Bruin Reseurch Bdlefin, Vol. 4, pp. 567-570. Printed in the U.S.A.

Determination of Picogram Levels of Brain Serotonin by a Simplified Liquid

Chromatographic Electrochemical Detection Assay1

JERRY J. WARSH*, ANDREW CHIU AND DAMODAR D. GODSE

Section of Neurochemistry

AND

DONALD V. COSCINA

Section of Biopsychology Clurke Institute of Psychiatry, University of Toronto, 250 College Street, Toronto, Ontario, Cunada M5T lR8

Received 23 March 1979

WARSH, J. J., A. CHIU, D. D. GODSE AND D. V. COSCINA. Defrrminafion ofpicogram levels ofhruin srrofonin by a

,simpli’ed liquid cllromatogruphic electrochemicul detection assay. BRAIN RES. BULL. 4(4) 567-570, 1979.---A simplified assay is described for measurement of picogram amounts of serotonin (5-HT) by high pressure liquid chromatog- raphy with electrochemical detection (LCEC). The procedure involves pre-purification of brain 5-HT by

adsorption on Amberlite CG-50. Serotonin is subsequently resolved and detected by LCEC on Zipax SCX resin. The present method gives working sensitivities of at least 100 pg, tissue recoveries of 95% and very low interassay variability (coefficient of variation=3%). Determination of rat brain area 5-HT by this LCEC method is described and compared to other high sensitivity methods.

Rat Brain areas Serotonin Electrochemical detection High pressure liquid chromatography Method

UNTIL recently, quantitation of biogenic amines in brain regions or nuclei could only be achieved by gas chromatography-mass spectrometric (GC-MS) or radioen- zymatic procedures. These techniques permit detection of tissue serotonin (5HT) in 0.3-0.5 pmol quantities [2,12]. While potentially very specific GC-MS procedures require chemical derivatization of 5-HT prior to analysis, are com- plex, and necessitate costly instrumentation. Radioenzyma- tic assay of 5-HT involves a two stage enzymatic procedure and final purification of the reaction products by thin layer chromatography [12]. This later technique is cumbersome and limited in sample processing capacity. Moreover, the radioenzymatic procedure may by QItbject to interference from other 5-HT metabolites (e.g. N-acetylserotonin) which could affect assay specificity [ 121.

Recently, the utility of high pressure liquid chromatog- raphy (HPLC) has been demonstrated for the measurement of biogenic amines [5]. Assay of rat brain 5-HT by HPLC _.

with fluorometric detection has achieved sensitivity of 1 pmol [lo]. HPLC with electrochemical detection (LCEC) ex- tends this sensitivity to as little as 0.1 pmol [14]. Determina- tion of 5-HT by LCEC in single brain areas has been de- scribed by Sasa and Blank [153 and Ponzio and Jonsson [Ill. Unfortunately problems exist with these methods. In the former, recovery of 5-HT was quite low (i.e. 25%). Jn the latter, 3-methoxytyramine was inadequately resolved from 5-HT on Vydac cation exchange resin. We report here a modified cation exchange LCEC procedure that circumvents the above difficulties. Additionally we demonstrate the ap- plication of this procedure to the determination of 5-HT in individual rat brain regions.

Apparatus METHOD

The HPLC (Bioanalytic Systems Inc., IN) was modified as described previously [51. A 500x2.1 mm i.d. glass column

‘This work was conducted during the first author’s tenure as an Ontario Mental Health Foundation Research Scholar and was supported by grants from the Medical Research Council of Canada (MA-6579) and the Ontario Mental Health Foundation (740-77179). We thank Mrs. Susan McNally and Miss Jill Hansen for typing the manuscript and Mrs. Jan Chang for her technical assistance.

‘Reprint requests to Dr. J. J. Warsh, Section of Neurochemistry, Clarke Institute of Psychiatry, University of Toronto, 250 College Street, Toronto, Ontario, Canada M5T 1 Rg.

Copyright 0 1979 ANKHO International Inc.-0361-9230/79/040567-04$00.90/O

packed with Zipax SCX resin (DuPont Instruments. WII- mington, DE) was used. The column. detector and controller amplifier were housed in a copper grid Faraday cage. The mobile phase consisted of citrate/acetate buffer, pH 5.2 II41 and the flow rate was 0.5 mlimin. The buffer was degassed under vacuum at 45°C immediately prior to use. The working electrode potential was maintained constant at + 0.5 V 1s. AgiAgCl reference electrode.

Deionized water was used throughout. Reagent grade chemicals were used without further purification. Amberlite CC-50 resin (H’ form, Type 1. 100-200 mesh. Sigma) was recycled by washing 100 g (dry weight) sequentially with 5x0.8 L H,O, 0.3 L 2M NaOH, 10x0.8 L H,O, 0.3 L 4 M formic acid and 10x0.8 L H?O. The treated resin was stored in 100 ml H,O at 4°C.

Stock solutions (100 /L&ml. free base) of dopamine (DA) hydrochloride (HCI) (Calbiochem). serotonin (5-HT) creatinine sulphate, (Sigma) and the internal standard.

epinine (EP) HCI (Aldrich) were prepared in 0.01 M HCI. Freshly degassed H,O was used for all aqueous solutions. Standard solutions were freshly diluted from their corre- sponding stock solutions on the day of the experiment.

Atrittrtrl Procwlttm

Male Wistar rats (Woodlyn Farm, Ont) weighing 18&200 g were housed communally in a temperature (22°C) and light (lights on 0800-2000 hr) controlled environment for I week before use. Rats were given food and water ad lib. Animals were killed at I100 hr by decapitation, the brains rapidly removed from the crania and dissected into brain regions over an ice water bath using previously described lines of demarcation [4,6]. Brain regions were frozen on dry ice and stored at -70°C. The area including the nuclei locus coeruleus and raphe (dorsal and median) was obtained by making a transverse section caudal to the anterior colliculus extending ventrally to just posterior to the mamillary bodies followed by a second parallel transverse section 2 mm caud- ally.

Brain regions were homogenized in 1.5 ml polypropylene centrifuge tubes containing 500 ~1 of 80% ethanol (v/v) and EP (20 ng). Homogenization was performed on ice for 30-60 set using a Biosonik ultrasonic probe homogenizer (Bronwill Scientific, Rochester, NY) at half speed. After adding an- other 500 ~1 of 80% ethanol, the homogenate was mixed and centrifuged at 56OOxg at 4°C for 20 min. A 100 ~1 aliquot of the supematant was diluted with 7 ml H,O in a glass tube. A peristaltic pump was used to pass the dilute supematants through the Amberlite columns (lOOx3mm i.d. glass tubing with a resin bed height of 20 mm) at a flow rate of 0.27 ml/min at 4°C. The columns were washed with 7 ml H,O and the amines eluted in 0.6 ml ethanol-2 M formic acid (I : 1. v/v) at 0.2 ml!min into a 3 ml Reacti-Vial (Pierce Chemical Co.. Rockford, IL). The eluates were evaporated to dryness a~ ambient temperature under a stream of nitrogen. Im- mediately prior to injection, the residue was reconstituted in 200 ~1 degassed citrate/acetate buffer and a 100 ~1 aliquot introduced into the LCEC through a Rheodyne IOO~I-loop valve injector. Calibration curves were generated by pro- cessing known concentrations of 5-HT (0. I-2.0 ng) contain- ing 2 ng EP. Quantitation was based on the ratio of

k-

z

w

CK

L11

3

0

DA

.HT

EP T 0.5nA

T I M E (Min. )

FIG. I. Representative LCEC chromatogram of a single rat brain sepal area. A recorder setting 10 nAUO0 mV full scale was used for SHT and DA. while a 5 nA;100 mV futt scale setting was used for

EP.

5-HT/EY peak heights. Tissue pellets were solubilized in 0.3 M NaOH for protein determination [RI.

In the present LCEC procedure 5-HT (as well as primary and secondary arylalkylamines not measured) were initially separated from neutral and acidic coumpounds in the brain homogenates by adsorption on Amberlite CG-50. S-HT was

BRAIN REGIONAL 5HT BY LCEC 569

TABLE 1

COMPARISON OF RAT BRAIN AREA 5-HT CONCENTRATIONS DETERMINED BY LCEC AND OTHER TECHNIQUES

Area Present Method Radioenzymatic

Assay j GC-MS$

Olfactory Tubercle Nucleus Accumbens Septal Region Frontal Cortex Hypothalamus Neostriatum Hippo~amp~ls Locus Coeruleus and Raphe Slice Cerebral Cortex Cerebellum

21.39 + 2.08 (6)* 26.1 ? 2.6 (18) 12.32 -f 1.16 (5) 14.7 + 1.2 (16) 14.62 + 1.76 (4) 27.71 ? 2.68 (6) 15.50 i- 1.44 (4) 13.31 2 1.68 (5)

9.51 + 1.02 (3)

7.90 t 1.36 (6) 11.0 2 1.0 (6) 7.75 ? 0.53 (12) 5.73 i 0.58 (6) 3.8 i 0.5 (11) 5.90 i- 0.17 (4) 6.39 i 0.56 (5) 3.17 + 0.33 (6) 7.3 f 0.9 (11) 1.17 i- 0.09 (6) 2.0 i- 0.9 (7)

*Results are expressed as the mean ng/mg protein * SEM. The number of animals is indicated in the parentheses. tData from Saavedra [ 131 r;Data from Koslow et al. [7] and Neckers et al. 191

then separated from other amines by HPLC on Zipax SCX resin.

Figure 1 shows a representative chromatogram obtained from analysis of a single rat brain septal area. In agreement with Sasa and Blank (141 DA, 5-HT and EP were all well resolved on Zipax SCX resin eluting with retention times (ta: [I]) of 5.8, 8.7 and 15 min, respectively. Norepinephrine, epinephrine and normetanephrine all eluted rapidly from the column along with the solvent front, while cY-methyl-5-HT (tH = 23 mitt), N-methly-IHT (ta = 25 min) and metanephrine (t,, =22 mitt) eluted after EP. In contrast to the method of Ponzio and Jonsson [ 11],6-hydroxyt~p~mine (tn = 13 min) and 3-methoxytryramine (t,(=49 mm) were well resolved from 5-HT. At a detector potential of 0.65 V there was no peak for tryptamine, tyramine or bufotenin as these com- pounds require an electrical potential of 1 .O V for oxidation. The small peak eluting before DA in the chromatograms of brain samples was not due to any endogenous brain com- pound as it was also present in the chromato~ams of reagent blank and standards.

Under the conditions described here, 3,4-dihy- droxybenzylamine (t,< = 3.5 min) eluted very close to the solvent front when processing brain samples. For this rea- son, and because 3,4-dihydroxybenzylamine elutes iso- graph~caIly with epinephrine from ZipaX SCX (141, this amine was not satisfactory as an internal standard in the present method. Although EP proved adequate as an internal standard, its use had the disadvantage of prolonging sample run duration.

Linear calibration curves for the peak height ratio of 5-HTTP vs. concentration were obtained from at least 100 pg to 2 ng of S-FIT (Fig. 2). This linear relationship was not altered by the presence of brain tissue. The intra and inter- assay variations for processed standards and tissue samples were small (coefficient of variation = 3% for 10 samples). - -- _--_. _.-_.__

The sensitivity of this LCEC method is dependent on two factors. First and most important, the ultimate sensitivity for LCEC separation and detection of S-HT using Zipax SCX is in the order of 12.5 pg (signal/noise: 5: 1). However, to utilize this level of sensitivity, initial sample extraction and purifi- cation procedures with high yield are essential. The acid butanol extraction procedure of Sasa and Blank [14J gave

Y

3 D-

FIG.

6,

s-

3’

2.

1 -

0 I” I I 1 0 0.1 0.2 0.5 1.0 20

5-HT (ng)

2. LCEC calibration curve of S-HTIEP peak height ratio vs. 5-HT concentration.

recoveries in the order of 25%. In contrast, the extraction and pre-pu~~cation process developed here gives yields of 2 least 80% and overall tissue recoveries of 94.1 + 3.9% (X + SD, N=5). The high yield of pre-purification was achieved with ethanol-formic acid in extraction and chromatography. Adsorption of 5-HT on Amberlite was much enhanced if the alcoholic content of homogenate supemanants was extensively diluted, e.g. to less than 10% (v/v). On the other hand, desorption of 5-HT from the Am- berlite columns was facilitated by adding high concentrations of ethanol to the eluting acid. This permitted complete elu- tion of S-HT in only 0.5 ml, which could be rapidly evapo-

570 WAKSH t-,7 /ii.

rated under nitrogen to minimize oxidative loss of S-HI‘ to about 10% during evaporation and reconstitution in buffer. As 5-HT was totally recovered in the ethanol-formic acid eluate. a small evaporative loss was the sole factor reducing S-HT yield in the extraction and pre-purification processing. Direct introduction of formic acid or ethanol-formic acid column eluates into the LCEC could not be done as broad solvent peaks occurred which interfered with 5-HT resolu- tion.

Amberlite was used in H I form to avoid formation of high salt concentrations in the eluting acid that otherwise oc- curred when the resin was used in the Na’ form. The former high salt concentrations contributed to greater variability in S-HT yields during pre-purification and adversely affected the consistency of the LCEC chromatograms.

Various antioxidants have been used by others to prevent oxidative degradation of catecholamines and 5-HT during

isolation and chromatography 15. IO. IJ I. We have t’ound that the use ofdegassed aqueous reagent5 and buffers effectiveI! avoids S-HT oxidation during analysis. In fact. in our pre- liminary experiments the presence of such antioxidants ;is ascorbic acid. thioglycolic acid or sodium mctabisulphitc 41 interfered with the resolution of .i-H-1’ by I,<‘EC

The concentrations of 5-HT in individual rat brain rugion\ (Table I) obtained with this method agree cioseiy kvith tho\e obtained by radioenzymatic or (X-MS ashay\. With the prz- sent technique it is possible to conveniently process 3.) sam- pies over a two day period using ;I Gngle HPLI’. Hccausc of the low cost and relatively maintcnancc-free operation of the IXEC it is quite practical to use two or three t.t’i<C’\ Gmut- taneously. thur permitting substanrially greater s;implc pro- cessing capacity. Finafiy, as shown in figure f tissue DA i4 also readily detected and may hc ~imtrltaneottsl~ quanttfied by this method (Warsh (‘t ol . unpttbfithed ohscrt ztion\!.

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