Coupling of single droplet micro-extraction with desorption electrospray ionization-mass spectrometry

Article abstract of DOI:10.1016/j.ijms.2010.07.024

Single droplet micro-extraction (SDME) is a powerful preconcentration and purification technique for trace chemical analysis, but the detection of the resulting single droplet extract is often time-consuming with traditional detection methods such as GC/MS or LC/MS. In this study, desorption electrospray ionization-mass spectrometry (DESI-MS) was coupled with SDME to serve as a fast detection method for the first time, demonstrated by the trace analysis of methamphetamine (MA) in aqueous solution and the detection of an organic reaction product from an ionic liquid (IL). In the former application, three-phase liquid SDME was conducted to enrich MA in aqueous solution to an organic solvent and then to back-extract the analyte to a single aqueous droplet. Subsequent DESI-MS analysis can be performed either to a single droplet or multiple droplets in a row. The average enrichment factor obtained for MA was 390-fold. In the latter case, two-phase liquid SDME was conducted to directly extract the product of an organic reaction performed in a room temperature ionic liquid (i.e., the nucleophilic addition of aniline to phenyl isothiocyanate to form N,N′-diphenylthiourea in 1-butyl-3-methyl- imidazolium tetrafluoroborate). The ionization of the resulting droplet extract by DESI allows one to directly examine the reaction product without interference from the ionic liquid. Such an analysis uses a few microliters of an organic solvent, achieving green chemistry. This combined SDME/DESI-MS method is characterized with high preconcentration efficiency, high throughput capability and minimizing organic solvent waste.

Full text of DOI:10.1016/j.ijms.2010.07.024

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International Journal of Mass Spectrometry 301 (2011) 102–108
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Coupling of single droplet micro-extraction with desorption electrospray
ionization-mass spectrometry
Xiaobo Sun, Zhixin Miao, Zongqian Yuan, Peter de B. Harrington∗, Jennifer Colla, Hao Chen∗∗
Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Single droplet micro-extraction (SDME) is a powerful preconcentration and purification technique for
trace chemical analysis, but the detection of the resulting single droplet extract is often time-consuming
with traditional detection methods such as GC/MS or LC/MS. In this study, desorption electrospray
ionization-mass spectrometry (DESI-MS) was coupled with SDME to serve as a fast detection method
for the first time, demonstrated by the trace analysis of methamphetamine (MA) in aqueous solution and
the detection of an organic reaction product from an ionic liquid (IL). In the former application, three-
phase liquid SDME was conducted to enrich MA in aqueous solution to an organic solvent and then to
back-extract the analyte to a single aqueous droplet. Subsequent DESI-MS analysis can be performed
either to a single droplet or multiple droplets in a row. The average enrichment factor obtained for MA
was 390-fold. In the latter case, two-phase liquid SDME was conducted to directly extract the product of
an organic reaction performed in a room temperature ionic liquid (i.e., the nucleophilic addition of aniline
to phenyl isothiocyanate to form N,Nꢀ-diphenylthiourea in 1-butyl-3-methyl-imidazolium tetrafluorob-
orate). The ionization of the resulting droplet extract by DESI allows one to directly examine the reaction
product without interference from the ionic liquid. Such an analysis uses a few microliters of an organic
solvent, achieving green chemistry. This combined SDME/DESI-MS method is characterized with high
preconcentration efficiency, high throughput capability and minimizing organic solvent waste.
© 2010 Elsevier B.V. All rights reserved.
Received 26 March 2010
Received in revised form 14 July 2010
Accepted 26 July 2010
Available online 6 August 2010
Dedicated to Prof. Michael L. Gross in the
occasion of his 70th birthday.
Keywords:
Desorption electrospray ionization-mass
spectrometry
Single droplet micro-extraction
Single droplet analysis
Methamphetamine
Room temperature ionic liquid
Green chemistry
1. Introduction
of organic solvents/acids to samples prior to ionization as required
by ESI-MS. Liquid DESI-MS was advantageous to a protein confor-
Desorption electrospray ionization-mass spectrometry (DESI-
MS), first introduced by Cooks and co-workers [1,2], is a powerful
detection method for a variety of analytes encompassing illicit
drugs, chemical warfare agents, pharmaceuticals, biological sam-
ples, etc. [3–22]. DESI-MS is characterized by no or little need
of sample pretreatment, satisfactory performance under ambi-
ent conditions, short analysis time (as short as a few seconds),
and equivalent specificity to other mass spectrometric methods.
Recently, in our and other laboratories, DESI-MS has been extended
to the direct ionization of liquid samples [23–28] such as biological
samples and drugs. It has been demonstrated that liquid DESI-MS
has the advantages of direct analysis of liquid samples as opposed
to drying the sample in conventional DESI-MS or adding additives
mation study [29] and coupled to an electrochemical cell for the
reduction of protein disulfide bonds [25]. Notably, liquid DESI-MS
is suitable for direct analysis of droplets or thin liquid films [23,28].
Single droplet micro-extraction (SDME) is a preconcentra-
tion/purification protocol used to preconcentrate or separate
analytes from complex sample matrices. As an efficient enrich-
ment and separation method, micro-extraction has been developed
for various applications ranging from environmental monitoring
[30,31], anti-doping screening [32], to forensic detection [33,34].
Among all the micro-extraction techniques, single droplet micro-
extraction (SDME) [33–36] has displayed advantages of low cost,
simplicity and effective preconcentration capability. Because of the
microliter volume of extraction solvent used, only a small extrac-
tion stock solution is needed. Moreover, a small volume of organic
solvent is consumed in the process, producing less amount of waste
than traditional extraction methods and enabling easy clean-up
[37]. A microliter sized droplet used in SDME offers a larger phase
ratio compared with solid phase micro-extraction, leading to a
highly efficient extraction of the analyte into the desired phase.
In the traditional two-phase liquid SDME experiments, a single
droplet is immersed into another liquid layer for analyte extrac-
∗
Corresponding author at: Department of Chemistry and Biochemistry, Ohio Uni-
versity, Athens, OH 45701, USA. Tel.: +1 740 994 0265.
∗∗
Corresponding author at: Department of Chemistry and Biochemistry, Ohio Uni-
versity, 136 Clippinger Laboratories, Athens, OH 45701, USA.
Tel.: +1 740 593 0719; fax: +1 740 597 3157.
E-mail addresses: peter.harrington@ohio.edu (P.d.B. Harrington),
chenh2@ohio.edu (H. Chen).
1387-3806/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijms.2010.07.024

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X. Sun et al. / International Journal of Mass Spectrometry 301 (2011) 102–108
103
tion. In a variant form of SDME such as three-phase liquid SDME, the
protocol involves a two-step extraction so that low concentration
analytes can be preconcentrated from a bulky solution to another
liquid phase and then into a single droplet of a suitable solvent.
In both cases, the resulting single droplet extract is subsequently
subjected to analysis. For this purpose, SDME was typically coupled
with various analytical methods such as capillary electrophore-
sis (CE) [38–40], high-performance liquid chromatography (HPLC)
[30,31,33,37], GC/MS [41] and fluorescence (FL) spectroscopy [38].
However, the detection of the resulting single droplet extract by
these methods involving separation is often either time-consuming
or limited to volatile/chromophoric compounds. It is necessary to
develop a rapid and general analysis platform for SDME.
a
water purification system (Nanopure Diamond Barnstead,
Thermo Scientific). High-performance liquid chromatography
grade methanol was purchased from GFS Chemicals (Columbus,
OH) and glacial acetic acid was purchased from Fisher Chemicals
(Fair Lawn, NJ). ACS grade hexane was purchased from J.T. Baker
Chemical Co. (Phillipsburg, NJ, USA). Reagent grade tetramethy-
lammonium chloride was supplied by Sigma Aldrich. Sodium
hydroxide was obtained from Spectrum Chemical MFG Corp.
(Gardena, CA, USA). Aniline (ACS reagent, purity >99.5%), phenyl
isothiocyanate (ACS reagent, purity of 99%), tetrachloromethane
(anhydrous, ACS reagent, purity of 99.9%) and acetonitrile (HPLC
grade) were supplied by Sigma-Aldrich. The room temperature
ionic liquid [Bmim][BF4] (HPLC grade) was from Fluka.
Methamphetamine (MA) [42] as a highly addictive and easy-to-
make drug has been commonly abused among a vast population
with the promotion of increasing production by illicit drug lab-
oratories worldwide [43]. MA abuse can cause impairments to
neurotransmitter systems distributed throughout the brain and
further damage cognitive ability [44]. Because of the reasons men-
tioned above, fast, convenient and economical detection methods
of MA are needed. For this reason, MA was selected as one of the
model compounds in this work to couple SDME with DESI-MS.
Green chemistry has become important with recent concerns of
climate change and overpopulation. Ionic liquids (ILs) have been
applied frequently as green solvents in organic chemistry because
of their special properties such as negligible vapor pressures and
simplicity in recycling and reuse [45–49]. It would be informative to
monitor the progress of a reaction by instrumental methods such as
MS during the reaction process with high chemical specificity and
sensitivity. Although matrix-assisted laser desorption/ionization
(MALDI) mass spectrometry can be used to detect peptides or pro-
teins from ILs directly [50], the direct detection of reaction products
in ILs using electrospray ionization (ESI) based mass spectrometric
methods is challenging because ILs are not amenable to ESI-MS. As
a result of the low volatility and the ionic nature of ILs, they may
either contaminate the MS instrument or suppress the ion signals of
product compounds. Therefore, new approaches for simplifying the
MS detection of the reaction products in ionic liquids are needed.
Because of the capability of direct analysis of nonvolatile
compounds in small volume of liquid films (sub-microliter) by
DESI-MS (even in a high throughput manner [23]) and the power
of SDME in preconcentration, it would be reasonable to combine
these two techniques for enhanced performance in analysis of
liquid samples. As demonstrated in this work, for the first time
SDME was coupled with DESI-MS to detect a trace amount of
MA in aqueous solution. In addition, a reaction product of N,Nꢀ-
diphenylthiourea (DPTU), generated from aniline addition onto
phenyl isothiocyanate in a room temperature ionic liquid (RTIL)
of 1-butyl-3-methyl-imidazolium tetrafluoroborate [Bmim][BF4],
was successfully extracted into a small organic droplet and
subsequently detected by DESI-MS. Such an analysis provides
higher chemical specificity in comparison with traditional reaction
monitoring techniques such as thin-layer chromatography (TLC).
This combination benefits both techniques because DESI-MS
allows fast chemical analysis of SDME extracts and SDME can be
an ideal preconcentration and separation protocol for DESI-MS.
The coupled SDME/DESI-MS will find useful applications in trace
chemical analysis as well as in the green chemistry field.
2.2. Three-phase SDME for trace analysis of MA
2.2.1. Sample preparation for MA extraction
All the MA samples were prepared from a 1.0 ␮g/mL stock
MA·HCl solution in water. The calibration series included 50., 100.,
200. and 800. ng/mL MA·HCl solutions in 5% acetic acid. The sus-
pended droplet used for SDME was 4 ␮L of a 5% acetic acid aqueous
solution.
2.2.2. Extraction procedure for MA
An extraction procedure from the literature [33] was followed
with some modifications. The extraction set-up is displayed in
Fig. 1(A). First 4.00 mL of 0.50 M NaOH solution was added to a
10 mL test tube followed by the addition of 4.0 ␮L of 1.0 ␮g/mL
MA·HCl aqueous solution. After mixing, the test tube was sealed
with aluminum foil. This solution served as a mimetic of 1. ng/mL
of MA in aqueous solution and the added base NaOH was for neu-
tralizing the MA molecules. For the 1st step extraction, 400.0 ␮L
of hexane was added and the test tube was shaken manually for
5 min. The test tube with a stirring bar (length: 10 mm; diame-
ter: 3 mm) then was mounted on a magnetic stirrer (Corning Inc.,
Model PC-220) and it was stirred for 15 min at the highest speed
(1100 rpm) for accelerating the hexane extraction of MA from the
bulk aqueous phase. After the stirring was stopped a 4 ␮L droplet
of 5% acetic acid in water was suspended in the organic layer of
hexane by using a 50 ␮L flat-top HPLC syringe (Hamilton) with the
needle replaced with 7 cm of a deactivated fused silica capillary
(i.d.: 0.25 mm; o.d.: 0.36 mm; Alltech Associates Inc., Deerfield, IL,
USA) after being inserted through the aluminum foil seal of the
test tube and fastened with a ring stand clamp. About 10 ␮L of air
above the droplet was kept in the syringe for the convenience of
handling the droplet. The solution then was stirred at the high-
est speed (1100 rpm) for 40 min to convey MA to the suspended
droplet while the test tube was sealed with aluminum foil. The
whole extraction procedure took about an hour. After extraction,
the hanging droplet was withdrawn back into the syringe and then
subjected to DESI-MS analysis as described below.
2.2.3. Calibration of DESI-MS detection of MA
A series of calibration solutions were used to calibrate the DESI-
MS detection of MA in aqueous solution. The concentrations of
the calibration solutions were 50., 100., 200. and 800. ng/mL of
MA·HCl in 5% acetic acid and the extractions and measurements
were conducted in triplicate. The experiment procedures contained
three parts. First, methanol/water/acetic acid (50:50:1 by volume)
was used as the DESI spray solvent and a blank solution of 5%
acetic acid in water was sampled for obtaining the background
spectrum for signal correction; secondly, 4 ␮L of the prepared MA
calibration solutions was sampled with the syringe used for micro-
extraction from which they were exposed to DESI-MS detection.
An internal standard of 1.0 ␮M tetramethylammonium chloride
was contained in the DESI spray solvent for correction of signal
2. Experimental
2.1. Chemicals and reagents
Methamphetamine hydrochloride was purchased from
Sigma–Aldrich (St. Louis, MO) and used as received. The de-
ionized water used for sample preparation was obtained using

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X. Sun et al. / International Journal of Mass Spectrometry 301 (2011) 102–108
Fig. 1. Schematics showing (A) the three-phase SDME set-up for MA analysis and (B) the two-phase SDME set-up for DPTU detection from an ionic liquid; (C) image showing
the direct desorption and ionization of the obtained single droplet extract contained in the syringe with a needle of the deactivated fused silica capillary.
fluctuation. The DESI-MS analyses of MA extracts were conducted
following the same procedure under the same conditions as the
calibration experiments. The calibration results were modeled by
linear regression analysis [51]. The 95% confidence intervals for the
calibration line, the slope, and the intercept were calculated from
the averages of the replicates for each standard concentration. The
average concentration for the single droplet sample containing the
extracted MA was calculated from three extraction replicates and
reported with a 95% confidence interval.
quantification of MA extracts, the spray solvent was doped with
1 ␮M tetramethylammonium chloride as an internal standard. The
ionization of the single droplet obtained from SDME occurred by
directing the charged microdroplet beam from DESI spray onto the
silica capillary of the syringe that was used in the micro-extraction
while the sample was expelled from the capillary. The resulting ions
were collected and detected by the mass spectrometer. As shown in
Fig. 1(C) the angle of the DESI sprayer with respect to the sampling
capillary needle was about 45◦ and the tips of these two capillaries
were approximately 1 mm apart.
2.3. Detection of the products of organic reactions performed in
ionic liquids
For MA detection a 4 ␮L droplet was expelled from the capillary
at 40 ␮L/min by a Chemyx Model F100 syringe pump (Huston, TX),
which took 4–6 s. Full MS scans were acquired in positive ion mode.
For the detection of the DPTU extract, similar DESI-MS parameters
were used with the differences listed below. First, acetonitrile with
0.5% acetic acid was used as the DESI spray solvent. The spray speed
was adjusted to 5 ␮L/min and the DPTU extract was expelled from
the syringe at a rate of 20 ␮L/min. A 2 ␮L droplet of the blank solvent
of CCl4 was detected under the same conditions to characterize
the background signal. Collision induced dissociation (CID) mass
spectra were also collected for the product identification.
The reaction procedure in Ref. [52] was followed with some
modifications. Briefly, 200 ␮L of [Bmim][BF4] was added to a 0.5 mL
plastic vial. Under stirring with two mini stir bars (diameter:
2.0 mm; length: 2.2 mm) at the highest speed (1100 rpm), 0.1 mmol
of each reactant (aniline and phenyl isothiocyanate) was added to
the ionic liquid solvent and the reaction was allowed to proceed for
20 min. As shown in Fig. 1(B), the same type of 50 ␮L HPLC syringe
(the metallic needle was also replaced with a 4 cm fused silica cap-
illary) as used in MA extraction was used to suspend a CCl4 droplet
of 3 ␮L in the layer of ionic liquid without stirring. Extraction times
of 1, 2 and 10 min were evaluated for the best signal intensity. To
prevent accidental withdrawing of the ionic liquid into the syringe,
only about 2 ␮L of the 3 ␮L CCl4 droplet was withdrawn into the
syringe. Subsequently, fused silica capillary was cut to a length of
ca. 0.5 cm after withdrawing the droplet into the syringe and prior
to DESI-MS analysis. This step was necessary to avoid the vibration
of the capillary during DESI ionization of the sample.
3. Results and discussion
3.1. SDME/DESI-MS for trace analysis of MA
3.1.1. Optimization of three-phase SDME for MA extraction
In our experiment for MA extraction, three-phase liquid extrac-
tion procedure was selected for preconcentration because with
two-phase micro-extraction of MA from an aqueous solution, the
acceptor phase should be an organic solvent, which gave a relatively
weaker mass spectrometric signal of MA than from an aqueous
solution. One approach to improve the extraction efficiency of an
extraction is to adjust the pH of aqueous phase by adding acid or
base. For MA as a model compound for controlled phenethylamine
drugs, this approach was adopted. In the process of preparing the
stock solution for extraction of MA, NaOH was added to reach a final
concentration of 0.50 M of NaOH. By adding NaOH, MA·HCl in water
was converted to the free-base form of MA, hence improving its sol-
ubility in the hexane layer. According to the literature, 0.50 M NaOH
is basic enough to convert all of the MA to its free-base form [33].
Subsequently, 5% acetic acid was used in the suspended droplet
2.4. Instrumentation
An ion trap mass spectrometer (Thermo Finnigan LCQ DECA, San
Jose, CA) was used in this work. The DESI source set-up is described
in detail elsewhere [23] with a slight modification as illustrated
in Fig. 1(C) for which no surface was used as in the conventional
DESI-MS. Briefly, for all the experiments a heated capillary tem-
perature of 150 ◦C, a spray voltage of +5.0 kV, and a nebulizing gas
(N2) pressure of 180 psi were maintained. For the MA detection a
typical DESI spray solvent consisted of methanol:water:acetic acid
(50:50:1 by volume) with an injection flow rate of 10.0 ␮L/min. For

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X. Sun et al. / International Journal of Mass Spectrometry 301 (2011) 102–108
105
to convert the MA to its protonated form; therefore the equilibria
would direct MA into the aqueous suspended droplet phase. Acetic
acid was used to acidify the acceptor droplet because it was also
used with the DESI spray. A concentration of 5% acetic acid was suf-
ficient to convert MA to its protonated form. The size of droplet was
selected to be 4 ␮L because a droplet of this size allowed a balance
between better extraction efficiency with larger sized droplets [33]
and the better preconcentration of MA with smaller sized droplets.
The smaller the volume of hexane, the better the extraction effi-
ciency; however, if the volume of hexane is too small, the organic
layer is not thick enough to separate the aqueous droplet from the
aqueous bottom layer. Therefore, 400 ␮L was chosen as the small-
est volume of hexane possible to give an enough thickness of the
organic layer to maintain the droplet.
3.1.2. Optimization of the DESI apparatus for MA detection
Two improvements were made to the DESI apparatus. In tradi-
tional DESI-MS, a surface is involved, serving as a sample desorption
platform. In this study no surface was used in the DESI set-up.
As seen in Fig. 1(C), the syringe was mounted on a syringe pump
and the tip of the capillary was placed in between the tip of
DESI sprayer and the MS inlet, similar to the apparatus that was
used to couple with an electrochemical cell in one of our previous
studies [25]. Another improvement was based on the observation
that conductive surface material such as metal or graphite may
cause neutralization of the charged droplets from the DESI sprayer,
reducing the ionization efficiency [53]. Although a surface was not
used in this experiment, the metallic syringe needle would act
as a metal surface that might cause partial neutralization of the
charged droplets, leading to the weakening of the MS signal. The
replacement of the syringe needle with the deactivated fused sil-
ica capillary avoided this problem. For the DESI-MS detection of
extracted MA in the acceptor droplet, because the needle of syringe
was already replaced with a capillary (7 cm for MA extraction to
match with the dimension of the test tube used for MA extraction)
before the extraction, there was no need to transfer the droplet
Fig. 2. Representative DESI-MS spectrum of an 800. ng/mL MA standard solution
for calibration. Inset: standard calibration line for the DESI-MS detection of single
droplet extract samples. The ratios of peak heights of m/z 150 to those of m/z 74 were
plotted versus the concentrations of standard MA solutions. (-) represents the upper
and lower boundaries of the 95% confidence intervals; (—) represents the calibration
line obtained from the average data points of three replicates of standard solutions.
from the syringe to a surface, a capillary or a vial. After the sus-
pended droplet was withdrawn into the syringe, the extract could
be directly detected after the fused silica capillary was cut to a
length of 0.5 cm to avoid vibration of the capillary.
3.1.3. Calibration of single droplet DESI-MS analysis for MA
All the calibration samples were in 5% acetic acid to match up
with the pH condition of the MA extract. Fig. 2 is the DESI-MS
spectrum of the standard solution containing 800. ng/mL of MA.
The observed peaks at m/z 74 and 150 correspond to the tetram-
Fig. 3. DESI-MS spectra of three extraction replicates (a single scan is displayed for each spectrum of three replicates A–C). Inset shows the CID MS/MS spectrum of the
protonated MA ion [MA+H]+ (m/z 150).