Evaporative derivatization of phenols with 2-sulfobenzoic anhydride for detection by matrix-assisted laser desorption/ionization mass spectrometry

Article abstract of DOI:10.1002/rcm.6816

RATIONALE Phenols are an important class of analytes, for example as bioactive environmental contaminants. Towards a goal of improving their detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or MALDI-tandem time-of-flight (TOF/TOF)-MS, we studied their derivatization with 2-sulfobenzoic anhydride (SBA). We chose SBA for this purpose since it is commercially available, inexpensive, and forms an anionic derivative. METHODS Under the selected conditions developed here for phenols, a reaction mixture of one or more of such compounds in acetonitrile containing SBA and 4-dimethylaminopyridine (DMAP) is evaporated to a solid, heated at 60 °C for 1 h, redissolved in 50% acetonitrile containing matrix, spotted onto a MALDI target, and subjected to negative ion MALDI-TOF/TOF-MS. RESULTS While conventional (solution-phase) reaction of 4-phenylphenol (model analyte) with SBA and DMAP only gave a 47% yield of SBA-tagged 4-phenylphenol, evaporative derivatization as above gave a 96% yield, and 25 pmol (4.3 ng) of 4-phenylphenol could be detected in this way by MALDI-TOF/TOF-MS at signal-to-noise ratio (S/N) = 260, whereas even 1 nmol of the nonderivatized phenol was not detected in the absence of derivatization. A wide range of responses was observed when a mixture of 15 phenols was derivatized, with the higher responses coming from phenols with a pK_a value above 9. Without derivatization, phenols with pK_a values below 5 were the most readily detected. CONCLUSIONS Evaporative derivatization with SBA (a convenient reagent) can improve the detection of phenols with relatively high pK _a values (above 9) by negative ion MALDI-TOF-MS, and accomplish this in the absence of post-derivatization reaction cleanup. Copyright 2014 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/rcm.6816

Research Article
Received: 25 September 2013
Revised: 23 December 2013
Accepted: 23 December 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 653–661
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6816
Evaporative derivatization of phenols with 2-sulfobenzoic anhydride
for detection by matrix-assisted laser desorption/ionization mass
spectrometry
*
Yuanyuan Yao, Poguang Wang and Roger Giese
Department of Pharmaceutical Sciences and Barnett Institute, Bouve College, Northeastern University, Boston, MA 02115, USA
RATIONALE: Phenols are an important class of analytes, for example as bioactive environmental contaminants. Towards
a goal of improving their detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) or MALDI-tandem time-of-flight (TOF/TOF)-MS, we studied their derivatization with 2-sulfobenzoic
anhydride (SBA). We chose SBA for this purpose since it is commercially available, inexpensive, and forms an
anionic derivative.
METHODS: Under the selected conditions developed here for phenols, a reaction mixture of one or more of such
compounds in acetonitrile containing SBA and 4-dimethylaminopyridine (DMAP) is evaporated to a solid, heated at
60 °C for 1 h, redissolved in 50% acetonitrile containing matrix, spotted onto a MALDI target, and subjected to negative
ion MALDI-TOF/TOF-MS.
RESULTS: While conventional (solution-phase) reaction of 4-phenylphenol (model analyte) with SBA and DMAP only
gave a 47% yield of SBA-tagged 4-phenylphenol, evaporative derivatization as above gave a 96% yield, and 25 pmol
(4.3 ng) of 4-phenylphenol could be detected in this way by MALDI-TOF/TOF-MS at signal-to-noise ratio (S/N) = 260,
whereas even 1 nmol of the nonderivatized phenol was not detected in the absence of derivatization. A wide range of
responses was observed when a mixture of 15 phenols was derivatized, with the higher responses coming from phenols
with a pKa value above 9. Without derivatization, phenols with pKa values below 5 were the most readily detected.
CONCLUSIONS: Evaporative derivatization with SBA (a convenient reagent) can improve the detection of phenols
with relatively high pKa values (above 9) by negative ion MALDI-TOF-MS, and accomplish this in the absence of
post-derivatization reaction cleanup. Copyright © 2014 John Wiley & Sons, Ltd.
As reviewed by Zaikin and Halket,[1] ionic tagging of small
molecules to increase their response by soft ionization
techniques such as electrospray ionization (ESI) and matrix-
assisted laser desorption/ionization (MALDI) is of increasing
interest. Other benefits can accrue such as increased
selectivity. One of the reagents which has been studied in this
way is 2-sulfobenzoic anhydride (SBA). While the compound
is commercially available and relatively inexpensive, it has
been explored to a limited degree in terms of the number of
publications reporting its analytical use; how much it
enhances analyte response; and its performance when
applied to a small amount of analyte.
In more detail, the analytical studies of SBA to date are as
follows. Peptides were tagged with SBA to improve their
fragmentation (for sequencing purposes) in the post-source
decay positive ion mode of MALDI.[2,3] SBA has been used
to derivatize a diversity of alcohols (including phenol,
glucose, and mandelic acid) for detection by negative
ion MALDI-time-of-flight mass spectrometry (TOF-MS).[4]
Multiple products were reported from multi-hydroxy
compounds, whose formation was attributed to steric effects.
The amount of each alcohol reacted (calculated per hydroxy
function) was 0.1 mmol. Yields were not defined. The limit
of detection (LOD) was reported as 2.5 pmol for SBA-tagged
methanol, based on testing a diluted reaction mixture,
assuming a reaction yield of 100%; apparently this value
was obtained by extrapolating from a measurement at a
higher concentration. It was not clear whether a blank sample
was derivatized to rule out contamination. Zu and coworkers
derivatized fatty alcohol ethoxylate non-ionic surfactants
at the 0.05 mmol level using SBA in the presence of 4-
dimethylaminopyridine (DMAP), followed by diluting the
reaction mixture and subjecting it to liquid chromatography
(LC)/ESI-MS or flow injection ESI-MS.[5] The yield was
reported to be about 95% based on the assumption that the
product (SBA-tagged alcohol) and starting alcohol gave the
same response by ESI-MS. SBA has also been used to modify
proteins[6,7] and industrial polymers.[8]
Towards a goal of improving the detection of phenols by
MALDI-MS, we have extended the work of others concerning
derivatization with SBA, and this manuscript reports our
progress. The detection of phenols is of broad interest mainly
because some are bioactive as biomolecules or xenobiotics.
* Correspondence to: R. Giese, Department of Pharmaceutical
Sciences and Barnett Institute, Bouve College, Northeastern
University, 206 The Fenway, 360 Huntington Ave., Boston,
MA 02115, USA.
E-mail: r.giese@neu.edu
Rapid Commun. Mass Spectrom. 2014, 28, 653–661
Copyright © 2014 John Wiley & Sons, Ltd.

Y. Y. Yao, P. G. Wang and R. Giese
Mass spectrometry is used commonly to detect phenols in
both of these roles, usually as part of a gas chromatography
(GC)/MS[9–12] or LC/MS[13–16] system.
Evaporative derivatization of 15 phenols with SBA
A mixture of 15 phenols (4PP, 4-nonylphenol, BPA, and a
12-component Phenols Calibration Mix) in 1 mL of ACN
was set up as follows: 5 × 10–4 M each component phenol,
5 × 10–3 M SBA, and 2% (w/w of total phenols) DMAP. A
50 μL aliquot was evaporated and heated at 60 °C for 1 h
followed by dissolving in 50 μL of ACN /H2O (1:1), and
1 μL was then combined with 10 μL of CCA for subsequent
analysis of 1 μL by MALDI-MS.
EXPERIMENTAL
Materials
4-Phenylphenol (4PP), 4-nonylphenol, bisphenol A (BPA),
2-sulfobenzoic anhydride (SBA), 4-dimethylaminopyridine
(DMAP), acetonitrile (ACN, anhydrous, 99.8%), and α-cyano-
4-hydroxycinnamic acid (CCA) were from Sigma-Aldrich
(St. Louis, MO, USA). CCA matrix was 5 mg/mL of CCA in
ACN/H2O (1:1, v/v). Phenol Calibration Mix (12 components
Cat. No. 31694) was purchased from Restek (Bellefonte, PA,
USA). Water (distilled, HPLC grade) and ACN (HPLC grade)
were purchased from Thermo Fisher Scientific (Waltham, MA,
USA). Glass vials with inserts (product No. 09-1200-101)
and screw thread caps (product No. 09-0040R) were
purchased from Microliter Analytical Supplies (Suwanee,
GA, USA).
Analysis by HPLC-UV
A 10 μL aliquot of the diluted sample after derivatization was
injected into a Hewlett Packard Series 1100 HPLC system.
The HPLC column was an AQUASIL (Thermo Fisher
Scientific) C18 column (150 × 2.1 mm, 5 μm) that was kept at
room temperature and operated at a flow rate of 0.21 mL/min.
Before each run, the column was equilibrated for 10 min with
2:98, v/v, ACN/H2O. The following solvent gradient was
applied: 2% ACN for 3 min, ramp to 50% ACN over 13 min, hold
for 4 min, ramp to 100% ACN over 5 min, and hold for 5 min.
Absorbance detection was at 256 nm.
Analysis by MALDI-MS (MALDI-TOF-MS or
MALDI-TOF/TOF-MS)
Non-evaporative derivatization of 4-phenylphenol with SBA
MALDI-MS analysis was performed on a model 5800
MALDI-TOF/TOF-MS instrument (AB SCIEX, Framingham,
MA, USA) in negative ion, reflectron mode with a delay time
of 100 ns, with 1200 laser pulses being averaged to generate a
spectrum. MS/MS was performed by TOF/TOF with the
metastable-suppressor on and the collision-induced
dissociation (CID) off. A mass resolution window of 400
(instrumental setting) was used. This setting (viz. 400)
combined with the precursor ion mass defines the mass
window for MS2 (done automatically by the software). The
relative laser intensity was set at ~2500 for MS mode and
~ 3500 for MS/MS mode. For the direct analysis of the
derivatized phenol mixture, a 1 μL aliquot of the redissolved
reaction mixture in ACN/H2O (1:1, v/v) was mixed with
10 μL CCA matrix, and 1 μL was deposited onto the MALDI
plate. For the analysis of the collected HPLC peak fraction for
SB-4PP after the derivatization reaction, 10 μL CCA matrix
was added to the dried fraction, and 1 μL was deposited
onto the MALDI plate.
The conditions of Zu and coworkers[5] for derivatization of
polyols with SBA were employed. Briefly, 4PP (8.5 mg,
0.05 mmol), SBA (92 mg, 0.5 mmol), and DMAP (0.1 mg,
8.2 × 10-4 mmol, 1% w/w of 4PP) were dissolved in 1 mL of
ACN. After sitting at room temperature for 1 h, the mixture
was kept at 80 °C for 2 h. The reaction was also conducted
at room temperature for 24 h. After the reaction, the
mixture was diluted 100-fold with ACN/H2O (20:80, v/v)
for HPLC analysis.
Evaporative derivatization of 4-phenylphenol with SBA
4PP was used as a model phenol to optimize the conditions
for evaporative derivatization. For the initial studies, one
equivalent of 4PP and ten equivalents of SBA were combined
in 2% (w/w of 4PP) of DMAP in 1 mL ACN to reach a
concentration of 4PP (5 × 10–2 M, 5 × 10–3 M, and 5 × 10–4 M).
A 50 μL aliquot was pipetted into a glass vial with an insert
and evaporated in a Speed Vac (Savant Instruments, Saroor
Nagar, Hyderbad, India; the manufacture-specified vacuum
was 0.6 Torr) at room temperature for 40 min. The resulting
solid was incubated at 60 °C in a sand bath (Reacti-therm,
Pierce, Rockford, IL, USA) for 1 h with the bottom of the
cap 4 mm above the sand. For subsequent HPLC-UV analysis
after derivatization, each reaction mixture was dissolved in
50 μL of 50% ACN. The reaction mixtures with initial 4PP
concentrations of 5 × 10–2 M, 5 × 10–3 M, and 5 × 10–4 M were
diluted, respectively, 100-fold, 10-fold, and 10-fold with
ACN/H2O (20:80, v/v) prior to HPLC analysis. In addition,
5 μL of a reaction mixture having 5 × 10–6 M 4PP along with
5 × 10–3 M SBA and 1.6 × 10–7 M DMAP was subjected to
evaporative derivatization prior to addition of 10 μL of
CCA matrix and deposition of 1 μL onto the target plate for
analysis by MALDI-MS.
Evaporative derivatization of pentachlorophenol with SBA
This reaction was conducted on 50 μL of 0.05
M
pentachlorophenol in the same way as the corresponding
sample of 4PP described above, except that, in addition to a
reaction time of 1 h at 60 °C, reaction temperatures of 80 °C
and 100 °C were studied. This was followed by HPLC-UV
analysis and, in turn, peak collection for analysis by
MALDI-TOF, as above for 4PP.
RESULTS AND DISCUSSION
To optimize the derivatization of phenols by SBA, we initially
tested 4-phenylphenol (4PP) as a model analyte, and relied on
HPLC-UV and/or MALDI-MS to assess the product mixture.
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Derivatization of phenols with 2-sulfobenzoic anhydride prior to MS
A scheme for the derivatization and analysis of this (and later
other phenols) by MALDI-MS is shown in Fig. 1. At first we
studied the two kinds of reaction conditions reported by Zu
and coworkers.[5] Since both the 4PP and the desired product,
SBA-tagged 4-phenylphenol (SB-4PP), have relatively simple
structures, and the HPLC chromatograms displayed no
significant side products, we relied on the disappearance of
the peak for 4PP to estimate the yield. (This way of assessing
the yield is validated further below.) By this strategy, the
yield of SB-4PP was about 52% (n = 3, coefficient of variance
(CV) = 6%) when the reaction was conducted by keeping it
at room temperature for 1 h prior to heating it at 80 °C (one
type of condition employed previously[5]). A representative
HPLC chromatogram from this experiment is shown in Fig. 2
(A), along with that from an unheated reaction at time zero
(inset of Fig. 2(A)). Peak 1 was confirmed to be SB-4PP by
collecting it and subjecting it to MALDI-TOF-MS and
MALDI-TOF/TOF-MS analysis (see below). Conducting the
reaction at room temperature for 24 h (second type of
condition reported previously[5]) gave a yield of 3% (n = 3,
CV = 0.17%; data not shown).
We next explored ’evaporative derivatization conditions’ to
potentially improve the yield (by increasing the concentration
of the reagent, and perhaps also by creating a different
reaction microenvironment), and also potentially to facilitate
or even avoid post-derivatization sample cleanup (by
minimizing the amounts of the derivatization reagents) when
4PP is reacted with SBA. The reaction mixture was
evaporated prior to incubating it at room temperature or at
an elevated temperature. We initially tested the following
concentrations of reactants in ACN (same as those tested
above) prior to evaporation: 5 × 10–2 M 4PP, 5 × 10–1 M SBA,
and 1.6 × 10–3 M DMAP. Evaporation of a 50 μL aliquot of
a
1 mL reaction solution was carried out at room
temperature for 40 min at 0.6 Torr, yielding a solid. Keeping
this solid at room temperature for 3 days gave a 48% yield
of SB-4PP based on HPLC-UV (the yield progressively
climbed to this level during this period of time; data not
shown). A longer reaction time at room temperature was
not studied.
The evaporative derivatization reaction was faster and the
yield was higher at an elevated temperature. After studying
various times and temperatures (see below), we selected 1 h
at 60 °C, which gave a yield of 96% (n = 3, CV = 1%), since
the other conditions tried (longer times and higher
temperatures) gave little improvement in yield, and a higher
temperature or longer time risks an increase in noise. A
representative HPLC chromatogram is shown in Fig. 2(B),
Figure 2. HPLC chromatograms of reaction mixtures of 4PP,
SBA and DMAP: (A) non-evaporative derivatization where
the reaction mixture was heated at 80 °C for 2 h and (B)
evaporative derivatization where the reaction mixture was
evaporated and then heated at 60 °C for 1 h. Peak 1: SB-4PP;
peak 2: 4PP. For HPLC conditions, see Experimental section.
Insets: unheated reactions at time zero.
along with that from an unheated reaction at time zero in
the inset. The early-eluting peak in Fig. 2(B) was 10% higher
when the phenol was not present in the reaction mixture
(unreacted or hydrolyzed reagents contributed to this peak,
so the peak was slightly increased in the blank since some
of these reagents had reacted with the phenol). This further
validated our way of assessing the yield. Examples of results
from other conditions are as follows (expressed as mean
CV) where the reaction time was 2 h and n = 3 in each case:
59 1% at 40 °C, 97 0.2% at 60 °C, and 98 0.3% at 80 °C
(data not shown). Direct analysis of the evaporative reaction
mixture (after addition of CCA matrix), using the condition
60 °C for 1 h, by MALDI-TOF-MS, gave the data shown in
Fig. 3(A), with a peak for deprotonated SB-4PP at m/z
353.050 (exact mass 353.048), and the data from a blank
reaction (no 4PP) in Fig. 3(B). (The isotope peak at m/z
354.050 in Fig. 3(A) falsely appears to be too high since the
corresponding nonisotopic peak is saturated.) Corresponding
data recorded by MALDI-TOF/TOF-MS are shown in Fig. 4
Figure 1. Scheme for detection of phenols by derivatization
with 2-sulfobenzoic anhydride (SBA) followed by mass
spectrometry, where MS designates MALDI-TOF-MS, and
MS/MS designates MALDI-TOF/TOF-MS, each in negative
ion mode.
Rapid Commun. Mass Spectrom. 2014, 28, 653–661
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Y. Y. Yao, P. G. Wang and R. Giese
353.050
354.050
100
90
80
70
60
50
40
30
20
10
0
8.0E+4
A
375.059
355.051
359.068
351.025
376.064
377.071
360.073
350.020
378.083
340
348
356
364
372
380
m/z
375.052
100
90
80
70
60
50
40
30
20
10
0
3.8E+4
B
351.016
359.061
376.057
377.063
353.017
355.069
352.020
360.065
354.214
378.073
350.011
340
348
356
364
372
380
m/z
Figure 3. Negative ion MALDI-TOF-MS spectra: (A) reaction mixture of 4PP and
SBA after 1 μL was combined with 10 μL CCA matrix and spotted onto the target
for MALDI-TOF-MS giving a peak for deprotonated SB-4PP at m/z 353.050 (exact
mass 353.048); the reaction was conducted by non-evaporative derivatization at
60 °C for 1 h with the initial reaction mixture of 5 × 10–2 M: 4PP, 5 × 10–1 M SBA,
and 1.6 × 10–3 M DMAP. (B) 1 μL of ACN combined with CCA matrix, showing
an absence of this peak.
169
100
90
80
70
60
50
40
30
20
10
0
A
4.0E+4
144
353
208
235
311
82
155
228
301
9
374
m/z
100
90
80
70
60
50
40
30
20
10
0
4.0E+4
B
144
353
311
208
235
9
82
155
228
301
374
m/z
Figure 4. Negative ion MALDI-TOF/TOF-MS spectra of: (A) same sample as in
Fig. 3(A) and (B) same sample as in Fig. 3(B), after selection of the deprotonated
SB-4PP precursor ion at m/z 353.
(A) (showing a peak for the 4-phenylphenolate product ion at
m/z 169 derived from the deprotonated molecule at m/z 353)
and in Fig. 4(B) (blank: no 4PP).
HPLC-UV that the yield of SB-4PP was unchanged even
when the following concentrations were set up prior to
evaporation: 5 × 10–4 M 4PP, 5 × 10–3 M SBA, and 1.6 × 10–5
M
The reaction of 4PP with SBA was next studied at lower
concentrations of 4PP under the selected reaction conditions
(60 °C, 1 h), while also lowering the concentration of SBA
and DMAP, but keeping the relative molar amounts of 4PP,
SBA, and DMAP constant (1:10:0.03). We observed by
DMAP. Lower concentrations (with the usual dilution after
the reaction) than this of 4PP could not be detected reliably by
HPLC-UV. However, SB-4PP product (at m/z 353.050) could
be detected by both MALDI-TOF-MS (Fig. 5(A)) and MALDI-
TOF/TOF-MS (peak at m/z 169 in Fig. 5(B)), even when the
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Derivatization of phenols with 2-sulfobenzoic anhydride prior to MS
Figure 5. (A) Negative ion MALDI-TOF-MS spectrum after evaporative derivatization
at 60 °C for 1 h with the initial reaction mixture of 50 μL 5 × 10–6 M 4PP, 5 × 10–3 M SBA,
and 1.6 × 10–7 M DMAP, showing a peak for deprotonated SB-4PP at m/z 353.050. (B)
Negative ion MALDI-TOF/TOF-MS spectrum of the same sample after selection of
the ion at m/z 353 for deprotonated SB-4PP, showing a peak at m/z 169 for the phenolate
product ion of SB-4PP (see Fig. 1). Inset of (B): Spectrum a: negative ion MALDI-TOF/
TOF-MS spectrum after evaporative derivatization 60 °C for 1 h of an initial reaction
mixture of 5 μL 5 × 10–6 M 4PP, 5 × 10–3 M SBA, and 1.6 × 10–7 M DMAP; the peak at
m/z 169 has a S/N of 260. Spectrum b: blank reaction (no 4PP).
following initial solution concentrations were tested: 5 × 10–6 M
the corresponding concentrations of SBA and DMAP were
5 × 10–3 and 1.6 × 10–5 M, respectively. The relative
4-PP, 5 × 10–3 M SBA, and 1.6 × 10–7 M DMAP. Since the volume
of this reaction solution prior to evaporation was 50 μL, about
43 ng of 4PP was detected in this way. While evaporating
5 μL of this reaction solution (containing 4.3 ng or 25 pmol)
did not yield a peak for the product by MALDI-TOF-MS,
detection by MALDI-TOF/TOF-MS was successful (S/N= 260
for m/z 169), as seen in spectrum a in the inset of Fig. 5(B), where
the spectrum from the blank reaction (no 4PP) is also shown
(spectrum b in the inset).
M
responses (relative to 4-nonylphenol; see below) of the
products were obtained by directly subjecting an aliquot
of the reaction mixture, after matrix addition, to MALDI-
TOF-MS and MALDI-TOF/TOF-MS, giving the data
shown in Fig. 6. This data is tabulated in Table 1, along
with the pKa values of the phenols. Note that the values
for relative response in the table reflect the combined
variation in both reaction yield and S/N by the two
MALDI techniques. The response of 4-nonylphenol was
arbitrarily assigned a value of 100 independently for
both the TOF and the TOF/TOF data since it gave the
highest S/N in each case. The nature of the cited ions is
defined generically in Fig. 1. Overall, it is clear that
phenols can differ widely in their response by the method
presented here.
Evaporative derivatization of a mixture of phenols
A mixture of 15 phenols (structures shown in Table 1) was
reacted with SBA under evaporative derivatization
conditions, where the concentration of each phenol was
5 × 10–4 M in the 50 μL solution prior to evaporation, and
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Y. Y. Yao, P. G. Wang and R. Giese
Table 1. Relative yield/response values by MALDI-MS (data from Fig. 6) for phenols after evaporative derivatization with
SBAa
Deprotonated molecule by TOF
R-Phenolate by TOF/TOF
Number
1
Structure
(m/z)
(m/z)
pKa
10.4
100 (403.158)
100 (219.17)
2
3
29.5 (353.048)
25.0 (305.048)
16.5 (169.07)
4.0 (121.07)
9.6
10.6
4
5
12.3 (324.994)
11.6 (411.090)
3.8 (141.01)
7.1 (227.11)
9.6
9.6, 11.3
6
7
8.6 (291.033)
6.4 (277.017)
1.1 (107.05)
1.0 (93.03)
10.3
10.0
8
0.3 (344.939)
0.1 (322.002)
0.1 (160.96)
0.2 (138.02)
7.9
7.2
9 and 10
11
N/D (381.003)
N/D (197.02)
4.3
4.1
12
13
N/D (366.987)
N/D (310.978)
N/D (183.00)
N/D (127.00)
8.6
(Continues)
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Derivatization of phenols with 2-sulfobenzoic anhydride prior to MS
Table 1. (Continued)
Deprotonated molecule by TOF
(m/z)
R-Phenolate by TOF/TOF
Number
14
Structure
(m/z)
pKa
N/D (378.900)
N/D (446.822)
N/D (194.92)
N/D (262.84)
6.1
15
4.7
aNames of compounds: 1) 4-nonylphenol; 2) 4-phenylphenol (4PP); 3) 2,4-dimethylphenol; 4) 4-chloro-3-methylphenol; 5)
bisphenol A (BPA); 6) o-cresol; 7) phenol; 8) 2,4-dichlorophenol; 9) 2-nitrophenol; 10) 4-nitrophenol; 11) 2-methyl-4,6-
dinitrophenol; 12) 2,4-dinitrophenol; 13) 2-chlorophenol; 14) 2,4,6-trichlorophenol; 15) pentachlorophenol.
bND: not detected or not readily detected.
Figure 6. Negative ion MALDI-TOF-MS spectra of: (A) reaction mixture of 15 phenols
(see Table 1) in which evaporative derivatization with SBA (5 × 10–3 M) and DMAP
(2% w/w of total phenols) was performed at 60 °C for 1 h, where each phenol was
5 × 10–4 M and (B) reaction blank (phenols not present).
There could be several reasons that eight of the phenols
(8–15) gave little or no response by MALDI-TOF-MS after
derivatization with SBA, such as incomplete derivatization,
instability of the derivatives in the MALDI step, and
variation in the efficiency of desorption. It is interesting that
all these compounds have relatively low pKa values (below 9).
We tested pentachlorophenol with reaction monitoring by
HPLC-UV, and HPLC-peak collection-MALDI-MS, to gain
some insight. The yield of SBA-tagged pentachlorophenol
was about 1% at 60 °C, about 2% at 80 °C, and about 25% at
100 °C after 2 h of an evaporative derivatization reaction, based
on disappearance of the peak for starting material. Longer
reaction times increased the formation of side products
without significantly increasing the yield. The HPLC
chromatogram from the latter reaction (100 °C, 2 h) is
shown in Fig. 7. The new peak (peak 1) was confirmed
to be SBA-tagged pentachlorophenol by collection and
analysis by MALDI-TOF-MS in positive ion mode, giving
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Y. Y. Yao, P. G. Wang and R. Giese
still off scale. Reducing the laser setting to 2200 brought the
peaks for 11 and 12 on scale (S/N = 1860 and 1760,
respectively) and lowered the S/N of compound 15 to 219.
We note that the highest responders (11, 12 and 15) have the
lowest pKa values (4.1–4.7). Overall, derivatization with
SBA therefore is somewhat complementary to detection by
negative ion MALDI-MS without derivatization, since
phenols giving little or no response when underivatized
(compounds 1, 2, 4, 5, 6) tended to be readily or highly
detected after derivatization (compounds 1–7).
2
1
CONCLUSIONS
(min)
2-Sulfobenzoic anhydride has some properties, either well-
known or introduced here, that encourage its further study
as an analytical derivatization reagent for phenols with a
relatively high pKa (above 9) to improve their detection by
negative ion MALDI-MS: commercial availability at low cost;
high reaction yield by evaporative derivatization; and
effectiveness under this condition without post-derivatization
sample cleanup. In the absence of derivatization, phenols
with pKa values below 5 were the most readily detected.
Figure 7. HPLC chromatogram of the reaction mixture of
pentachlorophenol, SBA and DMAP after evaporative deri-
vatization at 100 °C for 1 h. Peak 1: SBA-derivatized penta-
chlorophenol. Peak 2: underivatized pentachlorophenol. For
concentrations and HPLC conditions, see Experimental section.
an ion for [M–H+2Na]+ at m/z 492.810 (exact mass 492.801;
data not shown). The observed isotope pattern of this
deprotonated disodiated molecule matched the calculated
isotope pattern (data not shown).
Acknowledgements
In negative ion mode, no deprotonated molecule was
observed from the collected HPLC peak (peak 1 in Fig. 7) from
the above reaction of pentachlorophenol; instead peaks for
[C6Cl5O]– (m/z 264.839), [C6Cl5O+CCA–H+Na]– (475.853),
and [C6Cl5O+C6Cl5O+Na]– (552.651) were seen (data not
shown); peak ratio 7:6:2. This prompted us to re-evaluate the
MALDI-TOF-MS data related to pentachlorophenol from the
reaction mixture of 15 phenols. Indeed, peaks for [C6Cl5O]–
and [C6Cl5O+CCA–H+Na]– were seen. For pentachlorophenol,
one can speculate that the failure to readily detect the
derivatized compound in the reaction of 15 phenols, according
to the strategy summarized in Table 1, was mainly due to a
combination of low reaction yield (apparently from steric
and/or inductive effects), spread of the signal in MALDI-
TOF-MS over several ions including isotope peaks, and
pronounced instability of the deprotonated molecule in the ion
source. To different degrees, these same factors may contribute
to the behavior of the other phenols in Table 1 that gave little
or no response under the conditions employed. Overall, it is
seen that the phenols with relatively high pKa values (above 9,
compounds 1–7) are much more detectable (>10-fold) than
those with pKa values below 9 (compounds 8–15). The highest
responders are monophenols with a higher degree of alkyl or
aryl bulk, and a pKa above 9 (compounds 1–3).
This work was supported by NIH Grant P42ES017198
from NIEHS. This is contribution number 1040 from the
Barnett Institute.
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