Simultaneous measurement of serotonin, dopamine and their metabolites in mouse brain extracts by high-performance liquid chromatography with mass spectrometry following derivatization with ethyl chloroformate

Article abstract of DOI:10.1248/bpb.b12-00689

In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxy-phenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an effective derivatization method followed by high-perfor

Full text of DOI:10.1248/bpb.b12-00689

252
Regular Article
Biol. Pharm. Bull. 36(2) 252–258 (2013)
Vol. 36, No. 2
Simultaneous Measurement of Serotonin, Dopamine and Their
Metabolites in Mouse Brain Extracts by High-Performance Liquid
Chromatography with Mass Spectrometry Following Derivatization with
Ethyl Chloroformate
Ju-Young Park,a Seung-Woon Myung,b In-Soo Kim,c Dong-Kug Choi,c Soon-Jung Kwon,a and
,a
Sung-Hwa Yoon*
a Department of Molecular Science and Technology, Ajou University; San 5, Woncheon, Yeongtong, Suwon 443–749,
b
Republic of Korea: Department of Chemistry, Kyonggi University; San 94–6, Iui, Yeongtong, Suwon 443–760,
c
Republic of Korea: and Department of Biotechnology, Konkuk University; 268 Chungwondaero, Chungju 380–701,
Republic of Korea.
Received August 3, 2012; accepted November 18, 2012; advance publication released online November 29, 2012
In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxy-
phenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an
effective derivatization method followed by high-performance liquid chromatography (HPLC) coupled to
electrochemical ionization mass spectrometry was used. The derivatization reaction of biological samples
with ethyl chloroformate occurred rapidly at room temperature in aqueous conditions, and the resulting de-
rivatives were isocratically separated with good selectivity using a C18 reversed-phase column within 30min.
The study results showed that the new derivatization procedure offers an excellent means of simultaneous
determination of 5-HT, DA and their metabolites in mouse brain homogenates, which are important in a
number of physiological and behavioral functions.
Key words neurotransmitter; HPLC-MS; ethyl chloroformate; derivatization
Dopamine (DA) and serotonin (5-hydroxyltryptamine, neurotransmitters by a derivatization procedure,13) which was
5-HT), which are widely distributed as a monoamine neu- performed by benzylamine and 1,2-diphenylethylenediamine
rotransmitter in the central nervous system (CNS),1) play im- in a two-step reaction in weakly alkaline media and in the
portant physiological and behavioral roles such as the control presence of potassium hexacyanoferrate (III).14) However,
and regulation of affective behavior, sleep, reward system, since this protocol requires the vigorous reaction condition of
motor and cognitive functions.2) Changes of DA and 5-HT heating to 50°C for 20min, it is necessary to develop a sim-
basal levels have been linked to some CNS-related disorders pler method under mild reaction conditions.
such as depression, panic disorder, schizophrenia and Par-
HPLC coupled with MS, which can be used to identify
kinson’s disease (PD).2,3) PD is pathologically characterized analytes simply on the basis of their m/z (mass-to-charge) ra-
by the death of dopaminergic neurons in the substantia nigra tios,15,16) is a popular and powerful technique for analyzing
pars compacta4) and caused by neurotoxins such as 1-methyl- neurotransmitters in several biological fluids.17) However, it
4-phenyl-1,2,3,6-tetrahypyridine (MPTP), which produces a was to our interest that there was no report for the simultane-
clinical syndrome similar to PD. Because the administration ous determination of neurotransmitters containing different
of MPTP is known to affect the dopaminergic neurons in the ionizable groups such as carboxylic acid or amino groups
nigrostriatal system with subsequent depletion of DA and its in the biological samples by using the HPLC-MS method.
metabolites,5,6) the measurement of 5-HT, DA and their me- Thus, in order to determine a neurotransmitter containing
tabolites is necessary to discover and test new drugs for treat- an amino group, we have to use a positive ion polarity mode
ing these disorders.7)
of MS condition while using a negative ion polarity mode of
Various analytical methods have been widely investigated MS condition for determining a neurotransmitter containing a
for determining the level of monoamine neurotransmitters carboxylic acid group. As a HPLC-MS method, we previously
and their metabolites, including high-performance liquid chro- reported a new derivatization method using the HPLC-MS for
matography (HPLC) coupled with electrochemical detection the measurement of catecholamines (DA, norepinephrine and
(ECD), dual ECD, fluorescence detection (FLD), FLD/ECD epinephrine) in urine samples following derivatization with
system, chemiluminescence detection and mass spectrom- ethyl chloroformate.18) Our derivatization method has several
eter (MS) and gas chromatography (GC) coupled with MS advantages over other derivatization methods because it can
in biological samples such as plasma, rat brain, and mouse be performed in fast derivatization reaction time, in aqueous
brain and in vitro experiments.8–10) HPLC-ECD, which is the solutions and by utilizing inexpensive reagents. Additionally,
most common analytical technique used to determine mono- this method enables good chromatographic separation and
amines and their metabolites, has the advantages of low cost simultaneous analysis.
and fast speed11) but maintaining its routine use is problem-
As an extended effort to apply our derivatization technique
atic due to sudden noise problems.12) The HPLC-FLD method to neuroscience research, we herein describe our development
is used to measure fluorescent derivatives of monoamine of an optimized chromatographic condition using HPLC-
MS for the simultaneous determination of 5-HT, DA and
their metabolites (3,4-dihydroxyphenylacetic acid (DOPAC),
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: shyoon@ajou.ac.kr
© 2013 The Pharmaceutical Society of Japan

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253
Fig. 1. Derivatization of 5-HT, 3-MT, DHBA (IS; Internal Standard), DA, HVA and DOPAC with Ethyl Chloroformate
3-methoxytyramine (3-MT) and homovanillic acid (HVA)) U.S.A.) by mobile phases consisting of 40% solvent A (0.1%
(Fig. 1) through derivatization with ethyl chloroformate in formic acid in water, pH 3) and 60% solvent B (0.1% formic
bran tissue extracts from mouse samples, which can be useful acid in acetonitrile). The injection volume was 5µL and an
for measuring neurotransmitters quantitatively and effectively. analysis time of 30min was used for the separation of all
analytes at a flow rate of 0.2mL/min. The column oven was
held constant at 30°C, and all analytes were determined in the
positive ESI mode. MS conditions were optimized to obtain
MATERIALS AND METHODS
Chemicals DA, 3,4-dihydroxybenzylamine (DHBA), maximum sensitivity. The curve dissolution line temperature
DOPAC, 3-MT, HVA, 5-HT and ethyl chloroformate were was 250°C and heat block temperature was 200°C. Nitrogen
obtained from Sigma-Aldrich (Saint Louis, MO, U.S.A.). Ace- generated from Schmidlin Mistral nitrogen generator (Geneva,
tonitrile and ethanol were purchased from J. T. Baker (Phil- Switzerland) was used as the nebulizer gas at 1.5L/min.
lipsburg, NJ, U.S.A.). Pyridine and formic acid were sourced
Preparation of Stock Solution The stock standard solu-
from Acros Organics (Morris Plains, NJ, U.S.A.). De-ionized tions of the DA, its metabolites (DOPAC, 3-MT and HVA) and
water with an Ultima UP-900 (Guri, Gyeonggi, Korea) was 5-HT were prepared by dissolving their accurately weighted
used for all aqueous solutions.
amounts (10.0mg) in 0.1n perchloric acid solutions (1.0mL).
Chromatographic and Mass Spectrometric (MS) Con- All solutions were kept in darkness at −24°C. These solutions
ditions A Shimadzu LCMS-2010EV (Chiyoda-ku, Tokyo, were stable for at least 1 month and were diluted further with
Japan) equipped with an electrospray ionization (ESI) probe water to the required concentrations before use.
and QoQ system (Q-array-octupole-quadrupole mass analyzer)
Sample Preparation Procedure Standard solution or the
was used. The HPLC system consisted of two pumps (model filtrate obtained from brain homogenate (185µL) was added to
LC-20AD), a degasser (model DGU-20 A3), an autosampler water (265µL). The DHBA (200ngmL−1, 50µL) was added as
(model SIL-20A), a column oven (model CTO-20A) and an an internal standard. After ethanol–pyridine (4:1, v/v, 300µL)
UV/VIS photodiode array detector (model SPD-M20A). The and ethyl chloroformate (20µL) were added, the solution was
derivatized DA, DOPAC, 3-MT, HVA and 5-HT were sepa- shaken for 3min and diethyl ether (1mL) was then added. The
rated on a ZORBAX Eclipse C-18 column (5µm particle size, tube was shaken on a vortex mixer for 2min and then the re-
150 mm×4.6mm i.d.; Agilent Corporation, Palo Alto, CA, sulting solution was cooled to −40°C. The organic phase was

254
Vol. 36, No. 2
Fig. 2. Full Scan Mass Spectra Obtained after Derivatization with Ethyl Chloroformate
5-HT (panel 1), 3-MT (panel 2), DHBA (panel 3), DA (panel 4), HVA (panel 5) and DOPAC (panel 6).
transferred to another tube and the solvent was evaporated and
dried in a vacuum oven. The obtained residue was dissolved
in acetonitrile (50µL) and 5µL of the resulting solution was
injected into the HPLC-MS.
RESULTS
Optimization of the Derivatization Procedure In the
present method, as shown in Fig. 1, the derivatization reaction
Application to Mouse Brain Striatum Samples Male using ethyl chloroformate proceeded rapidly even at room tem-
C57BL/6 mice (23.6 1.5g, 8 weeks old) purchased from Cen- perature in aqueous solution and the derivatized compounds
tral Laboratory Animal Company (Seoul, Korea) were housed were then concentrated in acetonitrile. The amino (–NH2) or
five per cage with room temperature, and lit by artificial light hydroxyl (–OH) groups in 5-HT, DA and their metabolites
for 12h each day. The mice were housed in standard labora- were converted to the N(O)-alkoxycarbonyl form, whereas
tory cages and had free access to food and water throughout the carboxylic acid groups were changed to ethyl ester form
the study period. Mice received five intraperitoneal injections by the decarboxylation of the intermediate mixed anhydride
of MPTP hydrochloride (30mg/kg in saline, Sigma-Aldrich, form.19) The molecular ion obtained from the positive selected
U.S.A.) at 1d intervals or saline (equal volume injected in- ion monitoring mode in HPLC-ESI/MS was much stronger
traperitoneally (i.p.)) alone (control groups). Mice (n=3 per than that from the negative mode.20,21) The ethyl chlorofor-
group) were euthanized by cervical dislocation, and the dorso- mate derivatives of 5-HT, DA, 3-MT, DOPAC, HVA and
lateral striatum from both the left and right sides were dissect- DHBA (internal standard) provided various molecular cations
ed and frozen at −80°C. Brain tissue samples were weighed [M+H]+, [M+NH4]+ or [M+Na]+. The mass spectra obtained
and then homogenized in 700µL of ice-cold lysis buffer after ethyl chloroformate derivatization are presented in Fig.
containing 1× phosphate buffered saline (PBS), 1% NP-40 2. ESI mass spectra for derivatives of DHBA, 5-HT, DA and
(nonidet P-40), 0.5% sodium deoxycholate and 0.1% sodium their metabolites were dominated by the m/z 392 [M+Na]+ for
dodecyl sulfate (SDS). The homogenate was centrifuged at DA, the m/z 358 [M+ NH4]+ for DOPAC, the m/z 312 [M+H]+
9000rpm for 20min at 4°C and filtered through a 0.2µm filter for 3-MT, the m/z 300 [M+ NH4]+ for HVA, the m/z 321
(Whatman, Middlesex, England).
[M+H]+ for 5-HT and the m/z 378 [M+Na]+ for DHBA.
Selectivity To obtain good resolution and symmetric
peak shapes of the analytes, the chromatographic conditions,
such as the composition of mobile phase and total analysis

February 2013
255
time, were optimized through several trials. As a result, good
chromatographic separation was obtained using an Agi-
lent ZOBAX Eclipse C18 (150mm×4.6mm i.d., 5µm) column.
The composition of the mobile phase was 60% solvent B as
isocratic elution at a flow rate of 0.2mL/min and with a total
analysis time of 30min. A typical chromatogram for separa-
tion of DHBA, 5-HT, DA and their metabolites is shown in
Fig. 3. The retention times of the analytes were approximately
12.67min (5-HT), 14.25min (3-MT), 15.53min (DHBA),
16.65min (DA), 19.22min (HVA) and 23.42min (DOPAC),
respectively.
Linearity The calibration curve of each compound was
determined by plotting the ratio of peak area to DHBA (in-
ternal standard) versus concentration of the spiked standard
solution, and the equations are summarized in Table 1. The
calibration curves were linear in the concentration range from
1 to 700ng/mL for DA, from 1 to 100ng/mL for DOPAC,
from 1 to 50ng/mL for 3-MT, from 5 to 100ng/mL for HVA,
and from 1–50ng/mL for 5-HT from three replicated measure-
ments. A linear regression equation (y=ax+b) was evaluated,
where x is the concentration of the analytes and y is the peak
Fig. 3. The HPLC-MS Chromatogram Obtained from the Derivatized
Standard with Ethyl Chloroformate
The peaks are identified as follows: peak 1; 5-HT, peak 2; 3-MT, peak 3; DHBA,
peak 4; DA, peak 5; HVA, peak 6; DOPAC.
Precision, Accuracy and Recovery Table 2 summarizes
area ratio. The correlation coefficients (R2) of between 0.998 the intra- and inter-day precision and accuracy at two concen-
and 0.999 described good linearity in the corresponding con- trations. The intra-day precision and accuracy was monitored
centration (Table 1).
by analyzing three replicates of 20 and 50ng/mL of each
Limits of Detection (LOD) and Quantification (LOQ) analyte. Intra-day precisions (expressed as percent relative
Table 1 shows the limits of detection (LOD) and quantification standard deviation, R.S.D. %) of the method ranged between
(LOQ) of the analytes. The LODs were determined by ana- 0.99% for the lowest concentration of DOPAC and 4.72% for
lyzing the spiked standard solution of progressively lowering the lowest concentration of HVA. Inter-day precisions of this
concentrations until the signal of each analytes approached to assay ranged between 0.35% for the highest concentration
3-fold of the noise (S/N=3). The LODs in the present study of DOPAC and 3.08% for the lowest concentration of 3-MT.
indicated that the minimum detectable amount of 5-HT, DA The accuracy (expressed as a percentage) of the intra-day and
and DOPAC was 0.2ng/mL, while that of 3-MT and HVA was inter-day assays were −1.64 to 2.56% and −2.01 to 1.23%
0.1ng/mL. The LOQ corresponded to an S/N ratio of 10. The for concentration of 20 and 50ng/mL, respectively. The mean
LOQs of the assay were 1ng/mL for 5-HT, 3-MT, DA and recovery rates of each compound were above 90% at the mea-
DOPAC and 5ng/mL for HVA.
sured concentration.
Table 1. Linear Equations, Correlation Coefficients, LODs and LOQs for Quantification
Analyte
Equation
R2
LOD (ng/mL)
LOQ (ng/mL)
5-HT (1–50ng/mL)
3-MT (1–50ng/mL)
DA (1–700ng/mL)
HVA (5–100ng/mL)
DOPAC (1–100ng/mL)
y=0.018x+0.073
y=0.047x+0.013
y=0.103x−0.515
y=0.005x+0.025
y=0.101x−0.096
0.998
0.999
0.998
0.998
0.998
0.2
0.1
0.2
0.1
0.2
1
1
1
5
1
Table 2. Accuracy and Precision of Analytes (n=3)
Intra-day
Inter-day
Analyte
Amount added (ng/mL)
Bias (%)a)
Precision (%)
Bias (%)a)
Precision (%)
5-HT
3-MT
DA
20
50
20
50
20
50
20
50
20
50
1.14
0.62
2.56
−1.07
0.04
−0.44
1.30
−1.64
0.60
1.85
1.83
1.01
3.65
2.08
1.19
1.67
4.72
1.92
0.99
2.13
0.46
0.98
−0.78
−2.01
0.85
0.89
−0.94
1.16
2.20
0.79
3.08
1.68
0.99
0.63
1.58
2.70
1.94
0.35
HVA
DOPAC
−1.19
1.23
a) Bias (%)={(calculated concentration−found concentration)/calculated concentration}×100.

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Vol. 36, No. 2
Fig. 4. The HPLC-MS Selected-Ion Chromatograms Obtained from the Derivatized Monoamines in the C57BL/6 Mouse Samples
The peaks are identified as follows: peak 1; 5-HT, peak 2; 3-MT, peak 3; DHBA, peak 4; DA, peak 5; HVA, peak 6; DOPAC.
Table 3. Amount of the Monoamines in C57BL/6 Mice (n=3)
Measured concentration (ng/mg wet tissue weight, mean S.E.M.)
Subject
Reference
Method
DA
DOPAC
3-MT
HVA
5-HT
Control
Control
Control
Control
MPTP (7d)
Control
MPTP (1d)
MPTP (2d)
MPTP (7d)
20.06 1.13
78 2a)
0.75 0.04
5.8 0.2a)
12 2a)
0.73 0.02
0.15 0.02
5.01 0.22
0.86 0.09
0.84 0.17
0.91 0.03
1.15 0.06
6.8 0.4a)
12 2a)
1.34 0.04
0.48 0.04
7.05 0.26
2.79 0.52
2.30 0.50
3.18 0.14
26
27
28
30
30
HPLC-ECD
HPLC-ECD
HPLC-ECD
HPLC-ECD
HPLC-ECD
110 10a)
19 4a)
11.11 0.33
1.30 0.29
30.58 1.10
3.08 0.17
2.95 0.13
2.68 0.16
0.38 0.02
0.13 0.02
2.04 0.05
0.53 0.03
0.52 0.10
0.65 0.01
0.48 0.01
0.44 0.01
1.79 0.08
0.93 0.09
0.52 0.09
0.95 0.09
a) Because the levels of DA, DOPAC and HVA were presented as a bar graph in refs. 27 and 28, data were expressed as approximate values.
Application of the Methods to Real Biological Samples tissue weight for DA, 5.01 0.22ng/mg wet tissue weight for
The applicability of the derivatization method with ethyl DOPAC, 2.04 0.05ng/mg wet tissue weight for 3-MT and
chloroformate to determine the levels of 5-HT, DA, DOPAC, 7.05 0.26ng/mg wet tissue weight for HVA. Seven days after
3-MT and HVA in the C57BL/6 mice brain striatal tissue after MPTP treatment, the level of DA was decreased to 8.8% of
MPTP treatment was investigated. The derivatization reac- the control. The striatal levels of DA metabolites, DOPAC,
tions for DA, 5-HT and DA’s metabolites in brain tissue ex- 3-MT and HVA were also reduced in the MPTP-treated mice.
tracts were carried out under the same condition described for
the preparation of the individual standards and their mixtures.
All the analytes were confirmed as the ethyl chloroformate de-
rivatives of 5-HT, DA, DOPAC, 3-MT and HVA on the basis
DISCUSSION
MPTP, as a neurotoxin in several models of Parkinson’s
of their retention times and mass spectra by a comparison disease in non-human primates and some rodents, such as
with the peaks of the standard samples shown in Fig. 3. From C57BL/6 mice, is capable of selective destruction of dopa-
the HPLC-MS selected-ion chromatograms of the derivatized minergic neurons22) located in substantia nigra. As DA is
monoamines in the C57BL/6 mouse samples shown in Fig. 4, metabolized to DOPAC, 3-MT and HVA via monoamine
the amount of monoamines are calculated and presented as the oxidase and catechol-o-methyl transferase in the normal state,
mean S.E.M. in ng/mg wet tissue weight for mouse striata the damage of substantia nigral cells in MPTP-treated mice
(n=3) in Table 3. The basal levels from the strata were 1.79
is consistently accompanied by a decrease in striatal DA and
0.08ng/mg wet tissue weight for 5-HT, 30.58 1.10ng/mg wet its metabolites (DOPAC, 3-MT and HVA).23) Therefore, the

February 2013
257
levels of DA, DOPAC, 3-MT and HVA are reflected by the sensitivity with DA and 8 times more sensitivity with 5-HT
decreased activity of dopaminergic neurons.24)
than the HPLC-ECD method. When we compare the LODs
The HPLC-ECD method is the most commonly used for of the present method with those of the HPLC-MS method
detection of 5-HT, DA and their metabolites in MPTP-treated without derivatization, the present method revealed 32.5 times
C57BL/6 mice because it is a relatively simple and economic better sensitivity with DA and 3.9 times better sensitivity with
analytical system.25–28) However, the HPLC-ECD method has 5-HT than HPLC-MS without derivatization since the detec-
low sensitivity and serious limitations in the simultaneous de- tion limits of DA and 5-HT by HPLC-MS method without
termination of monoamines such as 5-HT, DA and DOPAC.29) derivatization were 6.5ng/mL and 0.78ng/mL, respectively.17)
Under the standard separation protocols, the HPLC-ECD Upon comparison of the present method with the HPLC-FLD
method requires two different mobile phases along with two method with derivitization, it was found that the LODs of DA
separated HPLC-ECD systems for measuring the levels of and 5-HT using HPLC-FLD method have approximately 67
5-HT, DA and their metabolites.25) In addition, as shown in times better sensitivity with DA and 38 times better sensitiv-
Table 3, the levels of DA, DOPAC, 3-MT, HVA and 5-HT ity with 5-HT than those using the present method. However,
in the control mouse striatum measured by the HPLC-ECD the HPLC-FLD method requires the vigorous reaction condi-
method revealed significantly different values in different ex- tions of heating to 50°C for 20min for the derivatization,
periments, which are possibly due to different electrochemical which are not desirable reaction conditions for biological
behaviors and effects of analytes under different applied po- samples.14) Moreover, the HPLC-FLD method cannot deter-
tentials and mobile phase compositions.26–28,30,31) For example, mine 3-MT and HVA due to failure of the derivatization with
the levels of DA reported by different workers were approxi- fluorescent moiety.3,14)
mately in the range of 11 to 110ng.
In contrast to the previously reported methods, this de-
Our results obtained from derivatization with ethyl chlo- rivatization method with ethyl chloroformate using the HPLC-
roformate using the HPLC-MS system showed that the level MS system offers the advantages of less time required for the
of striatal DA seven days after treatment with MPTP was derivatization process and the ability to be performed in aque-
decreased significantly (8.8% of control) compared to that of ous media.33) In addition, this derivatization demonstrated sat-
striatal DA in control mice, while MPTP-treated mice ex- isfactory derivatization efficiencies with good yield in a short
hibited 18.2, 31.9 and 45.1% depletion of DOPAC, 3-MT and reaction time and enhanced data collection34) because not only
HVA in the striatum, respectively (Table 3). In addition, when the levels of 5-HT, DA and their metabolites were simultane-
the levels of DA and its metabolites were determined 1, 2 and ously determined, but each peak area of 5-HT, DA and their
7d after the last MPTP injection, the results indicated that metabolites was significantly expanded and more sensitive
the levels of DA and its metabolites had gradually decreased than those generated when using the HPLC-ECD method.
for 7d. These results revealed patterns similar to those in a
previous report by Obuchowicz et al., who determined levels
of striatal DA, DOPAC, 3-MT and HVA in control mice and
MPTP-treated mice using the HPLC-ECD method. However,
CONCLUSION REMARKS
The present paper has described an HPLC method with MS
the levels of DA, HVA and 5- HT in this study were around 3 for simultaneous determination of endogenous 5-HT, DA and
times higher, and the levels of DOPAC and 3-MT were around their metabolites (DOPAC, 3-MT and HVA) in mouse brain
7 times higher than those measured by the conventional tissue. The advantages of this derivatization method are the
HPLC-ECD method.30) The HPLC-MS method generally has concurrent measurement of monoamines using inexpensive
higher selectivity than those of the HPLC-ECD method, but reagents in aqueous solution and the short derivatization re-
it is difficult to measure the 5-HT, DA and their metabolites action time. The method can be useful for estimating the ef-
at the same time when analytes possess different ionizable ficiency of new drugs targeting brain-related diseases such as
groups (carboxylic acid group in DOPAC and HVA, and Parkinson’s disease, depression and panic disorder.
amino group in 5-HT, DA and 3-MT). Therefore, without the
derivatization process, both positive and negative ion polarity
Acknowledgments This research was supported by the
mode have to be used separately for the determination of the Basic Science Research Program through the National Re-
levels of 5-HT, DA and their metabolites under the general search Foundation of Korea (NRF) funded by the Ministry of
HPLC-MS condition. However, our derivatization method en- Education, Science and Technology (Grant No. 2011-0009050),
ables us to determine these analytes at the same time under and was partially supported by the Post Brain Korea 21 pro-
one type of ion polarity mode of HPLC-MS condition.
gram.
The LODs for each analyte were also determined by ana-
lyzing derivatized standard solution lowering concentrations
until the signal of each analyte approached 3 folds of noise
(S/N=3).23) Although the LODs of the present method cannot
directly compare with those of the HPLC-ECD method due
to the absence of data using mouse brain, we can evaluate
the difference between the LODs of DA and 5-HT in this
study and those with the HPLC-ECD method using rat brain
reported by Vaarmann et al..32) The detection limits of DA and
5-HT in our study were both 0.2ng/mL, while Vaarman et
al. reported values of 0.7 and 1.6ng/mL in, respectively. This
result indicated that the present method has 3.5 times more
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