Journal of Chromatography A, 1305 (2013) 234243

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Comprehensive proling analysis of bioamines and their acidic

metabolites in human urine by gas chromatography/mass
spectrometry combined with selective derivatization
Na-Hyun Park, Joo Yeon Hong, Hyun Ju Shin, Jongki Hong
College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 25 March 2013
Received in revised form 24 June 2013
Accepted 1 July 2013
Available online 4 July 2013
Keywords:
Biogenic amines
Human urine
HMDS/MBHFBA derivatization
MCX SPE
GC/MS

a b s t r a c t
A comprehensive analytical method was developed for the proling of biogenic amines in human urine
using GC/MS in SIM mode. Biogenic amines and their acidic metabolites were converted into their volatile
O-trimethylsilyl/N-heptauorobutyryl (OTMS/-NHFBA) derivatives for GC/MS analysis. Dual hexamethyldisilazane (HMDS)/-N-methyl-bis-heptauorobutyramide (MBHFBA) derivatizations have been shown
to be quite effective, with high derivatization yields and the absence of side products for primary biogenic amines. In this study, selective derivatization conditions by HMDS/MBHFBA were optimized in
terms of the reagent amount, reaction temperature and reaction time period. The highest derivatization
reaction yield was obtained at 40 C for 10 min for OTMS derivatization and 80 C for 5 min for N-HFBA
derivatization. The use of MCX SPE cartridges with different SPE elution solvents was effective for the
pre-concentration and selective cleanup of the biogenic amines and their acidic metabolites in human
urine. The selection of appropriate ions in SIM mode provided reliable quantication and identication
and a reduction in background effects. The established method was validated in terms of linearity, limits
of detection (LOD), limits of quantication (LOQ), precision, and accuracy. The present method was linear
(r2 > 0.996), reproducible (relative standard deviation range 1.16.9%), and accurate (range 87.9111.9%),
with LOQs of 0.1717.84 ng/mL. The biogenic amine proling of human urine was successfully accomplished by GC/MS in SIM mode combined with selective HMDS/MBHFBA derivatization and MCX SPE
cleanup.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Biogenic amines, such as catecholamines and serotonin, and
their metabolites are important mammalian neurotransmitters
that are released by the adrenal glands and the sympathetic nervous
system [13]. The proling analysis of biogenic amines in biological
uids has provided valuable information regarding physiological
status and metabolic disorders that may be useful for diagnosing
neuronal diseases [4,5]. In some cases, the concentrations of the
major acidic metabolites in urine can serve as indirect biomarkers
for the diagnosis of metabolic disorders. However, both biogenic
amines and their metabolites should be quantied because physiological levels are inuenced by the concentrations of both the
biosynthetic and metabolic enzymes [6]. Thus, the development of
analytical methods for the determination of biogenic amines and

Corresponding author at: College of Pharmacy, Kyung Hee University, HoegiDong, Dongdaemoon-Ku, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9255;
fax: +82 2 962 9255.
E-mail address: jhong@khu.ac.kr (J. Hong).
0021-9673/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2013.07.003

their acidic metabolites has become an important subject in the

clinical diagnosis of neuronal diseases.
Several analytical methods, such as HPLC and capillary
electrophoresis combined with electrochemical detection [7], uorescence detection [8,9] or mass spectrometric (MS) detection [10],
GC/MS [1113], and LCMS/MS [14,7], have been developed for the
biogenic amine proling analysis of biological samples. Recently,
an LCMS/MS multiple ion reaction monitoring (MRM) method
provided high selectivity and sensitivity for the analysis of biogenic amines and/or their metabolites in biological samples with
complex matrixes [15,16]. The disadvantages of LCMS/MS methods applied toward the simultaneous determination of basic and
acidic endogenous compounds, apart from sample preparation and
mobile phase limitations, include several factors associated with
sensitivity drift [17], the application of dual polarity in electrospray ionization (ESI) according to the acidic and basic properties
of endogenous compounds, and the susceptibility of certain ionization techniques to matrix effects [18].
GC/MS has been widely used for the analysis of some biogenic
amines and their acidic metabolites in biological samples due to
its high peak capacity, high detection sensitivity, and positive peak

N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

235

conrmation. However, the GC/MS analysis of biogenic amines and

their acidic metabolites is relatively complicated due to the wide
range of structures and functional groups of the analytes and their
low physiological concentrations in biological samples. In addition
to these factors, other issues exist that complicate GC/MS analyses.
First, biogenic amines and their acidic metabolites require derivatization of their hydroxyl, amino and/or carboxyl groups to reduce
their polarity, to enhance the thermal stability and volatility, and
to improve detection sensitivity [7,14,15]. The derivatization of
multiple functional biogenic amines, especially compounds containing primary amines, often yields incomplete reactions, and
the formation of by-products, resulting in multiple derivatives
and leading to difculties in the quantication of the analytes
[19]. Second, the simultaneous extraction and cleanup of biogenic
amines and their acidic metabolites from human urine remains a
difcult task. Current extraction methods, including liquid-liquid
extraction (LLE) [20,21], immersion solid-phase microextraction
(SPME) [17,22] combined with ethyl chloroformate derivatization,
and solid-phase extraction (SPE) using various adsorbents [2325],
have been applied for the selective extraction of basic and acidic
bioamines from biological uids due to their unique chemical properties. Third, in many cases, the detection of trace amounts of
biogenic amines and their metabolites can be easily masked by
high-concentration biological constituents; thus, the analytes of
interest remain undetected. Due to these reasons, the proling
analysis of biogenic amines and their acidic metabolites in human
urine has not been fully investigated.
To overcome these analytical challenges, we have attempted
to apply a selective derivatization method using HMDS/MBHFBA
for the sensitive and selective detection of biogenic amines and
their acidic metabolites. It is well known that HMDS reagent is
an effective TMS reagent for selective reactions on hydroxyl and
carboxylic groups, and it does not react with amine groups [26].
MBHFBA was then applied for the selective derivatization of the
amine groups. Using these reagents, both acidic and basic biogenic
compounds were successfully derivatized without any side products. For the effective extraction and selective cleanup of biogenic
amines and their acidic metabolites from human urine, a mixed
cation exchange (MCX) SPE cartridge using different SPE eluents
was applied. Although MCX SPE cartridges have generally been
used for the extraction of basic compounds from biological samples,
this SPE method was also applied for the simultaneous extraction of
both biogenic amines and their acidic metabolites. GC/MS selected
ion monitoring (SIM) was then applied for the sensitive and selective detection of the targets of interest, selecting specic ions to
reduce any potential matrix effects.
The aim of this study was to develop an analytical method
for the reliable quantication of biogenic amines and their acidic
metabolites in human urine by GC/MS. This study included (i) the
development of a novel derivatization method that prevented the
formation of side products during the derivatization reaction to
improve detection sensitivity, (ii) the development of an extraction
of the biogenic amines and their acidic metabolites from urine to
effectively eliminate interferences using an MCX SPE cartridge, and
(iii) a demonstration of the effectiveness of the established method
applied toward a diagnostic clinical test.

(St. Louis, MO, USA). d,l-noradrenaline hydrochloride (NE),

3,4-dihydroxyphenylacetic acid (DOPAC), and 4-hydroxy-3
methoxymandelic acid (VMA) were obtained from Fluka (Buchs,
Switzerland). Serotonin (ST) hydrochloride and its metabolite
5-Hydroxyindole-3-acetic acid (5-HIAA) were also obtained from
Sigma Chemical (St. Louis, MO, USA). Deuterium-labeled internal
standards (HVA-d5 and DA-d3 ) were obtained from Cambridge
Isotope Laboratories (Woburn, MA, USA) and CDN Isotopes
(Vaudreuil, Quebec, Canada), respectively. The purities of these
chemicals were greater than 97%. The biosynthesis pathways of
these biogenic amines are depicted in Fig. 1.
The derivatization reagents N-methyl-N-(trimethylsilyl)triuoroacetamide (MSTFA) and hexamethyldisilazane (HMDS)
were obtained from SigmaAldrich Chemical Co. (Milwaukee,
WI, USA) and Supelco (Bellefonte, PA, USA), respectively. NMethyl-bis(heptauorobutyramide) (MBHFBA) was purchased
from Macherey-Nagel (Duren, Germany).
Ethyl acetate, methanol, and pyridine were obtained from J. T.
Baker (Rockford, IL, USA). Hydrochloric acid was obtained from
Merck (Darmstadt, Germany), and phenanthrene-d10 , used as a
recovery internal standard, was obtained from Supelco.

2. Materials and methods

2.4. Derivatization

2.1. Chemicals

An authentic standard mixture solution (each 1 g) containing

internal standard was dried under nitrogen in a silylated reaction
vial prior to derivatization. The vial was dried under vacuum for
5 min at room temperature. Trimethylsilylation (TMS) of the biogenic amines was achieved by the addition of 20 L of ethyl acetate
and 40 L of HMDS.

Authentic catecholamines, dopamine hydrochloride (DA),

homovanillic acid (HVA), 3,4-dihydroxy-l-phenylalanine (L -DOPA),
d,l-normetanephrine hydrochloride (NMN) and ()-epinephrine
hydrochloride (EP) were purchased from Sigma Chemical

2.2. Stock solutions

The biogenic amines and their metabolites were dissolved in
methanol at a concentration of 1 mg/mL. However, L -DOPA was dissolved in methanol with the addition of 2 L of 6 M HCl to improve
its solubility. All standard solutions were stable for several weeks at
4 C. The solutions were kept in amber vials to protect the material
from photooxidation.
2.3. Sample preparation and MCX SPE procedure
Urine samples from four healthy (two female and two male)
subjects aged 2431 years were collected without the addition
of preservatives. Samples were stored in a deep freezer at 80 C
until analysis. Prior to the experiments, the samples were thawed
at room temperature. The labeled internal standards (HVA-d5 for
acidic metabolites and DA-d3 for biogenic amines, each 0.1 g)
were added to 2 mL of urine sample, and then 150 L of 0.1 M
HCl was added. The resulting solution was heated at 90 C for
30 min. After the hydrolysis procedure, SPE was performed with
mixed cation exchange (MCX, 1 mL, 30 mg) cartridges obtained
from Waters Corporation (Wexford, Ireland). The cartridges were
conditioned with 1 mL of methanol, followed by 1 mL of distilled
water. After adsorption of the sample, the cartridge was washed
with 1 mL of 0.1 M HCl and dried for 1 min under vacuum to remove
the excess water. All analytes were retained on the solid surface
of MCX cartridge by two mechanisms: interactions with the
phenyl rings and ionic interaction with the sulfonic acid moieties.
Different pH values and volumes of the elution solutions were
tested. The elution of the acidic metabolites from the MCX SPE
cartridge was performed with 1 mL of methanol. The elution of
the biogenic amines was then performed with 1 mL of 5% NH4 OH
in methanol (v/v). The eluate was evaporated to dryness under a
gentle stream of nitrogen gas.

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N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

Fig. 1. Biosynthesis pathways of biogenic amines and their metabolites.

To optimize the TMS derivatization of the biogenic amines by

HMDS, the inuence of the reagent amounts, the reaction temperature and reaction time was investigated. The reaction mixture was
purged with argon for 30 s, tightly capped, and vortexed, and the
reaction temperature was tested at 0, 20, 30, 40, 60, 80, 120 C.
The reaction time course for the TMS derivatization of the biogenic
amines by HMDS at 40 C was performed with varying reaction
periods of 5, 10, 20, and 40 min. A total of 20 L of MBHFBA was
added to the solution to acylate the amine group, and the solution
was heated at 80 C for 5 min. After cooling the reaction mixture,
20 L of the internal standard phenanthrene-d10 (1 g/mL) was

added to the solution. The derivatized sample was injected into the
GC/MS system. The overall analytical procedure for the determination of biogenic amines and their acidic metabolites is depicted in
Fig. 2.
2.5. GC/MS conditions
An Agilent 5975 N (Palo Alto, CA, USA) mass spectrometer (EI
mode, 70 eV) connected to an Agilent 6890 gas chromatograph
equipped with a DB-5MS capillary column (15 m 0.25 mm i.d.,
0.25 m lm thickness, J & W Scientic, Folsom, CA, USA) was

Fig. 2. Overall analytical procedure for the analysis of biogenic amines and their acidic metabolites in urine by GC/MS SIM mode.

N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

237

Table 1
Retention times (min) and characteristic ions (m/z) of biogenic amines and their metabolite-OTMS,-NHFBA derivatives by GC/MS scan mode.
Compound

RT (min)

M.W.

EI-characteristic ion value (%)

HVA
DOPAC
DA
VMA
NMN
NEP
EP
L-DOPA
5-HIAA
ST
HVA-D5
DA-D3
phenanthrene-d10

5.64
6.29
6.44
6.94
7.02
7.45
7.89
8.65
10.30
10.35
5.61
6.41
5.26

326
384
493
414
523
581
595
609
407
444
331
496
188

209(100), 326(77), 179(55), 311(46), 267(42), 296(17), 149(15), 237(11)

179(100), 384(75), 267(73), 237(22), 207(12), 369(12), 193(4)
267(100), 193(52), 179(45), 493(41), 280(35), 478(5)
297((100), 147(6), 267(4), 371(3), 399(2), 414(0.7)
297((100), 298(16), 267(3), 508(2), 523(0.7)
355((100), 297(8), 265(3), 281(2), 147(2), 476(2) 581(0.8)
355((100), 284(5), 240(4), 580(2), 267(2), 147(2), 595(0.4)
267(100), 179(33), 396(5), 594(4), 609(3), 219(2)
218((100), 335(38), 292(10), 320(8), 407(2), 392(0.8)
218((100), 444(17), 231(11), 146(4), 429(0.8)
214(100), 331(83), 184(56), 316(45), 272(40), 301(15), 242(10)
270(100), 496(47), 182(46), 193(42), 283(33), 481(8)
188(100), 160(8), 133(3)

Bold: selected ion in SIM mode.

used for analysis. The samples were introduced via split (ratio
10:1) injection with the port heated at 280 C. Helium was used
as the carrier gas at a ow rate of 1.0 mL/min. The oven temperature, initially 120 C and held for 2 min, was ramped to 175 C at
a rate of 10 C/min and held for 1.5 min, raised to 250 C at a rate
of 15 C/min, then ramped to 280 C and held for 3 min. The mass
spectrometer interface temperature was set at 280 C. The manifold
temperature was maintained at 230 C. The mass spectrometer was
operated in scan mode from 70 to 650 amu. For the monitoring and
conrmation analysis, the selected ion monitoring (SIM) mode was
used. In GC/MS SIM mode, two characteristic ions for each biogenic
amine and acidic metabolite were used for peak identication, and
the most abundant ion was selected for quantication (Table 1).
The dwell time of each ion was set at 50 ms. The relative peak area
was obtained by dividing the integrated area of the base peak by
that of the internal standard.
2.6. Method validation
The established method was validated by determining the linearity, limits of detection (LOD), limits of quantication (LOQ),
precision and accuracy. The linearity of the calibration was evaluated by combining 2 mL of urine with the analyte standard solutions
at six different concentrations, subjecting the mixtures to the
SPE procedure, derivatization and analysis by GC/MS SIM. The
analyte/IS peak area ratios were plotted against the corresponding concentrations, and the calibration curves were constructed
by means of the least-squares method. The considered analyte
linearity ranges were 12000 ng/mL for the acidic metabolites,
101000 ng/mL for DA, NE, ST and NMN and 202500 ng/mL for
L -DOPA and EP. The LODs and LOQs were determined at signalto-noise (S/N) ratios of 3 and 10, respectively. The precision of
the developed method was evaluated using intra- and inter-day
variations. The relative standard deviation was determined as a
measurement of the precision. The intra- and inter-day repeatabilities were determined on triplicates within one day and over
three consecutive days, respectively. To evaluate the accuracy of
the analytical method, the recovery study was measured by spiking known amounts of standards in known real samples in triplicate
at three concentration levels, 50, 250 and 1000 ng/mL. The mixture
was analyzed using the method described in Section 2.4.
3. Results and discussion
3.1. Optimization of derivatization conditions by HMDS/MBHFBA
Six derivatization techniques were investigated in our previous
study to nd an effective derivatization method for biogenic
amine proling analysis by GC/MS [27]. Among them, the

two-step
derivatization
involving
O-silylation
and
Nperuoroacylation with MSTFA and MBHFBA, respectively,
possessed several advantages compared to single derivatization
techniques, including the production of thermally stable derivatives, improvements in the sensitivities of higher-mass fragment
ions with decreasing interference effects, and improvements in
the chromatographic properties.
However, the MSTFA/MBHFBA method still produced side products from the primary amine compounds, even though it was a
highly sensitive and selective derivatization. As seen in Fig. 3A and
Fig. 4A, biogenic amines containing primary amines, such as DA,
NE, NMN, L -DOPA and ST, produced signicant amounts of side
products when MSTFA/MBHFBA was used. Their corresponding
side products were identied as the biogenic amine-OTMS,-NTMS,NHFBA derivatives using GC/MS scan mode. The formation of the
resulting side products was most likely due to the favorable reactivity of the primary amines with MSTFA. The amounts of these side
products tended to increase as the reaction period and reaction
temperature increased. Thus, the irreproducible derivatization of
the primary amines could lead to unreliable quantication results.
To prevent the formation of side products, an HMDS reagent was
applied for the TMS derivatization of the biogenic amines. HMDS is
known to selectively derivatize hydroxyl or carboxylic acid groups,
but not amine groups [16] due to the strong basicity of the amine
group in HMDS. As seen in Fig. 4B, HMDS prevents the formation of
side product -NTMS derivatives (e) during the TMS derivatization
of the primary amines. In other words, the excess amount of HMDS
cannot further react with the biogenic primary amine compounds
to form any -OTMS,-NTMS derivatives because the basicity of the
HMDS and the reaction intermediate of HMDS (f) is greater than
the reaction products (b), as shown in Fig. 4B.
To optimize the TMS derivatization of the biogenic amine-OTMS
conditions, the amount of HMDS reagent, reaction temperature,
and reaction time were examined (Fig. 5). The overall reaction yield
for the biogenic amine-OTMS derivatives increased proportionally
as the amount of HMDS was increased to 40 L (Fig. 5A). However,
the reaction yield did not increase at volumes of HMDS greater
than 40 L. The reaction temperature (Fig. 5B) and reaction time
(Fig. 5C) of the HMDS derivatization of biogenic amines strongly
affected the kinetics of the TMS derivatization reaction. As shown
in Fig. 5B, the TMS derivatization yields were shown to decreased
at temperatures greater than 40 C and reaction times longer than
10 min. Temperatures greater than 40 C can lead to a retardation
of the TMS derivatization because HMDS is an endothermic reagent
(H = 42.40 kJ/mol) [28]. The longer reaction period produced a
white precipitate in the reaction solution that decreased the TMS
derivatization yield.
The N-HFBA derivatization by MBHFBA was performed at 80 C
for 5 min by using previously optimized conditions [27]. The alkyl

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N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

Fig. 3. TICs of biogenic primary amines with derivatization using (A) MSTFA/MBHFBA and (B) HMDS/MBHFBA by GC/MS scan mode. Peak identities as follow: (1)
phenanthrene-d10 , (2) dopamine-(OTMS)2 ,-NHFBA, 2 . DA-(OTMS)2 ,-NTMS,-NHFBA, (3) NMN-(OTMS)2 ,-NHFBA, 3 . NMN-(OTMS)2 ,-NTMS,-NHFBA, (4) NE-(OTMS)3 ,-NHFBA,
4 . NE-(OTMS)3 ,-NTMS,-NHFBA, (5) L-DOPA-(OTMS)3 ,-NHFBA, 5 . L-DOPA-(OTMS)3 ,-NTMS,-NHFBA, (6) ST-OTMS,-NHFBA and 6 . ST-OTMS,-NTMS,-NHFBA.

amine and primary amine groups of the biogenic amines were

readily reacted to form the N-HFBA derivative. For serotonin and 5HIAA, the amine group of the indole ring did not react with MBHFBA
due to low basicity of the indole amine. However, the indole amine
group of 5-HIAA did react with MSTFA to form theOTMS,-NTMS
derivative as a side product. Thus, in this study, a dual derivatization was successfully applied for the selective derivatization of
bioamine-OTMS,-NHFBA derivatives and their acidic metaboliteOTMS derivatives. As seen in Fig. 3B, the single peaks of the
derivatives were observed for the primary amine compounds of the

biogenic amines and their acidic metabolites, in contrast to the multiple peaks when derivatized with MSTFA/MBHFBA. The retention
times and characteristic ions of the biogenic amine-OTMS,-NHFBA
derivatives and the acidic metabolite-OTMS derivatives are summarized in Table 1.
To compare the reaction yields of the biogenic amine derivatives formed by HMDS/MBHFBA and MSTFA/MBHFBA, the relative
response factors were calculated by the peak area ratios of the
analytes versus the internal standard (phenanthrene-d10 ). The
relative response factors of the biogenic amine-OTMS,-NHFBA

Fig. 4. Chemical derivatization of biogenic compounds containing primary amines using (A) MSTFA/MBHFBA and (B) HMDS/MBHFBA reagents.

N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

239

Fig. 5. Optimization of derivatization conditions of bioamines containing primary amines using HMDS reagent followed by HFBA derivatization.

Table 2
Area ratio of biogenic amine-OTMS,-NHFBA derivatives formed with different
derivatization reagents by GC/MS scan mode. (n = 3).
MSTFA/MBHFBA
HVA
DOPAC
DA
VMA
NMN
NE
EP
L-DOPA
5-HIAA
ST

1.97
1.62
3.41
3.38
4.03
4.86
3.09
1.09
2.90
4.09

7.7
7.35
10.24
6.07
4.18
8.01
4.61
4.09
8.70
3.31

HMDS/MBHFBA
1.81
1.66
3.64
3.32
3.87
5.70
2.86
2.62
4.95
4.80

1.82
2.42
1.65
3.03
3.89
3.00
2.21
2.27
1.03
1.37

derivatives produced by HMDS/MBHFBA were equivalent or better

than those produced by MSTFA/MBHFBA, as indicated in Table 2.
Especially, the relative response factors of the bioamine O-TMS/NHFBA derivatives of the primary amines formed by HMDS/MBHFBA
were higher than those formed by MSTFA/MBHFBA. This result was
due to the HMDS/MBHFBA derivatization of the primary amine
compounds producing single products, but the MSTFA/MBHFBA
derivatization produced both bioamine-OTMS/-NHFBA as a major
product and -OTMS,-NTMS,-NHFBA derivatives as side products.
3.2. Sample cleanup by MCX SPE
In this study, the applicability of MCX SPE cartridges was examined for the cleanup of bioamines and their acidic metabolites
in urine. The MCX adsorbent, containing N-divinylpyrrolidone,
divinylbenzene, and sulfonic acid groups, can be useful for the
cleanup of acidic and basic components in biological samples via
hydrogen bonds, van der Waals interactions, and ionic interactions
when appropriate elution solvents are used. The mechanisms of
interactions between the biogenic amines and acidic metabolites
and the MCX sorbent are depicted in Fig. 6.

The elution patterns of the acidic metabolites were studied using

the MCX SPE with methanol solvent. After loading the sample onto
the MCX SPE cartridge, the carboxylic acid and benzene groups in
the acidic metabolites interact with the N-divinylpyrrolidone and
divinylbenzene of the MCX sorbent, respectively, through hydrogen bonding and van der Waals interaction, respectively. The acidic
metabolites retained on the MCX SPE cartridges were easily eluted
by methanol. For all the acidic metabolites, the elution recovery
with MCX SPE using 0.5 mL of methanol was found to be greater
than 93%.
The elution patterns of the biogenic amines were also investigated using the MCX SPE cartridge with 5% ammonium hydroxide
in methanol. During sample loading onto the MCX SPE at pH values less than at least 3, most of the biogenic amines are converted
into the protonated amine forms. Although L -DOPA has both carboxylic acid and amine groups, its amine group can be protonated in
acidic media. The positively charged amine groups of the biogenic
amines strongly interact with the sulfonic anion of the MCX sorbent. To elute the biogenic amines from the MCX SPE cartridges, a
basic 5% NH4 OH in MeOH eluent was used to cleave the electrostatic
interactions between the ammonium ion and sulfonic anion. Most
of the basic compounds, except for serotonin, were readily eluted
within 0.5 mL of 5% NH4 OH in methanol. Serotonin contains two
amine groups and interacted more strongly with the MCX sorbent
compared to the other basic compounds. Thus, to effectively elute
the basic compounds, the amount of elution solvent was increased
to 1 mL. For all the biogenic amines, the elution recovery with the
MCX SPE using 1.0 mL of 5% NH4 OH in methanol was found to be
greater than 92%.
The biogenic amines and their acidic metabolites in urine sample were successfully puried on MCX SPE cartridges using different
eluting solvents. MCX SPE cleanup method could effectively apply
for the proling analysis of biogenic amines and acidic metabolites,
respectively, in urine even though sample procedure was relatively
complicated by the addition of SPE procedure, compared with HPLC
based analytical methods [710].

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N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

Fig. 6. Three main interactions between MCX sorbent and (A) acidic metabolites and (B) biogenic amines.
Table 3
Calibration curves, correlation coefcients, LODs and LOQs of biogenic amine and acidic metabolites-OTMS, -NHFBA derivatives obtained by GC/MS SIM.
Compound

Linear range (ng/mL)

Slope

Intercept

Correlation coefcient (r2 )

LOD (ng/mL)

LOQ (ng/mL)

HVA
DOPAC
DA
VMA
NMN
NEP
EP
L-DOPA
5-HIAA
ST

12000
12000
101000
12000
101000
101000
202500
202500
12000
101000

0.104
0.0062
0.0272
0.0067
0.09
0.0038
0.0045
0.0005
0.0067
0.1412

0.1338
0.1321
1.4787
0.1187
1.466
0.0098
0.2449
0.0801
0.0564
2.4097

0.998
0.996
0.997
0.999
0.999
0.998
0.998
0.997
0.997
0.998

0.27
0.12
1.42
0.05
0.92
1.15
6.24
4.34
0.07
0.75

0.75
0.68
6.28
0.17
2.85
5.48
17.84
13.88
0.27
2.31

Table 4
Inter- and intra-day data for acidic metaboliteOTMS,-NHFBA derivatives obtained by GC/MS SIM.
Compound

Concentration (ng/mL)

Intra-day (n = 3)
Mean

Acuuracy (%)

RSD (%)

Inter-day (n = 3)
Mean

Acuuracy (%)

RSD (%)

VMA

50
250
1000

53.6
259.9
1049.3

107.3
103.9
104.9

3.5
3.9
2.3

55.9
262.4
1052.6

111.9
104.9
105.3

4.1
1.9
2.8

DOPAC

50
250
1000

51.6
249.4
964.5

102.1
100.8
96.5

1.4
3.7
1.5

52.1
251.7
951.9

104.2
100.7
95.2

3.4
3.0
2.3

HVA

50
250
1000

50.1
264.5
1039.8

100.1
105.8
103.9

2.7
3.4
3.3

53.9
266.7
1053.4

107.9
106.7
105.3

4.3
4.3
3.6

5-HIAA

50
250
1000

47.3
239.2
980.4

94.6
95.7
98.0

1.3
4.9
3.0

47.5
241.3
985.7

95.1
96.5
98.6

2.3
3.2
2.1

Table 5
Inter- and intra-day data for biogenic amineOTMS, -NHFBA derivatives obtained by GC/MS SIM.
Compound

Concentration (ng/mL)

Intra-day (n = 3)
Mean

Acuuracy (%)

RSD (%)

Inter-day (n = 3)
Mean
Acuuracy (%)

RSD (%)

DA

50
250
1000

48.6
238.3
963.7

97.1
95.3
96.4

4.8
5.1
2.1

49.3
240.2
970.6

98.6
96.1
97.1

1.7
5.4
3.6

L-DOPA

50
250
1000

50.2
242.5
978.3

100.4
97.0
97.8

3.0
3.5
2.7

46.1
238.6
996.2

92.3
95.4
99.6

3.5
4.9
2.5

NE

50
250
1000

53.5
218.3
879.1

107.0
87.9
87.9

4.0
3.9
2.9

52.9
241.6
899.8

105.9
96.7
89.9

3.0
5.5
3.5

NMN

50
250
1000

46.6
256.2
964.0

93.2
102.5
96.4

5.5
2.3
1.8

49.0
260.1
967.2

98.0
104.0
96.7

5.9
4.8
2.3

EP

50
250
1000

47.9
256.6
1004.8

104.4
102.6
100.5

6.9
3.4
1.9

48.5
254.2
978.3

96.9
101.7
97.8

5.1
3.9
1.8

ST

50
250
1000

49.5
247.6
990.5

99.0
95.6
99.0

5.8
2.2
2.2

51.4
239.4
994.5

99.6
98.2
98.2

3.0
2.5
1.1

N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

241

Fig. 7. Extracted ion chromatograms of (A) m/z 179 and 384 for DOPAC-(OTMS)3 derivative and (B) m/z 193 and 493 for DA-(OTMS)2 ,-NHFBA in human urine by GC/MS SIM
mode.

3.3. Method validation

To evaluate the linearity of the developed method, calibration spiked solutions (n = 3 each) with a concentration range of
12000 ng/mL urine (1, 50, 250, 500, 1000 and 2000 ng/mL urine)
for the acidic metabolites; of 101000 ng/mL urine (10, 50, 100,
250, 500 and 1000 ng/mL urine) for DA, NE, ST and NMN; and of
202500 ng/mL urine (20, 50, 250, 500, 1000 and 2500 ng/mL urine)
for EP and L -DOPA were prepared by spiking the stock solutions
diluted with human urine. The calibration curves were constructed
and calculated according to the method of least squares, relating
y (the peak area ratio of the biogenic amine-derivatives to the
internal standard) to x (the concentration of the biogenic amines
in g/mL). The calibration solutions were analyzed in triplicate.
Table 3 lists the data for the equations of the calibration curves,
correlation coefcients, and the limits of detection (LOD). The

calibration curves of the analytes showed good linearity within

the given concentration ranges, with correlation coefcients (R2 )
greater than 0.996.
In many cases during GC/MS analysis, the LOD can be affected
by several factors, such as GC injection volume, injection split
ratio, and nal solvent volume. Therefore, the sensitivity of
this developed method could be enhanced by adjusting these
factors. The LODs that were obtained for this method ranged
from 0.12 to 6.24 ng/mL and were lower or equivalent to those
obtained by other HPLC-based methods [12,22]. Although recently
published GC/MS method using O-ethoxycarbonyl (EOC)/tertbutyldimethylsilyl (TBDMS) derivatization [11] provided higher
sensitivity for acidic metabolites than this established method.
However, the previously developed method could not be applied
for the analysis of biogenic amines due to less reaction yield
of EOC for amine compounds. The established method provided

Fig. 8. TICs of (A) standard mixture of acidic metabolites (each 10 ng/mL) and (B) healthy human urine obtained by GC/MS SIM. Peak identities as follow: IS. phenanthrene-d10
(1) HVA-(OTMS)2 , 1 HVA-d5 -(OTMS)2 , (2) Dopac-(OTMS)3 , (3) VMA-(OTMS)3 , and (4) 5-HIAA-(OTMS)2 .

242

N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

Fig. 9. TICs of (A) standard mixture of biogenic amines (each 10 ng/mL) and (B) healthy human urine obtained by GC/MS-SIM mode. Peak identities as follow: IS. phenanthrened10 , (1) DA-(OTMS)2 ,-NHFBA, 1 . DA-d3 -(OTMS)2 ,-NHFBA, (2) NMN-(OTMS)2 ,-NHFBA, (3) NE-(OTMS)3 ,-NHFBA, (4) EP-(OTMS)3 ,-NHFBA, (5) L -DOPA-(OTMS)3 ,-NHFBA, and (6)
ST-OTMS,-NHFBA.

reasonable sensitivity on both biogenic amines and acidic metabolites for clinical test. It is important to note that clinically signicant
concentrations [9,29,30] of most target analytes (exept for EP and
L-DOPA) in urine are usually 10- to 100-fold higher concentrations
than the LODs achieved in this study. Thus, the established method
could be effectively applied for the proling analysis of biogenic
amines and their metabolites in human urine.
The relative standard deviations (RSDs) for the intra-and interday variations in precision of the acidic metabolites and biogenic
amines, are indicated in Tables 4 and 5. The recoveries of all the
analytes ranged from 87.9 and 111.9%, and the RSDs were less than
6.9%, which demonstrated that the present method was reproducible and accurate for determining biogenic amines and their
acidic metabolites in urine samples.

3.4. Method application

The analytical conditions were optimized for the determination of trace amounts of biogenic amines and their metabolites
in human urine. In this study, the GC oven temperature program
was optimized to sufciently separate the target compounds from
interfering peaks. The GC column length was also tested for the
separation of target analyte derivatives. The 15 m capillary column
showed better separation efciency for the biogenic amines and
their metabolite derivatives than the 30 m capillary column (see
Supplementary Fig. 1). Furthermore, the overall detection sensitivity of the biogenic amines and their metabolite derivatives obtained
by the 15 m column was much higher than that of the 30 m capillary
column. Thus, the use of a 15 m column for GC/MS provided more
rapid analyses, better sensitivity, and increased separation power
than using a 30 m column.
Following the method developed and described above, human
urine samples were extracted, puried, derivatized, and analyzed.
For the sensitive detection by GC/MS SIM, two abundant ions for
each biogenic amine and metabolite were selected for quantication and peak conrmation. The base peak for each compound
was typically used for quantication in SIM mode (Table 1). However, in certain cases, the base peak ion or second abundant ion
chromatograms could be interfered with other matrix peaks in the
SIM chromatogram. For example, the ion chromatogram of the base
peak at m/z 179 for dopac-(OTMS)3 exhibited higher baseline and

overlapped with other peaks, as shown in Fig. 7A. To eliminate

any possible interferences, an appropriate abundant ion (m/z 384),
instead of the base peak at m/z 179, was selected for quantitation
for the SIM analysis. As another example, the ion chromatogram
of m/z 193 as the second abundant ion for DA-(OTMS)2 ,-NHFBA
derivative exhibited a high baseline (Fig. 7B). By the selection of a
higher-mass ion at m/z 493 instead of m/z 193 or 179, the peak could
be clearly detected, providing positive the peak identication and
the precise quantication of DA. Fig. 7 shows two components that
were baseline separated from other co-extractive interferences by
the selection of appropriate higher mass ions. Although the peak
intensities of the two components were reduced by selecting the
appropriate abundant ions, the selectivity and specicity were signicantly improved.
The SIM chromatograms and extracted ion chromatograms of
the acidic metabolite-OTMS derivatives (Fig. 8) and biogenic amineOTMS,-NHFBA derivatives (Fig. 9) from human urine samples were
obtained using the developed method. The peak abundance of the
phenanthrene-d10 was used to estimate the overall recovery of
the analytes from the urine sample using the analytical procedure.
The deuterium-labeled HVA-d5 and DA-d3 compounds were used
as internal standards of the acidic metabolites and the biogenic
amines, respectively, which improved the precision and accuracy
of the quantication. In practice, the LOQs of most target analytes
were at least 10-fold lower than the clinically signicant concentrations [9,29,30]. In view of the recoveries and sensitivities of
the target analytes and the removal of interferences, the MCX SPE
cleanup and selective derivatization approach was effective for the
reliable proling analysis of bioamines and their acidic metabolites
in human urine.

4. Conclusion
A comprehensive analytical method for the determination of
bioamines and their acidic metabolites in urine was developed
using selective derivatization, MCX SPE cleanup and GC/MS in SIM
mode. A selective derivatization using HMDS/MBHFBA reagents
provided improvements in the chromatographic properties and
MS detection sensitivity without any side product formation, thus
enabling the reliable quantication of bioamines and their acidic
metabolites in human urine by GC/MS. The sample cleanup of

N.-H. Park et al. / J. Chromatogr. A 1305 (2013) 234243

human urine was effectively performed with an MCX SPE procedure

using methanol eluent for the acidic metabolites and 5% NH4 OH in
methanol eluent for the biogenic amines. The established method
enabled the effective extraction, purication and selective derivatization of both biogenic amines and acidic metabolites in urine.
The specicity of SIM mode through the selection of appropriate
ions provided an improvement in the analytical performance in
terms of linearity and sensitivity by the increase in the signal-tonoise ratios. The development of a reproducible analytical method
and the incorporation of isotopically labeled internal standards led
to improvements in accuracy and precision. Thus, this proposed
method can be used for the reliable diagnosis of neuronal diseases
through the measurement of both biogenic amines and their acidic
metabolites in biological samples, allowing for further applications
of clinical tests of neuronal diseases due to metabolic alterations.
Acknowledgments
This study was nancially supported by the National Research
Foundation of Korea (Development of analytical method for proling analysis of biogenic amines and their metabolites in biological
samples, No. 2011-0012671).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.chroma.2013.07.003.
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