Development of a liquid chromatographic method based on ultraviolet-visible and electrospray ionization mass spectrometric detection for the identification of nitrocatechols and related tracers in biomass burning atmospheric organic aerosol

Article abstract of DOI:10.1002/rcm.6170

Rationale: Studying the chemical composition of biomass burning aerosol (BBA) is very important in order to assess their impact on the climate and the biosphere. In the present study, we focus on the characterization of some newly recognized biomass burning aerosol tracers including methyl nitrocatechols, nitroguaiacols and 4-nitrocatechol, but also on nitrophenols, methyl nitrophenols and nitrosalicylic acids, using liquid chromatography tandem mass spectrometry. Methods: For the purpose of their separation and detection in atmospheric aerosol, a new chromatographic method was initially developed based on reversed-phase chromatography coupled with ultraviolet/visible (UV/Vis) detection. The method was afterwards transferred to a liquid chromatography/electrospray ionization linear ion trap mass spectrometry (LC/ESI-LITMS) system in order to identify the targeted analytes in winter aerosol from the city of Maribor, Slovenia, using their chromatographic retention times and characteristic (-)ESI product ion (MS²) spectra. Results: The fragmentation patterns of analytes obtained with LITMS are presented. Additional nitro-aromatic compounds (m/z 168 and 182) closely related to the targeted nitrocatechols and nitroguaiacols were detected in the aerosol. According to their MS² spectra these compounds could be attributed to methyl homologues of methyl nitrocatechols and nitroguaiacols. Conclusions: The proposed LC/MS method results in a better separation and specificity for the targeted analytes. Several nitro-aromatic compounds were detected in urban BBA. The LC/MS peak intensity of the newly detected methyl nitrocatechols and nitroguaiacols is comparable to that of the methyl nitrocatechols, which also qualifies them as suitable molecular tracers for secondary biomass burning aerosol.

Full text of DOI:10.1002/rcm.6170

Research Article
Received: 18 November 2011
Revised: 18 January 2012
Accepted: 18 January 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
wileyonlinelibrary.com) DOI: 10.1002/rcm.6170
(
Development of a liquid chromatographic method based on
ultraviolet–visible and electrospray ionization mass spectrometric
detection for the identification of nitrocatechols and related tracers
in biomass burning atmospheric organic aerosol
1
1
2†
2
3,4
Zoran Kitanovski *, Irena Grgić , Farhat Yasmeen , Magda Claeys and Alen Čusak
1
Laboratory for Analytical Chemistry, National Institute of Chemistry, Ljubljana, Slovenia
Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium
EN-FIST Center of Excellence, Ljubljana, Slovenia
Slovenian NMR Center, National Institute of Chemistry, Ljubljana, Slovenia
2
3
4
RATIONALE: Studying the chemical composition of biomass burning aerosol (BBA) is very important in order to assess
their impact on the climate and the biosphere. In the present study, we focus on the characterization of some newly recog-
nized biomass burning aerosol tracers including methyl nitrocatechols, nitroguaiacols and 4-nitrocatechol, but also on
nitrophenols, methyl nitrophenols and nitrosalicylic acids, using liquid chromatography tandem mass spectrometry.
METHODS: For the purpose of their separation and detection in atmospheric aerosol, a new chromatographic method
was initially developed based on reversed-phase chromatography coupled with ultraviolet/visible (UV/Vis) detection.
The method was afterwards transferred to a liquid chromatography/electrospray ionization linear ion trap mass spectro-
metry (LC/ESI-LITMS) system in order to identify the targeted analytes in winter aerosol from the city of Maribor,
2
Slovenia, using their chromatographic retention times and characteristic (ꢀ)ESI product ion (MS ) spectra.
RESULTS: The fragmentation patterns of analytes obtained with LITMS are presented. Additional nitro-aromatic com-
pounds (m/z 168 and 182) closely related to the targeted nitrocatechols and nitroguaiacols were detected in the aerosol.
2
According to their MS spectra these compounds could be attributed to methyl homologues of methyl nitrocatechols
and nitroguaiacols.
CONCLUSIONS: The proposed LC/MS method results in a better separation and specificity for the targeted analytes.
Several nitro-aromatic compounds were detected in urban BBA. The LC/MS peak intensity of the newly detected methyl
nitrocatechols and nitroguaiacols is comparable to that of the methyl nitrocatechols, which also qualifies them as suitable
molecular tracers for secondary biomass burning aerosol. Copyright © 2012 John Wiley & Sons, Ltd.
Biomass burning (BB) is considered as one of the greatest
primary sources of organic aerosols in the atmosphere.
and climate of Earth. Recently, methyl nitrocatechols were
proposed to be suitable tracers for highly oxidized secondary
[1]
[9]
The chemistry of compounds present in biomass burning
aerosol (BBA) is diverse and directly dependent on the chemi-
cal composition of the burning material and the combustion
BBA. These compounds are formed through photooxida-
tion, in the presence of NO , of m-cresol, which is emitted at
significant levels during BB.
related compounds to nitrocatechols, have already been
x
[9,10]
Nitrophenols, structurally
[
2]
conditions. A well-established tracer for primary BBA is
[e.g. 11–15]
levoglucosan (1,6-anhydro-b-anhydroglucose), which origi-
reported in the atmosphere.
Their atmospheric
[2–8]
nates from the pyrolysis of cellulose or hemicelluloses.
emission can originate from primary sources (e.g. traffic, coal
and wood combustion, industry) and predominates the
secondary emission (reactions of monoaromatic compounds
Secondary BBA, which is formed after physical and chemical
changes (aging) of primary BBA in the atmosphere, contains
more oxidized and polar compounds. An important class of
these secondary organic aerosol (SOA) compounds are nitro-
catechols, which are strong absorbers of ultraviolet and
visible light, and, therefore, could affect the radiative balance
[11–15]
x
with OH radicals and NO ), especially in urban areas.
The influence of nitrocatechols on the Earth’s climate and
the biosphere has not been measured yet, nor has their
toxicity toward living organisms (including humans) been
studied. Several studies so far indicate that nitrophenols are
[
11]
phytotoxic
and can induce estrogenic, anti-androgenic or
[16–18]
*
Correspondence to: Z. Kitanovski, Laboratory for Analytical
vasodilatory effects.
Chemistry, National Institute of Chemistry, Hajdrihova
The objectives of the present study were to characterize
nitrocatechols and related compounds in ambient aerosol
using mass spectrometric and chromatographic techniques
with a view to develop in a further phase suitable quantita-
tive methodology. The selected ambient aerosol samples were
collected during a cold winter episode from an urban site in
1
9, P.O. Box 660, SI-1001 Ljubljana, Slovenia.
E-mail: zoran.kitanovski@ki.siUniversity of Engineering
and Technology, Chemistry Department, Lahore, Pakistan
†
Present address: University of Engineering and Technology,
Chemistry Department, Lahore Pakistan
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
Copyright © 2012 John Wiley & Sons, Ltd.

Z. Kitanovski et al.
Maribor, Slovenia, which is influenced by heavy traffic and
substantial emissions from residential wood burning for
domestic purposes. In a first part of this study, we report
the synthesis of a major reference compound, i.e. 3-methyl-
Instrumentation
Two different LC/MS configurations were used in this study:
system 1 comprised an Agilent 1100 Series HPLC system
(degasser, quaternary pump, autosampler and diode-array
UV/Vis detector; Agilent Technologies, Waldbronn, Germany)
coupled to an Applied Biosystems/MDS Sciex 4000 QTRAP
LC/MS/MS system (Applied Biosystems/MDS Sciex, Ontario,
Canada), while system 2 comprised a Surveyor Plus system
(pump and autosampler) and a linear ion trap mass spectro-
meter (LXQ) equipped with an ESI source (Thermo Scientific,
San Jose, USA).
5-nitrocatechol, and its structural characterization using
nuclear magnetic resonance (NMR). In a second part, emphasis
is given to selecting a suitable LC technique and optimizing the
chromatographic conditions for separating methyl nitrocate-
chols and related nitro-aromatic compounds using diode array
UV/Vis and subsequently (ꢀ)ESI-MS detection. In a third part,
2
LC/(ꢀ)ESI-MS data are presented and interpreted for the
nitro-aromatic compounds that occur in ambient samples.
The triple quadrupole – linear ion trap hybrid mass spec-
trometer of system 1 (4000 QTRAP LC/MS/MS system)
was equipped with a TurboIonSpray (TIS) source, which is a
variation of an ESI source. A Harvard 11 plus syringe pump
EXPERIMENTAL
(
Harvard Apparatus, Holliston, MA, USA) was used for
direct infusion of standards into the TIS source at a flow rate
of 10 mL min . For the MS system, a central supply of high-
Reagents, standards and standard solutions
–1
Acetonitrile and methanol (Chromasolv gradient grade, for
purity nitrogen was used as nebulizer, drying and collision
2
HPLC, ≥99.9%; Sigma-Aldrich, St. Louis, USA), 2-propanol
gas. To obtain the MS product ion spectra of the deproto-
–
(LiChrosolv gradient grade, for HPLC, ≥99.9%; Merck,
nated molecules [M – H] of 4M5NC and 3M5NC in negative
Darmstadt, Germany), tetrahydrofuran (Chromasolv Plus,
for HPLC, ≥99.9%, inhibitor-free; Sigma-Aldrich), and high-
purity water (18.2 MΩꢁcm), supplied by a Milli-Q water pur-
ification system (Millipore, Bedford, MA, USA), were used for
the mobile phase and sample preparation. The following
additives for mobile phase or sample preparation were used:
glacial acetic acid (100% Suprapur; Merck), ammonium acet-
ate (Fractopur; Merck), formic acid (98–100% analysis grade;
Merck), ammonia solution (25% Suprapur; Merck), ammo-
nium formate (Puriss p.a., eluent additive for LC; Fluka,
Buchs, Switzerland), and ammonium bicarbonate (Fluka).
The following standard substances were used: 4-methyl-5-
nitrocatechol (4M5NC, Santa Cruz Biotechnologies, USA),
ion ESI, the following values for the MS parameters were
used: –4500 V for the TIS capillary voltage, 10.0 psi for the
–
5
curtain gas, ’high’ setting (vacuum: 4.5–5.0 ꢃ 10 Torr) for
the collision gas, 20.0 psi for the nebulizer gas, –49.0 V for
the declustering potential, and ꢀ5.0 V for the collision cell exit
potential. The spectra recorded for each compound were
summed spectra obtained by ramping the collision energy from
ꢀ120 V to ꢀ5 Vusing 1 V per spectrum step. This approach for
spectra recording was followed in order to obtain a final spec-
trum that is rich in characteristic product ions. A Chemstation
for LC 3D systems Rev. B.03.02 (Agilent Technologies) and
Analyst 1.5 Software (Applied Biosystems/MDS Analytical
Technologies Instruments) were used for acquisition and
analysis of the LC and (ꢀ)ESI-MS/MS data.
3
-methyl-4-nitrophenol (3M4NP, Acros Organics, Geel,
Belgium), 4-nitrocatechol (4NC), 2-methyl-4-nitrophenol
2M4NP), 2-nitrophenol (2NP), 4-nitrophenol (4NP), 2,4-
dinitrophenol (2,4DNP), 4-nitroguaiacol (4NG), 2-methoxy-
-nitrophenol (5-nitroguaiacol, 5NG), 3-nitrosalicylic acid
3NSA), 2-hydroxy-5-nitrobenzoic acid (5-nitrosalicylic acid,
NSA) and 3-methylcatechol were purchased from Sigma-
System 2 also incorporated a data system using Xcalibur
version 2.0 software (Thermo Scientific). The LXQ linear ion
trap was operated under the following conditions: sheath
(
–1
5
(
5
gas flow (nitrogen), 0.75 L min ; auxiliary gas flow (nitrogen),
–
1
1.5 L min ; source voltage, –4.5 kV; capillary temperature,
ꢂ
350 C; and maximum ion injection time, 200 ms. The
Aldrich. All standards had purity higher than 95% and were
used without further purification. 3-Methyl-5-nitrocatechol
ion optics of the LXQ instrument were optimized for max-
imum [M – H] signal intensity using direct introduction
–
(
3M5NC) and 3-methyl-6-nitrocatechol (3M6NC) were synthe-
of a solution of 4-nitrocatechol in methanol/water (3/7; v/v)
–1
sized from 3-methylcatechol by procedures described in the
literature (details about the synthesis are provided in the Sup-
porting information).
containing 10 mM ammonium acetate (pH 3) of 50 mg mL at
–
1
a flow rate of 200 mL min . For collision-induced dissociation
[9,19,20]
2
Individual standard stock solutions
(CID) experiments (MS ) on system 2, the ions of interest were
of the studied nitro compounds were prepared at concentra-
activated by applying a fraction of an 80-V supplementary
a.c. (alternating current) potential to the exit rods of the
linear ion trap at the resonance frequency of the selected
ion. The instrument settings were as follows: activation q
value, 0.25; normalized collision energy, 35%; isolation
width, 2 m/z units; and activation time, 30 ms. Helium
was introduced as damping and collision gas at a flow
–1
tions of 500 mg L in methanol. A composite standard solution
–1
with a concentration of 2 mg L of each nitro compound in
methanol/water (3/7; v/v) mixture containing 10 mM ammo-
nium formate buffer pH 3 was initially used for the optimiza-
tion of the chromatography of the nitro-aromatic compounds.
For the optimization of the (ꢀ)ESI-MS/MS conditions on
system 1 (see below) the individual dilute standards of
–
1
rate of 1.0 mL min .
–1
1
1
mg L in methanol/water (30/70; v/v) mixture containing
0 mM ammonium formate buffer pH 3 were used. Before
The chromatographic columns used were two Atlantis T3
(150 mm long, 3 mm particle size, with 3.0 mm or 2.1 mm i.d.)
and an XBridge BEH Amide (100 mmꢃ 3.0 mm i.d., 3.5 mm
particle size), and were all purchased from Waters (Milford,
MA, USA). An external thermostated water bath was used to
keep the column temperature constant.
injecting aliquots into the LC or LC/MS systems, the standards
were filtered through a PTFE membrane filter (pore size 0.2 mm,
Iso-Disk, Supelco, Bellefonte, PA, USA). All standard solutions
ꢂ
were stored at 4 C and were stable for at least 1 month.
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 793–804

LC/MS method for identification of nitro-aromatics in ambient aerosol
1
13
H-NMR,
synthesized 3M5NC were recorded on a Varian Unity Inova
00 MHz NMR spectrometer using acetone-d as solvent.
For H spectra, 100 scans were acquired with relaxation delay
C-NMR and 2D correlation spectra of the
ambiguity with regard to the identification of 3M5NC and
3M6NC, the identification of the major synthesized product
as 3M5NC (and not 3M6NC) by NMR techniques is also2
discussed here. In addition, we present direct (ꢀ)ESI-MS
data for 3M5NC and 4M5NC, which could be obtained in
pure form, and compare them with data obtained for an
ambient winter PM10 sample from Maribor, Slovenia.
3
6
1
13
of 10.0 s. For C spectra, 4000 scans and relaxation of 5.0 s
were used.
The pH of the aqueous buffers used during the LC method
optimization and application was measured using a pH
meter (Mettler Toledo MP 220), which was calibrated with
commercially available buffer solutions with pH 4.0 and pH
Nitration of 3-methylcatechol in aqueous solution using a
NaNO /H SO system (the synthesis procedure is given in
2
2
4
the Supporting information) led to the preferential formation
[20]
7
.0 (Merck).
of 3M5NC (as reported previously by Palumbo et al. ). The
ACD/LC simulator (Advanced Chemistry Development,
product was isolated as a yellow solid and its structure deter-
1
13
Inc.) software was used to aid the reversed-phase (RP) chro-
matography method development for the selected nitro-
aromatic compounds.
mined by H-NMR, C-NMR and 2D correlation spectro-
scopy. By careful inspection of the H-2 splitting pattern which
1
appears as an apparent doublet of doublets in H-NMR at
4
7
.63 ppm we were able to deduce a long-range J coupling
constant between H-1 and H-2 with a value of 0.7 Hz
Aerosol sample collection and preparation
4
(
Table 1). In addition, the J coupling constant between H-2
The filter samples containing particulate matter PM10 (parti-
cles with aerodynamic diameter below 10 mm) and control
blank filters were provided by the Environmental Agency of
the Republic of Slovenia. PM10 samples were collected on
quartz fibre filters (QMA 47 mm diameter, Whatman) at an
urban location in Maribor (ca. 110 000 inhabitants), Slovenia,
using low-volume sampling. The measurement site has the
typical character of a street canyon and is located on the main
road leading to the city centre. Due to heavy traffic, the cho-
sen location is one of the most polluted ones in Slovenia.
and H-3 has a value of 2.7 Hz which falls within the range
of meta-coupling in aromatic protons. Accordingly, proton
H-3 which resonates at 7.60 ppm appears as an apparent
4
13
doublet with a J coupling constant of 2.7 Hz. C-NMR ana-
lysis reveals a quaternary carbon C-5 bearing the nitro group
which resonates at 140.7 ppm and C-6 which resonates at
108.9 ppm. Carbon C-4 is slightly more deshielded in respect
to C-6 and resonates at 119.0 ppm (Table 1). This structural
assignment of 3-methyl-5-nitrocatechol is additionally sup-
ported by HSQC 2D correlation spectra. Analysis of COSY
spectra clearly displays a correlation between aromatic H-2
and aliphatic H-1 protons due to long-range coupling. Based
on the HMBC correlation from H-2 to C-3, C-5 and C-6 and
from H-3 to C-5 and C-4, the quaternary carbon C-5 bearing
a nitro group is connected to C-4 and C-6 (Supplementary
Fig. S1, see Supporting information).
3
–1
The air flow through the sampler was 2.3 m h and the sam-
pling time was 24 h. Prior to sampling the filters were heated
ꢂ
at 500 C for 3 h to remove organic contaminants. The weigh-
ing of the filters was done before and after sampling, after
conditioning for 48 h at a relative humidity of 50 ꢄ 5% and
ꢂ
a temperature of 20 ꢄ 1 C. Samples from winter (February)
2
2
010 were chosen for this study. The average daytime tem-
Figure 1 presents the m/z 168 product ion MS spectra for
ꢂ
perature during February was about ꢀ1 C. PM filters and
3M5NC and its positional isomer 4M5NC, which were
obtained using direct infusion on system 1. It can be seen that
both product ion spectra contain ions corresponding to the
neutral loss of NO (m/z 138), the combined loss of NO and a
1
0
ꢂ
blank filters were stored at ꢀ18 C until analysis. Before
extraction, they were equilibrated to room temperature under
controlled ambient conditions. The filters were extracted fol-
lowing the aerosol sample preparation procedure described
[21]
in our previous article.
Briefly, the filters were extracted
three times with 15 mL of methanol in an ice-cooled ultraso-
nic bath for 15 min. The total extract was then evaporated
to dryness at 25 C using a gentle stream of nitrogen. The resi-
Table 1. NMR data of 3-methyl-5-nitrocatechol in acetone-d
6
ꢂ
due was redissolved in 2 mL of methanol/water (30/70; v/v)
mixture containing 10 mM ammonium formate buffer pH 3
(the same buffer type used in the final method; see below).
RESULTS AND DISCUSSION
Synthesis and identification of methyl nitrocatechols by
NMR and direct (ꢀ)ESI-MS techniques
1
13
2
H-NMR: d
H
[ppm], mult,
C-NMR:
Carbon
J [Hz]
d
C
[ppm]
Three different positional isomers are possible for the methyl
nitrocatechols, i.e. 4M5NC, 3M5NC, and 3M6NC, which all
occur in ambient atmospheric aerosol with 3M5NC being pre-
sent at high relative abundance (see below). Of these methyl
nitrocatechols, 4M5NC was commercially available, while
C-1
C-2
C-3
C-4
C-5
C-6
C-7
144.8
151.1
125.7
119.0
140.7
108.9
16.0
7.63, app dd, 0.7, 2.7
3
M5NC was synthesized in pure form, and 3M6NC was also
7.60, app d, 2.7
2.28, s
obtained synthetically as a minor product together with
[9,20]
3
M5NC following reported procedures.
As there was
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z. Kitanovski et al.
evaluated for their separation. 4NC and methyl nitrocatechols
are weak acids in aqueous solution with the first pKa values
[
19]
between 6 and 8,
while 2NP, 4NP and 2,4DNP have pKa
[23]
values of 7.23, 7.15 and 4.07, respectively.
All these com-
pounds are polar and can be transformed into a more polar
state by dissociation to monoanions, i.e. nitrocatecholate or
nitrophenolate, respectively. These dissociated forms should
be suitable for separation under HILIC conditions. This
hypothesis was first tested using an XBridge Amide BEH
column and a mobile phase consisting of acetonitrile and
aqueous ammonium bicarbonate buffer of pH 9.0 and
1
0 mM concentration in the final mobile phase. 4NC standard
and the LC part of system 1 were used for the development of
the separation mode. Under HILIC conditions, 4NC gave a
retained, very broad and tailing peak, suggesting that these
conditions are not optimal for its chromatography, even when
the chromatographic conditions were further optimized. In
the next experiments, a mobile phase consisting of acetoni-
trile/ammonium acetate buffer pH 5.0 or acetonitrile/ammo-
nium formate buffer pH 3.0 was tested for HILIC of 4NC.
Neither sufficient symmetrical peak shape nor better reten-
tion was achieved; the loss of retention at pH 3.0 and 5.0 for
4
NC is logical and can be explained by the presence of
analyte molecules in undissociated form on the column. As
a conclusion, HILIC using the amide stationary phase is not
suitable for nitrocatechol analysis. It is worth mentioning that
previous studies also reported difficulties for obtaining
proper peak shape and retention of catecholamines (biogenic
compounds that are structurally related to nitrocatechols)
[24,25]
under HILIC conditions using a bare silica column.
2
However, a satisfactory separation of several catecholamines
on the same HILIC column as employed in our study
Figure 1. Direct infusion ESI ꢀ MS product ion spectra of m/z 168
for 3-methyl-5-nitrocatechol (A) and 4-methyl-5-nitrocatechol
B) standards obtained on the 4000 QTRAP from system 1.
[25]
(
(XBridge Amide BEH) was recently reported.
Dos Santos
[26]
Pereira et al.
suggested a new approach for overcoming
the difficulties with the separation of catecholamines under
HILIC using so-called ’per-aqueous LC’ (or PALC). Briefly,
PALC uses a 100% aqueous mobile phase and a polar column
stationary phase (like bare silica). The suggested mechanism
of retention is a reversed-phase (RP)-like interaction of the
undissociated polar analytes from the mobile phase and silox-
ane bridges on the surface of the silica packing. The retention
of the analytes lowers as the percentage of the organic modi-
fier (added to the aqueous mobile phase) increases (a typical
RP mechanism of retention). A very good and improved
separation of the catecholamines under PALC using a silica
column and aqueous 50 mM ammonium formate buffer as
mobile phase could be obtained. The findings of dos Santos
Pereira et al. prompted us to evaluate the PALC separation
technique. Using aqueous buffers (ammonium acetate pH 5.0
and ammonium formate pH 3.0) as mobile phase and the
amide column in PALC gave better results for 4NC only
when ammonium formate pH 3.0 was used as mobile phase.
However, even though the retention factor (k) of 4NC was
hydrogen radical (m/z 137), the loss of NO
combined loss of NO and CO (m/z 109) from the deproto-
nated molecule, as well as an ion at m/z 46 (NO ), although
2
with different relative abundances. The same m/z 168 product
ions were also obtained for the PM10 sample (not shown),
indicating that methyl nitrocatechols are present; however,
at this stage the isomeric composition could not be inferred
with certainty. In addition, the m/z 168 product ion spectrum
of the ambient sample contained very weak ions at m/z 153,
2
(m/z 122), the
–
1
23 and 95 that are characteristic of nitroguaiacols, which
[9]
have been reported in ambient aerosol. Thus, in order to
confirm the presence or absence of certain isomeric methyl
nitrocatechols or nitroguaiacols an adequate chromato-
graphic separation is needed. Hence, an effort was made to
develop systematically a suitable chromatographic method
for the separation of isomeric methyl nitrocatechols and
related nitro-aromatic compounds that are expected to be
present in ambient BBA.
[26]
<1, its peak showed significant tailing and thus smaller
Development of a LC method for separation of methyl
nitrocatechols and related compounds
separation efficiency, which was substantially influenced by
the ionic strength of the buffer in the mobile phase. A higher
ionic strength of the buffer (e.g. 50 mM) gave better peak
shape and efficiency. Together with the column temperature
it remains the only parameter that can be optimized under
PALC conditions in order to obtain proper retention or
separation. Addition of methanol or acetonitrile to the
aqueous mobile phase significantly decreased the retention
Selection of the separation mode
Initially, the study was focused on some selected nitro-
aromatic compounds, i.e. 4NP, 2NP, 4NC, 2,4DNP, 4M5NC,
3
M5NC and 3M6NC. In a first series of experiments hydro-
[22]
philic interaction liquid chromatography (HILIC)
was
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 793–804

LC/MS method for identification of nitro-aromatics in ambient aerosol
of the 4NC peak under these conditions. Taking into account
the structural similarity of the nitrocatechols, nitrophenols,
and methyl nitrocatechols, together with the limited chroma-
tography parameters that could be changed under PALC
to further optimize their separation, we did not proceed
with further experiments using this mode. Hence, in a next
series of experiments reversed-phase chromatography (RPC)
was evaluated and optimized for the separation of nitrocate-
chols, nitrophenols and methyl nitrocatechols. Previous stu-
dies on nitro-aromatics have shown the suitability of RPC
[e.g. 11,13–16]
in the analysis of nitrophenols,
nitrocatechols and
[
9,13,14]
4
-nitroguaiacol.
Influence of buffer type and pH under reversed-phase
chromatography
In order to obtain the best conditions concerning symmetrical
peak shapes and a high efficiency and resolution among
peaks, different buffers and pH values were evaluated initi-
ally for the separation of nitro-aromatics. Ammonium acetate
buffer pH 5.0 used in the mobile phase together with metha-
nol gave non-symmetrical and badly tailing peaks for nitroca-
techols, while ammonium formate at pH 3.9 and 3.4 gave
fronting peaks for nitrocatechols and symmetrical peaks for
nitrophenols. The reasons for peak fronting could be differ-
ent, i.e. formation of column void, buffer/pH mismatch
between the injected solutions and the mobile phase, a ther-
mally non-equilibrated column, overloading of the column,
or eventually a wrongly chosen mobile phase buffer and/or
[
27]
pH conditions.
Formation of column void as a cause for
the observed fronting was ruled out because all the peaks
should have been equally affected, which was not the case.
In a next experiment, the column was thermally equilibrated
ꢂ
at constant temperature (25 C) and the final standard solu-
tions were prepared in the mobile phase (methanol/water/
2
00 mM ammonium formate buffer; 30:50:20; v/v/v) in order
to exclude the possibility for peak fronting owing to a ther-
mally non-equilibrated column or a buffer/pH mismatch.
The overloading of the column was ruled out as the cause
for peak fronting, because the mass of the analytes injected
on column was around 100 ng. The only remaining logical
explanation for the observed peak effect is the non-optimal
buffer and/or pH used in the final mobile phase. Indeed,
when ammonium formate buffer pH 3.0 was used in the
mobile phase, the problem with fronting nitrocatechol peaks
was alleviated. Peak symmetry and column efficiency were
slightly influenced by the ionic strength of the final mobile
phase. It was found that for optimal peak shape and
efficiency, a concentration of 40 mM ammonium formate is
needed in the final mobile phase.
Figure 2. Chromatograms of standard containing: 4-
nitrocatechol (1), 4-methyl-5-nitrocatechol (2), 4-nitrophenol
Subsequently, 0.1% (w/w) formic and 0.1% (w/w) acetic
acid were evaluated as additives in the mobile phase. The
pH values of the aqueous 0.1% formic and 0.1% acetic acid
(
3), 3-methyl-6-nitrocatechol (4), 2,4-dinitrophenol (5), 2-
nitrophenol (6) and 3-methyl-5-nitrocatechol (7), obtained
under following conditions: methanol/water/1.0% formic
acid = 44/46/10 (v/v/v) (A), methanol/water/1.0% acetic
acid = 44/46/10 (v/v/v) (B), methanol/water/200 mM
ammonium formate pH 3.0 = 44/36/20 (v/v/v) (C). For
all chromatograms – column: Atlantis T3 (150 x 3.0 mm
ꢂ
are 2.7 and 3.3 (at 25 C), respectively. Figure 2 shows the
chromatograms of a standard mixture containing nitrophe-
nols, 4-nitrocatechol and methyl nitrocatechols studied,
obtained under the same chromatographic conditions, differ-
ing only in the acid used. When formic or acetic acid was
used in the mobile phase (with final mobile phase concentra-
tion of 0.1% w/w, see Figs. 2(A) and 2(B)) there was no
change in selectivity and all peaks showed significant tailing,
which was more pronounced when acetic acid was used. The
–
1
i.d, 3 mm particle size), flow rate: 0.4 mL min , column
ꢂ
temperature: 30 C, injection volume: 5 mL, detection: UV
(
345 nm).
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z. Kitanovski et al.
chromatogram obtained using a 40 mM final mobile phase
concentration of ammonium formate buffer pH 3.0 (Fig. 2(C))
shows a different selectivity and a much better separation and
peak shape for the analytes compared to the previous ones
(Figs. 2(A) and 2(B)). Increasing the concentration of acetic or
formic acid in the final mobile phase from 0.1% to 0.2%, 0.3%
or 0.5% gradually improved the peak shape to some extent,
but did not completely alleviate the problem with peak tailing.
Hence, ammonium formate buffer pH 3.0 was chosen for the
RPC optimizations reported in the next section.
Optimization of isocratic separation using methanol as organic
modifier
Isocratic mobile phases with a fixed ionic strength (40 mM) of
ammonium formate pH 3.0, but a different percentage of
methanol in the final composition (30, 40, 50, and 60%), were
ꢂ
used at four different temperatures (25, 30, 35, and 40 C) to
obtain the retention data for all compounds. The resolution
map for the software-aided simultaneous optimization of %
organic modifier B (methanol) and column temperature is
given in Supplementary Fig. S2 (see Supporting information).
In order to obtain a better prediction for analyte retention
and separation, only the theoretical points within the rectan-
gular area limited and populated by the experimental points
on the map were taken into consideration. Within this area,
the theoretical points that show sufficient separation among
peaks and reasonable run times (< 20 min) have ’coordinates’
ꢂ
around 45% for methanol and around 30 C for column
temperature. The most optimal separations according to the
software predictions and the real experiment are shown in
Supplementary Fig. S3 (see Supporting information). As a
conclusion, the best separation for the targeted analytes can
be achieved using isocratic elution with 44% of methanol
and ammonium formate pH 3.0 (final mobile phase concen-
Figure 3. Chromatograms of PM10 sample from the city of
Maribor, Slovenia (February 2010). Conditions: isocratic
elution, methanol/water/200 mM ammonium formate pH
ꢂ
3
.0 = 44/36/20 (v/v/v), column T: 32 C (A) and isocratic
ꢂ
tration of 40 mM) and at temperature of 32 C. A wintertime
elution, methanol/acetonitrile/water/200 mM ammonium
PM10 sample analyzed under these optimized conditions is
shown in Fig. 3(A). For the purpose of identification, reten-
tion times (RTs) and UV/Vis spectra (range: 200–800 nm) of
the sample peaks were compared with those of standards.
Several nitro-aromatic compounds were detected in the
winter PM10 sample from Maribor, Slovenia (Fig. 3(A)), i.e.
ꢂ
formate pH 3.0 = 30/11/39/20 (v/v/v/v), column T: 30 C
(B). For both chromatograms – column: Atlantis T3 (150 x
–
1
3.0 mm i.d., 3 mm particle size), flow rate: 0.4 mL min , injec-
tion volume: 20 mL, detection: UV (345 nm).
phase organic modifier. For that purpose, methanol in the
optimized mobile phase (44%) was substituted by another
organic solvent giving an equivalent eluotropic strength
(34% for acetonitrile, 25% for tetrahydrofuran, and 22% for
2-propanol), while the rest of the mobile phase (up to 100%)
was ammonium formate pH 3.0 with a 40 mM final mobile
phase concentration. Substituting methanol with acetonitrile
4
2
NC, 4M5NC, 3M5NC, and 4NP. The peak assigned to
,4DNP, based on chromatographic RT matching, turned
out to be a different compound because its UV/Vis spectrum
significantly differed from that of 2,4DNP (not shown).
Change of selectivity by addition of different organic modifiers
(
Fig. 4(A) compared to Fig. 2(C)) and tetrahydrofuran gave
A substantial change in the selectivity of separation can be
accomplished by a change in the composition of the station-
ary phase and/or mobile phase, as well as of the column
a substantial change in the selectivity of the separation, while
2-propanol did not result in a substantial change. Further,
using different blends of methanol/acetonitrile, methanol/
tetrahydrofuran or methanol/2-propanol in the mobile
phase, an effort was made to optimize the composition of
the ternary mobile phase that would give the best (baseline)
separation for the targeted compounds (Figs. 4(B) and 4(C)).
The mobile phase containing a blend of methanol and aceto-
nitrile (30/11; v/v) as strong solvent was used for further
analysis (Fig. 4(B)). This mobile phase composition gave a
satisfactory chromatographic separation and also a lower
back pressure compared to the mobile phases containing
methanol/tetrahydrofuran or methanol/2-propanol blends.
[
27–29]
temperature.
Because only one stationary phase was
used in our experiments and taking into account the narrow
optimal temperature range, changing the composition of the
mobile phase should be the strongest tool for altering the
selectivity. For ionizable analytes, changing the mobile phase
pH could substantially influence the selectivity of separa-
[
29]
tion.
However, the change of pH and buffer type is very
limited because of the peak shape problems encountered
when the pH is greater than 3. Hence, a practical tool for
altering the selectivity could be the change of the mobile
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 793–804

LC/MS method for identification of nitro-aromatics in ambient aerosol
based on its RT, elutes now at 12.91 min, and no longer co-
elutes with the dinitrophenol (RT 13.41 min) and is at this
stage unknown.
Adaptation of the LC/UV–vis method to LC/(ꢀ)ESI-MS
The experiments performed for adapting the LC/UV–vis
method outlined above to LC/(ꢀ)ESI-MS analysis were made
using system 2. For the purpose of LC/(ꢀ)ESI-MS analysis,
some modifications of the developed LC/UV–vis method
were necessary in order to accommodate the chromato-
graphic conditions for (ꢀ)ESI-MS detection. The ionic
strength of the mobile phase was decreased from 40 mM to
5
mM (keeping the pH at 3.0) to avoid excessive ion suppres-
sion in the ion source as well as clogging problems in transfer
lines and the ion source. Due to the decrease in the ionic
strength of the mobile phase, a lowering of the overall
chromatographic resolution occurred. However, a sufficient
resolution (>2) is still kept among the methyl nitrocatechol
isomers under the new chromatographic conditions.
At this stage, two nitroguaiacols (4- and 5-nitroguaiacol:
4
NG and 5NG), two nitrosalicylic acids (3- and 5-nitrosa-
licylic: 3NSA and 5NSA) and two methyl-nitrophenols
2-methyl-4-nitrophenol and 3-methyl-4-nitrophenol: 2M4NP
(
and 3M4NP) were additionally included in the method. The
separation of all analytes was achieved using the optimized
mobile phase containing methanol/acetonitrile/aqueous
ammonium formate buffer pH 3 = 30/11/59 (v/v/v), with
5
mM salt concentration in the final mobile phase mixture.
Other chromatographic parameters were not modified. The
–1
mobile phase flow was decreased to 0.2 mL min because of
the narrower column (2.1 mm i.d.) used for the corresponding
LC/MS analysis. LC/MS chromatographic data for the same
wintertime PM10 sample (previously analyzed with system 1)
is shown in Fig. 5. The two most intense peaks in the ambient
sample at RTs 7.08 and 16.30 min correspond to 4NC (Fig. 5(B))
and 3M5NC (Fig. 5(C)), respectively. 4M5NC (Fig. 5(C))
and 4NP (Fig. 5(D)) also show intense peaks (RTs 10.74 and
11.08 min, respectively), while 3M6NC (Fig. 5(C)) is present
as a minor peak (RT 12.04 min). In addition, the PM10 sample
from Maribor also revealed the presence of 3NSA and 5NSA
(
2
Fig. 5(E); RTs 5.24 and 7.45, respectively), and 3M4NP and
M4NP (Fig. 5(F); RTs 18.01 and 22.88 min, respectively).
Characterization of methyl nitrocatechols and related
2
tracers using LC/(ꢀ)ESI-MS
2
The experiments for obtaining LC/(ꢀ)ESI-MS data were
Figure 4. Chromatograms of the same standard mixture as in
carried out with system 2. In this section, we only deal with
Fig. 2, obtained under following conditions: acetonitrile/water/
2
LC/(ꢀ)ESI-MS data for nitro-aromatic compounds that
200 mM ammonium formate pH 3.0 = 34/46/20 (v/v/v) (A),
methanol/acetonitrile/water/200 mM ammonium formate pH
.0 = 30/11/39/20 (v/v/v/v) (B), methanol/tetrahydrofuran/
water/200 mM ammonium formate pH 3.0 = 30/15/35/20
occur in ambient atmospheric aerosol.
Figure 6 presents [M–H] product ion MS spectra for 4NP
ꢀ
2
3
(Fig. 6(A)), 4NC (Fig. 6(B)), 3M5NC (Fig. 6(C)), 3M6NC (Fig. 6
(
v/v/v/v) (C). Other chromatographic parameters are the
(
D)), and 4M5NC (Fig. 6(E)). Under the selected CID condi-
same as in Fig. 2.
tions (i.e. 35% normalized collision energy) the deprotonated
molecules of 4NP and 4NC are quite stable and only result in
a weak loss of NO, affording an ion at m/z 108 and 124,
respectively. Furthermore, the precursor ions have a weak
satellite ion one unit higher at m/z 139 and 155, respectively,
which can be explained by the addition of a hydrogen radical,
Figure 3(B) presents a chromatogram obtained from the
same urban PM10 sample as in Fig. 3(A); it can be seen that
using the ternary mobile phase (methanol/acetonitrile/buffer
pH 3.0) gave better separation and specificity for the peaks in
the PM10 sample chromatogram. The relatively intense peak
at RT 9.49 min in Fig. 3(A), which was assigned to 2,4DNP
[30,31]
a reaction that is known to occur in the ion trap.
It is
worth noting that the extent of loss of NO (m/z 138) is quite
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z. Kitanovski et al.
Figure 5. Base peak (BPC) (A) and extracted ion current (EIC) chromatograms (B–F) of PM10 sample from city of Maribor,
Slovenia (February 2010). Conditions: 0–13 min: isocratic methanol/acetonitrile/8.5 mM ammonium formate pH 3.0=
3
0/11/59 (v/v/v), 13–19 min: linear gradient methanol: 30! 50% and buffer: 59! 39%, 19–35 min: isocratic methanol/
acetonitrile/8.5 mM ammonium formate pH 3.0 = 50/11/39 (v/v/v), column: Atlantis T3 (150 x 2.1 mm i.d., 3 mm particle
ꢂ
–1
size), column T: 30 C, flow rate: 0.2 mL min , injection volume: 10 mL, detection: LIT-MS (scan m/z 50–500).
different among the three methyl nitrocatechol isomers. The
deprotonated form of 4M5NC reveals the most extensive loss,
that of 3M5NC the smallest one, and that of 3M6NC a loss
that is intermediate between that observed for 4M5NC
and 3M5NC. The m/z 138 also shows a small satellite ion at
m/z 139 due to the addition of a hydrogen radical, as already
outlined above for deprotonated 4NP and 4NC. In addition,
deprotonated 3M6NC (Fig. 6(D)) leads to minor ions at
m/z 150, due to the loss of a molecule of water (an ortho
effect); m/z 123, corresponding to the combined loss of NO
and a methyl radical, and at m/z 94, corresponding to a loss
of 74 units that cannot be readily explained. Thus, the three
methyl nitrocatechol isomers can be differentiated on the
2
basis of the MS product ion spectra of their deprotonated
[32]
forms. With regard to the observed NO loss, Levsen et al.
reported that nitro-aromatic compounds show characteristic
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 793–804

LC/MS method for identification of nitro-aromatics in ambient aerosol
2
Figure 6. LC/MS product ion spectra of 4-nitrophenol (A), 4-nitrocatechol (B), 3-methyl-5-nitrocatechol (C), 3-methyl-
6
-nitrocatechol (D), and 4-methyl-5-nitrocatechol (E) (Fig. 5).
•
–
•
–
[
M–H–NO] and [M–H–NO
2
]
ions in the product ion mass
and a peak at m/z 138 due to the loss of NO. The formation of
–
–
spectra of their deprotonated molecules [M–H] , with the loss
of NO being favored over the loss of NO
a prominent product ion [M–H
2
] in negative ESI has been
•
•
[34]
2
, owing to the
reported by Attygalle et al.
for 2,4-dihydroxybenzaldehyde
preferential formation of a thermodynamically more stable
(2,4-DHBA). By using deuterated 2,4-DHBA and high-resolution
MS, it was established that the coexistence of a hydroxyl and an
aldehyde group in the ortho-position on the aromatic ring is a
prerequisite for H2 elimination. Taking this behavior into
account the investigated m/z 168 compound should have the
2-hydroxybenzaldehyde moiety (salicylaldehyde group). Since
the more oxidized form, i.e. salicylic acid, has already been
•
–
•
–
product ion ArO over the less stable product ion Ar
in the case of deprotonated Ar-NO , where Ar denotes an
(
2
aromatic moiety). It is noted that in the case of 4NP, 4NC, and
•
the methyl nitrocatechols discussed above, the loss of NO from
2
the deprotonated molecule was not observed at a normalized
•
–
collision energy of 35%. The formation of ArO corresponds
to a rearrangement reaction involving a 1,2-phenyl shift
•
(
Ar-N(O)O ! Ar-O-NO) followed by the loss of NO .
It can be seen in the m/z 168 extracted ion chromatogram
EIC) (Fig. 5(C)) that in addition to the three methyl nitrocate-
(
chol isomers there are other m/z 168 compounds eluting earlier
–
2
at RTs of 5.97 and 9.26 min. The [M–H] product ion MS spec-
tra for the latter compounds is given in Figs. 7(A) and 7(B),
respectively. The compound eluting at 9.26 min (Fig. 7(B)) could
be assigned to 4NG, 5NG or 6-nitroguaiacol (6NG, 2-methoxy-
6-nitrophenol), based on the loss of 15 units corresponding to
the loss of a methyl radical that is characteristic for an aromatic
[
9,33]
2
methoxy group.
However, subsequent LC/(ꢀ)ESI-MS
experiments with 4NG and 5NG reference compounds allowed
us to rule out 4NG and 5NG, which elute at RTs 11.97 and
10.76 min, respectively. Since 6NG is not available commer-
cially we cannot at this stage unambiguously confirm or reject
its presence in our sample. However, if we take into account
the possible nitration of guaiacol in ortho- or para-position with
respect to the hydroxyl group, 6NG is a very probable candi-
[
33]
date.
.97 min (Fig. 7(A)) shows an abundant product ion at
m/z 166 (formal loss of 2 units), likely due to the loss of H
The deprotonated form of the compound eluting at
2
5
Figure 7. LC/MS product ion spectra of m/z 168 compounds
2
at RT = 5.97 min (A) and RT = 9.26 min (B) (Fig. 5).
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z. Kitanovski et al.
[
35,36]
reported in the atmosphere,
this appears a reasonable
shown), and a pattern similar to that observed for the depro-
n
possibility. However, this nitro-aromatic compound remains
only partially characterized.
tonated form of 4NP (Fig. 6(A)). These (ꢀ)ESI-MS (n = 2 and
3) data allowed us to conclude that these two compounds
With respect to the m/z 182 compounds, the EIC reveals
several peaks in the 4–30 min RT region (Fig. 5(E)). The MS
contain a carboxyl, a hydroxy and an aromatic nitro
2
[32,37,38]
group.
This led us to consider nitrosalicylic acid iso-
product ion spectra of the three compounds eluting at 5.24,
mers, i.e. 3NSA and 5NSA, which have been reported by
[35]
7
.45, and 19.41 min are presented in Figs. 8(A), 8(B) and 8
C), respectively, while those of the later-eluting compounds
are shown in Figs. 8(D) (RT = 20.01 min), 8(E) (RT = 21.79 min),
(F) (RT = 24.38 min) and 8(G) (RT = 26.82 min). It is worth
noting that the m/z 182 compounds (i.e. RTs 20.01 and
6.82 min) have a relative abundance that is comparable to
those of the methyl nitrocatechols (i.e. RTs 10.74 and
6.30 min); hence, an effort was made to characterize them.
van Pinxteren and Herrmann in atmospheric aerosol using2
capillary electrophoresis/MS. Subsequent LC/(ꢀ)ESI-MS
experiments with 3NSA and 5NSA reference compounds
allowed us to assign the m/z 182 compounds eluting at RTs
5.24 and 7.45 min to 3NSA and 5NSA, respectively. The
m/z 182 compound eluting at RT 19.41 min (Fig. 8(C)) shows
a characteristic loss of a methyl radical (m/z 167) consistent
with the presence of an aromatic methoxy group, as observed
for deprotonated nitroguaiacol (Fig. 7(B)). Other losses
observed for this m/z 182 compound include the loss of NO
(m/z 152) and the combined loss of NO and a methyl radical
(m/z 137). This fragmentation behavior allows us to partially
(
8
2
1
The two first-eluting compounds show a fragmentation beha-
vior that is distinctly different from that of the later-eluting
m/z 182 compounds. The two first-eluting compounds (Figs. 8
(
A) and 8(B)) both reveal the loss of 44 units, corresponding to
the loss of CO (m/z 138) consistent with the presence of a
carboxylic group.
2
characterize this compound as a methyl homolog of 6NG.
[
37]
[33]
Subsequent fragmentation of m/z 138
In this regard Iinuma et al.
reported an m/z 182 compound
leads to the loss of NO resulting in an ion at m/z 108 (not
containing aromatic nitro, methoxy and hydroxyl groups in
2
Figure 8. LC/MS product ion spectra of m/z 182 compounds at RT = 5.24 min (A), RT = 7.45 min (B), RT = 19.41 min (C),
RT = 20.01 min (D), RT = 21.79 min (E), RT = 24.38 min (F), and RT = 26.82 min (G) (Fig. 5).
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 793–804

LC/MS method for identification of nitro-aromatics in ambient aerosol
on the basis of the structural characterization using NMR for
the 3-methyl-5-nitrocatechol synthesized in pure form. The pro-
posed LC/MS method has the advantage compared to a pre-
[9]
viously published one in that it results in a better separation
and specificity for the targeted analytes. For example, a better
separation is obtained for the MW 169 compounds (m/z 168).
In addition, a satisfactory separation is achieved for homolo-
gous MW 183 nitro-aromatic compounds (m/z 182), some of
which are also quite abundant in ambient aerosol. Comparison
2
of LC/(ꢀ)ESI-MS data of available reference compounds and
compounds observed in ambient aerosol allowed us to unam-
biguously confirm the presence of 4-nitrophenol, 2-methyl-
4
-nitrophenol, 3-methyl-4-nitrophenol, 4-nitrocatechol, 3-nitro-
salicylic acid, 5-nitrosalicylic acid, 3-methyl-5-nitrocatechol,
-methyl-6-nitrocatechol, and 4-methyl-5-nitrocatechol. Further-
3
2
more, interpretation of LC/(ꢀ)ESI-MS data for other nitro-
aromatic compounds present in ambient aerosol led to the
complete or partial characterization of methyl 6-nitroguaiacol
and dimethyl nitrocatechols, which could serve as additional
useful SOA tracers. The characterization of these tracers is a
prerequisite to develop in a further stage a quantitative method
and apply it to BBA. This should enable us to compare the con-
centrations of the SOA tracers with those of the well-established
primary BB tracer, levoglucosan.
2
Figure 9. LC/MS product ion spectra of m/z 152
compounds: 3-methyl-4-nitrophenol at RT = 18.01 min (A)
and 2-methyl-4-nitrophenol at RT = 22.88 min (B) (Fig. 5).
aerosol obtained by combustion of pine wood with green
needles. On a basis of its MS fragmentation patterns they
2
attributed this m/z 182 compound to 4-methyl-6-nitroguaiacol
(2-methoxy-4-methyl-6-nitrophenol).
Concerning the m/z 182 compounds eluting between 20
SUPPORTING INFORMATION
and 27 min (5 peaks), all the m/z 182 precursor ions reveal
the loss of NO (m/z 152), consistent with the presence of an
aromatic nitro group. These compounds were partially char-
acterized as methyl homologs of the methyl nitrocatechols,
for which six positional isomers are possible. However, with-
out reference compounds no unambiguous attributions of the
dimethyl nitrocatechols can be made. It can be seen from the
mass spectral data for two of the five isomers (Figs. 8(D) and
Additional supporting information may be found in the
online version of this article.
Acknowledgements
This work was supported by the Slovenian Research Agency
(
Contract Nos. P1-0034-0104 and BI-BE/11-12-F-012). The
8
(F)) that the stability of the m/z 182 precursor ion upon CID
authors would like to thank mag. Tanja Bolte from the Envir-
onmental Agency of Republic of Slovenia for providing the
PM10 samples. We thank also Aleksandar Gaćeša from the
Slovenian NMR Center (National Institute of Chemistry,
Ljubljana) for the help during the NMR analysis and for
useful discussions.
is similar, i.e. the relative abundance of m/z 152 corresponding
to the loss of NO is quite comparable.
A last set of compounds that could be characterized in the
ambient PM10 sample from Maribor were m/z 152 compounds.
These compounds were assigned to isomeric methyl nitrophe-
nols, i.e. 3-methyl-4-nitrophenol (3M4NP: RT 18.01 min) and
2-methyl-4-nitrophenol (2M4NP; RT 22.88 min), based on com-
parison of chromatographic and mass spectral data with refer-
ence compounds (Figs. 9(A) and 9(B)). These nitro-aromatic
compounds have been reported in rainwater by Ganranoo
et al. and in BBA produced by pine wood combustion. Both
isomers reveal a weak ion at m/z 122 due to the loss of NO, while
REFERENCES
[15]
[13]
[1] A. Laskin, J. S. Smith, J. Laskin. Molecular characterization of
nitrogen-containing organic compounds in biomass burning
aerosols using high-resolution mass spectrometry. Environ.
Sci. Technol. 2009, 43, 3764.
2] B. R. T. Simoneit. Biomass burning – a review of organic tra-
cers for smoke from incomplete combustion. Appl. Geochem.
2002, 17, 129.
3] B. R. T. Simoneit, J. J. Schauer, C. G. Nolte, D. R. Oros,
V. O. Elias, M. P. Fraser, W. F. Rogge, G. R. Cass. Levoglucosan,
a tracer for cellulose in biomass burning and atmospheric
particles. Atmos. Environ. 1999, 33, 173.
4] V. O. Elias, B. R. T. Simoneit, R. C. Cordeiro, B. Turcq. Evaluat-
ing levoglucosan as an indicator of biomass burning in Carajás,
Amazônia: A comparison to the charcoal record. Geochim.
Cosmochim. Acta 2001, 65, 267.
3M4NP shows an additional weak ion at m/z 134 due to the loss
of water; the latter can be explained by an ortho effect.
[
CONCLUSIONS
[
A new LC/MS method based on reversed-phase chromatogra-
phy and negative ion electrospray ionization mass spectro-
metric detection has been systematically developed for the
separation and characterization of methyl nitrocatechols and
related nitro-aromatic compounds, including 4-nitrophenol,
[
4-nitrocatechol, 4- and 5-nitroguaiacols, 3- and 5-nitrosalicylic
acids, and 2- and 3-methyl-4-nitrophenols in atmospheric aero-
sols. The ambiguity concerning the identification of 3-methyl-
[
5] V. Pashynska, R. Vermeylen, G. Vas, W. Maenhaut, M. Claeys.
Development of a gas chromatographic/ion trap mass spec-
trometric method for the determination of levoglucosan and
5-nitrocatechol and 3-methyl-6-nitrocatechol has been clarified
Rapid Commun. Mass Spectrom. 2012, 26, 793–804
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z. Kitanovski et al.
saccharidic compounds in atmospheric aerosols. Application
to urban aerosols. J. Mass Spectrom. 2002, 37, 1249.
6] M. Mochida, K. Kawamura, P. Fu, T. Takemura. Seasonal
variation of levoglucosan in aerosols over the western North
Pacific and its assessment as a biomass-burning tracer. Atmos.
Environ. 2010, 44, 3511.
7] D. Hoffmann, A. Tilgner, Y. Iinuma, H. Herrmann. Atmo-
spheric stability of levoglucosan: A detailed laboratory
and modeling study. Environ. Sci. Technol. 2010, 44, 694.
8] L.-J. Kuo, B. E. Herbert, P. Louchouarn. Can levoglucosan be
used to characterize and quantify char/charcoal black carbon
in environmental media? Org. Geochem. 2008, 39, 1466.
9] Y. Iinuma, O. Böge, R. Gräfe, H. Herrmann. Methyl-
nitrocatechols: Atmospheric tracer compounds for biomass
burning secondary organic aerosols. Environ. Sci. Technol.
[22] A. J. Alpert. Hydrophilic-interaction chromatography for
the separation of peptides, nucleic acids and other polar
compounds. J. Chromatogr. A 1990, 499, 177.
[
[23] D. R. Lide (Ed.). CRC Handbook of Chemistry and Physics, (87th
edn.), CRC Press, Taylor & Francis Group, Boca Raton, 2006.
[24] D. V. McCalley. Evaluation of the properties of a superfi-
cially porous silica stationary phase in hydrophilic interac-
tion chromatography. J. Chromatogr. A 2008, 1193, 85.
[25] A. Kumar, J. P. Hart, D. V. McCalley. Determination of
catecholamines in urine using hydrophilic interaction chro-
matography with electrochemical detection. J. Chromatogr.
A 2011, 1218, 3854.
[26] A. dos Santos Pereira, F. David, G. Vanhoenacker, P. Sandra.
The acetonitrile shortage: Is reversed HILIC with water an
alternative for the analysis of highly polar ionizable solutes?
J. Sep. Sci. 2009, 32, 2001.
[
[
[
2
010, 44, 8453.
[
10] J. L. Kelly, D. V. Michelangeli, P. A. Makar, D. R. Hastie,
[27] L. R. Snyder, J. J. Kirkland, J. W. Dolan. Introduction to
Modern Liquid Chromatography, (3rd edn.), John Wiley &
Sons, Hoboken, New Jersey, 2010.
M. Mozurkewich, J. Auld. Aerosol speciation and mass pre-
diction from toluene oxidation under high NO conditions.
x
Atmos. Environ. 2010, 44, 361.
[28] U. D. Neue, J. E. O’Gara, A. Méndez. Selectivity in reversed-
phase separations – Influence of the stationary phase.
J. Chromatogr. A 2006, 1127, 161.
[29] U. D. Neue, A. Méndez. Selectivity in reversed-phase
separations: General influence of solvent type and mobile
phase pH. J. Sep. Sci. 2007, 30, 949.
[30] R. Szmigielski, J. D. Surratt, R. Vermeylen, K. Szmigielska,
J. H. Kroll, N. L. Ng, S. M. Murphy, A. Sorooshian,
J. H. Seinfeld, M. Claeys. Characterization of 2-methyl-
glyceric acid oligomers in secondary organic aerosol
formed from the photooxidation of isoprene using
trimethylsilylation and gas chromatography/ion trap
mass spectrometry. J. Mass Spectrom. 2007, 42, 101.
[31] P. A. Demirev. Generation of hydrogen radicals for reac-
tivity studies in Fourier transform ion cyclotron reso-
nance mass spectrometry. Rapid. Comm. Mass Spectrom.
2000, 14, 777.
[
[
[
11] M. A. J. Harrison, S. Barra, D. Borghesi, D. Vione, C. Arsene,
R.I. Olariu. Nitrated phenols in the atmosphere: a review.
Atmos. Environ. 2005, 39, 231.
12] A. Cecinato, V. Di Palo, D. Pomata, M.C.T. Scianò,
M. Possanzini. Measurement of phase-distributed nitrophe-
nols in Rome ambient air. Chemosphere 2005, 59, 679.
13] D. Hoffmann, Y. Iinuma, H. Herrmann. Development of a
method for fast analysis of phenolic molecular markers in
biomass burning particles using high performance liquid
chromatography/atmospheric pressure chemical ionisation
mass spectrometry. J. Chromatogr. A 2007, 1143, 168.
14] Y. Y. Zhang, L. Müller, R. Winterhalter, G. K. Moortgat,
T. Hoffmann, U. Pöschl. Seasonal cycle and temperature
dependence of pinene oxidation products, dicarboxylic
acids and nitrophenols in fine and coarse air particulate
matter. Atmos. Chem. Phys. 2010, 10, 7859.
[
[
15] L. Ganranoo, S. K. Mishra, A. K. Azad, A. Shigihara,
P. K. Dasgupta, Z. S. Breitbach, D. W. Armstrong, K. Grudpan,
B. Rappenglueck. Measurement of nitrophenols in rain and
air by two-dimensional liquid chromatography-chemically
active liquid core waveguide spectrometry. Anal. Chem.
[32] K. Levsen, H. M. Schiebel, J. K. Terlouw, K. J. Jobst,
M. Elend, A. Preib, H. Thiele, A. Ingendoh. Even-electron
ions: a systematic study of the neutral species lost in the
dissociation of quasi-molecular ions. J. Mass Spectrom.
2007, 42, 1024.
2
010, 82, 5835.
[33] Y. Iinuma, E. Brüggemann, T. Gnauk, K. Müller,
M. O. Andreae, G. Helas, R. Parmar, H. Herrmann. Source
characterization of biomass burning particles: The combustion
of selected European conifers, African hardwood, savanna
grass, and German and Indonesian peat. J. Geophys. Res.
2007, 112, D08209.
[34] A. B. Attygalle, J. Ruzicka, D. Varughese, J. B. Bialecki, S. Jafri.
Low-energy collision-induced fragmentation of negative ions
derived from ortho-, meta-, and para-hydroxyphenyl carbalde-
hydes, ketones, and related compounds. J. Mass Spectrom.
2007, 42, 1207.
[35] D. van Pinxteren, H. Herrmann. Determination of functio-
nalized carboxylic acids in atmospheric particles and cloud
water using capillary electrophoresis/mass spectrometry.
J. Chromatogr. A 2007, 1171, 112.
[36] P. Fu, K. Kawamura, J. Chen, L. A. Barrie. Isoprene, mono-
terpene, and sesquiterpene oxidation products in the high
Arctic aerosols during late winter to early summer. Environ.
Sci. Technol. 2009, 43, 4022.
[
16] K. Noguchi, A. Toriba, S. W. Chung, R. Kizu, K. Hayakawa.
Identification of estrogenic/anti-estrogenic compounds in
diesel exhaust particulate extract. Biomed. Chromatogr. 2007,
2
1, 1135.
[
17] S. Taneda, Y. Mori, K. Kamata, H. Hayashi, C. Furuta, C. Li,
K. Seki, A. Sakushima, S. Yoshino, K. Yamaki, G. Watanabe,
K. Taya, A.K. Suzuki. Estrogenic and anti-androgenic activity
of nitrophenols in diesel exhaust particles (DEP). Biol. Pharm.
Bull. 2004, 27, 835.
18] K. Seki, Y. Noya, Y. Mikami, S. Taneda, A. K. Suzuki, Y. Kuge,
K. Ohkura. Isolation and identification of new vasodilative
substances in diesel exhaust particles. Environ. Sci. Pollut.
Res. 2010, 17, 717.
19] D. H. Rosenblatt, J. Epstein, M. Levitch. Some nuclearly
substituted catechols and their acid dissociation constants.
J. Am. Chem. Soc. 1953, 75, 3277.
20] A. Palumbo, A. Napolitano, M. d’Ischia. Nitrocatechols
versus nitrocatecholamines as novel competitive inhibi-
tors of neuronal nitric oxide synthase: Lack of the
aminoethyl side chain determines loss of tetrahydrobiopterin-
antagonizing properties. Bioorg. Med. Chem. Lett. 2002,
[
[
[
[37] J. Dron, G. Eyglunent, B. Temime-Roussel, N. Marchand,
H. Wortham. Carboxylic acid functional group analysis
using constant neutral loss scanning-mass spectrometry.
Anal. Chim. Acta 2007, 605, 61.
1
2, 13.
[
21] Z. Kitanovski, I. Grgić, M. Veber. Characterization of
carboxylic acids in atmospheric aerosols using hydrophilic
interaction liquid chromatography tandem mass spectrometry.
J. Chromatogr. A 2011, 1218, 4417.
[38] J. Dron, E. Abidi, I. El Haddad, N. Marchand, H. Wortham.
Precursor ion scanning-mass spectrometry for the determi-
nation of nitro functional groups in atmospheric particulate
organic matter. Anal. Chim. Acta 2008, 618, 184.
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 793–804