Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization

Article abstract of DOI:10.1021/ac200842e

Reaction kinetics studied by mass spectrometry (MS) has previously been limited to millisecond time resolution. This paper presents the development of a submillisecond time-resolved mass spectrometric method for fast reaction kinetic study, based on the capability of desorption electrospray ionization (DESI) for direct and fast ionization of a high-speed liquid jet stream. The principle underlying this methodology is that two reactant solutions undergo rapid mixing to produce a free liquid jet which is ionized by DESI at different positions corresponding to different reaction times. Due to the high velocity of the liquid jet, high time resolution can be achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol (DCIP) and l-ascorbic acid (L-AA) was chosen as an example to demonstrate this concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 μs. The good agreement of the measured value of (116 ± 3) s ^-1 with that measured by the stopped-flow optical method (105 ± 2) s^-1 validates the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.

Full text of DOI:10.1021/ac200842e

LETTER
pubs.acs.org/ac
Development of Submillisecond Time-Resolved Mass Spectrometry
Using Desorption Electrospray Ionization
Zhixin Miao, Hao Chen,* Pengyuan Liu, and Yan Liu
Department of Chemistry and Biochemistry, Center for Intelligent Chemical Instrumentation, Ohio University, Athens, Ohio 45701,
United States
S Supporting Information
b
ABSTRACT: Reaction kinetics studied by mass spectrometry (MS) has previously
been limited to millisecond time resolution. This paper presents the development of a
submillisecond time-resolved mass spectrometric method for fast reaction kinetic
study, based on the capability of desorption electrospray ionization (DESI) for direct
and fast ionization of a high-speed liquid jet stream. The principle underlying this
methodology is that two reactant solutions undergo rapid mixing to produce a free
liquid jet which is ionized by DESI at different positions corresponding to different
reaction times. Due to the high velocity of the liquid jet, high time resolution can be
achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol
(
DCIP) and L-ascorbic acid (L-AA) was chosen as an example to demonstrate this
concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 μs. The good
ꢀ
1
ꢀ1
agreement of the measured value of (116 ( 3) s with that measured by the stopped-flow optical method (105 ( 2) s validates
the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or
labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide
applications in fast (bio)chemical reaction investigations.
inetic studies play a central role in the elucidation of
chemical and biochemical reaction mechanisms. Generally,
bio)chemical kinetic studies are carried out by optical methods
detection of the radical cation intermediate as well as the
1
22,23
K
isolated biomolecules at ambient conditions.
Recently,
(
besides examining solid analytes on surfaces, DESI has been
extended to directly analyze liquid samples in our and other
such as absorption or fluorescence spectroscopy with the time
resolution as low as femtoseconds. However, these methods
require chromophoric substrates or labeling. Mass spectrometry
2
4ꢀ29
laboratories.
It was shown that, owing to its direct
sampling capability, liquid sample DESI is useful in coupling
2,3
(
MS)-based approaches to study kinetics have arisen, particu-
25,30,31
33
32
MS with electrochemical cells,
chromatography,
4
larly with the advent of electrospray ionization (ESI). The
striking advantages of the use of MS as a detector over spectro-
scopic detection are that MS has high chemical specificity and
does not require chromophoric substrates. Notably, the time-
resolved ESI-MS methods based on elegant designs for reactant
solution mixing have been established by Konermann and other
27
microfluidics, and microextraction, as well as probing protein
26
conformation in solution.
Herein, we report a novel time-resolved MS-based method for
fast reaction kinetic study with the time resolution down to
submilliseconds, based on the capability of liquid sample DESI in
directly ionizing analytes in a “flying” liquid jet. The principle
underlying the method is that two reactant solutions undergo fast
mixing to produce a free liquid jet which is sampled by a DESI
probe at different positions corresponding to different reaction
times. Due to the high velocity of the liquid jet, high time
resolution can be achieved (e.g., 0.1 mm distance interval
between two spots in a liquid jet of 100 m/s would correspond
5
ꢀ11
groups,
allowing the monitoring of reaction kinetics with the
12
time resolution from seconds to milliseconds. Nevertheless,
it is necessary to further improve the time resolution of MS
methods.
Desorption electrospray ionization (DESI), introduced by
13
Cooks and co-workers, is a recently established technique that
allows direct ionization of analytes from surfaces with little or
no sample preparation. Various novel DESI-MS applications
have been reported, ranging from drug detection to tissue
34
to 1 μs difference in the reaction time ). Such an experiment
would be difficult to perform using traditional ESI techniques
because ESI is generally not tolerant to high flow-rate liquid
samples which would “flood” the ion source. Also, it is not
convenient to ionize jet samples by ESI. In this study, the classic
1
4ꢀ19
imaging.
In terms of kinetic study, DESI-MS has been used
to monitor solid-phase reaction kinetics with the time resolution
2
0
of hours. Taking advantages of the short time scale during
DESI ionization (<2 ms), transient reaction intermediates can be
Received: April 2, 2011
Accepted: May 3, 2011
Published: May 03, 2011
21
intercepted and detected in reactive DESI experiments. Also,
the extractive electrospray ionization (EESI) has been used in the
r 2011 American Chemical Society
3994
dx.doi.org/10.1021/ac200842e
|Anal. Chem. 2011, 83, 3994–3997

Analytical Chemistry
LETTER
Figure 1. (a) Scheme and (b) real image showing the apparatus of the submillisecond time-resolved DESI-MS.
Scheme 1. Equation for the Reduction Reaction of DCIP and
L-AA
jet final pH was 2.0 as measured by a SevenEasy pH meter). The
DESI spray solvent used was 20 mM aqueous NH OAc buffer
4
with the pH of 7.0. All sample solutions were prepared fresh daily.
For comparison purposes, the stopped-flow spectroscopic mea-
surement of the rate constant was performed using a stopped-
flow UVꢀvis spectrometer (Applied Photophysics, Model
SX.18MV). This measurement was carried out using the same
experimental conditions as those used in the DESI-MS measure-
ment, including the sample concentrations, pHs, and solvents.
Briefly, two reactant solutions, 4.0 mM L-AA in 20 mM HCl and
fast reaction with known kinetics, the rapid reduction of 2,
6-dichlorophenolindophenol (DCIP, MW 267 Da) by L-ascorbic
0.2 mM DCIP in H O, were pneumatically driven from two
2
acid (L-AA), was chosen as an example to demonstrate the
concept and to validate the feasibility of this new submillisecond
time-resolved DESI-MS.
5.0 mL Hamilton syringes into a mixing cell. The decrease in the
characteristic absorbance by the reactant DCIP at 524 nm was
monitored, and the correlation curve of absorbance response vs
time was fitted to the exponential trend to calculate the reaction
rate constant.
In this experiment, the apparatus of the submillisecond
time-resolved DESI-MS was home-built, mainly consisting of
2
5
a DESI source, which was the same as described previously,
a
In the DESI-MS experiments, as the height of the Tee mixer
Tee mixer assembly (a Tee junction, Supelco), and a LCQ
DECA mass spectrometer (Thermo Finnigan, San Jose, CA,
USA; Figure 1). Two reactant solutions were mixed in the Tee
mixer (the third outlet of the Tee mixer was inserted with a
for producing the jet (L ) was adjustable using a micrometer,
jet
the jet was sampled at the positions of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
and 4.0 cm away from the Tee mixer outlet tip by DESI,
corresponding to the reaction times of 2.45, 2.75, 3.04, 3.33,
3.62, 3.91, and 4.20 ms, respectively, with the time interval of ca.
300 μs. The reaction time for each sampling position was
calculated on the basis of the dead mixing time spent in the
Tee mixer and the jet flight time. With increased reaction time, it
is evident that the concentration of the oxidized DCIP decreases,
as indicated by the drop in the intensity of the corresponding
protonated oxidized DCIP (m/z 268) shown in the acquired
DESI mass spectra (Figure 2).
In order to calculate the reaction rate constant, the con-
centration of the remaining reactant DCIP at each reaction
time needs to be quantified. The acquired DESI mass spectra
provide the abundance ratios of m/z 268 vs 270 (denoted as R).
The peak at m/z 268 corresponds to the ion of the remaining
reactant DCIP while the peak at m/z 270 observed in the DESI
spectra is the combination of the reduced DCIP product ion
and the isotopic ion of m/z 268 (as the reactant DCIP carries
two chlorine atoms, the m/z 268 has two additional isotopic
peaks at m/z 270 and 272, and their intensity ratio follows
9:6:1). Thus, we can relate the concentration of the remaining
oxidized DCIP (denoted as c) to R, using the following
equation:
3
.2 cm deactivated fused silica capillary of i.d. 50 μm) via
infusion injection by HPLC pumps at a total flow rate of
.0 mL/min, leading to a high speed liquid jet (17.1 m/s). Tee
mixers were employed before for the reaction kinetics
2
1
0,11,35ꢀ37
monitoring.
The jet was subject to ionization via
interaction with the charged microdroplets generated by the
DESI spray, and the ionized species were detected by the mass
spectrometer. To evaluate the capability of DESI for ionizing
the jet samples, amino acids, peptides, and protein samples
were tested, and all analytes (even proteins in low concentra-
tions such as 1 μM lysozyme) were readily ionized and clearly
observed in the acquired DESI mass spectra (Figure S-1,
Supporting Information).
L-AA, the principal form of vitamin C, is a water-soluble
antioxidant present naturally in a wide range of foods. DCIP is
the most commonly used reagent for the determination of
3
8
L-AA (Scheme 1 shows the reaction equation). Because of
its fast reaction kinetics, this reaction was often used to test the
39
performance of stopped-flow apparatuses. In our experiment,
to measure the rate constant of DCIP reduction by L-AA using
mass spectrometry, the concentration of L-AA was kept 20 times
higher than that of the oxidized DCIP so that the reaction takes
place as a pseudofirst-order reaction. A solution of 4.0 mM L-AA
ð9=16Þ ꢁ c ꢁ i
in 20 mM HCl was mixed with 0.2 mM DCIP in H O to form a
2
¼ R
ð1Þ
ð6=16Þ ꢁ c ꢁ i þ ð9=16Þ ꢁ ðc0 ꢀ cÞ
jet by pumping each solution at the flow rate of 1.0 mL/min (the
3
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dx.doi.org/10.1021/ac200842e |Anal. Chem. 2011, 83, 3994–3997

Analytical Chemistry
LETTER
Figure 2. Time-resolved DESI MS spectra of the reaction between 0.1 mM oxidized DCIP and 2.0 mM ascorbic acid (pH = 2.0) sampled at (a) 2.45 ms,
b) 3.04 ms, (c) 3.62 ms, and (d) 4.20 ms, respectively.
(
where c is the remaining oxdizied DCIP concentration; c is the
0
initial concentration of the oxidized DCIP, which is 0.1 mM;
c ꢀ c is the concentration of the reduced DCIP product; and i
0
is the relative ionization efficiency factor of the oxidized DCIP
over the reduced DCIP.
In eq 1, the numerator represents the abundance of m/z 268.
The denominator represents the abundance of m/z 270 observed
in the DESI spectra, which is the sum of m/z 268 isotopic peak
abundance (the first term in the denominator) and the product
ion abundance (the second term in the denominator). In a
separate experiment, it was found that the relative ionization
efficiency factor of the oxidized DCIP vs the reduced DCIP in the
jet (denoted as i) is 1.09 ( 0.08 (Figure S-2, Supporting
Information; the slightly higher ionization efficiency of the
oxidized DCIP over the reduced DCIP is probably due to the
fact that the oxidized DCIP carries one more carbonyl group
which enhances its proton affinity). Thus, this relative ionization
efficiency factor was also taken into account in eq 1.
0
Figure 3. Plot of ln(c/c ) vs time (ms) deriving the pseudofirst-order
1
ꢀ
reaction rate constant to be (116 ( 3) s . The error bars stand for the
standard deviations in triplicate measurements.
mixer to adjust the jet position to be sampled. In addition,
there is a probability that the reduction reaction does not stop
during the DESI ionization and it is difficult to practically
measure or calculate this portion of reaction time. However, it
is believed that the continuation of the reaction during DESI
ionization has limited effect on our rate constant measurement
for the following reasons. First, this portion of reaction time is
the same for all of the different jet spots that were sampled by
DESI. As the liquid DESI ionization time scale (i.e., from the DESI
sampling to the time when ions are generated inside the mass
Using eq 1, the concentrations of the remaining reactant at
different reaction times are calculated and the reaction rate
constant k can be derived by plotting ln(c/c ) vs time (ms). A
0
2
straight line was obtained (R = 0.9957), and the slope of the line
ꢀ1
gives a k value of 116 ( 3 s (Figure 3). The reproducibility is
quite good as indicated by the small error bars shown in Figure 3.
In comparison, we carried out the stopped-flow measurement of
the reaction under the same reaction condition as the DESI
experiment, which gave the pseudofirst-order reaction rate
constant of 105 ( 2 s (Figure S-3, Supporting Information).
Therefore, the good agreement on reaction rate measurements
between the DESI-MS approach and the stopped-flow optical
method validates this novel time-resolved DESI-MS method. In
particular, the time resolution of 300 μs obtained in this study is
unprecedented for MS study, which should broaden the MS
applications to the fast kinetic study.
21,26
spectrometer) is short and estimated to be less than 2 ms,
the
continuation of the reaction during DESI ionization, if occurring, is
analogous to an added small dead time for the total reaction time
scale. More importantly, in our experiment, we used 20 mM
ammonium acetate buffer (pH 7) as the DESI spray solvent. It is
likely that the secondary microdroplets resulting from the pick-up
of the jet sample by the primary DESI sprayed microdroplets has a
pH that is very different from the low pH of the jet due to the buffer
present in the primary spray. It is known that the reaction rate of
DCIP reduction by L-AA will be reduced by over 100 times with
ꢀ
1
40
We also noted that, in order to obtain reproducible results,
it is important to keep the ionization conditions the same for
each sampling position of the jet. This includes keeping the
DESI probe in a fixed position and vertically moving the Tee
a pH change from 2 to 7. Thereby, the pH change during the
DESI ionization process quenches the rapid reduction reaction
and thus limits the post-DESI-sampling reaction effect on our
measurements.
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dx.doi.org/10.1021/ac200842e |Anal. Chem. 2011, 83, 3994–3997

Analytical Chemistry
LETTER
In conclusion, this study reports a time-resolved MS method
with submillisecond time resolution (300 μs) in the kinetic study
for the first time. The preliminary application to the fast reaction
kinetics of the reduction of DCIP by L-AA shows good agree-
ment in reaction rate constant measurements by MS and
stopped-flow optical methods. Further time resolution improve-
ment is possible by increasing the jet speed or decreasing the jet
sampling distance interval. Also, using a Tee mixer with a small
dead volume and an outlet with a small i.d. for generating a slim
and long jet will benefit this method in many ways: (1) it will
shorten the dead mixing time; (2) it will consume less amount of
samples (in our current triplicate measurements, it consumed ca.
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(
4
1
42
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ASSOCIATED CONTENT
Supporting Information. Additional information, with
(
S
b
regard to the DESI ionization of biomolecules in jet, the
ionization efficiency comparison between reduced and oxidized
DCIP, and the stopped-flow experimental results. This material
is available free of charge via the Internet at http://pubs.acs.org.
(
Society for Mass Spectrometry Conference on Mass Spectrometry, Denver,
CO, June 1ꢀ5, 2008.
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2
’
AUTHOR INFORMATION
(
Corresponding Author
Chem. 2008, 80, 6131–6136.
*
Tel: 740-593-0719. Fax: 740-597-3157. E-mail: chenh2@ohio.
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’
ACKNOWLEDGMENT
This work was supported by NSF (CHE-0911160). The
(
1
authors are grateful to Professor Kenneth L. Brown for access
to his stopped-flow instrument and to Professor Michael Jensen
for his helpful suggestions on the kinetics study.
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dx.doi.org/10.1021/ac200842e |Anal. Chem. 2011, 83, 3994–3997