Hydrazide and hydrazine reagents as reactive matrices for MALDI-MS to detect gaseous aldehydes

Article abstract of DOI:10.1002/jms.3408

The reagents 19 hydrazide and 14 hydrazine were examined to function as reactive matrices for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect gaseous aldehydes. Among them, two hydrazide (2-hydroxybenzohydrazide and 3-hydroxy-2-naphthoic acid hydrazide) and two hydrazine reagents [2-hydrazinoquinoline and 2,4-dinitrophenylhydrazine (DNPH)] were found to react efficiently with carbonyl groups of gaseous aldehydes (formaldehyde, acetaldehyde and propionaldehyde); these are the main factors for sick building syndrome and operate as reactive matrices for MALDI-MS. Results from accurate mass measurements by JMS-S3000 Spiral-TOF suggested that protonated ion peaks corresponding to [M + H]⁺ from the resulting derivatives were observed in all cases with the gaseous aldehydes in an incubation, time-dependent manner. The two hydrazide and two hydrazine reagents all possessed absorbances at 337 nm (wavelength of MALDI nitrogen laser), with, significant electrical conductivity of the matrix crystal and functional groups, such as hydroxy group and amino group, being important for desorption/ ionization efficiency in MALDI-MS. To our knowledge, this is the first report that gaseous molecules could be derivatized and detected directly in a single step by MALDI-MS using novel reactive matrices that were derivatizing agents with the ability to enhance desorption/ionization efficiency. Copyright 2014 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/jms.3408

Application note
Received: 8 May 2014
Revised: 1 June 2014
Accepted: 3 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3408
Hydrazide and hydrazine reagents as reactive
matrices for MALDI-MS to detect gaseous
aldehydes
Yasushi Shigeri,a* Shinya Ikeda,a Akikazu Yasuda,a Masanori Ando,a
Hiroaki Satob and Tomoya Kinumic
The reagents 19 hydrazide and 14 hydrazine were examined to function as reactive matrices for matrix-assisted laser desorption/
ionization mass spectrometry (MALDI-MS) to detect gaseous aldehydes. Among them, two hydrazide (2-hydroxybenzohydrazide
and 3-hydroxy-2-naphthoic acid hydrazide) and two hydrazine reagents [2-hydrazinoquinoline and 2,4-dinitrophenylhydrazine
(DNPH)] were found to react efficiently with carbonyl groups of gaseous aldehydes (formaldehyde, acetaldehyde and
propionaldehyde); these are the main factors for sick building syndrome and operate as reactive matrices for MALDI-MS. Results
from accurate mass measurements by JMS-S3000 Spiral-TOF suggested that protonated ion peaks corresponding to [M + H]+ from
the resulting derivatives were observed in all cases with the gaseous aldehydes in an incubation, time-dependent manner. The
two hydrazide and two hydrazine reagents all possessed absorbances at 337 nm (wavelength of MALDI nitrogen laser), with,
significant electrical conductivity of the matrix crystal and functional groups, such as hydroxy group and amino group, being
important for desorption/ionization efficiency in MALDI-MS. To our knowledge, this is the first report that gaseous molecules
could be derivatized and detected directly in a single step by MALDI-MS using novel reactive matrices that were derivatizing
agents with the ability to enhance desorption/ionization efficiency. Copyright © 2014 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: gaseous aldehyde; hydrazide; hydrazine; MALDI-MS; reactive matrix
thiophene compounds were found to be good matrices for
MALDI-MS, and 2-[5-(2,4-dichlorobenzoyl)-2-thienyl] acetic acid
Introduction
Matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS) is a representative, soft ionization technique in mass
spectrometry.[1–3] MALDI-MS that is suitable for the analysis of
synthetic polymers and biomolecules has several characteristics,
such as an easy operation, capacity to handle many samples
and applicability to samples with high levels of impurities. But
compounds with absorption at the wavelength of the MALDI
laser, such as 337 nm (nitrogen laser), 349 nm (Nd:YLF laser)
and 355 nm (Nd:YAG laser), are required, as MALDI matrices
and mixed crystal of matrices and desalted samples should
be formed perfectly. Although DHB (2,5-dihydroxybenzoic acid)
and cinnamic acid-related compounds, such as CHCA (α-cyano-
4-hydroxycinammic acid) and sinapinic acid, have been developed
as standard matrices for MALDI-MS, they exhibit matrix-derived ion
peaks at smaller values of m/z (about 100–500), and this hampers
the measurement of lower molecular weight molecules.[4,5]
Because these well-known MALDI matrices were developed in the
early 1990s, there was clearly room for improvement. To make
MALDI-MS a more versatile form of mass spectrometry, it is neces-
sary to develop highly functional matrices that could be used with
new samples, such as sugar, carbohydrate and large proteins with
lower desorption/ionization efficiency.
(DCBTA) provided an important clue to measure the electrical
conductivity of the matrix crystal.[6] In the process of our screen-
ing, we encountered that some hydrazide and hydrazine re-
agents reacted with the carbonyl groups of measurement
samples and that their resulting derivatives (hydrazones) could
be effectively detected in MALDI-MS. The findings suggested that
these reagents might operate as reactive matrices for MALDI-MS
and derivatizing agents that might enhance desorption/ioniza-
tion efficiency.
Gaseous aldehydes have received a great deal of attention as
hazardous substances among carbonyl compounds. For example,
formaldehyde and acetaldehyde, included in adhesive agents
and coating materials at residential homes, are thought to be
*
Correspondence to: Yasushi Shigeri, Health Research Institute, National Institute
of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda,
Osaka, 563-8577, Japan. E-mail: yasushi.shigeri@aist.go.jp
a Health Research Institute, National Institute of Advanced Industrial Science
and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan
b Research Institute for Environmental Management Technology, National Insti-
tute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa,
Tsukuba, Ibaraki, 305-8569, Japan
In our previous studies, we utilized a peptide sample
(hypertensin, [Asn1, Val5]-angiotensin II) and a diverse chemical
library containing approximately 13 000 compounds to look for
highly functional matrices for MALDI-MS and unravel the detailed
desorption/ionization mechanism of MALDI-MS. As a result, many
c
National Metrology Institute of Japan, National Institute of Advanced Indus-
trial Science and Technology (AIST), Tsukuba Central 3, 1-1-1 Umezono,
Tsukuba, Ibaraki, 305-8563, Japan
J. Mass Spectrom. 2014, 49, 742–749
Copyright © 2014 John Wiley & Sons, Ltd.

Reactive matrices to detect gaseous aldehydes
major factors for sick building syndrome.[7] So far, these gaseous
aldehydes have been collected by cartridge devices containing
solid sorbents coated with DNPH, a representative hydrazine
reagent and eluted by organic solvent (acetonitrile). The resulting
solution has been analyzed by HPLC or LC-MS.[8] DNPH forming the
2,4-dinitrophenylhydrazones (DNPhydrazones) is one of the most
common derivatization reagents for aldehydes and the most impor-
tant qualitative and quantitative method for analyzing carbonyl
compounds.[8,9] However, a consumption of relatively large amounts
of organic solvents by HPLC and a cumbersome procedure might be
a problem, and analytical error occurs due to the formation of
E-stereoisomers and Z-stereoisomers of DNPhydrazones. There-
fore, the development of new methods to detect gaseous
aldehydes easily, quickly and measurably has been needed.
In the present study, we examined 19 hydrazide and 14 hydra-
zine reagents and found two hydrazide (2-hydroxybenzohydrazide,
3-hydroxy-2-naphthoic acid hydrazide) and two hydrazine reagents
[2-hydrazinoquinoline, 2,4-dinitrophenylhydrazine (DNPH)] that
could efficiently react with gaseous aldehydes, such as formalde-
hyde, acetaldehyde and propionaldehyde. To our knowledge, this
is the first report that gaseous molecules could be detected directly
by MALDI-MS using novel reactive matrices.
put in a plastic container (Tight box No. 1, 260 ml, Chopla, Aichi,
Japan). Eppendorf tube was still open, while the lid of a plastic
container was closed. After either 15, 30 or 60 min incubation
times at room temperature, the sample plate was taken from
the plastic container. The MALDI-MS analysis was performed on
a MicroflexAI (Bruker Daltonics, Bremen, Germany) with a pulsed
nitrogen laser (337 nm) in the linear positive ion mode, and
100 laser shots were summed. In the case of accurate mass
measurement, JMS-S3000 Spiral-TOF (JEOL, Tokyo, Japan) with a
pulsed Nd:YLF laser (349 nm) was utilized; PEG 200 and PEG
400 were used as proofreading samples. To measure the concen-
trations of gaseous aldehydes (formaldehyde, acetaldehyde and
propionaldehyde) alternatively, we utilized the following gas
detecting tubes: formaldehyde detector tube (GASTEC, No. 91M,
Kanagawa, Japan), acetaldehyde detector tube (Komyo Rikagaku
Kogyo K.K., No. 133A, Kanagawa, Japan) and propionaldehyde
detector tube (GASTEC, No. 151L, Kanagawa, Japan). The con-
centrations of formaldehyde, acetaldehyde and propionaldehyde
were 43 + 8, 9000 + 1500 and 3196 + 677 ppm (mean SD, n = 3),
respectively after 15 min incubation times. The crystal on the
sample plate was also observed directly with a stereomicroscope
(Nikon, SMZ800).
Spectroscopic analyses
Experimental
Spectroscopic analyses of hydrazide and hydrazine reagents
were performed with a DU800 UV/Visible spectrophotometer
(Beckman Coulter, CA, USA) using wavelengths 200 to 800 nm.
The molar extinction coefficients of these reagents at 337 nm
(wavelength of MALDI nitrogen laser) were calculated.
Chemicals
The following materials were obtained from Tokyo Chemical
Industry (Tokyo, Japan): 19 hydrazide reagents (benzohy-
drazide, 2-chlorobenzohydrazide, 2-fluorobenzohydrazide,
2-nitrobenzohydrazide, 2-hydroxybenzohydrazide, 3-chloroben-
zohydrazide, 3-nitrobenzohydrazide, 3-methoxybenzohydrazide,
4-chlorobenzohydrazide, 4-nitrobenzohydrazide, 4-methoxyben-
zohydrazide, 4-tert-buthylbenzohydrazide, 4-aminobenzohydrazide,
4-hydroxybenzohydrazide, 2-pyridine carboxylic acid hydrazide,
nicotinic acid hydrazide, isonicotinic acid hydrazide, 1-naphthohydrazide
and 3-hydroxy-2-naphthoic acid hydrazide), 14 hydrazine re-
agents (phenylhydrazine hydrochloride, o-tolylhydrazine hydro-
chloride, 2-nitrophenylhydrazine hydrochloride, m-tolylhydrazine
hydrochloride,
4-methoxyphenylhydrazine hydrochloride, 4-nitrophenylhydrazine
hydrochloride, 4-chlorphenylhydrazine hydrochloride, 2,4-
dichlorophenylhydrazine hydrochloride, 1-naphthylhydrazine
hydrochloride, 2-hydrazinopyridine, 2-hydrazinoquinoline, 2-
hydrazinobenzothiazole and DNPH), polyethylene glycol 200
(PEG 200) and polyethylene glycol 400 (PEG 400). Formalde-
hyde solution (5–10%) in methanol, acetaldehyde (about
90%) and propionaldehyde were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan).
Electrochemical measurements
Disposable electrical printed chips (DEP-SP-N) were obtained
from BioDevice Technology, Co. (Ishikawa, Japan). The matrix
solution (10 mg/ml, 2 μl) in 50% acetonitrile containing 0.1%
TFA was dropped onto the DEP chip and dried in vacuo.
This operation was repeated at least five times. The resulting
DEP chip was connected to a Keithley 2400 SourceMeter, and
the electrical conductivity of the crystal was measured as
described previously.[6]
3-nitrophenylhydrazine
hydrochloride,
Results and discussion
MALDI-MS measurements of the hydrazide and hydrazine
reagents with gaseous aldehydes
We previously utilized a diverse chemical library containing
approximately 13 000 compounds and found many thiophene-
containing compounds that were good matrices for MALDI-MS.
Moreover, we suggested that the electrical conductivity of the
matrix crystals might be an important factor to explore as a
characteristic of good matrices for MALDI-MS.[6] In the process
of our screening, we found that some hydrazide and hydrazine
reagents functioned as reactive matrices for MALDI-MS, serving
as derivatizing agents with the ability to enhance desorption/
ionization efficiency. In general, hydrazide and hydrazine re-
agents react with carbonyl compounds, such as aldehydes and
ketones, and promote condensation and derivatization reactions,
as shown in Fig. 1.
MALDI-MS analysis
The following procedure was performed for the reaction and
detection of gaseous aldehydes by MALDI-MS. Hydrazide
(10 mg/ml) and hydrazine reagent solutions (10 mg/ml) were
prepared using 50% acetonitrile containing 0.1% TFA. In the case
of 3-hydroxy-2-naphthoic acid hydrazide and DNPH, 4.76 and
1.67 mg/ml saturated solutions were used, respectively. A sample
of each solution (1 μl) was applied to a stainless-steel sample
plate, followed by drying at room temperature. The sample plate
and a 1.5-ml Eppendorf tube containing the stock solution of
either formaldehyde, acetaldehyde or propionaldehyde were
Indoor air quality, especially in new dwellings, should be well
maintained for the health and safety of the occupants; otherwise,
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Y. Shigeri et al.
peaks obtained by MicroflexAI MALDI-MS in detail, accurate
mass measurement was also performed by JMS-S3000 Spiral-
TOF (JEOL, Tokyo, Japan). In principle, we described the accurate
m/z values in Figs. 4–6 and S1–S9, obtained alternatively by JMS-
S3000 Spiral-TOF. In the case of the 2-hydroxybenzohydrazide,
the ion peak of m/z 153.0660, corresponding to protonated 2-
hydroxybenzohydrazide ([M + H]+), was observed without the
incubation with formaldehyde, acetaldehyde or propional-
dehyde (Fig. 4(a), 5(a) and 6(a)). After a 15 min incubation
with formaldehyde, intense protonated molecule peaks of 2-
hydroxybenzohydrazide and the resulting derivative
(hydrazone) at m/z = 153.0660 and 165.0658 could be observed
at high resolution (Fig. 4(b)). After a 60-min incubation, the peak
height of [M + H]+ from 2-hydroxybenzohydrazide decreased, as
compared to that from its hydrazone (Fig. 4(c)). Next, we utilized
2-hydroxybenzohydrazide and another gaseous aldehyde, acet-
aldehyde, and performed a similar experiment. After a 15-min
incubation with acetaldehyde, the protonated molecular peak
of 2-hydroxybenzohydrazide at m/z 153.0660, disappeared,
while the ion peak of m/z 179.0824, corresponding to proton-
ated hydrazone, appeared (Fig. 5(b)). We obtained a similar
spectrum after a 60-min incubation with acetaldehyde, as well as
after a 15-min incubation (Fig. 5(c)). In the case of the reaction of
2-hydroxybenzohydrazide with the other gaseous aldehyde,
propionaldehyde, protonated molecule peaks of 2-hydroxyben-
zohydrazide and its hydrazone at m/z = 153.0660 and 193.0966,
respectively, could be observed after a 15-min incubation; this
was also the case for formaldehyde (Fig. 6(b)). But, after a 60-min
incubation with propionaldehyde, the protonated molecular peak
from its hydrazone did not increase prominently, as compared to
that from 2-hydroxybenzohydrazide. Taken together, these results
suggest that 2-hydroxybenzohydrazide could react with all three
gaseous aldehydes and function as a reactive matrix for MALDI-
MS, while the reaction efficiency, reaction rate and desorption/
Figure 1. Condensation reactions of hydrazide and hydrazine derivatives
with gaseous aldehydes (formaldehyde, acetaldehyde and propionaldehyde).
sick building syndrome may occur.[7] Among carbonyl com-
pounds, gaseous aldehydes have been considered as hazardous
substances and the main factors associated with sick building
syndrome. Therefore, as shown in Figs. 2 and 3, we obtained 19
hydrazide and 14 hydrazine reagents that were commercially
available and examined their activities as reactive matrices
for MALDI-MS to detect gaseous aldehydes. Consequently, two
hydrazide reagents (2-hydroxybenzohydrazide and 3-hydroxy-2-
naphthoic acid hydrazide) and two hydrazine reagents (2-
hydrazinoquinoline and DNPH) could efficiently react with
gaseous aldehydes, such as formaldehyde, acetaldehyde and
propionaldehyde, as shown in Tables 1 and 2.
To analyze the reaction mechanism in detail, gaseous alde-
hydes (formaldehyde, acetaldehyde and propionaldehyde) were
reacted with two hydrazide reagents (2-hydroxybenzohydrazide
and 3-hydroxy-2-naphthoic acid hydrazide) and two hydrazine
reagents (2-hydrazinoquinoline and DNPH) for 0, 15 and 60 min,
and the MALDI-MS measurement was subsequently performed.
To confirm the m/z values of spectra and characterization of ion
Figure 2. Chemical structures of hydrazide derivatives used in this study.
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Reactive matrices to detect gaseous aldehydes
Figure 3. Chemical structures of hydrazine derivatives used in this study.
Table 1. Hydrazide derivatives as MALDI-MS reactive matrices to detect gaseous aldehydes
Compounds
HCHO
CH3CHO
CH3CH2CHO
Molar extinction coefficient
Monoisotopic
mass (calculated)
(at 337 nm, molÀ1cmÀ1l)
benzohydrazide
++
+
+
+
0
0
136.0637
170.0247
154.0542
181.0487
152.0586
170.0247
181.0487
166.0742
170.0247
181.0487
166.0742
192.1263
151.0746
152.0586
137.0589
137.0589
137.0589
186.0793
202.0742
2-chlorobenzohydrazide
2-fluorobenzohydrazide
2-nitrobenzohydrazide
+
+
0
+
+
370
60
0
2-hydroxybenzohydrazide
3-chlorobenzohydrazide
3-nitrobenzohydrazide
+++
+
+++
+
+++
+
+
495
0
3-methoxybenzohydrazide
4-chlorobenzohydrazide
4-nitrobenzohydrazide
+
+
+
+
0
+
++
++
+
525
71
2
4-methoxybenzohydrazide
4-tert-buthylbenzohydrazide
4-aminobenzohydrazide
4-hydroxybenzohydrazide
2-pyridine carboxylic acid hydrazide
nicotinic acid hydrazide
isonicotinic acid hydrazide
1-naphthohydrazide
+
+
++
+
++
+
57
84
0
+
+
+
+
53
359
29
1458
++
+
+
++
+++
3-hydroxy-2-naphthoic acid hydrazide
++
+++
Desorption/ionization efficiency (+++, high), (++, middle), (+, low) and (–, no ability).
ionization efficiency varied among gaseous aldehydes. Similar
results using 3-hydroxy-2-naphthoic acid hydrazide (Figs. S1–S3),
2-hydrazinoquinoline (Figs. S4–S6) and DNPH (Figs. S7–S9) were
shown in supporting information. All these compounds reacted
with carbonyl groups of gaseous aldehydes (formaldehyde, acetal-
dehyde and propionaldehyde) and operated as reactive matrices
for MALDI-MS and derivatizing agents with the ability to enhance
desorption/ionization efficiency, because the protonated molec-
ular peaks of their resulting derivatives (hydrazones) were all mark-
edly detected. These data are summarized in Tables 1 and 2.
Among 19 hydrazide and 14 hydrazine reagents, at least 2-
hydrazide possessed hydroxy groups, as shown in Fig. 2. As well
as the case of DHB, general-purpose matrix for MALDI-MS, hydroxy
group seemed to work as a source of protons and facilitate the ion-
ization efficiency in these cases. Furthermore, toluene and xylene
have been used as solvents of coating materials and resins of
building materials, and thought as the causative substances
for sick building syndrome. Although they do not have carbonyl
groups that are essential for condensation (dehydration) reac-
tion with hydrazide and hydrazine reagents, we used toluene
and xylene and made similar MALDI-MS experiments instead
of gaseous aldehydes. Unfortunately, two hydrazide (2-
hydroxybenzohydrazide and 3-hydroxy-2-naphthoic acid
hydroxybenzohydrazide
and
3-hydroxy-2-naphthoic
acid
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Y. Shigeri et al.
Table 2. Hydrazine derivatives as MALDI-MS reactive matrices to detect gaseous aldehydes
Compounds
HCHO
CH3CHO
CH3CH2CHO
Molar extinction coefficient
Monoisotopic
mass (calculated)
(at 337 nm, molÀ1cmÀ1l)
phenylhydrazine hydrochloride
o-tolylhydrazine hydrochloride
2-nitrophenylhydrazine hydrochloride
m-tolylhydrazine hydrochloride
3-nitrophenylhydrazine hydrochloride
4-methoxyphenylhydrazine hydrochloride
4-nitrophenylhydrazine hydrochloride
4-chlorphenylhydrazine hydrochloride
2,4-dichlorophenylhydrazine
++
À
À
+
À
À
À
À
À
+
52
86
108.0687
122.0844
153.0538
122.0844
153.0538
138.0793
153.0538
142.0298
175.9908
2405
37
À
+
2154
0
À
+
À
À
À
11 191
21
À
89
hydrochloride
1-naphthylhydrazine hydrochloride
2-hydrazinopyridine
++
++
+++
+
+
52
829
158.0844
109.0640
159.0796
165.0361
198.0389
+
2-hydrazinoquinoline
+++
+++
+++
+++
+++
8174
0
2-hydrazinobenzothiazole
DNPH (2,4-dinitrophenylhydrazine)
À
19 973
Desorption/ionization efficiency (+++, high), (++, middle), (+, low) and (À, no ability).
Figure 4. Condensation reactions of 2-hydroxybenzohydrazide with formaldehyde. The protonated ions marked with asterisks, (*) and (**), were
derived from 2-hydroxybenzohydrazide and the resulting hydrazone, respectively.
hydrazide) and two hydrazine reagents [2-hydrazinoquinoline
and 2,4-dinitrophenylhydrazine (DNPH)] could not detect
toluene or xylene in MALDI-MS (data not shown). These were
quite reasonable results because both toluene and xylene do
not have carbonyl groups.
protonation, deprotonation and/or adduct formation. Therefore,
we measured absorption spectra of 19 hydrazide and 14 hydra-
zine reagents with wavelengths from 200 to 800 nm, and
the molar extinction coefficients of these reagents at 337 nm
(wavelength of MALDI nitrogen laser) were calculated. As
shown in Tables 1 and 2, molar extinction coefficients of 2-
hydroxybenzohydrazide, 3-hydroxy-2-naphthoic acid hydrazide,
2-hydrazinoquinoline and DNPH at 337 nm were 60, 1458, 8174
and 19 973 (molÀ1cmÀ1L), respectively. It is well-known that
sufficient optical absorption of the matrix at the laser wave-
length is important for spectrum quality in MALDI-MS. However,
once the absorption exceeds a certain value, differences
tend to level out.[3,10] The molar extinction coefficients of these
hydrazide and hydrazine reagents at 337 nm did not correlate
with desorption/ionization efficiency in MALDI-MS, but at
Physical properties of hydrazide and hydrazine reagents with
gaseous aldehydes
The characteristics that all matrices for MALDI-MS should share
include the following.[10] First, they should cocrystalize well with
analytes under investigation. Second, they should absorb the
laser light efficiently by excitation of electronic singlet states.
Third, they should produce reactive ionic species that form the
basis for an efficient ionization of analyte molecules by
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Reactive matrices to detect gaseous aldehydes
Figure 5. Condensation reactions of 2-hydroxybenzohydrazide with acetaldehyde. The protonated ions marked with asterisks, (*) and (**), were
derived from 2-hydroxybenzohydrazide and the resulting hydrazone, respectively.
Figure 6. Condensation reactions of 2-hydroxybenzohydrazide with propionaldehyde. The protonated ions marked with asterisks, (*) and (**), were
derived from 2-hydroxybenzohydrazide and the resulting hydrazone, respectively.
least they all possessed prominent absorbance at 337 nm
(nitrogen laser), as well as 349 nm (Nd:YLF laser) and 355 nm
(Nd:YAG laser) (data not shown). In principle, molar extinction
coefficients of the resulting hydrazones at 337 nm, products
of condensation reaction with gaseous aldehydes, should
be calculated. However, we could not measure them due to
their low solubility and the products of the condensation
(dehydration) reaction.
Next, we observed the crystal on the MALDI sample plate by
stereomicroscope. When solid-state matrices are used in MALDI-
MS, there is often an associated sweet-spot effect, such as a
position-dependent variation derived from an inhomogeneous
crystallization with matrices and analytes.[3,10] For example, DHB
is apt to form large crystal needles that produce sweet spots.
As shown in Fig. 7, an acicular portion could be observed in the
case of 2-hydroxybenzohydrazide with formaldehyde, while
the other three reagents showed typical crystals. Compared
to DHB, we did not observe any sweet-spot effect in the case of
2-hydroxybenzohydrazide with formaldehyde. With regard to
the color, as shown in Fig. 7, longer incubation with formalde-
hyde increased the yellow color of the 2-hydrazinoquinoline
crystal, suggesting the progress in a certain type of reaction, such
as condensation, but we could not find a marked color change in
the cases of 2-hydroxybenzohydrazide, 3-hydroxy-2-naphthoic
acid hydrazide and DNPH. Similar results using acetaldehyde
(Fig. S10) and propionaldehyde (Fig. S11) were shown in
supporting information.
We previously reported that the electrical conductivity of
the matrix crystals might be associated with the performance of
matrices for MALDI-MS.[6] Therefore, we measured the electrical
conductivity of the crystals. As shown in Table 3, both the two
hydrazide and the two hydrazine reagents exhibited similar levels
of electrical conductivity as seen with CHCA, sinapinic acid and
super-DHB.
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Y. Shigeri et al.
a
b
c
d
Figure 7. Photographs of crystalline hydrazide and hydrazine derivatives after incubation with formaldehyde. (a) 2-hydroxybenzohydrazide, (b) 3-hydroxy-
2-naphthoic acid hydrazide, (c) 2-hydrazinoquinoline and (d) DNPH (2,4-dinitrophenylhydrazine). Incubation times were 0, 15, 30 and 60 min.
derivatized and detected directly in a single step by MALDI-MS
using novel reactive matrices.
Table 3. Electrochemical properties of hydrazide and hydrazine
derivatives
Compounds
Current (pA)
128 + 79
Conclusions
2-hydroxybenzohydrazide
3-hydroxy-2-naphthoic acid hydrazide
2-hydrazinoquinoline
DNPH (2,4-dinitrophenylhydrazine)
CHCA
63 + 15
In this study, two hydrazide (2-hydroxybenzohydrazide and
3-hydroxy-2-naphthoic acid hydrazide) and two hydrazine
[2-hydrazinoquinoline and 2,4-dinitrophenylhydrazine (DNPH)]
reagents, which are derivatizing agents with the ability to enhance
desorption/ionization efficiency, were found to react with gaseous
aldehydes (formaldehyde, acetaldehyde and propionaldehyde)
and function as reactive matrices for MALD-MS. Further studies
are needed to improve our detection in a quantitative way and elu-
cidate a mechanism of reactive matrices, because many unreacted
hydrazide and hydrazine reagents could affect desorption/ioniza-
tion efficiency in MALDI-MS.
130 + 59
170 + 53
100–200 Ref[6]
100–200 Ref[6]
150–250 Ref[6]
Sinapinic acid
Super-DHB
All measurements were carried out using DEP chips at room
temperature and under a constant voltage at 20 V as described
previously. Data are the mean + SD from ten independent
experiments.
Acknowledgements
To date, reactive matrices for MALDI-MS, derivatizing
agents with the ability to enhance desorption/ionization efficiency,
have been often reported, such as DNPH for steroids,[11] DMNTH (4-
dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine)
for carbonyl compounds,[12] DAA [9-(3,4-diaminophenyl)acridine]
for α-dicarbonyl compounds,[13] and DNPH for phospholipids and
related oxidation products.[14,15] In these cases, though, all analytes
were liquid substances, and the derivatizing procedures were per-
formed by mixing substances on the MALDI sample plate or in
the tubes at room temperature. On the other hand, gaseous mole-
cules, such as small aldehydes in cigarette smoke, were detected by
MALDI-MS.[16] Only conventional procedures, such as extraction
and derivatization by diphenylamine and 2,5-dihydroxybenzoic
acid, and subsequent detection by MALDI-FTICR-MS using DHB, a
representative MALDI matrix, were performed. To our knowledge,
this is the first report showing that gaseous molecules could be
We thank Prof. Leslie Sargent Jones (Appalachian State University)
for her careful reading of the manuscript. This work was supported
by an AIST research grant, the Japan Society for the Promotion of
Science (JSPS) KAKENHI Grant number 24619012 to T. K. and
Platform for Drug Discovery, Informatics and Structural Life Science
and Technology from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
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Supporting information
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Additional supporting information may be found in the online
version of this article at the publisher’s web site.
J. Mass Spectrom. 2014, 49, 742–749
Copyright © 2014 John Wiley & Sons, Ltd.
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