Dmhn

Na

b

a

ARRAA

KHGcMM

1

ittrlcsmtt(

0d

Analytica Chimica Acta 645 (2009) 48–55

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

etermination of monoamine neurotransmitters and theiretabolites in a mouse brain microdialysate by coupling

igh-performance liquid chromatography with goldanoparticle-initiated chemiluminescence

a Lia, Jizhao Guoa, Bo Liua, Yuqi Yua, Hua Cuia,∗, Lanqun Maob, Yuqing Linb

Department of Chemistry, University of Science and Technology of China (USTC), JinZhai Road No: 96, 230026 Hefei, Anhui, PR ChinaBeijing National Laboratory for Molecular Sciences, Institute of Chemistry, the Chinese Academy of Sciences (CAS), 100080 Beijing, PR China

r t i c l e i n f o

rticle history:eceived 17 February 2009eceived in revised form 30 April 2009ccepted 30 April 2009vailable online 7 May 2009

eywords:igh-performance liquid chromatographyold nanoparticle-initiatedhemiluminescenceonoamine neurotransmitters

a b s t r a c t

Our previous work showed that gold nanoparticles could trigger chemiluminescence (CL) between lumi-nol and AgNO3. In the present work, the effect of some biologically important reductive compounds,including monoamine neurotransmitters and their metabolites, reductive amino acids, ascorbic acid, uricacid, and glutathione, on the novel CL reaction were investigated for analytical purpose. It was found thatall of them could inhibit the CL from the luminol–AgNO3–Au colloid system. Among them, monoamineneurotransmitters and their metabolites exhibited strong inhibition effect. Taking dopamine as a modelcompound, the CL mechanism was studied by measuring absorption spectra during the CL reaction andthe reaction kinetics via stopped-flow technique. The CL inhibition mechanism is proposed to be dueto that these tested compounds competed with luminol for AgNO3 to inhibit the formation of luminolradicals and to accelerate deposition of Ag atoms on surface of gold nanoparticles, leading to a decrease

ouse brain microdialysates in CL intensity. Based on the inhibited CL, a novel method for simultaneous determination of monoamineneurotransmitters and their metabolites was developed by coupling high-performance liquid chromatog-raphy with this CL reaction. The new method was successfully applied to determine the compounds ina mouse brain microdialysate. Compared with the reported HPLC–CL methods, the proposed method issimple, fast, and could determine more analytes. Moreover, the limits of linear ranges for NE, E, and DAusing the proposed method were one order of magnitude lower than the luminol system without goldnanoparticles.

. Introduction

Monoamine neurotransmitters, including catecholamines andndoleamines, play important functions in the peripheral and cen-ral nervous systems [1]. The determination of these amines andheir metabolites is important for the studies of their physiologicalole and the diagnosis of several mental diseases [2,3]. Many ana-ytical methods have been reported for the determination of theseompounds [2,4–8]. The methods without prior on-line separationuffer from poor selectivity because these monoamine neurotrans-

itters and their metabolites with similar structure often occur

ogether and have similar spectral and electrochemical proper-ies [1,9]. In recent years, high-performance liquid chromatographyHPLC) combining with various detection technologies, such as

∗ Corresponding author. Tel.: +86 551 3606645; fax: +86 551 3600730.E-mail address: hcui@ustc.edu.cn (H. Cui).

003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2009.04.050

© 2009 Elsevier B.V. All rights reserved.

electrochemical detection [10,11], fluorescent detection [12], massspectrometry [13,14] and chemiluminescent (CL) detection [15,16],become very attractive due to their powerful separation abil-ity and high sensitivity so that monoamine neurotransmittersand their metabolites can be determined simultaneously. Up todate, for HPLC assay of monoamine neurotransmitters and theirmetabolites, several CL reactions, including luminol [16], peroxy-oxalate [17–21], 6-aminomethylphthalhydrazide (6-AMP) [22–24]and potassium permanganate CL systems [15], have been reported.In peroxyoxalate- and 6-AMP-based methods, the analytes deriva-tized chemically by pre-column or post-column can be detected bypost-column CL reaction. Potassium permanganate- and luminol-based methods are on-line reaction systems. The analytes separated

by HPLC can be directly detected by on-line CL reaction. However,these methods could detect either catecholamines or indoleamines,and none of the HPLC–CL methods could simultaneously deter-mine catecholamines and indoleamines. For neurochemical andphysiological studies, it is necessary to determine simultaneously

N. Li et al. / Analytica Chimica Acta 645 (2009) 48–55 49

ne ne

cta

[lHtCttncttbplscScmttcrettdted(tmpi

Other reagents were of analytical grade. All the solutions were

Fig. 1. Chemical formulae of monoami

atecholamines and indoleamines because both of them occur inissues and body fluids such as urine, blood and cerebrospinal fluid,nd play an important role in life activity [2].

Recently, a novel CL system was found in our previous work25]. Silver nitrate is a relatively weak oxidant and no CL betweenuminol and silver nitrate can be detected in general conditions.owever, we found that luminol could react with silver nitrate in

he presence of gold nanoparticles to produce a strong and flashL at 425 nm. The CL reaction mechanism was proposed to be dueo that Au nanoparticles as nucleation centers catalyzed the reduc-ion of AgNO3 to Ag atoms by luminol to yield Au/Ag core/shellanoparticles; meanwhile, luminol was oxidized to luminol radi-al, which further reacted with the dissolved oxygen, giving riseo light emission. The previous work reported the new CL reac-ion and its mechanism. The new CL system has the merits of lowackground and good stability, which is well suitable for analyticalurpose. Therefore, in present work, the analytical application of

uminol–AgNO3–gold nanoparticle system was explored. Primarytudies showed that reductive compounds such as ascorbic acidould inhibit the CL from the luminol–AgNO3–Au colloid system.ubsequently, the effect of some biologically important reductiveompounds, including monoamine neurotransmitters and theiretabolites, reductive amino acids (AA), uric acid (UA), and glu-

athione, on the novel CL reaction was examined. It was foundhat all of them could inhibit the CL from the luminol–AgNO3–Auolloid system to some extent. Among them, monoamine neu-otransmitters and their metabolites exhibited strong inhibitionffect. Considering that none of HPLC–CL methods could simul-aneously determine catecholamines and indoleamines, based onhe inhibited CL, a new CL procedure coupled with HPLC waseveloped for simultaneous determination of monoamines neuro-ransmitters and their metabolites including norepinephrine (NE),pinephrine (E), dopamine (DA), 5-hydroxytryptamine (5-HT), 3,4-ihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid

5-HIAA) and homovanillic acid (HVA) as shown in Fig. 1. Andhe applicability of the present method for the determination of

onoamine neurotransmitters and their metabolites in real sam-le mouse brain microdialysates was explored. In addition, the CL

nhibition mechanism was also studied by virtue of absorbance

urotransmitters and their metabolites.

spectra and stopped-flow kinetic experiments during the CL reac-tion.

2. Experimental

2.1. Chemicals and solutions

A stock solution of luminol (1.0 × 10−2 mol L−1) was preparedby dissolving luminol (Sigma, America) in sodium hydroxide solu-tion (0.10 mol L−1). Gold colloids with a diameter of 8 nm weresynthesized by the citrate reduction method [26]. The size andshape of the synthesized nanoparticles were characterized by amodel H-800 transmission electron microscope (TEM, Hitachi). Sta-tistical analysis of TEM data revealed that the average diametersof the gold colloids were 8.0 ± 2.5 nm. The Au concentration inthe as-prepared colloids was 3.0 × 10−4 mol L−1. 8-nm Au colloidsolutions with various concentrations used during the detectionwere prepared by diluting the as-prepared Au colloid with appro-priate amount of ultra-pure water. AgNO3, HAuCl4·4H2O (48%,w/w), sodium citrate, ethylenediamine tetraacetic acid disodiumsalt (EDTA), sodium dihydrogen phosphate dihydrate and AA wereobtained from Shanghai Reagent (Shanghai, PR China). Heptane-sulphonic acid sodium salt in HPLC grade was purchased fromYuwang Chemical Reagent (Shandong, PR China). NE, E, DA, 5-HT, DOPAC, HVA and 5-HIAA were purchased from Sigma. Thestock solutions (0.1 mg mL−1) of compounds were prepared freshlyby dissolving them in phosphoric acid (1.2 mmol L−1) and werestored in dark at 4 ◦C. UA was purchased from Fluka. Amino acidsand glutathione were purchased from Solarbio (Beijing, PR China).For HPLC–CL detection, the working solutions of the compoundswere obtained by diluting their stock solutions with appropri-ate amount of ultra-pure water. Methanol was of HPLC grade.

prepared using ultra-pure water prepared by a Direct-Q 3 UVwater purification system (Millipore, USA). The HPLC mobile phaseswere fresh daily prepared, filtered through a 0.22 �m membranefilter (Xinya Company, Shanghai), and then degassed prior touse.

50 N. Li et al. / Analytica Chimica

Fbs

2

tIipc

icw(Awta

2

PsmS2sScd13das

2

plpmv3otib

ig. 2. Schematic diagram of FI-CL detection for investigating the effect of someiologically important reductive compounds on the luminol–AgNO3–Au colloid CLystem.

.2. CL detection

The CL detection was conducted on a flow injection (FI) CL sys-em (Ruimai Electronic Science Co., PR China), including a modelFFM-D flow injection system, a model IFFS-A luminometer, a mix-ng tee, a glass coil (used as reaction coil and detection cell), ahotomultiplier (CR105 Bingsong Electronic Co., PR China) and aomputer.

The FI-CL detection system shown in Fig. 2 was used fornvestigating the effects of some biologically important reductiveompounds on the luminol–AgNO3–Au colloid CL system. Wateras first mixed with AgNO3 (3.5 × 10−5 mol L−1), then with luminol

2 × 10−4 mol L−1), finally with 8-nm Au colloid (3 × 10−5 mol L−1).nalytes were carried by water and the volume of sample loopas 100 �L. The light emission was monitored by the photomul-

iplier tube. The high potential of the photomultiplier tube was sets −550 V.

.3. Optical measurement

UV–vis absorption spectra were obtained on a model UV-2401C spectrophotometer (Shimadzu, Japan). The stopped-flow kinetictudy was carried out on a Bio-Logic SFM300/S stopped-flow instru-ent in absorbance mode coupled with MOS-250 spectrometer.

FM300/S equipped a model FC-15 flow cell (typical dead time.6 ms) and a three-syringe (10 mL) containing three independenttep-motor-driven syringes (S1, S2 and S3). In our experiments,1, S2 and S3 were used to transfer 2.0 × 10−5 mol L−1 AgNO3ontaining 0.01 mol L−1 NaOH, water (blank) or 1.0 × 10−6 mol L−1

opamine or 2.0 × 10−4 luminol mol L−1 or solution containing.0 × 10−6 mol L−1 dopamine and 2.0 × 10−4 mol L−1 luminol, and.0 × 10−5 mol L−1 8-nm Au colloid, respectively. For absorbanceetection, Au colloid was introduced into mixture of solution S1nd S2 and the absorbance of the system at 520 nm was mea-ured.

.4. HPLC–CL detection

HPLC–CL detection system consisted of HPLC system andost-column CL detection system. The HPLC system was Agi-

ent 1100 series (Agilent Technologies, USA), including a binaryump, a thermostat column compartment, a diode array andultiple wavelength detector (DAD), a manual sample injection

alve with a 100 �L loop, and an analytical column (Zorbax ODS,

.0 mm × 250 mm i.d., 5 �m; Agilent technologies). The manifoldf post-column CL detection was the same as that in FI-CL sys-em. Knotted reactors and smaller diameter tubes were employedn the CL detection system in order to decrease chromatographicandbroadening. Monoamine neurotransmitters and their metabo-

Acta 645 (2009) 48–55

lites were separated by a Zorbax ODS-C18 column at 25 ◦C withmobile phase at a flow rate of 0.5 mL min−1. The column effluentfrom DAD was first mixed with AgNO3 solution at a mixing tee, andthen combined with luminol solution. Finally, the combined streammet gold colloid at a mixing tee, accompanying strong CL emission.The light emission in flow cell was detected by the photomultipliertube. The detection by diode array was performed simultaneouslyat 281 nm. The identification of the monoamine neurotransmittersand their metabolites was carried out by comparing their retentiontime and UV–vis spectra to those of available standards. The quan-tification was performed according to an external standard method.The quantitative determination was based on the relative CL inten-sity �I, where �I = −(IS − I0), IS is the CL intensity in the presenceof analytes and I0 (blank signal) is the CL intensity in the absenceof analytes.

2.5. Sample solution preparation

The procedures for animal surgery and for in vivo micro-dialysis were similar to those described in an early work [27].Briefly, adult male Sprague–Dawley rats (250–300 g) purchasedfrom Health Science Center, Peking University were housed on a12:12 h light–dark schedule with food and water ad libitum. Theanimals were anesthetized with chloral hydrate (345 mg kg−1, i.p.)and put onto a stereotaxic frame. The microdialysis guide cannu-las (BAS/MD-2250, BAS) were implanted into the hippocampus(AP = 5 mm, L = 5 mm from bregma, V = 4.5 mm from dura) usingstandard stereotaxic procedures. The guide cannula was kept inplace with three skull screws and dental cement. Stainless steeldummy blockers were inserted into the guide cannula and fixeduntil the insertion of the microdialysis probe. Throughout thesurgery, the body temperature of the animals was maintainedat 37 ◦C with a heating pad. Immediately after surgery, the ratswere placed into a warm incubator individually until they recov-ered from the anesthesia. The rats were allowed to recover forat least 24 h before in vivo microdialysis sampling. Artificial cere-brospinal fluid (aCSF) used as the perfusion solution for in vivomicrodialysis was prepared by mixing NaCl (126 mM), KCl (2.4 mM),KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4(0.5 mM), and CaCl2 (1.1 mM) into doubly distilled water. Whencollecting samples for off-line analysis, the microdialysis probes(BAS; dialysis length, 2 mm; diameter, 0.24 mm) were implantedinto rat hippocampus and were perfused with aCSF solution at1 �L min−1 for at least 90 min for equilibration. The sampleswithin the first 90 min from each rat were discarded from anal-ysis. Subsequently, the samples were collected and immediatelyfrozen. Before HPLC–CL analysis, the sample was unfrozen at 4 ◦Cand was diluted two times with phosphoric acid (1.2 mmol L−1)prior to injection. The quantitation of the monoamine neuro-transmitters and their metabolites in mouse brain microdialysatescarried out by external standard method. The recovery exper-iments were carried out by spiking the known amounts ofmonoamine neurotransmitters and their metabolites in samplesolutions.

3. Results and discussion

3.1. CL inhibition of biologically important compounds on goldnanoparticle-initiated CL

When Au colloids were met with the mixture of luminol andAgNO3, a strong CL was detected. The effects of monoamine neuro-transmitters and their metabolites such as NE, E, DA, 5-HT, 5-HIAA,DOPAC and HVA, reductive amino acids such as methionine (Met),lysine (Lys), histidine (His), phenylalanine (Phe), tyrosine (Tyr),

N. Li et al. / Analytica Chimica Acta 645 (2009) 48–55 51

Table 1Inhibition effect of biologically important compounds on luminol–AgNO3–gold colloids CL system.

Analytes I Quenching (%)a Analytes I Quenching (%)a

Blankb 16,465NEc 8,622 47.6 Lys 12,248 25.6Ec 10,517 36.1 His 9,599 41.7DAc 5,577 66.1 Phe 13,601 17.4DOPACc 6,585 60.0 Tyr 14,374 12.75-HIAAc 8,226 50.0 Trp 14,818 10.0HVAc 13,580 17.5 Met 14,514 11.95-HTc 7,460 54.7 Cysteine 2,058 87.5AA 6,586 60.0 Homocysteine 11,525 30.0UA 7,739 53.0 Glutathione 10,735 34.8

a The percentage of quenching was calculated as 100 − I/I0.absen

( 1); moU e (1 ×

tAncltcrecD6tsiiawtcittf

3

lclcmpbsdtgsAatAbieb

Therefore, the experimental results suggested that dopamine com-peted with luminol for AgNO3 and inhibited the oxidation ofluminol to luminol radicals, leading to a decrease in CL inten-sity. On the other hand, dopamine accelerated the deposition ofAg when dopamine was added to the luminol–AgNO3–Au col-

b The blank CL signal I0 obtained by luminol–AgNO3–gold colloids system in the0.2 mol L−1); AgNO3 (3.5 × 10−5 mol L−1); 8-nm Au colloid solution (3 × 10−5 mol L−

A (4 × 10−8 g L−1); other amino acids (1 × 10−5 g L−1); homocysteine and glutathionc Monoamine neurotransmitters and their metabolites.

ryptophane (Trp), cysteine (Cys) and homocysteine, glutathione,A, and UA, on the gold nanoparticle-triggered CL between lumi-ol and AgNO3 were investigated as shown in Table 1. The testedompound carried by water was first mixed with AgNO3, then withuminol, and finally with gold colloid. It was observed that all of theested compounds could inhibit CL signal from luminol–AgNO3–Auolloid system to some extent. Among them, monoamine neu-otransmitters and their metabolites, ascorbic acid, and uric acidxhibited strong inhibition effect. Even at as low as 4 × 10−8 g mL−1

oncentration, the percentages of quenching (100 − I/I0) for DA,OPAC, 5-HT, 5-HIAA, NE, E, HVA, AA, and UA reached to 66.1%,0.0%, 54.7%, 50.0%, 47.6%, 36.1%, 17.5%, 60.0% and 53.0%, respec-ively. Glutathione and reductive amino acids except Tyr and Trphowed the CL inhibition at higher concentrations, whereas nonhibiting signal at 4 × 10−8 g mL−1 although cysteine also exhib-ted strong CL inhibiting signal at 1 × 10−5 g mL−1. From analyticalpplication point of view, it is necessary to couple the CL reactionith separation tools because the CL reaction could respond to all of

he tested compounds. Considering that none of HPLC–CL methodsould simultaneously determine catecholamines and indoleaminesn neurotransmitters, the CL reaction could just respond to thesewo kinds of compounds. Accordingly, for analytical purpose, theested monoamine neurotransmitters and their metabolites wereurther studied in the following work.

.2. CL inhibition mechanism

All the tested monoamine neurotransmitters and their metabo-ites in our experiments are reductants. In the luminol–AgNO3–goldolloid CL system, luminol (LH−) as a reductant was oxidized touminol radicals by AgNO3 under the catalysis of gold nanoparti-les. In the presence of monoamine neurotransmitters and theiretabolites, we supposed that the reductive ability of these com-

ounds might be stronger than that of luminol and AgNO3 maye reduced dominantly by these compounds to Ag. The conver-ion of luminol to luminol radicals was inhibited, resulting in aecrease in CL intensity. According to the hypothesis, the reac-ivity of dopamine as an example with AgNO3 in the presence ofold nanoparticles was studied by the absorption spectra. Fig. 3hows the absorption spectra of AgNO3–dopamine system (a),u colloid system (b), Au colloid–AgNO3–dopamine system (d)nd the algebraic sum of absorbance (c) from Au colloid sys-em and AgNO3–dopamine system. The absorption spectra of

gNO3–dopamine–Au colloid (d) were not equal with the alge-raic sum of absorbance (c) from Au colloid and AgNO3–dopamine,

ndicating that dopamine could react with AgNO3 in the pres-nce of gold nanoparticles. It was reported that the SPR absorptionand of gold nanoparticles at ca. 520 nm had an increase and

ce of analytes was 16,465. Conditions: luminol (2 × 10−4 mol L−1) in NaOH solutionnoamine neurotransmitters and their metabolites (4 × 10−8 g L−1); Tyr, Trp AA and10−6 g L−1).

blue shift with the deposition of Ag on the surface of goldnanoparticles [28,29]. In Fig. 3, Au colloid (b) had a SPR absorp-tion band at 520 nm, while the addition of AgNO3–dopamineinto Au colloid led to an obvious shift of the SPR absorbancefrom 520 to 506 nm and an increase in absorbance, revealingthat AgNO3 was reduced to Ag on the surface of gold nanoparti-cles.

Furthermore, the comparative study was carried out for the reac-tion rate of three systems, including dopamine–AgNO3–Au colloid,luminol–AgNO3–Au colloid and luminol–dopamine–AgNO3–Aucolloid. A spectrophotometer with stopped-flow equipment wasused to monitor the changes of the SPR absorption band at 520 nmof the three systems during the reactions, i.e. the formation ofAg. The results as shown in Fig. 1s (Supporting Information)demonstrated that the increase in the SPR absorbance in thedopamine–AgNO3–gold nanoparticle system (a) was more thanthat in the luminol–AgNO3–gold nanoparticle system (c), reveal-ing that the reduction rate of AgNO3 to Ag by dopamine was fasterthan that by luminol. Moreover, the reduction rate of AgNO3 toAg in the presence of both dopamine and luminol was medium.

Fig. 3. Absorption spectra of AgNO3–dopamine (a) (dash line), gold colloid (b) (dotline), gold colloid–AgNO3–dopamine (solid line) (d) at 2 min after mixing and thealgebraic sum of absorbance (dash dot line) of gold colloid and AgNO3–dopamine (c).Conditions: dopamine, 1 × 10−6 g mL−1; AgNO3, 4 × 10−5 mol L−1; 8-nm Au colloidsolution, 3 × 10−5 mol L−1.

5 imica

lq[r

3c

mndahafpttsispaostcm

oaimtnmcpoH

4gaWpt5

tsoorrw

ttprsrr1

metabolites with DAD at 281 nm and with CL detection are shownin Fig. 4(A) and (B), respectively. The retention time of NE, E, DOPAC,DA, 5-HIAA, HVA and 5-HT recorded by the CL detection was 3.32,3.87, 4.50, 6.07, 8.20, 10.42 and 14.69 min, respectively. The cali-

Fig. 4. Chromatograms of a mixture of four monoamine neurotransmitters andtheir three metabolites with DAD at 281 nm (a) and with CL detection (b). Peaks:(1) norepinephrine (NE) (4 × 10−8 g mL−1); (2) epinephrine (E) (4 × 10−8 g mL−1);(3) 3,4-dihydroxyphenylacetic acid (DOPAC) (4 × 10−8 g mL−1); (4) dopamine(DA) (4 × 10−8 g mL−1); (5) 5-hydroxyindoleacetic acid (5-HIAA) (4 × 10−8 g mL−1);

2 N. Li et al. / Analytica Ch

oid system. Au/Ag core/shell nanoparticle was conformed to be auite weaker catalyst than gold nanoparticle for the CL reaction25] and fast deposition of Ag caused a decrease in CL reactionate.

.3. Optimization of chromatographic conditions and CL reactiononditions

Based on the inhibition effects of monoamine neurotrans-itter and their metabolites on the luminol–AgNO3–8-nm gold

anoparticle CL system, a post-column HPLC simultaneousetection for these compounds was developed. The HPLC sep-ration of monoamine neurotransmitter and their metabolitesas been documented [30,31]. The mobile phases used includedcetonitrile–water or methanol–water containing one or moreollowing components: buffers of citrate, phosphate or acetate; ion-air reagent; EDTA and sodium chloride. For HPLC–CL detection,he mobile phase of HPLC is not only suitable for the separa-ion of analytes but also compatible with the CL reaction. Primarytudies showed that the mobile phase methanol–water contain-ng phosphate buffer, heptanesulphonic acid sodium, EDTA andodium citrate, was suitable for the separation of these com-ounds. In order to obtain complete separation, best sensitivitynd short analytical time, HPLC conditions, including the contentf methanol, the pH of mobile phase, the concentration of heptane-ulphonic acid sodium salt, and CL reaction conditions, includinghe order of CL reagents, the flow rate of CL reagents and theoncentration of NaOH, luminol, AgNO3, and Au colloid, were opti-ized.Gold nanoparticles stabilized by citrate are sensitive to the pH

f solution. They deposited more easily in acidic solution than inlkaline solution. Although the order of CL reagents had no obviousnfluence on the CL intensity in our experiments, it was recom-

ended that the analytes were first mixed with AgNO3 solution,hen with luminol solution, and finally with gold colloid since lumi-ol was solved in NaOH solution, which could neutralize acidicobile phase (pH 5.1) to prevent gold nanoparticles stabilized by

itrate from deposition. If the analytes carried by acidic mobilehase mixed with gold colloid before luminol, the deposition wouldccur and the aggregates in gold colloid would block tubes inPLC–CL systems.

The effect of the pH of the mobile phase was investigated from.5 to 5.5. When the pH of mobile phase was 4.5, the chromato-raphic peak of 5-HIAA overlapped with 5-HT. When the pH wasdjusted to 4.8, the peak of DA overlapped with that of DOPAC.hen the pH was 5.1, the baseline separation was obtained. When

H was over 5.1, E and DA could not be separated completely. Thus,he pH of mobile phase in subsequent experiments was chosen as.1.

The flow rates of NaOH, luminol, AgNO3, and Au colloid solu-ions were same in this case because they were controlled by theame pump and the same diameter of pump tubing. The effectf flow rate on the relative CL intensity was studied in the rangef 1.0–2.5 mL min−1. The relative CL intensity increased with flowate over the range 1.0–2.5 mL min−1, but the consumption of theeagent also increased. In order to save CL reagents, 2.0 mL min−1

as considered as an appropriate value of flow rate.The effects of the concentration of various reagents on the rela-

ive CL intensity were also examined. The content of methanol andhe concentration of heptanesulphonic acid sodium salt in mobilehase were studied over the range 8–12% and 0.20–0.30 mmol L−1,

espectively. The concentration of post-column reaction reagents,uch as, luminol, AgNO3, and Au colloid was tested over theange 0.05–0.30 mmol L−1, 15–45 �mol L−1 and 15–45 �mol L−1,espectively. The pH of luminol was studied over the range2.7–13.5. The concentrations of these reagents and the pH of

Acta 645 (2009) 48–55

luminol required for the maximum of the relative CL intensitywere different for different analytes. Therefore, these reactionconditions were chosen as a compromise to make the relativeCL intensity of most of the analytes reaching the maximumand that of others be acceptable. The recommended mobilephase was 9% (v/v) methanol solution containing 0.02 mol L−1

sodium citrate, 0.05 mol L−1 sodium dihydrogen phosphate (pH5.1), 0.2 mmol L−1 EDTA, and 0.25 mmol L−1 heptanesulphonic acidsodium. The selected CL reaction conditions were 0.20 mmol L−1

luminol (pH 13.3), 35 �mol L−1 AgNO3, and 30 �mol L−1 Au col-loid.

3.4. Post-column HPLC detection of monoamineneurotransmitters and their metabolites

Under the optimal conditions, typical chromatograms of amixture of four monoamine neurotransmitters and their three

(6) homovanillic acid (HVA) (4 × 10−7 g mL−1); (7) 5-hydroxytryptamine (5-HT) (4 × 10−8 g mL−1). Chromatographic conditions: mobile phase containing 9%methanol, 2.5 × 10−4 mol L−1 heptanesulphonic acid sodium, 0.02 mol L−1 sodiumcitrate, 0.05 mol L−1 sodium dihydrogen phosphate, 0.2 mmol L−1 EDTA (pH 5.1).Post-column CL reaction conditions: 0.20 mol L−1 NaOH, 3.5 × 10−5 mol L−1 AgNO3,2.0 × 10−4 mol L−1 luminol, and 3 × 10−5 mol L−1 8 nm Au colloid.

N. Li et al. / Analytica Chimica Acta 645 (2009) 48–55 53

Table 2Parameters of regression and detection limit of monoamine neurotransmitters and their metabolites.

Analytes Regression equation Linear range (ng mL−1) Correlation coefficient, r Limit of detection (ng mL−1)

NE �I = 197.97C + 254.77 0.4–28 0.9985 0.25E �I = 993.09C + 253.40 0.8–48 0.9987 0.40DA �I = 126.12C + 432.06 0.8–48 0.9986 0.40DOPAC log �I = 0.791 log C + 9.453 1–160 0.9963 0.605-HIAA log �I = 0.774 log C + 9.147 5–400HVA log �I = 0.756 log C + 8.079 50–40005-HT log �I = 1.067 log C + 10.974 10–400

Table 3Precision for analytes.

Analytes Concentration(ng mL−1)

Intra-day R.S.D.(%) (n = 7)

Inter-day R.S.D.(%) (n = 21)

NE 80 2.7 5.3E 80 4.3 5.6DOPAC 80 2.0 5.0DA 80 2.2 4.75-HIAA 200 2.0 3.0H5

bctio0rtpannm

tadbfdd

results for two samples are present in Table 5. Good recover-

TC

C

P

P

O

VA 2000 3.7 5.4-HT 200 2.7 6.1

ration curves of chromatographic peak height of HPLC–CL versusoncentration of the compounds were obtained. The parameters ofhe regression equations and the limit of detection (LOD) are listedn Table 2. Linear ranges of the CL detection are about two ordersf magnitude and the regression coefficients (r) are greater than.994 for all the curves. The detection limits at a signal-to-noiseatio of three (S/N = 3) for four monoamine neurotransmitters andheir three metabolites are in the range of 0.25–25 ng mL−1. Therecisions of the determination are summarized in Table 3. The rel-tive standard deviations (R.S.D.) within a day (intra-day precision,= 7) are less than 4.3%; the R.S.D. in 3 days (inter-day precision,= 21) less than 6.1%. The method exhibited good analytical perfor-ance.Compared with the reported HPLC–CL methods in Table 4,

he proposed method could simultaneously detect catecholaminesnd indoleamines, whereas the previous methods could onlyetect either catecholamines or indoleamines. In peroxyoxalate-

ased methods, different derivatization agents were neededor catecholamines and indoleamines. For example, for theetection of catecholamines such as NE, E and DA, ethylene-iamine was used as a derivatization reagent [17,19,21]; for

able 4L reactions used for HPLC–CL detection of monoamine neurotransmitters and their meta

hemiluminescence reagents Detectio

NE

re-column derivatizations6-Aminomethylphthalhydrazide

4-Dimethylaminobenzylamine (post-column peroxyoxalate CL)1,2-Bis(3-chlorophenyl)ethylenediamine (post-column peroxyoxalate CL) 0.007

ost-column derivatizationsEthylenediamine (post-column peroxyoxalate CL) 0.008

0.0300.03

n-line reactionsPermanganateLuminol–iodine 0.71This methodLuminol–AgNO3–gold nanoparticle 0.25

a The results from references used different units, which were changed to the same uni

0.9953 2.50.9982 250.9942 6.0

the detection of indoleamines such as 5-HT and 5-HIAA, 4-dimethylaminobenzylamine as a derivatization reagent [18]. In6-AMP-based methods, catecholamines and indoleamines neededdifferent derivatization conditions [22,23] so that no methodcould simultaneously detect both of them. In luminol- andpotassium permanganate-based on-line reaction systems, luminol-based method [16] could only detect catecholamines such asNE, E, and DA, while potassium permanganate-based method[15] could only determine the metabolites of monoamine suchas HVA and 5-HIAA. In summary, the proposed method couldsimultaneously detect 7 analytes, whereas the previous meth-ods could only detect 3–4 analytes among 7 analytes studied inpresent work. Moreover, the limit of detection of this method iscomparable with other on-line reaction methods, and the lowerlimits of linear ranges for NE, E, and DA using the proposedmethod are one order of magnitude lower than the luminol–I2system.

3.5. Detection of monoamine neurotransmitters and theirmetabolites in a mouse brain microdialysate

In order to validate the applicability of the proposed methodin real samples, monoamine neurotransmitters and their metabo-lites in mouse brain microdialysates were analyzed by the HPLC–CLmethod. The typical chromatograms of a microdialysate and thesample spiked with all the analytes are shown in Fig. 5. The peaksat 8.13 and 10.19 min were identified as 5-hydroxyindoleacetic acid(5-HIAA) and homovanillic acid (HVA), respectively. The analytical

ies from 85% to 106% were obtained for the seven monoamineneurotransmitters and their metabolites, indicating that the pro-posed HPLC–CL method was applicable for the quantification ofmonoamine neurotransmitters and their metabolites in mouse

bolites.

n limits (ng mL−1)a References

E DA DOPAC HVA 5-HT 5-HIAA

0.02 51 0.46 [22]0.022 0.035 [23]

0.002 [24]0.002 0.002 [18]

0.003 0.06 [20]

0.003 0.010 [17]0.011 0.073 [19]0.02 0.06 [21]

18.2 3.8 [15]0.26 0.73 [16]

0.40 0.40 0.60 25.0 6.0 2.5

t ng mL−1 in the table for a comparison.

54 N. Li et al. / Analytica Chimica Acta 645 (2009) 48–55

Table 5Results for determination of monoamine neurotransmitters and their metabolites in mouse brain microdialysate by using the HPLC–CL method.

Original (ng mL−1)a Added (ng mL−1) Found (ng mL−1)a Recovery (%)

Sample 1NE ND 25 23 ± 1 91E ND 25 23 ± 2 92DOPAC ND 25 26 ± 3 104DA ND 25 21 ± 1 855-HIAA 30 ± 4 10 40 ± 2 95HVA 70 ± 1 50 120 ± 9 1015-HT ND 25 24 ± 2 98

Sample 2NE ND 25 23 ± 1 92E ND 25 24 ± 1 94DOPAC ND 25 25 ± 2 100DA ND 25 22 ± 1 895-HIAA 35 ± 7 10 43 ± 2 88HVA 49 ± 8 50 93 ± 3 86

N

bnnd

Fpia

5-HT ND 25

D: not detected.a Mean value ± S.D. (n = 3).

rain microdialysates. Therefore, the present method provides aovel and alternative method for the detection of monoamineeurotransmitters and their metabolites in mouse brain micro-ialysates.

ig. 5. (a) HPLC chromatogram of a mouse brain microdialysate sample. The sam-le solution was diluted two times with phosphoric acid (1.2 mmol L−1) prior to

njection. (b) Chromatogram of the mouse brain microdialysate spiked with all thenalytes. HPLC and CL reaction conditions are same as that in Fig. 4.

26 ± 1 106

4. Conclusions

It was found that monoamine neurotransmitters and theirmetabolites, reductive amino acids, ascorbic acid, uric acid, and glu-tathione could inhibit the gold nanoparticle-initiated CL betweenluminol and AgNO3. Among them, monoamine neurotransmittersand their metabolites, including NE, E, DOPAC, DA, 5-HIAA, HVAand 5-HT exhibited strong inhibition. The CL inhibition mechanismis likely due to that these compounds competed with luminol forAgNO3 to inhibit the formation of luminol radicals and to accel-erate deposition of Ag atoms on surface of gold nanoparticles,leading to a decrease in CL intensity. Based on the inhibition, anew HPLC post-column detection method was successfully estab-lished for the determination of monoamine neurotransmitters andtheir metabolites in a mouse brain microdialysate. The proposedmethod is simple, fast, and could determine more analytes. Andthe limits of linear ranges for NE, E, and DA using the proposedmethod were one order of magnitude lower than the luminol sys-tem without gold nanoparticles. To the best of our knowledge, thisis the first time to use successfully gold nanoparticle-involved CLreaction for HPLC post-column detector. Moreover, for other morechallenging samples such as urine, a HPLC–CL strategy, involvingthe pretreatments of samples (such as solid-phase extraction), theluminol–AgNO3–Au colloid CL reaction and more analytes (suchas vanilmandelic acid, 3-methoxy-4-hydroxyphenylethyleneglycoland so on), is under further investigations.

Acknowledgements

The financial support of the research by the National Natural Sci-ence Foundation of PR China (Grant Nos. 20573101 and 20625517)and the Overseas Outstanding Young Scientist Program of ChinaAcademy of Sciences are gratefully acknowledged. Additionally, wethank Dr. S.Y. Liu and Z.Y. Zhu at University of Science & Technologyof China for stopped-flow experiments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.aca.2009.04.050.

References

[1] F. Xu, M.N. Gao, G.Y. Shi, L. Wang, W. Zhang, J. Xue, L.T. Jin, J.Y. Jin, Anal. Chim.Acta 439 (2001) 239.

[2] F.C. Cheng, J.S. Kuo, J. Chromatogr. B 665 (1995) 1.

imica

[[

[[

[

[

[

[

[

[[[

[[[[[

N. Li et al. / Analytica Ch

[3] W. Zhang, X.N. Cao, F.L. Wan, S. Zhang, L.T. Jin, Anal. Chim. Acta 472 (2002)27.

[4] M. Tsunoda, Anal. Bioanal. Chem. 386 (2006) 506.[5] X.E. Zhao, Y.R. Suo, Talanta 76 (2008) 690.[6] Z.D. Yang, X.Z. Zhang, H. Guo, Y.R. Gan, Anal. Lett. 39 (2006) 1837.[7] Q.F. Weng, G.W. Xu, K.L. Yuan, P. Tang, J. Chromatogr. B 835 (2006) 55.[8] M.C. Jung, G.Y. Shi, L. Borland, A.C. Michael, S.G. Weber, Anal. Chem. 78 (2006)

1755.[9] S. Zhang, Q. Xu, W. Zhang, L.T. Jin, J.Y. Jin, Anal. Chim. Acta 427 (2001) 45.10] J.X. Lu, S. Zhang, A.B. Wang, W. Zhang, L.T. Jin, Talanta 52 (2000) 807.11] M.A. Vicente-Torres, P. Gil-Loyzaga, F. Carricondo, M.V. Bartolome, J. Neurosci.

Methods 119 (2002) 31.12] A.T. Wood, M.R. Hall, J. Chromatogr. B 744 (2000) 221.13] M.E.P. Hows, L. Lacroix, C. Heidbreder, A.J. Organ, A.J. Shah, J. Neurosci. Methods

138 (2004) 123.14] A. Tornkvist, P.J.R. Sjoberg, K.E. Markides, J. Bergquist, J. Chromatogr. B 801

(2004) 323.15] J.L. Adcock, N.W. Barnett, J.W. Costin, P.S. Francis, S.W. Lewis, Talanta 67 (2005)

585.16] E. Nalewajko, A. Wiszowata, A. Kojlo, J. Pharm. Biomed. 43 (2007) 1673.

[[

[[[

Acta 645 (2009) 48–55 55

17] K. Takezawa, M. Tsunoda, K. Murayama, T. Santa, K. Imai, Analyst 125 (2000)293.

18] J. Ishida, M. Takada, N. Hitoshi, R. Iizuka, M. Yamaguchi, J. Chromatogr. B 738(2000) 199.

19] K. Takezawa, M. Tsunoda, N. Watanabe, K. Imai, Anal. Chem. 72 (2000) 4009.20] G.H. Ragab, H. Nohta, K. Zaitsu, Anal. Chim. Acta 403 (2000) 155.21] M. Tsunoda, K. Takezawa, T. Yanagisawa, M. Kato, K. Imai, Biomed. Chromatogr.

15 (2001) 41.22] T. Yakabe, H. Yoshida, H. Nohta, M. Yamaguchi, Anal. Sci. 18 (2002) 1375.23] J. Ishida, T. Yakabe, H. Nohta, M. Yamaguchi, Anal. Chim. Acta 346 (1997) 175.24] T. Yakabe, J. Ishida, H. Yoshida, H. Nohta, M. Yamaguchi, Anal. Sci. 16 (2000) 545.25] H. Cui, J.Z. Guo, N. Li, L.J. Liu, J. Phys. Chem. C (2008) 11319.26] C.K. Kim, R.R. Kalluru, J.P. Singh, A. Fortner, J. Griffin, G.K. Darbha, P.C. Ray,

Nanotechnology 17 (2006) 3085.

27] M.N. Zhang, K. Liu, L. Xiang, Y.Q. Lin, L. Su, L.Q. Mao, Anal. Chem. 79 (2007) 6559.28] J.H. Hodak, A. Henglein, M. Giersig, G.V. Hartland, J. Phys. Chem. B 104 (2000)

11708.29] X.H. Ji, L.Y. Wang, H. Yuan, L. Ma, Y.B. Bai, T.J. Li, Acta. Chim. Sin. 61 (2003) 1556.30] P.Y. Leung, C.S. Tsao, J. Chromatogr.-Biomed. Appl. 576 (1992) 245.31] M.H. Mills, D.C. Finlay, J. Chromatogr.-Biomed. Appl. 564 (1991) 93.