DBDA as a Novel Matrix for the Analyses of Small Molecules and Quantification of Fatty Acids by Negative Ion MALDI-TOF MS

Article abstract of DOI:10.1007/s13361-017-1881-y

Matrix interference ions in low mass range has always been a concern when using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze small molecules (<500 Da). In this work, a novel matrix, N1,N4-dibenzyli

Full text of DOI:10.1007/s13361-017-1881-y

B American Society for Mass Spectrometry, 2018
J. Am. Soc. Mass Spectrom. (2018)
DOI: 10.1007/s13361-017-1881-y
RESEARCH ARTICLE
DBDA as a Novel Matrix for the Analyses of Small
Molecules and Quantification of Fatty Acids by Negative Ion
MALDI-TOF MS
Ling Ling,1 Ying Li,1 Sheng Wang,1 Liming Guo,1 Chunsheng Xiao,2 Xuesi Chen,2
Xinhua Guo1,3
1State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, 130012,
China
2Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun,
130022, China
3Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, College of Life Science, Jilin
University, Changchun, 130012, China
Abstract. Matrix interference ions in low mass range has always been a concern
when using matrix-assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF MS) to analyze small molecules (<500 Da). In this work, a novel
matrix, N1,N4-dibenzylidenebenzene-1,4-diamine (DBDA) was synthesized for the
analyses of small molecules by negative ion MALDI-TOF MS. Notably, only neat ions
([M–H]-) of fatty acids without matrix interference appeared in the mass spectra and
the limit of detection (LOD) reached 0.3 fmol. DBDA also has great performance
towards other small molecules such as amino acids, peptides, and nucleotide.
Furthermore, with this novel matrix, the free fatty acids in serum were quantitatively
analyzed based on the correlation curves with correlation coefficient of 0.99. In
addition, UV-Vis experiments and molecular orbital calculations were performed to explore mechanism about
DBDA used as matrix in the negative ion mode. The present work shows that the DBDA matrix is a highly
sensitive matrix with few interference ions for analysis of small molecules. Meanwhile, DBDA is able to precisely
quantify the fatty acids in real biological samples.
Keywords: MALDI-TOF MS, Matrix, Small molecules, Fatty acids, Quantification, Negative ion mode
Received: 29 May 2017/Revised: 18 December 2017/Accepted: 21 December 2017
TOF MS is still limited because the commonly used organic
matrices, such as ɑ-cyano-4-hydroxycinnamic acid (CHCA)
and 2,5-dihydroxybenzoic acid (DHB), always produce strong
matrix-associated ions in the low-mass range [6, 7], which
interfere or even suppress the analyte signals.
To solve this problem, efforts have been made to reduce the
interference ions in low-mass range. A number of inorganic
matrices, such as porous silicon [8], carbon based materials
(graphite, graphene, carbon nanotubes, carbon nanodots, etc.)
[9–13], and other metal nanoparticles [14] are reported continu-
ously. However, the synthesis of these materials could be com-
plicated or expensive. In addition, several organic matrices with
less interference ions have been developed for small molecules
analysis. For instance, 9-aminoacridine (9AA) [15], N-(1-
naphthyl) ethylenediamine dinitrate [16], N-(1-naphthyl)
ethylenediamine dihydrochloride (NEDC) [17], and 1,5-
Introduction
atrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) is a powerful
M
tool for the characterization of large biomolecules [1–5], such
as proteins, peptides, oligosaccharides, oligonucleotides, as
well as synthetic polymers because of its high throughput, soft
ionization, high salt tolerance, and high sensitivity. Neverthe-
less, the analysis of small molecules (<500 Da) by MALDI-
Electronic supplementary material The online version of this article (https://
doi.org/10.1007/s13361-017-1881-y) contains supplementary material, which
is available to authorized users.
Correspondence to: Chunsheng Xiao; e-mail: xiaocs@ciac.ac.cn, Xinhua Guo;
e-mail: guoxh@jlu.edu.cn

L. Ling et al.: MALDI Matrix for Small Molecules
diaminonaphthalene hydrochloride [18] were reported to be
efficient for analysis of various small molecules. Furthermore,
superbasic matrices such as 1,8-bis(dimethylamino)naphthalene
(DMAN) [19], 1,8-di(piperidinyl)naphthalene (DPN) [20] opti-
mized from DMAN, and azahelicene superbases [21] were
reported by Svatoš et al. The authors suggested that superbasic
matrices captured the proton from acidic analyte in solution
according to Brønsted-Lowry acid base theory during sample
preparation. They called this ionization process Bmatrix-assisted
ionization/laser desorption^ (MAILD) [22]. Those previous re-
searches indicated that the matrices function depends on both
properties of analytes and their own, and additional matrices for
MALDI-MS analysis of small molecules are still needed on one
hand to enhance analytical efficiency and sensitivity and on the
other hand to understand ionization mechanism of MALDI-MS.
In this study, we designed and synthesized six Schiff base
compounds and screened out a most efficient matrix, N,N′-
dibenzylidenebenzene-1,4-diamine (DBDA) for analysis of
small molecules. The performance of DBDA towards various
small molecules (amino acids, peptide, nucleotide, fatty acid)
was demonstrated by comparison with 9AA, DMAN, and
NEDC in the negative ion mode.
Free fatty acids (FFAs), either saturated or unsaturated
aliphatic carboxylic acids, serve as the raw materials of lipo-
protein, grease, phospholipid, and glycolipid or functional
compounds for human health or diseases [23–26]. Hence, with
DBDA in hand, we further explored its application in analysis
of FFAs in serum. Under optimized conditions, serum FFAs
were accurately quantified using DBDA matrix. Our results
demonstrated that DBDA is an excellent matrix for small
molecules, particularly for the analysis of fatty acids FAs.
The possible mechanisms related to great performance of
DBDA were discussed based on experiments and theoretical
calculation.
and benzaldehyde were mixed at the mole ratio of 1:3 in
anhydrous ethanol and the obtained solution was refluxed for
6 h at 90 °C. Then the mixture was treated with suction
filtration and recrystallization using anhydrous ethanol three
times (other Schiff base compounds were carried out using a
method similar to that used for DBDA); 1H NMR (500 MHz,
DMSO) and (ppm); 8.70 (s, 2H), 7.98–7.94 (m, 4H), 7.54 (dd,
J = 5.1, 2.1 Hz, 6H), 7.37 (s, 4H) (Supplementary Figure S1).
UV-Vis Analysis
The Schiff base compounds were prepared at concentration
of 4.5 μg/mL in methanol/chloroform (1:1, v/v), and 1 mL of
the matrix solution was transferred to cuvette for UV-Vis
absorption. A cuvette equipped with 1 mL of methanol/
chloroform (1:1, v/v) was used as the reference.
Matrix and Sample Preparation
The matrix DBDA was prepared at 10 mg/mL in methanol/
chloroform (1:1, v/v). 9AA and NEDC were dissolved in
isopropanol/acetonitrile (1:1, v/v) and water/acetonitrile (3:7,
v/v) respectively, both at 10 mg/mL. DMNA was prepared in
ethanol at the same concentration as the analytes. The stored
solutions of pure FAs were prepared at 5 mM in chloroform.
The amino acids, glutathione, and 5'-adenylic acid were dis-
solved in water at 1 mM. For MALDI-MS analysis, the analyte
and matrix solutions were mixed in equal volumes, and 0.4 mL
of the obtained solution was spotted on target plate and dried at
room temperature. For the quantitative analysis of FFAs in
serum, the Bseeds^ sample deposition method was adopted
[27]. First, matrix solution was diluted five times, and 0.4 μL
matrix was placed on the target as seeds. After solvent evapo-
ration, the 2 μL sample solution was mixed with 2 μL matrix
solution. Subsequently, 0.4 μL of the mixed solution was
placed on the pre-deposited crystal and followed by air drying
before MALDI-MS analysis.
Crude Extract of Serum FFAs
Experimental
Three hundred μL of freshly thawed serum solution was pipet-
ted into a centrifuge tube. Then 1 mL of methanol was added,
and the mixture was vortexed for perfect mixing and stored at –
18 °C overnight. The overall solution was centrifuged at
19,000 rpm for 30 min at 4 °C. Then the supernatant was
transfered into another centrifuge tube, to which 1 mL of n-
hexane and 1 mL of Milli-Q water were added. The resultant
mixture was vortexed for 5 min and centrifuged at 5000 rpm for
10 min. Finally, the hexane layer with the serum FFAs was
pipetted into another tube, air-dried, and then stored at –18 °C.
Chemicals and Reagents
Amino acids, glutathione, 5'-adenylic acid, standard FAs
including myristic acid (C14:0), palmitic acid (C16:0),
heptadecenoic acid (C17:1), stearic acid (C18:0), oleic acid
(C18:1), linoleic acid (C18:2), arachidic acid (C20:0), arachi-
donic acid (C20:4), behenic acid (C22:0), docosahexaenoic
acid (C22:6), 9AA, DMAN, NEDC, 1,4-diaminobenzene,
and benzaldehyde were purchased from J&K Chemical (Bei-
jing, China). The solvents, including chloroform, methanol,
isopropanol, acetonitrile, ethanol, all analytical grade, were
purchased from Beijing Chemicals (Beijing, China). The serum
samples were obtained from Dr. Yang Xu at the Jilin Univer-
sity. The water was made by Milli-Q system.
Quantification and Method Validation
Heptadecenoic acid (C17:1) was used as the internal standard
(IS) to improve quantitative accuracy [28]. Each standard FAs
was diluted to a series of concentrations and the FAs solutions
were mixed with IS (40 μg/mL). The obtained mixtures were
analyzed by MALDI-MS. The calibration curves of each FAs
were constructed by the intensity ratios of FAs to IS versus the
Synthesis of Schiff Base Compounds
The DBDA was synthesized by the Schiff base reaction
(Supplementary Scheme S1). Briefly, 1,4-diaminobenzene

L. Ling et al.: MALDI Matrix for Small Molecules
Figure 1. The structures and molecular weights of Schiff base compounds investigated in this study
concentrations of respective FAs. For the quantification of
FFAs in serum, crude extract of serum FFAs were dissolved
in 150 μL chloroform containing 40 μg/mL of IS and analyzed
by MALDI-MS. The concentrations of serum FFAs were cal-
culated based on the calibration curve equations of each stan-
dard FAs.
sample 2: C16:0, C18:0, C18:1, C18:2, and C20:4 with the
concentrations of 50 μg/mL, C22:0 and C22:6 with the
concentration of 10 μg/mL; sample 3: C16:0, C18:0,
C18:1, C18:2, and C20:4 with the concentrations of 100
μg/mL, C22:0 and C22:6 with the concentration of 20 μg/
mL). Then the FAs were extracted from the serum samples
according to the above-mentioned method.
The reproducibility was estimated by detection of the
same samples that were handled using the same procedure.
The relative standard deviations (RSD) were acquired from
six parallel experiments. The limit of detection (LOD) was
calculated according to the lowest concentration of analyte
detected as [M–H]- with signal-to-noise ratio (S/N) ≥3. The
spike-and-recovery experiments were carried out as follows:
three serum samples were randomly selected and each was
divided in duplicate; one portion of each sample was mixed
with IS (20 μg/mL). Another portion of three samples were
spiked with different concentration of FAs and fixed con-
centration of IS (20 μg/mL). (Three different concentrations
of the spiked FAs are listed below: sample 1: C16:0, C18:0,
C18:1, C18:2, and C20:4 with the concentrations of 10 μg/
mL, C22:0 and C22:6 with the concentration of 5μg/mL;
Molecular Orbital Calculations Approach
The Gaussian 09 program was used with the B3LYP func-
tional density approach at the B3LYP/6-311+G (d.p) level of
theory to calculate the proton affinities, ionization energies, and
electron affinities of DBDA, 9AA, DMAN, CHCA, and DHB
in the gas phase. The basis sets have been proven to be able to
obtain similar thermochemical properties of traditional matri-
ces with the individual experimental value [29].
Instrumentation
The MALDI-TOF mass spectra were recorded on a Bruker
Autoflex speed TOF/TOF mass spectrometer (Bruker
Daltonics, Bremen, Germany). Its laser device is a pulsed
Figure 2. The MALDI-MS spectra of saturated and unsaturated FAs using DBDA as matrix in the negative ion mode; (a) C14:0, (b)
C16:0, (c) C18:0, (d) C18:1, (e) C18:2, (f) C20:4, (g) C22:0, (h) C22:6

L. Ling et al.: MALDI Matrix for Small Molecules
Table 1. The LOD of FAs Using DBDA as Matrix by MALDI-MS in the
Negative Ion Mode
(355 nm). Hence, the UV-Vis absorbances of six com-
pounds were tested first and the results are shown in
Supplementary Figure S2. DBDA has maximal absorption
at 355 nm, and it has a large mole absorption coefficient of
ε = 2.3 × 104 M-1. The absorption of compound S4 is
slightly lower than that of DBDA. Next, the mixtures of
saturated FAs (C14:0, C16:0, C18:0, C20:0, C22:0) were
analyzed using synthesized Schiff base compounds as ma-
trices. Interestingly, the five FAs were detected with sig-
nificantly high S/N (>1000) in the negative ion mode using
DBDA (Supplementary Table S1). With the compounds S2
and S4 (analogues of DBDA), as matrices, five FAs were
detected but with lower S/N than DBDA. Moreover, the
mass spectrum of DBDA is very clean in positive ion
mode of MALDI-MS (Supplementary Figure S3). This is
attributed to the strong ability of DBDA to seize a proton.
With this great performance matrix DBDA, eight saturated
and unsaturated FAs were individually analyzed (Figure 2)
and the limit of detection (LOD) of these FAs were ob-
tained by diluting gradually. As shown in Figure 2, hardly
any of the matrix-related ions were observed in the spectra.
There are only deprotonated ions of FAs ([C14:0-H]−, m/z
= 227.2; [C16:0-H]−, m/z = 255.2; [C18:0-H]−, m/z =
283.3; [C18:1-H]−, m/z = 281.2; [C18:2-H]−, m/z =
279.2; [C20:4-H]−, m/z = 303.2; [C22:0-H]−, m/z =
339.3; [C22:6-H]−, m/z = 327.2) in the mass spectra. Also,
FAs could be detected in the 10th femtomole range using
DBDA as MALDI matrix (Table 1). These results demon-
strated that DBDA is a potential matrix for small mole-
cules. So DBDA was selected to following experiments.
FAs
C14:0 C16:0 C18:0 C18:1 C18:2 C20:4 C22:0 C22:6
0.31 0.28 0.28 0.28 0.66 0.23 0.61
LOD/fmol 0.18
Nd:YAG with the wavelength of 355 nm. A ground steel
sample target (Bruker Daltonics, Bremen, Germany) with
384 spots was used. All analytes mass spectra were ob-
tained in negative ion mode with the delayed extraction
time of 150 ns and the acceleration voltage of 19 kV. All
mass spectra were processed by the Flex Analysis 3.0
software (Bruker Daltonics). The UV spectra were mea-
sured using a UV-2550 spectrophotometer (SHIMADZU,
Kyoto, Japan).
Results and Discussion
Screening of Schiff Base Compounds for Great Performance
Matrix
Six Schiff base compounds (S1–S6) with conjugated
structure were synthesized and tested to see whether they
are potential MALDI matrices for small molecules in the
negative ion mode. Their structures are listed in Figure 1.
According to the photoionization mechanism [30], elec-
tronic excitation of matrices is the initial ionization step;
thus it is important that MALDI matrices have strong
ultraviolet absorption at the instrument’s laser wavelength
Figure 3. The MALDI-MS spectra of (a) glutamic acid, (b) glutathione, (c) 5'-adenylic acid, and (d) C20:0 (200 pmol on plate each)
using different matrices; DBDA, 9AA, DMAN, and NEDC

L. Ling et al.: MALDI Matrix for Small Molecules
indicated that DBDA is a promising matrix for small mole-
cules not only for acidic compounds.
Quantification of Serum FFAs Using DBDA Matrix
In order to explore the application of DBDA for real world
sample, DBDA matrix was used for the analysis of FFAs in
serum. The serum sample is simply pretreated and analyzed
by MALDI-MS using DBDA matrix in the negative ion
mode. Seven FFAs, including C16:0, C18:0, C18:1, C18:2,
C20:4, C22:0, and C22:6 can be detected clearly. These
seven FFAs were further quantified. To eliminate the effect
of various factors on the detection and improve accuracy and
reproducibility of the analysis, C17:1 was used as the IS
based on the following two reasons; first, there is no C17:1
in serum, thus its content is completely determined by the
amount added. Second, its property and molecular weight are
similar to serum FFAs. Hence, the crude FFAs solution
containing 40 μg/mL IS was mixed with DBDA and ana-
lyzed by MALDI-MS. The representative mass spectrum is
shown in Figure 4. During the analysis, two sample prepara-
tion methods, dried-droplet and Bseeds^, were compared to
improve the reproducibility. As shown in Figure 5, the
MALDI-MS image which used the method of Bseeds^ great-
ly enhanced the crystal uniformity and therefore improved
the reproducibility of the quantitative analysis. Afterwards,
the standard curves of these seven FAs were constructed
based on the intensity ratio of the individual standard FAs
to the IS versus their respective concentration, and the cor-
responding equations are listed in Table 2. With our method,
the standard curves were obtained with correlation coeffi-
cient R
Figure 4. The MALDI-MS spectrum of serum FFAs including
IS using DBDA as matrix in the negative ion mode
Evaluate the Performance of DBDA for Small
Molecules
To evaluate the versatility of DBDA to various small mole-
cules, three amino acids (glutamic acid, phenylalanine, ly-
sine), glutathione, 5'-adenylic acid, and C20:0 were analyzed
by comparison with the commonly used matrix 9AA [15,
31], superbasic matrix DMAN [19, 22], and organic salts
matrix NEDC [17], which are reported for the analysis of
metabolites. The representative spectra of acidic analytes are
shown in Figure 3; using DBDA, glutamic acid, glutathione,
5'-adenylic acid, especially the C20:1, were detected with
higher S/N than the results that using the 9AA, DMAN, and
NEDC as matrices. Also, there are hardly any matrix inter-
ferences in mass spectra using DBDA matrix. 9AA and
NEDC produced several matrix-related ions; particularly
the ion of 9AA was always detected as base peak in the mass
spectra. Additionally, the non-acidic amino acids phenylal-
anine and lysine were analyzed using these four matrices
(Supplementary Figure S4). We found that DBDA has the
greatest ionization efficiency for phenylalanine and lysine
compared with 9AA, DMAN, and NEDC. These results
2 > 0.99. The linear dynamic ranges are more than two
orders of magnitude and the relative standard deviation
(RSD) of individual FAs are lower than 15% (Table 2).
Based on the equations of each FAs, the concentrations of
FFAs in the five different serum samples were acquired and
are listed in Supplementary Table S2. To further validate this
method, the spike-and-recovery experiments were per-
formed (details were given in the Experimental section) and
recoveries of each FAs ranged from 87.2% to 108%
(Supplementary Table S2). These results indicated that using
Figure 5. The MALDI-MS images of C16:0 using different sample preparation methods in the negative ion mode; dried-droplet (left)
and Bseeds^ (right)

L. Ling et al.: MALDI Matrix for Small Molecules
Table 2. Standard Curve Equation, Correlation Coefficient (R2), Linear Range, and Relative Standard Deviation (RSD) of Each FAs
FAs
Standard curve equation (n = 6)
R2
Linear range (μg/mL)
RSD (%) (n = 6)
10 μg/mL
50 μg/mL
100 μg/mL
C16:0
C18:0
C18:1
C18:2
C20:4
C22:0
C22:6
y=0.0530x+0.7556
y=0.0717x+1.4163
y=0.0403x+0.0475
y=0.0502x+0.1848
y=0.0137x+0.0615
y=0.1411x-0.3940
y=0.0736x-0.0637
0.995
0.994
0.9996
0.9993
0.994
0.9991
0.995
1–650
10–650
5–800
5–750
1–550
5–650
1–350
13
10
10
12
11
10a
10a
9
9
12
9
13
15
13c
15c
13
12
11
12
12b
13b
The Ba^, Bb^, and Bc^ represent concentration 5 μg/mL, 10 μg/mL and 20 μg/mL, respectively
DBDA matrix and with our optimized method, the concen-
trations of the FFAs in serum could be accurately quantified.
Table 3, the PA of DBDA, 9AA, and DMAN are obviously
stronger than the acidic matrices CHCA and DHB. In other
words, alkaline matrices combine with protons more easily
than acidic matrices in the gas phase. For DBDA, it captured
the proton of FAs and was itself protonated, and the
deprotonated FAs was detected in negative ion mode. FAs
could not be detected using acidic matrices CHCA and DHB
whether in the possitive ion mode or in the negative ion mode
[22]. In addition, DBDA is high sensitive for the analysis of
FAs (LOD reached 0.3 fmol), which may be attributed to its
strong ionization capacity. The smaller difference between the
IE and the EA of each matrix, the easier primary ionization step
could occur in MALDI MS [29]. The (IE-EA) of DBDA is
obviously lower than other matrices, which indicated that
DBDA is easier to ionize and transfer energy more effectively;
this maybe explain why using DBDA to detect FAs can obtain
a lower detection limit.
Possible Related Mechanism
Using superbasic matrix, ionization of acidic analytes occurs in
the solution phase, according to the acid-base theory developed
by Brønsted-Lowry [22]. The analyte signal is highest when
the matrix and analyte concentration is at mole ratio of 1:1.
This ionization model was denoted as Bmatrix-assisted
ionization/laser desorption^ (MAILD). In order to explore the
ionization mechanism of DBDA, we compared the UV-Vis
absorption curve of DBDA with the mixture of C16:0 and
DBDA at mole ratio of 1:1 in the same condition. However,
we found that addition of C16:0 to DBDA solution could not
change the UV-Vis absorption of DBDA (Supplementary
Figure S5). From this experiment result, we speculated that
the ionization step occurs in the gas phase but not in the
solution phase. Furthermore, we analyzed C16:0 by mixing it
with different concentrations of DBDA by MALDI-MS in the
negative ion mode, and the results are shown in Supplementary
Figure S6. When the mole ratio of DBDA/C16:0 increased, the
signal of C16:0 rose continuously. It suggests that the ioniza-
tion mechanism for DBDA is based on the conventional model
BMALDI^ and is different from DMAN.
Conclusions
This work demonstrated that DBDA as a novel matrix can
commendably detect the small molecules (amino acids, pep-
tides, nucleotide, FAs) with hardly any matrix interference by
MALDI-MS in the negative ion mode. Using DBDA matrix,
the FAs were analyzed with low LOD of 0.3 fmol. Further-
more, this study also indicated that DBDA can be applied for
rapid and accurate quantitative analysis of serum FFAs by
negative ion MALDI-MS. The strong UV absorption at instru-
ment laser wavelength (355 nm) and the great ionization capa-
bility make DBDA perform an excellent matrix for the analysis
of small molecules. Further applications of DBDA for meta-
bolomics are currently in progress in our laboratory.
We compared the gas-phase parameters of DBDA related
with ionization with the commonly used matrix 9AA and
superbasic matrix DMAN. Molecular orbital calculations were
carried out to obtain proton affinities (PA), ionization energies
(IE), and electron affinities (EA) of the matrices in the gas
phase. Also, the parameters of commonly used matrices CHCA
and DHB in the positive mode were calculated. As shown in
Table 3. Proton Affinities (PA), Ionization Energies (IE), Electron Affinities
(EA), and (IE-EA) of Five Matrices; DBDA, 9AA, DMAN, CHCA, and DHB
Matrices
PA (kJ/mol)
IE (eV)
EA (eV)
IE-EA (eV)
Acknowledgments
DBDA
9AA
DMAN
CHCA
DHB
976
6.87
6.93
6.28
8.36
8.13
1.35
0.80
–0.14
1.65
0.29
5.52
6.13
6.42
6.71
7.84
1026
1029
855
This work was financially supported by the National Natural
Science Foundation of China (51273080, 21675060, and
51203153) and International Collaboration Program of Jilin
province (20160414010GH).
852

L. Ling et al.: MALDI Matrix for Small Molecules
negative ion MALDI-TOF MS analysis of small molecules. J. Am. Soc.
Mass Spectrom. 23, 1454–1460 (2012)
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