Detection Limits of Electron and Electron Capture Negative Ionization-Mass Spectrometry for Aldehydes Derivatized with o-(2,3,4,5,6-Pentafluorobenzyl)-Hydroxylamine Hydrochloride

Article abstract of DOI:10.1016/j.jasms.2009.12.009

In contrast to common expectations, the differences in limits of detection (LODs) between electron capture negative ionization (ECNI) and electron ionization (EI) mass spectrometry (MS) were found to be insignificant for a wide range of aldehydes derivati

Full text of DOI:10.1016/j.jasms.2009.12.009

Detection Limits of Electron and Electron
Capture Negative Ionization-Mass
Spectrometry for Aldehydes Derivatized with
o-(2,3,4,5,6-Pentafluorobenzyl)-Hydroxylamine
Hydrochloride
Josef Beránek,a Darrin A. Muggli,b and Alena Kubátováa
a Department of Chemistry, University of North Dakota, Grand Forks, North Dakota, USA
b Department of Chemical Engineering, University of North Dakota, Grand Forks, North Dakota, USA
In contrast to common expectations, the differences in limits of detection (LODs) between electron
capture negative ionization (ECNI) and electron ionization (EI) mass spectrometry (MS)
were found to be insignificant for a wide range of aldehydes derivatized with o-(2,3,4,5,6-
pentafluorobenzyl)-hydroxylamine hydrochloride. Comparison of the two ionization methods
based on LOD confidence intervals revealed that a traditional presentation of the LOD or limit of
quantitation (LOQ) as a single value may over/underestimate the significance of obtained results.
LODs were between 20 and 150 pg injected for the majority of tested derivatized carbonyls using
both ionization methods. ECNI-MS improved LODs by ϳ10- to 20-fold only for two derivatized
aldehydes, 4-hydroxybenzaldehyde and 5-(hydroxymethyl)furfural. Selectivity of ECNI did not
appear to be beneficial when analyzing a wood smoke particulate matter (WS-PM) extract,
possibly because the majority of interferences were removed during sample preparation (i.e.,
liquid–liquid extraction). The impact of four different data acquisition modes of transmission
quadrupole (TQ)-MS on LODs and their precisions was also investigated. As expected, LODs in
the selected ion monitoring (SIM) were ϳtwo to four times lower than those obtained using total
ion current (TIC) mode. More importantly, TQ-MS in the selected ion-total ion (SITI) mode (i.e.,
acquiring SIM and TIC data in a single analysis) provided signal-to-noise ratios and precisions,
which were comparable to SIM alone. (J Am Soc Mass Spectrom 2010, 21, 592–602) © 2010
American Society for Mass Spectrometry
ldehydes, as products of the lipid peroxidation
reactions and various atmospheric processes,
are often detected in biological and environ-
aldehyde). In contrast to high-energy (i.e., hard ioniza-
tion) EI-MS, soft ionization (e.g., ECNI) does not typi-
cally induce extensive fragmentation of a molecular ion.
A negative-charge molecular ion (i.e., [M]Ϫ•) may ap-
pear with a high abundance in an ECNI spectrum
facilitating the analyte identification as well as increas-
ing sensitivity [18]. It was observed, however, that the
fragmentation of PFBHA-aldehyde negative-charge
molecular ions (namely C6–C10 linear aldehydes,
glyoxal, methylglyoxal, and benzaldehyde) in ECNI
was as extensive as that of positive-charge molecular
ions in EI, perhaps due to the dominating dissociative
electron capture mechanism of ionization [1, 11]. As a
consequence, the identification of unknown PFBHA-
aldehydes is not facilitated using ECNI. The main ions
observed in both EI and ECNI mass spectra originated
from the derivatization agent. EI mode showed patterns
with a base peak of m/z 181, which is [C6F5CH2]ϩ [1].
Similarly, the base peaks observed in the derivatized
aldehyde ECNI mass spectra represented the ions of
[C6F4CH2O]Ϫ (m/z 178), [C6F5CH2]Ϫ (m/z 181), and
[C6F5CHO]Ϫ (m/z 196) [1]. A significant [M Ϫ 20] ion
(perhaps [M Ϫ HF]Ϫ) was observed in the mass spectra
A
mental matrices [1–15]. Derivatization of aldehydes is
typically employed before the analysis to improve chro-
matographic separation and detection limits [1–15].
One of the suitable derivatization agents for alde-
hydes, recommended by the U.S. Environmental Pro-
tection Agency (EPA), is o-(2,3,4,5,6-pentafluorobenzyl)-
hydroxylamine hydrochloride (PFBHA) [11, 16]. Re-
action of aldehydes with PFBHA provides thermally
stable and volatile oximes amenable to gas chromato-
graphic (GC) analysis [16, 17]. Mass spectrometry (MS),
using either electron ionization (EI) or electron capture
negative ionization (ECNI), is often employed for de-
tection of PFBHA-aldehydes.
Use of selective and sensitive ECNI is enabled due to
the strong electron-withdrawing effect of five fluorine
atoms in the molecule of derivatized aldehyde (PFBHA-
Address reprint requests to Dr. A. Kubátová, Department of Chemistry,
University of North Dakota, 151 Cornell Street Stop 9024, Grand Forks, ND
58202, USA. E-mail: akubatova@chem.und.edu
Published online January 4, 2010
© 2010 American Society for Mass Spectrometry. Published by Elsevier Inc.
1044-0305/10/$32.00
doi:10.1016/j.jasms.2009.12.009
Received June 18, 2009
Revised December 17, 2009
Accepted December 17, 2009

J Am Soc Mass Spectrom 2010, 21, 592–602
EI AND NCI-MS OF PFBHA-DERIVATIZED ALDEHYDES
593
of C3–C12 saturated and unsaturated aliphatic alde-
hydes and some aromatic aldehydes in ECNI spectra
[1, 11].
PFBHA-Aldehydes Preparation
Individual standard stock solutions of aldehydes were
prepared in methanol at a concentration of ϳ20 mg
mLϪ1 and stored at Ϫ18 °C. The derivatization method
has been described elsewhere and was adopted from
the EPA 556 method [16, 23]. Triplicates of PFBHA-
derivatized calibration standards (10–600 ␮g LϪ1) were
prepared by spiking the appropriate amounts of alde-
hyde mixture into purified water. Two internal stan-
dards (IS), butanal-d2 and benzaldehyde-d6, were added
to this solution in the final concentrations of 0.500 mg
Limits of detection (LODs) obtained using ECNI and
EI have been previously compared for only a few
PFBHA-aldehydes (linear saturated and unsaturated
aldehydes, glyoxal, methylglyoxal, and benzaldehyde),
which showed ECNI to provide slightly lower LODs
than EI [1, 19]. However, neither the method of preci-
sion nor the description of LOD calculation were re-
ported [1, 7, 10, 19–22]. EI and ECNI experiments were
performed using different instrumentation [1, 19];
therefore, it is not clear whether the ECNI signal-to-
noise ratio (i.e., detectability) improved solely due to a
lower LODs of ECNI for PFBHA-aldehydes.
In this work, we have evaluated and compared the
ECNI and EI-MS based on LOD confidence intervals for
33 aldehydes derivatized with PFBHA. The evaluation
was performed using two GC/MS instruments (3D
quadrupole ion trap and transmission quadrupole) each
equipped with interchangeable CI and EI sources. We
have also investigated the performance of transmission
quadrupole (TQ)-MS in four data acquisition modes.
L
Ϫ1. PFBHA solution (1.7 ϫ 104 mg LϪ1) was prepared
fresh in purified water before the derivatization. A
quantitative reaction of aldehydes was assured by add-
ing PFBHA in excess (at least 10-fold based on molar
ratios). To adjust the pH to 3.5, a few drops of an acid
(1 ϫ 10Ϫ3 mol LϪ1 H2SO4) or base (1 ϫ 10Ϫ3 mol LϪ1
NaOH) were added. The final volume of the aqueous
calibration standard solution was 5.0 mL.
Solutions were set at room temperature in the dark
for 12 h to complete the reaction [16]. To avoid potential
interferences from the excess of PFBHA derivatization
agent during the GC analysis, a few drops of concen-
trated sulfuric acid were added after the reaction. An
extraction of PFBHA-aldehydes into 1.0 mL of DCM
was then employed. The extraction was repeated three
times [16]. The test-tube was hand-shaken for 1 min
before withdrawing the DCM layer; the DCM fractions
were combined (total volume was 3 mL) and filtered
through anhydrous Na2SO4 to remove residual water
[16]. The resulting DCM solutions were concentrated
under a gentle stream of nitrogen to 0.4 mL and
analyzed using GC/MS.
Experimental
Chemicals and Materials
Standards of aldehydes were of 95% and higher purity
(unless stated otherwise). Acetaldehyde, propanal, bu-
tanal, isobutanal, n-pentanal, n-hexanal, n-heptanal, n-
octanal, n-nonanal, n-decanal, n-undecanal, n-dodecanal,
acrolein, trans-2-pentenal, trans-2-hexenal, trans-2-
nonenal, trans,trans-2,4-nonadienal (Ͼ85%), glyoxal
(40% wt. in water), methylglyoxal (40% wt. in wa-
ter), glutaraldehyde (50% wt. in water), benzalde-
hyde, o-tolualdehyde, m-tolualdehyde, phenylace-
taldehyde (Ͼ90%), hydrocinnamaldehyde (Ͼ90%),
2,5-dimethylbenzaldehyde, 2-furaldehyde, and 5-
(hydroxymethyl)furaldehyde were purchased from
Sigma-Aldrich (Milwaukee, WI, USA). Formaldehyde
(36.6% wt. in water), crotonal, 2-hydroxybenzaldehyde,
4-hydroxybenzaldehyde, and p-anisaldehyde were pur-
chased from Chem Service (West Chester, PA, USA).
Benzaldehyde-d6 (99.6 atom % D) and butanal-2,2-d2
(99.4 atom % D) were purchased from CDN Isotopes
(Pointe-Claire, QC, Canada). PFBHA (Ͼ99%) was
purchased from Alfa-Aesar (Ward Hill, MA, USA).
Ultra high purity helium (99.9995%) and methane
(99.999%) were purchased from Airgas (Grand Forks,
ND, USA). Methanol (LC/MS optima grade) and
dichloromethane (DCM) of GC/MS quality were pur-
chased from Fisher Scientific (Pittsburg, PA, USA).
Water was purified using a Direct-Q3 water purifica-
tion system with an incorporated dual wavelength
UV lamp (Millipore, Billerica, MA, USA) for low total
organic carbon content (the manufacturer’s claimed
purity is less than 5 ng gϪ1).
Wood smoke particulate matter was extracted using
hot pressurized water, which is described in detail
elsewhere [23–25]. Aqueous extracts with aldehydes
were prepared for GC/MS analysis as calibration
standards.
GC with ECNI- and EI-TQ-MS
An Agilent 6890 GC coupled to Agilent 5975C inert XL
EI/CI MSD (Agilent Technologies, Inc., Wilmington,
DE, USA) and Gerstel MPS2 autosampler (Gerstel,
Baltimore, MD, USA) were used. MSD ChemStation
D.03.00.611 software was employed for the data acqui-
sition and integration of chromatographic peak areas.
The data obtained were further processed in Microsoft
Office Excel.
The automated tune and m/z calibration using the
instrument’s Autotune file were performed after ev-
ery change of a source or at least once a week. No
significant adjustments of EI or ECNI-MS parameters
were observed among individual tunes. Pentafluoro-
benzylbutylamine (PFTBA) was used as a mass cali-
brant for the EI mode and perfluoro-5,8-dimethyl-
3,6,9-trioxydodecane (PFDTD) for the ECNI mode.
The MS instrument was equilibrated overnight after

594
BERÁNEK ET AL.
J Am Soc Mass Spectrom 2010, 21, 592–602
Table 1. The parameters of analysis and quantitation employed for PFBHA-derivatized aldehydes including: quantitation and
confirmation ions (m/z) used in EI and ECNI for both TQ-MS and QIT-MS, ISs employed for the quantitation of particular analytes,
number of ions scanned, and scanning rates (s–1) in SIM and SITI (applicable for TQ-MS only). Scanning rate in SITI mode is reduced
due to the switching between TIC and SIM. The dwell time was 25 ms for all ions in SIM and SITI. SIM acquisition ions were the
same as quantitation and confirmation ions
EI-MS
Molecular
weight of
derivative
Internal
standard
used for
Number of ion
ions scanned
in SIM group
PFBHA derivatized
aldehyde
Quantitation
ion m/z
Confirmation
(g · molϪ1
)
quantification
ion m/z
Aliphatic saturated aldehydes
Formaldehyde
Acetaldehyde
Propanal
Isobutanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Undecanal
225
239
253
267
267
281
295
309
323
337
351
365
379
Butanal-d2
Butanal-d2
Butanal-d2
Butanal-d2
181
181
181
181
239
181
181
181
181
181
181
239
181
195, 225
209, 239
223, 236
250
4
3
3
2
4
3
3
3
3
2
3
5
2
Butanal-d2
226
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
207, 239
239, 295
207, 239
239, 323
239
239, 351
181, 345
239
Dodecanal
Aliphatic unsaturated aldehydes
Acrolein
Crotonal
251
265
279
293
335
333
Butanal-d2
Butanal-d2
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
181
181
181
181
181
181
221, 251
195, 250
250, 279
250, 293
250, 335
276, 333
3
3
3
3
5
3
trans-2-Pentanal
trans-2-Hexenal
trans-2-Nonenal
trans, trans-2,4-nonadienal
Aromatic aldehydes
Benzaldehyde
m-Tolualdehyde
o-Tolualdehyde
2,5-Dimethylbenzaldehyde
Phenylacetaldehyde
Hydrocinnamaldehyde
p-Anisaldehyde
2-Hydroxybenzaldehyde
4-Hydroxybenzaldehyde
2-Furfural
301
315
315
329
315
329
331
317
317
291
321
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
301
181
181
181
181
181
331
181
181
291
181
271
4
4
4
5
4
5
5
3
3
3
5
91, 315
91, 315
286, 329
91, 315
271, 329
181, 288
300, 317
274, 317
248
5-(Hydroxymethyl)furfural
Dialdehydes
291, 321
Glyoxal
Methylglyoxal
Glutaraldehyde
448
462
490
Benzaldehyde-d6
Benzaldehyde-d6
Benzaldehyde-d6
181
181
181
418, 448
432, 462
293, 490
5
3
3
changing the EI to the CI source. The ion gauge
pressure readings were 7.5 ϫ 10Ϫ6 Torr in EI and 1.7
ϫ 10Ϫ4 Torr in ECNI.
MS transfer line was kept at 280 °C. The EI source
temperature was 230 °C and the ionization energy was 70
eV. The CI source temperature in negative mode was
evaluated within a range of 155–250 °C. The optimal
temperature was found to be 155 °C. The ECNI buffer gas
(methane) was ionized using 230 eV; its flow was opti-
mized within 0.5–2 mL minϪ1. The optimal flow rate was
found to be 1.5 mL minϪ1 (optimization is further dis-
cussed in the Results section).
To compare the sensitivity using EI and ECNI,
data were acquired in the selected ion monitoring
(SIM) mode using a unit mass resolution (i.e., de-
noted as a low-resolution in the acquisition software).
Injections were performed in a splitless mode for 0.5
min at 250 °C. Injection volume was 1 ␮L. The separa-
tion was performed on a 30-m long, 0.25 mm i.d., 0.25
␮m film thickness fused silica DB-5MS column (J and W
Scientific, Folsom, CA, USA). A constant carrier gas
flow rate of 1 mL minϪ1 was maintained during the
analysis. The following temperature program was used:
50 °C held for 2 min, followed by a 3 °C minϪ1 gradient
to 230 °C, then a 35 °C minϪ1 gradient to 320 °C. The final
temperature was held for 5 min. The temperature of the

J Am Soc Mass Spectrom 2010, 21, 592–602
EI AND NCI-MS OF PFBHA-DERIVATIZED ALDEHYDES
595
Table 1. Continued
EI-MS
NCI-MS
Acquisition
rate in SIM
Acquisition
rate in SITI
Number of ion
ions scanned
in SIM group
Acquisition
rate in SIM
Acquisition
rate in SITI
Quantitation
ion m/z
Confirmation
(sϪ1
)
(sϪ1
)
ion m/z
(sϪ1
)
(sϪ1
)
6.20
8.10
8.10
11.80
6.20
8.10
8.10
8.10
8.10
11.80
8.10
4.90
11.80
3.97
4.78
4.78
5.93
4.02
4.78
4.78
4.78
4.74
5.93
4.74
3.40
5.93
181
181
181
178
247
178
178
178
178
178
178
345
178
178, 225
178, 197
178, 233
181, 247
220, 267
181, 261
181, 275
181, 289
276, 303
317, 337
196, 331
318
3
3
3
3
6
3
3
3
3
3
3
4
3
8.10
8.10
8.10
8.10
4.20
8.10
8.10
8.10
8.10
8.10
8.10
6.20
8.10
4.87
4.87
4.87
4.87
3.07
4.87
4.87
4.87
4.87
4.87
4.87
4.08
4.87
196, 359
8.10
8.10
8.10
8.10
4.90
8.10
4.78
4.78
4.78
4.78
3.40
4.74
231
245
259
273
178
196
178, 201
178, 215
178, 229
178, 243
315
4
3
3
3
5
3
6.20
8.10
8.10
8.10
4.90
8.10
4.08
4.87
4.87
4.87
3.45
4.87
283, 313
6.20
6.20
6.20
4.90
6.20
4.90
4.90
8.10
8.10
8.10
4.90
4.02
3.97
3.97
3.40
3.97
3.40
3.40
4.74
4.74
4.78
3.40
281
295
295
309
178
178
311
136
297
241
271
251
4
3
3
3
3
5
4
3
3
3
3
6.20
8.10
8.10
8.10
8.10
4.90
6.20
8.10
8.10
8.10
8.10
4.08
4.87
4.87
4.87
4.87
3.45
4.08
4.87
4.87
4.87
4.87
167, 265
167, 265
167, 279
204, 268
279, 309
178, 281
196, 280
178, 267
178, 271
196, 255
4.90
8.10
8.10
3.40
4.74
4.74
267
281
178
167, 196
167, 196
197, 450
3
3
3
8.10
8.10
8.10
4.87
4.87
4.87
To evaluate the sensitivity in various TQ-MS modes,
data acquisition was performed in a total ion current
(TIC) in the m/z range of 50–600, SIM, and selected
ion-total ion (SITI) mode (i.e., acquiring SIM and TIC
data in a single analysis). Table 1 shows the acquisi-
tion ions (i.e., quantitation and confirmation ions) as
well as data acquisition rates and a number of
scanned ions in each SIM group (in SIM alone and in
SITI). Analytes eluting close to each other from the
GC column were acquired in one SIM group. In-
creased number of acquisition ions (e.g., five ions
scanned for p-anisaldehyde in EI versus four ions in
ECNI for the same compound) did not have a signif-
icant effect on LOD or precision. The fragmentation
patterns of PFBHA-aldehydes obtained in both EI
and ECNI resembled those previously published [1].
EI and ECNI spectra of selected analytes (to our
knowledge not previously reported) are provided in
the supplementary data, Figure S2, which can be
found in the electronic version of this article.
In addition, TIC was performed using two scanning
speeds. In this paper, one is referred as normal TIC
(n-TIC), with a spectral acquisition rate of ϳ2.66 sϪ1 in
the m/z range of 50–600 (providing ϳ20–24 data points
across chromatographic peaks with half-widths of
ϳ7–8 s), and the other is fast TIC (f-TIC), ϳ13.24 sϪ1 in
the same mass range (providing ϳ100 data points
across chromatographic peaks with half-widths of
ϳ6–8 s). The mass acquisition threshold was set to 50
counts for all methods (i.e., f-TIC, n-TIC, SIM, SITI).

596
BERÁNEK ET AL.
J Am Soc Mass Spectrom 2010, 21, 592–602
GC with ECNI- and EI-QIT-MS
3.3 ϫ sY
m
LOD ϭ
(1)
k
A Trace GC equipped with Polaris-Q 291 3D quadru-
pole ion trap (QIT)-MS (further in the text referred as
QIT-MS) with both EI and CI options and AS3000
autosampler (Thermo Fisher Scientific, Inc., Waltham,
MA, USA) was used to confirm the trends of ECNI/
EI LODs for PFBHA-aldehydes observed using GC/
TQ-MS. Xcalibur 1.3 software was used for the data
acquisition and integration of chromatographic peak
areas. The data obtained were further processed in
Microsoft Office Excel.
where k is a slope of the calibration curve and sy is the
standard deviation of the linear regression residuals,
which is obtained as a square root of residual mean
square (MSRES) representing the unbiased estimate of a
calibration curve variance [27].
A confidence interval for MSRES was obtained using
eq 2 [27],
The automated tune and m/z calibration using the
instrument’s Autotune file were performed after every
change of the source or at least once a week. No
significant adjustments of EI or ECNI-MS parameters
were observed among individual tunes. PFTBA was
used as a mass calibrant for both EI and ECNI modes.
The CI ion volume was inserted into the MS ionization
source for the ECNI experiment without breaking the
vacuum. The instrument was equilibrated overnight.
The ion gauge pressure readings were 2.0 ϫ 10Ϫ5 Torr
in EI and 1.7 ϫ 10Ϫ4 Torr in ECNI.
Injection set-up, flow rate, column, oven tempera-
ture program, and transfer line temperature were the
same as for GC/TQ-MS (see above). Ion source
temperature during the EI experiment was 230 °C
and the ionization energy was 70 eV. The ionization
source temperature for ECNI was evaluated within a
range of 155–250 °C. The optimal temperature was
155 °C. The ECNI buffer gas (methane) was ionized
using 230 eV; its flow was optimized within 0.5–3 mL
minϪ1. The optimal flow rate was 3 mL minϪ1 (opti-
mization is further discussed in the Results and
Discussion section). Data were acquired in the TIC
mode (mass range from m/z 50 to 600) for both ECNI
and EI (see Table 1 for quantitation and confirma-
tion ions) experiments evaluating LODs of PFBHA-
derivatized aldehydes. QIT-MS acquisition parame-
ters were set for both EI and ECNI as follows: number
of microscans was 2, maximum ion time was 25 ms,
and automatic gain control was turned on. The aver-
age number of data points acquired across chromato-
graphic peaks with half-widths of ϳ6–8 s was ϳ20–
24. The mass acquisition threshold was set to 50
counts.
(n Ϫ c)MSRES
(n Ϫ c)MSRES
Յ MSRES Յ
(2)
ͳ
ʹ
2
2
␹
␹
a ⁄ 2,nϪc
(1Ϫa) ⁄ 2,nϪc
where (n Ϫ c) represents a number of degrees of
freedom (n is a number of data points, c is a number of
independent variables), ␹ is a chi-square distribution
coefficient at a decision level ␣ ϭ 0.05 and degrees of
freedom (n Ϫ c), where c ϭ 2. The square root of upper
and lower MSRES gives the sy critical limits.
The confidence interval for k was obtained using
eq 3 [27],
2
͗k Ϫ ta ⁄ 2,nϪcsk Յ k Յ k ϩ ta ⁄ 2,nϪcsk͘
(3)
where t is a coefficient of Student’s t-distribution at a
decision level ␣ ϭ 0.05 and degrees of freedom (n – c),
where c ϭ 2, sk is a standard error of k obtained from the
regression analysis.
The uncertainty of mLOD was estimated by propaga-
tion of errors eq 4 [assuming mLOD as a function of sy
and k (eq 1)].
2
2
Ѩ(3.3sY ⁄ k)
ѨsY
Ѩ(3.3sY ⁄ k)
dmLOD(sY, k) ϭ
dsY
ϩ
dk
ͱ
ͩ
ͪ ͩ ͪ
Ѩk
(4)
Solving and simplifying eq 4 assuming infinitesimal
changes of sy and k (i.e., dsy ϭ ⌬sy and dk ϭ
⌬k) provided the uncertainty of mLOD described in
eq 5.
Statistical Data Evaluation
2
2
(3.3)
Ϫ3.3sY
⌬mLOD(sY, k) ϭ
⌬sY 2 ϩ
⌬k2
(5)
LODs in pg (a minimal mass injected enabling the
analyte detection) and their confidence intervals for
individual PFBHA-aldehydes were computed using
data points from three calibration standard sets,
which were within the linear range and one order of
magnitude of suspected LOD [26]. A least-squares
linear regression analysis using Microsoft Office Ex-
cel was performed to obtain calibration curve param-
eters. Average mass LODs (mLOD) were calculated
using eq 1,
ͱ
ͩ ͪ ͩ ͪ
k
k2
Then the true value of mLOD lies within the 95%
confidence interval (eq 6).
͗mLOD Ϫ (⌬mLOD ⁄ 2) Յ mLOD Յ mLOD ϩ (⌬mLOD ⁄ 2)͘
(6)
This confidence interval may be directly associated with
the LOD precision. Calculations are graphically depicted

J Am Soc Mass Spectrom 2010, 21, 592–602
EI AND NCI-MS OF PFBHA-DERIVATIZED ALDEHYDES
597
in Figure 1a and b, (calibration curve parameters for all
compounds and methods are provided in Table S1).
Finally, the hypothesis whether the average LODs
obtained using EI and ECNI were different for a group
of derivatized aldehydes (saturated and unsaturated
aliphatic, aromatic aldehydes and dialdehydes) was
tested using a two-tail paired t-test. Results were ex-
pressed as a probability (i.e., P value). LODs obtained
for EI and ECNI were considered non-different if P Ͼ
0.05 (with 95% confidence).
Results and Discussion
Optimization of ECNI Parameters
The ECNI parameters (buffer gas flow and source
ionization temperature) were optimized for both GC/
ECNI-QIT-MS and GC/ECNI-TQ-MS systems before
evaluating the detection sensitivity.
The optimum ion source temperature of 155 °C for
both MS instruments corresponded to the general man-
ufacturers’ recommendations. Increased ion source
temperature (175–250 °C) negatively affected responses
of quantitation ions (listed in Table 1) due to the
extensive fragmentation at higher source temperatures
(Figure S1 demonstrates the decrease of m/z 178 re-
sponse for derivatized heptanal with the increasing ion
source temperature).
Unlike the temperature, the optimal flow rate of
buffer gas (methane) was different for GC/ECNI-
QIT-MS and GC/ECNI-TQ-MS. Responses of quantita-
tion ions increased linearly with increasing flow rate up
to 1.5 mL minϪ1 for GC/ECNI-TQ-MS. A signal drop
for all PFBHA-aldehydes was observed when the buffer
gas flow rate was above 1.5 mL minϪ1. By contrast, the
analyte responses increased linearly with increasing
flow rate up to 3 mL minϪ1 for GC/ECNI-QIT-MS. A
buffer gas flow rate of 1.5 mL minϪ1 and 3.0 mL minϪ1
was selected as optimal for GC/ECNI-TQ-MS and
GC/ECNI-QIT-MS, respectively.
A finding that optimal buffer gas flow rates vary for
different instruments (i.e., manufacturers) implies that
the design of ion source may affect the ECNI efficiency;
therefore, optimization of ECNI parameters may be a
crucial step in trace concentration analysis.
Minimum Detectability Using ECNI and EI
The minimum detectability of ECNI and EI was evalu-
ated using LODs of PFBHA-aldehydes expressed as
95% confidence intervals (calculation described in the
experimental section). This approach allows for evalu-
ating the LOD or LOQ precision, which is otherwise
neglected when LODs/LOQs are reported as single
values. The effect of data scattering at individual cali-
bration levels on the LOD precision is graphically
depicted in Figure 1a and b. A broad confidence inter-
val for nonanal (Figure 1b), compared with crotonal
(Figure 1a), was caused by decreased reproducibility at
low calibration levels.
Absence of peaks representing negative-charge mo-
lecular ions in spectra obtained using ECNI (due to the
dissociative electron capture ionization mechanism)
perhaps contributed to the LOD being similar for EI
and ECNI-MS for the majority of derivatized aldehy-
des employing both GC/TQ-MS (Figure 2) and GC/
QIT-MS (Figure 3).
Figure 1. The effect of reproducibility in individual calibration
levels on the LOD and its confidence interval for (a) crotonal and
(b) nonanal, the width of 95% confidence interval for crotonal was
11–26 pg, whereas nonanal interval was broader, 47–115 pg, due
to the low reproducibility in individual calibration levels (tabu-
lated LOD intervals for all aldehydes and modes of acquisition are
provided in supplementary Table S2). Data were acquired using
ECNI-TQ-MS. Insets show data points, which were used for
statistical calculations (sy, k, and LOD confidence interval). AA/
AIS is abundance of analyte/abundance of internal standard.

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BERÁNEK ET AL.
J Am Soc Mass Spectrom 2010, 21, 592–602
two PFBHA-derivatized hydroxylated aromatic alde-
hydes, 4-hydroxybenzaldehyde and (5-hydroxymethyl)
furfural. Their LODs were in the same range (20–150 pg)
as for other aldehydes using ECNI. However in EI, they
were undetected or provided a weak response within the
tested concentration range ϳ10–600 pg (Figure 2 and
Figure 3). In contrast to 4-hydroxybenzaldehyde, 2-
hydroxybenzaldehyde was detected with comparable
LODs in both ionization modes (Figure 2 and Figure 3).
This suggests that the position of a functional group
(ϪOH) in a molecule may affect the EI efficiency.
In summary, the individual data acquisition meth-
ods mainly affected the precision of LOD but not the
Figure 2. Comparison of LODs in pg (graphically depicted as
circles and squares) with their 95% confidence interval (graphi-
cally depicted as lines) for EI-SIM-TQ-MS (open circle) and
ECNI-SIM-TQ-MS (open square). Tabulated LOD intervals for all
aldehydes and modes of acquisition are provided in the supple-
mentary Table S2. Confidence intervals may be directly related to
a method of precision at low detection levels. Two-tail paired t-test
Pvalues show whether there is a significant difference in LODs
between ECNI-SIM-TQ-MS and EI-SIM-TQ-MS for a group of alde-
hydes (saturated and unsaturated aliphatic, aromatic, and dialde-
hydes), the P value Ͻ 0.05 signifies that methods are different.
N.D. ϭ not detected, within the tested range of 50–600 pg.
A small improvement (2–3 times) was observed for
derivatized alkenals using ECNI-TQ-MS when evaluat-
ing the average LOD values (depicted as squares in
Figure 2). However, based on the paired t-test, the two
ionization methods (i.e., ECNI and EI) were not statis-
tically different (P value ϭ 0.074) with 95% confidence
(Figure 2). This trend was confirmed by QIT-MS anal-
ysis of derivatized alkenals where LODs obtained using
ECNI and EI were nearly identical (Figure 3). Appar-
ently only LOD precisions were slightly improved
using ECNI-TQ-MS but average LODs remained similar
to EI (Figure 2 and Figure 3).
Figure 3. Comparison of LODs in pg (graphically depicted as
circles and squares) with their 95% confidence interval (graphi-
cally depicted as lines) for EI-TIC-QIT-MS (open circle) and
ECNI-TIC-QIT-MS (open square). Tabulated LOD intervals for all
aldehydes and modes of acquisition are provided in the supple-
mentary Table S2. Confidence intervals may be directly related to
a method precision at low detection levels. Two-tail paired t-test
P value shows whether there is a significant difference in sensi-
tivity between ECNI-TIC-QIT-MS and EI-TIC-QIT-MS for a group
of aldehydes (saturated and unsaturated aliphatic, aromatic, and
dialdehydes), the lower P value the greater probability that
methods are different. N.D. ϭ not detected within the tested range
of 50–600 pg.
The only significant improvement in LODs using
ECNI (for both TQ-MS and QIT-MS) was observed for

J Am Soc Mass Spectrom 2010, 21, 592–602
EI AND NCI-MS OF PFBHA-DERIVATIZED ALDEHYDES
599
detectability itself. It appeared that the LOD confidence
intervals were a more appropriate representation of
results than single LOD values, especially when the
differences between two methods were relatively small.
Consequently, comparing two techniques based only
on single LOD values may over/underestimate the
significance of obtained results.
Selectivity of ECNI Versus EI in Analysis of Wood
Smoke Particulate Matter Extract
To investigate whether the selective ECNI may reduce/
eliminate background noise from complex-matrices, the
extract of wood smoke particulate matter (WS-PM) was
analyzed using GC/MS using both ECNI and EI (Figure
4a–c).
As expected, chromatographic peaks representing
methoxyphenols, polycyclic aromatic hydrocarbons
(phenanthrene, fluoranthene), esters of carboxylic acids
and some other compound types were observed in the
EI chromatogram (Figure 4a) but not detected using
ECNI (Figure 4b), which does not efficiently ionize
these compounds. However, the overall chromatogram
“baseline noise” of quantitation ions (listed in Table 1)
was not improved in ECNI. Both ionization techniques
provided similar signal-to-noise ratio for PFBHA-
aldehydes detected in WS-PM extract.
Based on the comparison of EI (Figure 4a) and ECNI
(Figure 4b) chromatograms of derivatized and underi-
vatized WS-PM extract (Figure 4c), it appeared that the
majority of interferences were removed during sample
preparation (including PFBHA derivatization and LLE).
Effects of Data Acquisition Modes on LODs and
Their Precisions
Results discussed in the section “Minimum Detectabil-
ity Using ECNI and EI” demonstrated that QIT-MS
operated in TIC mode provided similar average LODs
(in both EI and ECNI) for derivatized aromatic alde-
hydes and only slightly higher LODs for some aliphatic
aldehydes than those obtained using TQ-MS in SIM
(Figures 2, 3, and Table S2). The use of QIT-MS may
thus be preferred in some cases (e.g., a complete char-
acterization of sample is desired apart from the quan-
titation of targeted analytes) over SIM in TQ-MS be-
cause it provides full spectral information.
Figure 4. The chromatographic analysis of the wood smoke
particulate matter extract obtained (a) after PFBHA derivatization
using EI-TIC-TQ-MS, (b) after PFBHA derivatization using ECNI-
(TIC)-TQ-MS, and (c) using EI-(TIC)-TQ-MS (no derivatization
applied), no aldehydes could be identified in this chromatogram
due to the insufficient chromatographic resolution as well as
strong interfering signal of other analytes.
Recent advancements in quadrupole analyzer tech-
nologies (i.e., shortening the settling times between
ion-scans and improving signal stability) have enabled
additional data acquisition modes (besides TIC and
SIM). New acquisition modes include SITI (i.e., acquir-
ing SIM and TIC data in a single run) and fast scanning
TIC (f-TIC). Due to these developments, TQ-MS may
offer similar performance as QIT-MS.
teria (Figure 5 and Figure 6). Average LODs obtained
by both n-TIC and f-TIC were not statistically different
for the majority of analytes (Figure 5). For several
compounds, including derivatized acrolein, trans-2-
nonenal, trans,trans-2,4-nonadienal, and dodecanal data
acquisition in f-TIC resulted in a higher LOD than in
n-TIC. The precision of LODs was also low when using
f-TIC (Figure 5). Chromatographic peaks at low con-
centration levels acquired using f-TIC were noisy and
We evaluated the efficiency of TQ-MS data acquisi-
tion modes including f-TIC, n-TIC, SITI, and SIM using
LODs (i.e., signal-to-noise ratio) and precisions as cri-

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BERÁNEK ET AL.
J Am Soc Mass Spectrom 2010, 21, 592–602
Figure 5. Comparison of LODs in pg (graphically depicted as
circles, squares, and triangles) with their 95% confidence interval
(graphically depicted as lines) for EI-SIM-TQ-MS (open circle),
EI-(n-TIC)-TQ-MS (open square), and EI-(f-TIC)-TQ-MS (inverted
open triangle). Tabulated LOD intervals for all aldehydes and
modes of acquisition are provided in the supplementary Table S2.
Confidence intervals may be directly related to a method precision
at low detection levels. Two-tail paired t-test P value shows
whether there is a significant difference in sensitivity between
EI-(n-TIC)-TQ-MS and EI-(SIM)-TQ-MS for a group of aldehydes
(saturated and unsaturated aliphatic, aromatic, and dialdehydes),
the lower P value the greater probability that methods are
different. N.D. ϭ not detected within the tested range of 50–600 pg.
distorted when compared with n-TIC (Figure 6a and b).
Even though data smoothing was applied, peak areas
were irreproducible resulting in a large data variability
and lower f-TIC detectability for some compounds.
A large LOD variability was observed for low-
molecular weight derivatized formaldehyde, acetalde-
hyde, and acrolein using all TQ-MS acquisition modes
as well as QIT-MS in TIC (Figures 2, 3, and 5). This was,
however, attributed to their random evaporative losses
during a controlled sample evaporation, which often
must be employed to re-concentrate trace amounts of
aldehydes in environmental samples [23]. A suitable
Figure 6. The reconstructed ion chromatograms (RIC) of m/z of
181 of heptanal (syn/anti stereoisomers) acquired using (a) EI-
(f-TIC)-TQ-MS, (b) EI-(n-TIC)-TQ-MS, (c) EI-(SITI)-TQ-MS, where
SIM signal was used, and (d) EI-(SIM)-TQ-MS. The peak half-
widths were ϳ6 s; f-TIC (a) provided ϳ100, n-TIC (b) ϳ21, SITI (c)
ϳ40, and SIM (d) ϳ70 data points across the chromatographic
peak. A low signal-to-noise ratio in (a) caused the decrease of
detectability and reproducibility compared with (b). SITI (c) and
SIM (d) had similar signal-to-noise ratio, thus the detectability and
reproducibility (peak shape) of the two scanning methods were
comparable. The amount of analyte was 150 pg, which was near
LOD in TIC mode.

J Am Soc Mass Spectrom 2010, 21, 592–602
EI AND NCI-MS OF PFBHA-DERIVATIZED ALDEHYDES
601
way to account for this discrepancy, and thus im-
prove the precision, would be to use the correspond-
ing isotopically-labeled internal standards [23].
LODs and peak shapes (i.e., precision) in SITI (based
on the SIM data) were not compromised compared with
a simple SIM for all tested PFBHA-aldehydes (Figure 6c
and d) despite the decreased number of data points
acquired across the chromatographic peaks (e.g., from
ϳ70 data points in SIM to ϳ40 in SITI for PFBHA-
heptanal, Figure 6c and d). TQ-MS in SITI mode may be
equally or more efficient than QIT-MS in TIC.
Acknowledgments
The authors thank Dr. E. Kozliak and D. Stahl for valuable
comments while writing and editing this manuscript. This work
was sponsored by the North Dakota EPSCoR through NSF grants
ATM-0747349 and EPS-0814442, and through the DOE grant
DE-FG02-06ER46292, and by the National Center for Research
Resources (NCRR), a component of the National Institutes of
Health (NIH), through the grant P20RR17699-05; DSM gratefully
acknowledges support from NSF CBET 0651058. The contents of
this article are solely the responsibility of the authors and do not
necessarily represent the official views of NSF, DOE, and NIH.
As expected, the lowest LODs were obtained using
SIM (or using SIM data from SITI mode). Average
LODs were ϳ2 to 4 times lower than with n-TIC with
significant improvement of the LOD precision as well
(Figure 5 and Figure 6). The P values obtained from a
paired t-test of SIM and n-TIC (Figure 5) show that the
two methods were statistically different in sensitivity
comparing groups of derivatized aldehydes (satura-
ted and unsaturated linear, aromatic aldehydes, and
dialdehydes).
Appendix A
Supplementary Material
Supplementary material associated with this article
may be found in the online version at doi:10.1016/
j.jasms.2009.12.009.
References
1. Le Lacheur, R. M.; Sonnenberg, L. B.; Singer, P. C.; Christman, R. F.;
Charles, M. J. Identification of Carbonyl Compounds in Environmental
Samples. Environ. Sci. Technol. 1993, 27, 2745–2753.
2. Culleré, L.; Cacho, J.; Ferreira, V. Analysis for Wine C5-C8 Aldehydes
Through the Determination of Their O-(2,3,4,5,6-Pentafluorobenzyl)
Oximes Formed Directly in the Solid Phase Extraction Cartridge. Anal.
Chim. Acta 2004, 524, 201–206.
3. Liggio, J.; McLaren, R. An Optimized Method for the Determination of
Volatile and Semivolatile Aldehydes and Ketones in Ambient Particu-
late Matter. Int. J. Environ. Anal. Chem. 2003, 83, 819–835.
4. Pacolay, B. D.; Ham, J. E.; Wells, J. R. Use of Solid-Phase Microextraction
to Detect and Quantify Gas-Phase Dicarbonyls in Indoor Environments.
J. Chromatogr. A 2006, 1131, 275–280.
5. Frankel, E. N.; Hu, M. L.; Tappel, A. L. Rapid Headspace Gas Chroma-
tography of Hexanal as a Measure of Lipid Peroxidation in Biological
Samples. Lipids 1989, 24, 976–981.
6. Spiteller, P.; Kern, W.; Reiner, J.; Spiteller, G. Aldehydic Lipid Peroxi-
dation Products Derived from Linoleic Acid. Biochim. Biophys. Acta Mol.
Cell. Biol. Lipids 2001, 1531, 188–208.
7. Selley, M. L. Determination of the Lipid Peroxidation Product (E)-4-
Hydroxy-2-Nonenal in Clinical Samples by Gas Chromatography-
Negative-Ion Chemical Ionization Mass Spectrometry of the O-
Pentafluorobenzyl Oxime. J. Chromatogr. B 1997, 691, 263–268.
8. Teixido, E.; Santos, F. J.; Puignou, L.; Galceran, M. T. Analysis of
5-Hydroxymethylfurfural in Foods by Gas Chromatography-Mass
Spectrometry. J. Chromatogr. A 2006, 1135, 85–90.
Conclusions
In contrast to general expectation, EI and ECNI-MS
offered similar LODs for 31 of 33 tested PFBHA-
derivatized aldehydes. LODs were significantly de-
creased (ϳ10- to 20-fold) using ECNI only for two spe-
cific polar analytes, derivatized 4-hydroxybenzaldehyde
and (5-hydroxymethyl)furfural. Assuming comparable
LODs, EI may be suggested as a more suitable tech-
nique than ECNI due to the availability of EI-MS
libraries, and predictability of fragmentation patterns.
This is an important consideration when, in addition to
the quantitation of targeted compounds, the identifica-
tion of unknown peaks is required.
Comparing ECNI and EI based on their single-LOD
values, without any consideration for precision, may
over/underestimate the statistical significance of the
obtained results. To overcome this problem, we derived
an approach allowing for calculation of LOD with its
confidence interval from the calibration curve. The
statistical calculation also has a large potential for
determining the limit of quantitation (LOQ) confidence
intervals upon the proper modification of eqs 1, 4, and
5. Obtaining the LOQ confidence intervals may be
critical when establishing/employing methods for
quality control of regulated substances in various
industries.
Finally, it was confirmed that signal-to-noise ratios
and precisions for PFBHA-aldehydes in the SITI mode
(acquisition of SIM and TIC data in a single analysis)
were the same as in the SIM mode alone. TQ-MS in SITI
was thus demonstrated to be a good alternative to
QIT-MS in TIC for a sensitive analysis of complex
samples.
9. Spiteller, G.; Kern, W.; Spiteller, P. Investigation of Aldehydic Lipid
Peroxidation Products by Gas Chromatography-Mass Spectrometry.
J. Chromatogr. A 1999, 843, 29–98.
10. Luo, X. P.; Yazdanpanah, M.; Bhooi, N.; Lehotay, D. C. Determination of
Aldehydes and Other Lipid Peroxidation Products in Biological Sam-
ples by Gas Chromatography-Mass Spectrometry. Anal. Biochem. 1995,
228, 294–298.
11. Hsu, F. F.; Hazen, S. L.; Giblin, D.; Turk, J.; Heinecke, J. W.; Gross, M. L.
Mass Spectrometric Analysis of Pentafluorobenzyl Oxime Derivatives
of Reactive Biological Aldehydes. Int. J. Mass Spectrom. 1999, 185/
186/187, 795–812.
12. Müller, K.; Haferkorn, S.; Grabmer, W.; Wisthaler, A.; Hansel, A.;
Kreuzwieser, J.; Cojocariu, C.; Rennenberg, H.; Herrmann, H. Biogenic
Carbonyl Compounds Within and Above a Coniferous Forest in Ger-
many. Atmos. Environ. 2006, 40, 81–91.
13. Rubio, M. A.; Zamorano, N.; Lissi, E.; Rojas, A.; Gutierrez, L.; Von Baer,
D. Volatile Carbonylic Compounds in Downtown Santiago, Chile.
Chemosphere 2006, 62, 1011–1020.
14. Jakober, C. A.; Charles, M. J.; Kleeman, M. J.; Green, P. G. LC-MS
Analysis of Carbonyl Compounds and Their Occurrence in Diesel
Emissions. Anal. Chem. 2006, 78, 5086–5093.
15. Nagy, K.; Pollreisz, F.; Takats, Z.; Vekey, K. Atmospheric Pressure
Chemical Ionization Mass Spectrometry of Aldehydes in Biological
Matrices. Rapid Commun. Mass Spectrom. 2004, 18, 2473–2478.
16. U.S. Environmental Protection Agency. Determination of Carbonyl
Compounds in Drinking Water by Pentafluorobenzylhydroxylamine
Derivatization and Capillary Gas Chromatography with Electron Cap-
ture Detection. Method 556. Washington, DC.
17. Destaillats, H.; Charles, M. J. Henry’s Law Constants of Carbonyl-
Pentafluorobenzyl Hydroxylamine (PFHBA) Derivatives in Aqueous
Solution. J. Chem. Eng. Data 2002, 47, 1481–1487.

602
BERÁNEK ET AL.
J Am Soc Mass Spectrom 2010, 21, 592–602
18. Bezabeh, D. Z.; Bamford, H. A.; Schantz, M. M.; Wise, S. A. Deter-
mination of Nitrated Polycyclic Aromatic Hydrocarbons in Diesel
Particulate-Related Standard Reference Materials by Using Gas Chro-
matography/Mass Spectrometry with Negative Ion Chemical Ioniza-
tion. Anal. Bioanal. Chem. 2003, 375, 381–388.
19. Sugaya, N.; Sakurai, K.; Nakagawa, T.; Onda, N.; Onodera, S.; Morita,
M.; Tezuka, M. Development of a Headspace GC/MS Analysis for
Carbonyl Compounds (Aldehydes and Ketones) in Household Products
After Derivatization with o-(2,3,4,5,6-Pentafluorobenzyl)-Hydroxylamine.
Anal. Sci. 2004, 20, 865–870.
20. Sugaya, N.; Nakagawa, T.; Sakurai, K.; Morita, M.; Onodera, S. Analysis of
Aldehydes in Water by Head Space-GC/MS. J. Health Sci. 2001, 47, 21–27.
21. Wichard, T.; Poulet, S. A.; Pohnert, G. Determination and Quantification
of a,b,g,d-Unsaturated Aldehydes as Pentafluorobenzyl-Oxime Deri-
vates in Diatom Cultures and Natural Phytoplankton Populations:
Application in Marine Field Studies. J. Chromatogr. B 2005, 814, 155–161.
22. Li, Z.; Jacobus, L. K.; Wuelfing, W. P.; Golden, M.; Martin, G. P.; Reed, R. A.
Detection and Quantification of Low-Molecular-Weight Aldehydes in
Pharmaceutical Excipients by Headspace Gas Chromatography. J.
Chromatogr. A 2006, 1104, 1–10.
23. Beránek, J.; Kubátová, A. Evaluation of Solid-Phase Microextraction
Methods for Determination of Trace Concentration Aldehydes in Aque-
ous Solution. J. Chromatogr. A 2008, 1209, 44–54.
24. Kubatova, A.; Steckler, T. S.; Gallagher, J. R.; Hawthorne, S. B.; Picklo,
M. J. Toxicity of Wide-Range Polarity Fractions from Wood Smoke and
Diesel Exhaust Particulate Obtained Using Hot Pressurized Water.
Environ. Toxicol. Chem. 2004, 23, 2243–2250.
25. Hawthorne, S. B.; Kubatova, A. Hot (Subcritical) Water Extraction. In
Comprehensive Analytical Chemistry, 1st ed.; Barcelo, D., Eds. Elsevier:
New York, NY, 2002; pp 587–608.
26. Pawliszyn, J. Solid Phase Microextraction. In Comprehensive Analytical
Chemistry; Barcelo, D., Ed. Elsevier: New York, NY, 2002; pp 389–478.
27. Montgomery, D. C.; Peck, E. A.; Vinning, G. G. Introduction to Linear
Regression Analysis, 4th ed.; John Wiley and Sons: Hoboken, NJ, 2006; pp
25–145.