka Uni-

versity) 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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