2,3,4,5-Tetrakis(3′,4′-dihydroxylphenyl)thiophene: A new matrix for the selective analysis of low molecular weight amines and direct determination of creatinine in urine by MALDI-TOF MS

Article abstract of DOI:10.1021/ac3021278

Small organic matrixes are still the most commonly used ones in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) because of their advantages of high sensitivity, convenience, and cost-effectiveness. However, due to the matrix interference in the low mass region, the direct analysis of low molecular weight amines in complex surroundings with conventional organic matrixes remains a challenge. Here, a new Br?nsted-Lowry acid compound 2,3,4,5-tetrakis(3′,4′- dihydroxylphenyl)thiophene (DHPT) was designed, synthesized, and applied as a matrix for analysis of low molecular weight amines by MALDI-TOF MS. DHPT displays good selectivity in the analysis of amines without matrix-related interference and the low picomole/femtomole limit-of-detection was obtained in positive ion mode. With DHPT, the metabolites including creatinine, glycine, alloxan, allantoin, and 3-hydroxyhippuric acid in human urine were directly analyzed by MALDI-TOF MS. The identity of these metabolites was confirmed by tandem mass spectrometry. Furthermore, the urine creatinine was quantitatively determined using isotope-labeled internal standard. This DHPT-assisted LDI MS method provides a general approach for both qualitative and quantitative analysis of low molecular weight amines.

Full text of DOI:10.1021/ac3021278

Article
pubs.acs.org/ac
2,3,4,5-Tetrakis(3′,4′-dihydroxylphenyl)thiophene: A New Matrix for
the Selective Analysis of Low Molecular Weight Amines and Direct
Determination of Creatinine in Urine by MALDI-TOF MS
Suming Chen,† Li Chen,‡ Jianing Wang,† Jian Hou,† Qing He,† Jian’an Liu,§ Jiyun Wang,†
Shaoxiang Xiong,§ Guoqiang Yang,‡ and Zongxiu Nie*,†,§
†Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of
Chemistry, Chinese Academy of Sciences, Beijing 100190, China
‡Beijing National Laboratory for Molecular Sciences, Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China
§Beijing Center for Mass Spectrometry, Beijing 100190, China
S
* Supporting Information
ABSTRACT: Small organic matrixes are still the most
commonly used ones in matrix-assisted laser desorption/
ionization mass spectrometry (MALDI MS) because of their
advantages of high sensitivity, convenience, and cost-
effectiveness. However, due to the matrix interference in the
low mass region, the direct analysis of low molecular weight
amines in complex surroundings with conventional organic
matrixes remains a challenge. Here, a new Brønsted−Lowry
acid compound 2,3,4,5-tetrakis(3′,4′-dihydroxylphenyl)-
thiophene (DHPT) was designed, synthesized, and applied
as a matrix for analysis of low molecular weight amines by MALDI-TOF MS. DHPT displays good selectivity in the analysis of
amines without matrix-related interference and the low picomole/femtomole limit-of-detection was obtained in positive ion
mode. With DHPT, the metabolites including creatinine, glycine, alloxan, allantoin, and 3-hydroxyhippuric acid in human urine
were directly analyzed by MALDI-TOF MS. The identity of these metabolites was confirmed by tandem mass spectrometry.
Furthermore, the urine creatinine was quantitatively determined using isotope-labeled internal standard. This DHPT-assisted
LDI MS method provides a general approach for both qualitative and quantitative analysis of low molecular weight amines.
rapid, high through-put, and sensitive analytical method for
ow molecular weight (LMW) amines (<1000 Da) are a
Llarge family of compounds which play important roles in detection and analysis of LMW amines and their relatives is
very necessary.
physiological processes, medicament, and industrial applica-
tions. Endogenous LMW amines in living organisms (e.g.,
amino acids, oligopeptides, nucleobase, biogenic amines, and
their derivatives) are closely related to their physiological
activities and healthy level, and the monitoring of the amines
metabolites is essential for metabolomics research1 and disease
diagnostics.2,3 The exogenous LMW amines (e.g., synthetic
amine drugs, aromatic amines dyes) are also very important and
widely used in our daily life, but the abuse or exposure to these
compounds could harm our health. For instance, β-agonists are
a group of synthetic phenylethanolamines drugs, which were
illegally used in animal feed because they could improve growth
rate and reduce carcass fat.4,5 However, β-agonists accumulated
in animal tissues can cause acute poisoning when consumed by
humans.6−8 Melamine is another example of abuse, which was
found in pet food and milk products and induced renal failure
and even death in pets and several infants.9,10 Aromatic amines
and their derivatives are also a type of LMW amines which were
regarded as carcinogens, and human exposure to them may
have the risks of inducing cancer.11 Due to all these facts, a
Matrix-assisted laser desorption/ionization mass spectrome-
try (MALDI MS)12−14 seems the suitable technique that can
meet these demands. However, the application of MALDI MS
in LMW molecules analysis is still limited. The main reason is
that the background interference of conventional small organic
matrixes15−17 can suppress and overlap with the analyte signals
in the low mass region (<500 Da) very much. To overcome this
problem, different approaches were proposed to minimize the
low mass background, for example, the matrix-free18−21 and
material-enhanced methods, such as titania particles,22,23 silicon
nanoparticles,24 and carbon based materials (e.g., graphite,25,26
carbon nanotubes,27,28 fullerene,29 graphene30,31). These
materials can give low background noise at LMW region and
produce signals for some analytes, but their sensitivity is
relatively low compared to that of the conventionally used
Received: August 7, 2012
Accepted: November 1, 2012
Published: November 1, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of the Matrix 2,3,4,5-Tetrakis(3′,4′-dihydroxylphenyl)thiophene (DHPT)
matrixes. At the same time, the ion suppression effect is still a
big problem in the analysis of complex samples due to their
poor selectivity.32−34 On the other hand, finding new organic
matrixes35,36 is still widely performed because of the
accessibility, high sensitivity, and excellent performance in
MALDI MS imaging.37−39 Although there are so many efforts,
efficient matrixes for LMW amines analysis by MALDI MS are
very scarce. To the best of our knowledge, only 2-
TOF MS. Creatinine is an end product of muscle metabolism,
and its concentration in urinary excretion is an important
diagnostic index for renal, muscular, and thyroid function.40
Compared with previously reported spectrophotometric,41
enzymatic,42 liquid chromatographic (LC),43 LC-MS/MS,44
and electrochemical methods,45 DHPT-assisted LDI MS
displayed advantages of simplicity, rapidness, interference
tolerance, and high through-put. This method may provide a
general approach for both qualitative and quantitative analysis
LMW amine metabolites in complex biological system.
̌
naphthalenesulfonic acid was mentioned by Svatos for the
analysis of several amines previously;35 hence the exploration of
new matrixes is necessary for the wider applications.
Herein, a new Brønsted−Lowry acid compound 2,3,4,5-
tetrakis(3′,4′-dihydroxylphenyl)thiophene (DHPT, Scheme 1)
was designed and synthesized as the matrix of MALDI. It
contains four catechol units with thiophene as the core, and
exhibits good performance for analysis of LMW amines in
positive ion mode. Compared with a conventional matrix (e.g.,
α-cyano-4-hydroxycinnamic acid, CHCA), DHPT exhibits a
clean background in the low mass range (Figure 1), which
EXPERIMENTAL SECTION
■
Materials. The isotope internal standard creatinine-
(methyl-13C) was purchased from Cambridge Isotope Labo-
ratories, Int. (Andover, MA) and used as received. Other
chemicals were purchased from Sigma-Aldrich (St. Louis, MO)
unless otherwise stated. The chemicals were used without
further purification. Solvents were purchased and were at least
analytical grade. The deionized water was obtained from a
Milli-Q system (Millipore, Billerica, MA).
Synthsis of DHPT. The matrix can be readily synthesized
by a two-step reaction starting from a Suzuki coupling reaction
(Scheme 1), and the detailed synthesis steps and character-
ization data are described in Supporting Information.
Matrix and Sample Preparation. The stock solutions of
matrix DHPT (10 mg/mL) were prepared in ethanol
containing 0.1% trifluoroacetic acid (TFA, v/v) and stored in
refrigerator at 4 °C. The usage period of the solution is less
than a week. CHCA and DHB matrixes were prepared as a
saturated solution in CH3CN/water (2:1, v/v) containing 0.1%
TFA. Stock solutions of all the analytes were made in respective
analytical and HPLC grade solvents at 5 mM. Typically, 1 μL of
the analyte was premixed with 1 μL of the matrix in a centrifuge
tube, and then 1 μL of the resulting mixture was pipetted on
the MALDI target plate and air-dried for MALDI-TOF MS
analysis. The solution of DHPT was diluted for the low
concentration of analytes, and let the molar ratio of matrix to
analyte become less than 500.
Figure 1. Positive ion LDI-TOF mass spectra of (A) α-cyano-4-
hydroxycinnamic acid and (B) DHPT. 90 pulsed laser shots were
applied under a laser fluence set at 60 and 80 μJ for CHCA and
DHPT, respectively.
Mass Spectrometry Analysis. MALDI-TOF MS analysis
was performed on a Bruker BIFLEX III mass spectrometer
(Bruker Daltonics, Germany) equipped with a nitrogen laser
(337 nm wavelength, 3 ns laser pulse duration) with reflectron
and positive ion mode. The laser power energy was adjusted
between 0% and 100% to provide laser pulse energy between 0
and 100 μJ per pulse. The mass spectra were typically recorded
at an accelerating voltage of 19 kV and a reflection voltage of 20
kV with the laser pulse energy of 80 μJ. Unless otherwise stated,
each mass spectrum was acquired as an average of 90 laser shots
at 1 Hz frequency. A mixture of PEG 200 and PEG 600 was
used for positive mode mass calibration.
renders it capable of giving reliable analysis results for LMW
molecules. It exhibits good selectivity to amines over other
types of compounds, which could effectively avoid the ion
suppression effect32−34 and interference from the analytes in
MALDI MS. A wide range of LMW amines were attempted and
analyzed using DHPT as matrix, such as amino acids, peptides,
vitamine B, β-agonists, and alkaloid and aromatic amines, and
the low femtomole limit-of-detection (LOD) was obtained.
Finally, DHPT was applied for the analysis of the amine
metabolites in urine. Moreover, the creatinine was quantitative
determined with isotope internal standard method by MALDI-
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Health safety notes: DHPT could be harmful if swallowed.
Use gloves and eye protection when working with this
substance. In case of contact with the eyes, rinse immediately
with plenty of water and seek medical advice.
RESULTS AND DISCUSSION
■
Characterization of the New Matrix. A Brønsted−Lowry
acid is a molecule or ion that is able to lose, or “donate”, a
proton. As shown in Scheme 1, DHPT is a Brønsted−Lowry
acid that possesses a structure of eight phenol hydroxyls, and
thus is anticipated to transfer proton into alkaline amines in the
ionization process. The UV−vis absorption spectrum of DHPT
was measured both in ethanol solution and in the solid state
(Figure S1 in Supporting Information). The DHPT molecule
has a large molar extinction coefficient of ε = 1.5 × 104 M−1
cm−1 at the wavelength of 337 nm in ethanol solution, which
facilitated strong absorption of the photon energy and efficient
energy transfer to the analytes in MALDI-TOF MS. The solid
state absorption spectrum of DHPT has a very similar pattern
to that in solution, except the slight blue shift of maximum
absorption wavelength from 297 to 292 nm. The DHPT shows
a good solubility in ethanol (about 15 mg/mL at room
temperature), and ethanol is a good solvent for analyte−matrix
cocrystallization to yield the strongest ion signals.
Figure 1 shows the LDI-TOF mass spectra of CHCA and
DHPT in positive ion mode. As compared with CHCA, DHPT
gives a simple mass spectrum at low mass range. Except the
[Na]+ and [K]+ at m/z 23.0 and 39.0, only a single peak at m/z
516.1 corresponding to the DHPT radical cation was observed.
The deprotonated ion at m/z 515.1 was also produced in the
negative ion LDI-TOF mass spectrum with a clean background
(Figure S2). Therefore, this new matrix can avoid the
background interference in the low mass range (m/z < 500),
which is the key obstacle of conventional matrixes for analyzing
LMW molecules. The acidic nature and easily formed
deprotonated ion of DHPT implies the proton transfer from
matrix to analyte may occur before or during the laser
desorption process.35
Analysis of Amino Acids and Peptides. Amino acids are
the basic structural units that make up proteins. Here, four
common amino acids arginine (Arg), histidine (His), leucine
(Leu), and tryptophan (Trp) were first chosen as representa-
tives for MALDI-TOF MS analysis by employing DHPT as the
matrix (Figure 2). As can be seen, hardly any of the matrix-
related background ions were produced in this condition.
Protonated signals were observed for each amino acid, which
indicates the good performance of DHPT. Just like the previous
reports,18,30 the multiple alkali metal adduct ions (m/z 197.1,
[Arg + Na]+; m/z 178.0, [His + Na]+; m/z 154.1, [Leu + Na]+;
m/z 170.0, [Leu + K]+; m/z 176.1, [Leu − H + 2Na]+; m/z
192.0, [Leu − H + Na + K]+; m/z 227.1, [Trp + Na]+; m/z
243.0, [Trp + K]+; m/z 249.0, [Trp − H + 2Na]+; m/z 265.0,
[Trp − H + Na + K]+) were also detected. Good sensitivity was
obtained for amino acid and peptide analysis. The LOD of Arg
and the dipeptide glycine-histidine (Gly-His) is lower than 2.5
pmol (S/N = 5:1, Figure S3A,B in Supporting Information).
In addition, the analyses of oligopeptides and polypeptides
were attempted. As shown in Figure S4A, the oligopeptides
Gly-Arg-Gly-Asp-Thr-Pro (MW. 601.3) and Tyr-Leu-Glu-Asp-
Tyr (MW. 701.3), even the 10 peptides and 13 peptides (Thr-
Leu-Ser-Arg-Arg-Thr-Arg-Phe-His-Thr, MW. 1273.7; Gly-Phe-
Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys, MW. 1603.0), were
clearly detected as [M + H]+ and alkali metal adducts ions.
Figure 2. MALDI-TOF mass spectra for different amino acids with
DHPT as matrix: (A) arginine m/z 175.1, [M + H]+; m/z 197.1, [M +
Na]+; (B) histidine m/z 156.1, [M + H]+; m/z 178.0, [M + Na]+; (C)
leucine m/z 132.1, [M + H]+; m/z 154.1, [M + Na]+; m/z 170.0, [M +
K]+; m/z 176.1, [M − H + 2Na]+; m/z 192.0, [M − H + Na + K]+;
(D) tryptophan m/z 205.1, [M + H]+; m/z 227.1, [M + Na]+; m/z
243.0, [M + K]+; m/z 249.1, [M − H + 2Na]+; m/z 265.0, [M − H +
Na + K]+. The amounts are 2.5 nmol.
Subsequently, the peptides mixture from tryptic digestion of β-
casein was also subjected to MALDI-TOF MS analysis, and a
maximum signal at m/z 1532.0 was obtained (Figure S4B).
Meanwhile, DHPT exhibits good analytical capability for the
mixture solution of Arg, His, and Gly-Pro-Arg-Pro tetrapeptide
when compared with CHCA and DHB. Figure S5 indicates that
both the amino acids and the peptide could be analyzed as [M
+ H]+ ions with DHPT, CHCA, and DHB, but the background
interferences of CHCA and DHB may confuse the identi-
fication. Only DHPT-assisted laser desorption/ionization mass
spectrum gives distinctive target peaks without interferences.
Analysis of Vitamins B. Vitamins B (VB) are a group of
water-soluble vitamins that play important roles in cell
metabolism. All of the VB compounds belong to amines that
we anticipate being protonated by DHPT in MALDI process.
The protonated positive ions were indeed observed for VB
compounds (Figure 3) except for thiamine (VB1, Figure 3A),
which is already a cation. The fragmentation of C−N bond
between ethyl and nitrogen cation in VB1 is observed, and gives
two fragment peaks at m/z 122.1 and 144.0 (Figure S6A). In
the meantime, the fragmentation of the C−N bond also occurs
for riboflavin (VB2, Figure S6B), and the elimination of 2,3,4,5-
tetrahydroxyl-1-pentene molecule leads to the formation of
little ion at m/z 243.1. Nevertheless, intact protonated signals
were obtained for nicotinamide (VB3, m/z 123.0, Figure 3C)
and pyridoxine (VB6, m/z 170.1, Figure 3D), which indicates
the fragmentation mainly depends on the unique structure of
individual molecule. The detection of VB3 is lower than 25
pmol (S/N = 5:1, Figure S3C).
Analysis of β-Agonists. As described above, the fast
detection of β-agonists is necessary due to the abuse. So, three
of most commonly used β-agonists, ractopamine, sulbutamol,
and terbutaline, were chosen as representatives to test the
applicability of this DHPT-assisted MALDI-TOF MS method.
Figure 4 shows the mass spectra of the three compounds with
DHPT as the matrix. Each of them was detected as the [M +
H]+ ions (ractopamine, m/z 302.2; sulbutamol, m/z 240.2;
terbutaline, m/z 226.1) with little background interference, and
the cation peak of DHPT (m/z 516.1) was deeply suppressed.
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(Figure S8A). This further indicates that the fragmentation
behaviors were mainly ascribed from the frangibility of
compounds in the energy transfer process. Nevertheless,
other interference ions were present while CHCA was used.
By using DHPT, matrix-related ion interference was nearly
eliminated, so DHPT is a suitable matrix for the detection of β-
agonists.
Analysis of Other LMW Amines. The use of DHPT for
the desorption/ionization of analytes was further investigated
using other types of amines. Guanine is one of the four main
nucleobases found in the nucleic acids DNA and RNA; the
protonated ion, sodium adduct, and potassium adduct ions
were observed in the mass spectrum (Figure 5A). Likewise, the
Figure 3. MALDI-TOF mass spectra for vitamins B compounds with
DHPT as matrix: (A) thiamine m/z 265.1, [M]+; m/z 122.1 and 144.0
corresponds to the fragmentation products of C−N bond between
ethyl and nitrogen cation; (B) riboflavin m/z 377.1, [M + H]+; m/z
399.1, [M + Na]+; m/z 415.1, [M + K]+; m/z 243.1 corresponds to
loss of one 2,3,4,5-tetrahydroxyl-1-pentene molecule from [M + H]+,
(C) nicotinamide m/z 123.0, [M + H]+, (D) pyridoxine m/z 170.1,
[M + H]+. The amount of each compound is 2.5 nmol.
Figure 5. MALDI-TOF mass spectra of (A) guanine m/z 152.0, [M +
H]+; m/z 174.0, [M + Na]+; m/z 190.0, [M + K]+; (B) dopamine m/z
154.1, [M + H]+; m/z 137.1, [M + H − NH3]+; (C) caffeine m/z
195.1, [M + H]+; m/z 179.0, [M + H − CH4]+; (D) melamine m/z
127.1, [M + H]+; (E) 3,3′-diaminobenzidine: m/z 214.1, [M]·+; (F) p-
toluidine m/z 107.1, [M]·+ with DHPT as matrix. The amounts are 2.5
nmol.
important neurotransmitter dopamine was also detected as a
protonated ion (Figure 5B), as well as the ion of loss of amine
group (Figure S9A). But this is not the case for caffeine, which
is a common alkaloid that usually acts as a stimulant drug.
Figure 5C shows the sensitive detection result of caffeine using
MALDI-TOF MS. Interestingly, the ion of loss of one CH4 (m/
z 179.0) from protonated ion is the predominant peak with a
S/N of about 505, and [M + H]+ ion is relatively low (S/N =
30) in the mass spectrum. The possible fragmentation
mechanism was proposed in the Supporting Information
(Figure S9B).
In addition, melamine and aromatic amine compounds were
also analyzed by this method. As shown in Figure 5D,
melamine can be sensitively detected as [M + H]+ using
MALDI MS with DHPT matrix, and the LOD is lower than 10
pmol (S/N = 5:1, Figure S3D). However, strong background
interferences were present while CHCA was used as the matrix
(Figure S10 in Supporting Information), which greatly
obscured the identification of melamine due to the peak at
m/z 128.1. Aromatic amines and their derivatives are regarded
as carcinogens, and human exposure to them may have the risks
of inducing cancer.11 Herein, 3,3′-diaminobenzidine and p-
toluidine were selected as representatives to test the detection
performances of DHPT to aromatic amines. As shown in
Figure 5E,F, both of the two compounds exhibit radical cation
signals in the MALDI-TOF mass spectra. The molecular ion
Figure 4. MALDI-TOF mass spectra for 2.5 nmol of β-agonists with
DHPT as matrix: (A) ractopamine m/z 302.2, [M + H]+; m/z 286.3,
[M + H − CH4]+; (B) sulbutamol m/z 240.2, [M + H]+; m/z 222.1,
[M − H2O + H]+; m/z 166.1, [M − H2O − CCH2(CH3)2 + H]+; m/z
148.1, [M − 2H2O − CCH2(CH3)2 + H]+; (C) terbutaline m/z 226.1,
[M + H]+; m/z 152.1, [M − H2O − CCH2(CH3)2 + H]+. Peaks at m/
z 516.1 denote the radical cation of DHPT.
In addition, few fragment ion peaks were also observed due to
the in-source decay of relatively weak chemical bonds of
analytes, especially the elimination of water (m/z 222.1, m/z
148.1) and tertiary carbon group (m/z 166.1, m/z 152.1). In
fact, the fragment ions could be the supplemental evidence for
the identification of the analytes with no need for MS/MS
analysis. The possible mechanisms of the fragmentation were
proposed in Figure S7.
Subsequently, the mixture solution of β-agonists containing
ractopamine, sulbutamol, and terbutaline was also subjected to
MALDI-TOF MS analysis using CHCA and DHPT as matrix,
respectively. As shown in Figure S8, all of the three compounds
can be sensitively detected with their protonated signals, and
the similar fragmentation patterns (e.g., the break of C−N
bonds) were observed using these two matrixes, but the
mixture themselves keep intact in the absence of matrixes
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Figure 6. MALDI-TOF mass spectrum of LMW metabolites in urine in low mass range. The identified peaks and possible metabolites were labeled
in the spectrum.
may ascribe to the loss of one electron from the lone pair
electron of nitrogen. In the analysis of the mixture solution of
3,3′-diaminobenzidine and p-toluidine, CHCA was employed
again for comparison (Figure S11). As expected, DHPT
displayed good analytical results over CHCA with very little
background interference.
To further investigate the analytical performance of DHPT
as a matrix to aliphatic amines, triethylamine, diisopentylamine,
tributylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
were selected. As shown in Figure S12, all of the four small
amines gave [M + H]+ signal without interference ions, and the
LOD of DBU is less than 500 fmol.
It must be pointed out that the analysis of some small
molecules using MALDI MS with a conventional organic matrix
(e.g., CHCA) sometimes can also be achieved under certain
elaborate experimental conditions, such as carefully choosing
molar ratio of matrix and analyte. But the slight change of the
condition may cause the matrix-related interference (Figures
S5, S8, S10, and S11 in Supporting Information). However, this
problem can be avoided by desorption/ionization mass
spectrometry with DHPT as the matrix, with no need to
worry about the interference peaks in the low mass range. For
the MALDI MS analysis of LMW amines compounds, sensitive
and distinct results can be obtained with the assist of DHPT
matrix.
Selectivity of the Matrix. According to the Brønsted−
Lowry acid−base theory, phenol is a typical Brønsted−Lowry
acid, so the polyphenol compound DHPT is also a Brønsted−
Lowry acid. It has high tendency to donate protons in the
presence of Brønsted−Lowry base (e.g., amines). Thus, we
anticipated that DHPT should display selectivity to amines. In
order to further explore the supposal, four types of compounds
dibenzylamine, benzophenone, 1-(benzyloxymethyl)benzene,
and diphenylmethanol were tested using DHPT as matrix. As
shown in Figure S13, only dibenzylamine gives distinct
protonated ion peak at m/z 198.1, which shows the selectivity
of DHPT to amine. In fact, other compounds including glucose
(Figure S13E), fatty acids, and lipids were also tested in our
experiments, and only amines can give related signals.
Therefore, DHPT could also be used as a matrix for the
selective analysis of LMW amines.
Analysis of LMW Metabolites in Urine. As described in
the above experiments, DHPT was proved to be a quite
efficient candidate matrix for the analysis of a wide range of
LMW amines in MALDI-TOF MS. This feature implies the
potential application of the matrix in metabolomics research.
Here, the human urine was taken as an example to test the
applicability of DHPT, because it is an important component of
human metabolome. Numerous small molecules including
amines were involved in the urine metabolites in the excretory
process, and some can indicate the pathological change. To
date, the direct MALDI MS analysis of metabolites in urine
remains a challenge due to the lack of suitable matrix in low
mass range.
In this experiment, fresh human urine from a male volunteer
was collected and first diluted with a 2-fold amount of water,
and then centrifuged to remove the solid particles. No other
pretreatment is needed. Subsequently, the urine sample was
subjected to MALDI-TOF MS analysis with DHPT as matrix.
Figure 6 shows the analytical results of the sample. Most of the
peaks are located at low mass range of m/z < 200, and agrees
with previous NASA contractor report of human urine
composition.46 The identity of the peaks was confirmed by
collision-induced dissociation (CID) experiments (see Support-
ing Information) and by comparison of the MS/MS spectra
observed to those reported in Human Metabolome Database
(HMDB).47 It needs to be pointed out that the peaks are not a
one-to-one correspondence between ESI (Figure S14) and the
MALDI-TOF mass spectra, because of the different ionization
methods and the ion suppression effects in electrospray
ionization. However, peaks of the main metabolites could
undoubtedly match together with protonated and alkali metal
adducts forms. Ten peaks in MALDI-TOF mass spectrum were
identified through CID experiments (Figure 6 and Figure S14,
Table S1), including the high concentration metabolites urea
and creatinine and their alkali metal adduct ions (m/z 60.0,
83.0, 99.0 and m/z 114.1, 136.0, 152.0). Besides these, other
metabolites of relatively low concentration were also observed
([Gly + H]+ at m/z 76.0; [alloxan + H]+ at m/z 143.0;
[allantoin + H]+ at m/z 159.0; [3-hydroxyhippuric acid + H]+
at m/z 196.1) and confirmed. In addition, some of the
unidentified peaks were further sought for confirmation from
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previous reports and HMDB, and the possible metabolites were
listed in Table S2 in Supporting Information.
the analytical demand of creatinine in healthy and abnormal
human urine samples.48 Furthermore, the addition of internal
standards can efficiently avoid the matrix effects, which allows
us to precisely quantify the level of creatinine in the biological
medium. With the three calibration curves, creatinine in three
untreated human urine samples was directly determined with
an error within 3% (Table 1). The results show that the
creatinine concentrations of these samples are in the normal
healthy range. The relative standard deviation among the three
groups of determination results are very low (<2%), which
indicates the availability of any one of the curves. The
determined results were also compared with those provided
by two-point Jaffe’s kinetic method.41 It is shown that the
values measured by the proposed DHPT-assisted LDI MS
method are in good agreement with the data provided by Jaffe’s
method within 5% error, demonstrating the great potential for
practical application of this rapid and high through-put method
for the clinical creatinine detection.
Quantitative Determination of Creatinine. The DHPT
matrix was also adopted for the direct determination of
metabolites in urine, and creatinine was taken as an example
here. The urine samples were collected without any pretreat-
ment, and the isotope creatinine-(methyl-13C) was added as an
internal standard. As shown in Figure 7, both creatinine and
CONCLUSIONS
■
In summary, a new compound DHPT was designed,
synthesized, and applied as an efficient MALDI matrix to
analyze a wide range of low molecular weight amines. These
results demonstrate the great advantages of DHPT over other
conventional matrixes, which include sensitivity, selectivity,
wider applicability, and interference-free background in low
mass range MS. Further experiments show its great perform-
ance in quantitative MS analysis of creatinine in urine. The new
matrix is ready to be used directly and can be extended to
analyze low molecular weight amines in other biological
systems, and may play an important role in metabolomics
and MALDI MS imaging. Further applications are currently in
progress in our laboratories.
Figure 7. Typical MALDI-TOF mass spectrum of for direct detection
of creatinine in urine with DHPT as matrix: m/z 114.1, [creatinine +
H]+; m/z 115.1, [creatinine-(methyl-13C) + H]+; m/z 136.0,
[creatinine + Na]+; m/z 137.0, [creatinine-(methyl-13C) + Na]+; m/
z 152.0, [creatinine + K]+; m/z 153.0, [creatinine-(methyl-13C) + K]+.
Other peaks including m/z 114.9 are ascribed from other components
in urine. Inset is the calibration curves used for quantitative
determination of creatinine according to the ion intensity ratio of
+
13
+
■
[creatinine + H] and [creatinine-(methyl- C) + H] ( ), [creatinine
+
13
+
+
●
+ Na] and [creatinine-(methyl- C)+Na] ( ), and [creatinine + K]
and [creatinine-(methyl-13C) + K]+
(
). The concentrations of
▲
creatinine are 0, 1, 5, 10, 20, 30, 40, and 50 mM, and the internal
standard added is 10 mM. The contributions from the natural isotopes
of creatinine to the internal standard at m/z 115.1, 137.0, and 153.0
were subtracted.
ASSOCIATED CONTENT
* Supporting Information
Synthesis of DHPT, analytical methods of urine sample, and
additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
creatinine-(methyl-13C) gave signals of [M + H]+, [M + Na]+,
and [M + K]+ in good signal-to-noise ratio, which provides
several options for quantitation. The peak at m/z 114.9 from
unknown substance in urine has a little superposition with m/z
115.1 form the isotope internal standard, but it may not be the
interference for quantification because of the enough mass
resolution. Three calibration curves (see the inset of Figure 7)
were plotted according to the ion intensity ratio of [creatinine
+ H]+ and [creatinine-(methyl-13C) + H]+, [creatinine + Na]+
and [creatinine-(methyl-13C) + Na]+, and [creatinine + K]+ and
[creatinine-(methyl-13C) + K]+ with the regression equations of
y = 0.0993x − 0.0025, y = 0.0930x + 0.0337, and y = 0.0984x +
0.0147, respectively. Good linearity was obtained with γ2 >
0.999 in the concentration range 1−50 mM, which could meet
AUTHOR INFORMATION
Corresponding Author
*E-mail: znie@iccas.ac.cn. Phone/Fax: +86-10-62652123.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from Chinese Academy of
Sciences and the National Natural Sciences Foundation of
China (Grants 21127901, 20927006, 21175139, and
21205123).
Table 1. Determination Results of Creatinine in Three Urine Samples Using the Developed Method
a
b
c
sample no.
[M + H]+ (mM)
[M + Na]+ (mM)
[M + K]+ (mM)
RSD (%)
Jaffe’s method (mM)
1
2
3
6.28 0.18
6.90 0.11
6.18 0.12
6.32 0.12
6.97 0.09
6.21 0.14
6.17 0.13
6.78 0.15
6.06 0.10
1.24
1.40
1.29
6.36 0.08
6.98 0.06
6.32 0.08
a
b
Concentration determined by the intensity ratio of [creatinine + H]+ and [creatinine-methyl-13C + H]+. Concentration determined by the
c
intensity ratio of [creatinine + Na]+ and [creatinine-methyl-13C + Na]+. Concentration determined by the intensity ratio of [creatinine + K]+ and
[creatinine-methyl-13C + K]+.
10296
dx.doi.org/10.1021/ac3021278 | Anal. Chem. 2012, 84, 10291−10297

Analytical Chemistry
Article
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