Direct mass spectrometry analysis of biofluid samples using slug-flow microextraction nano-electrospray ionization

Article abstract of DOI:10.1002/anie.201408338

Direct mass spectrometry (MS) analysis of biofluids with simple procedures represents a key step in the translation of MS techniques to clinical and point-of-care applications. The current study reports the development of a single-step method using slug-flow microextraction and nano-electrospray ionization for MS analysis of organic compounds in blood and urine. High sensitivity and quantitation precision have been achieved in the analysis of therapeutic and illicit drugs in 5 μL samples. Real-time chemical derivatization has been incorporated for analyzing anabolic steroids. The monitoring of enzymatic functions has also been demonstrated with cholinesterase in wet blood. The reported study encourages the future development of disposable cartridges, which function with simple operation to replace the traditional complex laboratory procedures for MS analysis of biological samples.

Full text of DOI:10.1002/anie.201408338

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DOI: 10.1002/anie.201408338
Mass Spectrometry
Hot Paper
Direct Mass Spectrometry Analysis of Biofluid Samples Using Slug-
Flow Microextraction Nano-Electrospray Ionization**
Yue Ren, Morgan N. McLuckey, Jiangjiang Liu, and Zheng Ouyang*
Abstract: Direct mass spectrometry (MS) analysis of biofluids
with simple procedures represents a key step in the translation
of MS techniques to clinical and point-of-care applications.
The current study reports the development of a single-step
method using slug-flow microextraction and nano-electrospray
ionization for MS analysis of organic compounds in blood and
urine. High sensitivity and quantitation precision have been
achieved in the analysis of therapeutic and illicit drugs in 5 mL
samples. Real-time chemical derivatization has been incorpo-
rated for analyzing anabolic steroids. The monitoring of
enzymatic functions has also been demonstrated with chol-
inesterase in wet blood. The reported study encourages the
future development of disposable cartridges, which function
with simple operation to replace the traditional complex
laboratory procedures for MS analysis of biological samples.
5 mL. More importantly, we demonstrated how to incorporate
a variety of different processes using a simple device,
including liquid–liquid extraction, internal standard (IS)
incorporation, chemical derivatization or even enzymatic
reactions, which are necessary for a high performance mass
analysis.
A disposable glass capillary of 0.8 mm i.d. (Figure 1a)
with a pulled tip for nanoESI was used to perform the entire
sampling ionization process. Two adjacent liquid plugs were
formed by sequentially injecting 5 mL organic solvent and
M
ass spectrometry (MS) has been demonstrated to be
a powerful tool for chemical and biological analysis. The high
specificity, high sensitivity, and high precision in quantitation
are achieved traditionally in the laboratory by eliminating the
matrix effect through sample extraction and chromatographic
separation prior to the MS analysis. The development of
ambient ionization,[1] especially with the recent demonstra-
tion of using a paper spray,[2] has indicated a promising future
for direct MS analysis with high quantitation performance but
with highly simplified procedures that consume ultrasmall
amounts of samples. This would be extremely important for
the translation of the MS analysis to field applications,
especially point-of-care (POC) diagnosis. The underlying
principle for a successful development along this direction is
to minimize the sample consumption and to achieve high
efficiency in an integrated process for analyte extraction and
ionization. In this study, we developed a new method that uses
slug-flow microextraction (SFME) and nanoESI (electro-
spray ionization) to perform a one-step analysis of biofluid
samples. Excellent sensitivity and high quantitation precision
have been obtained with blood and urine samples of only
Figure 1. a) In-capillary sample extraction using slug-flow microextrac-
tion and b) the subsequent MS analysis with nanoESI. MS/MS spectra
for c) 10 ngmLꢀ1 methamphetamine in 5 mL urine and d) 50 ngmLꢀ1
benzoylecgonine in 5 mL urine. e) Impact of the number of SFME
cycles on the extraction of the analytes, intensities of the MS/MS
product ions monitored for methamphetamine (m/z 150!91), nico-
tine (m/z 163!130), and benzoylecgonine (m/z 290!168), each at
50 ngmLꢀ1 in urine samples. 2 kV used for nanoESI.
[*] Y. Ren, M. N. McLuckey, J. Liu
Weldon School of Biomedical Engineering, Purdue University
West Lafayette, IN 47907 (USA)
5 mL urine or blood sample into the capillary. Liquid–liquid
extraction of the analytes from the biofluid into the organic
solvent is expected, but at a fairly low efficiency because of
the small interfacing area. However, the extraction speed
could be significantly increased with the slug flows induced by
the movements of the two liquid plugs, which can be
facilitated by tilting the capillary (Figure 1a) or by applying
a push-and-pull force through air pressure (see Figure S1 in
the Supporting Information). Due to friction with the
Prof. Z. Ouyang
Weldon School of Biomedical Engineering, Purdue University
West Lafayette, IN 47907 (USA)
E-mail: ouyang@purdue.edu
Homepage: http://engineering.purdue.edu/BioMS
[**] This research was supported by the Nation Institutes of Health
(1R01GM106016).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408338.
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capillary wall,[3] internal circulations are formed inside each
plug (Figure 1a) and transfer the analytes to and away from
the liquid–liquid interface, therefore significantly improving
the extraction efficiency. After the extraction process, the
organic solvent plug can be simply pushed to the tip of the
capillary, a stainless-steel wire then inserted through the
biofluid sample to reach the organic solvent plug, and a high
voltage applied to generate the nanoESI for MS analysis
(Figure 1b). The selection of the organic solvent is critical. It
needs to be immiscible with the biofluid samples, have good
solubility for the target analytes and be suitable for nanoESI.
Several organic solvents were tested (see the Supporting
Information) and ethyl acetate, which has a weak polarity, was
found to provide the optimal performance for analyzing
a broad range of chemical compounds in urine (Figure 1c,d)
and blood samples (see Figure S3 in the Supporting Informa-
tion).
benzoylecgonine in raw urine samples might not be the
absolute amount or concentration of the benzoylecgonine,
but the interference by the matrix effects, such as the
ionization suppression because of the high concentrations of
salts in the urine sample.[4] An efficient separation of
benzoylecgonine from the salts was achieved in the SFME
process. Even with a lower benzoylecgonine concentration in
the extraction phase, the ionization efficiency and the overall
sensitivity of the analysis were improved significantly.
In addition to the sensitivity, adequate precision in
quantitation is often mandatory for clinical and POC
applications.[5] Simple means for accurate incorporation of
internal standards are important[6] but can be challenging for
samples of small volumes taken by minimally invasive
methods. By using the SFME-nanoESI technique, the internal
standard compounds could be spiked in the extraction phase
(Figure 2, inset) and subsequently mixed with the analyte
The extraction process with the slug flows has been shown
to be very efficient, as demonstrated for extracting metham-
phetamine, nicotine, and benzoylecgonine (a main metabolite
of cocaine) from urine samples. The equilibrium was reached
after tilting the capillary five times (Figure 1e). Limits of
detection (LODs) as low as 0.05 ngmLꢀ1 for verapamil have
been obtained for whole blood samples using SFME-nanoESI
(Table 1). Fewer extraction cycles were needed to reach
Table 1: Limits of detection (LODs) of analytes in urine and/or whole
blood samples using SFME-nanoESI for MS analysis.
Analyte
Sample Derivatization[a] Sample vol- LOD
ume [mL]
[ngmLꢀ1
]
Figure 2. Quantitative analysis of whole blood spiked with metham-
phetamine (1–100 ngmLꢀ1). The blood samples were diluted 10 times
to decrease the viscosity. [D8]Methamphetamine (2 ngmLꢀ1) in ethyl
acetate was used as the extraction solvent.
methamphetamine
urine
blood
urine
blood
blood
blood
urine
urine
NA
NA
NA
NA
NA
NA
5
5
5
5
5
5
5
5
0.03
0.1
0.1
1
0.05
0.08
0.7
0.6
benzoylecgonine
LOD
verapamil
amitriptyline
epitestosterone
6-dehydrocholeste-
none
5a-androstan-
3b,17b-diol-16-one
stigmastadienone
during the slug-flow extraction process. This method was
tested for the quantitation of methamphetamine in bovine
blood samples with [D8]methamphetamine spiked in ethyl
acetate at 2 ngmLꢀ1 as the internal standard. The blood
samples were diluted 10 times and then analyzed using the
SFME-nanoESI and multiple reaction monitoring (MRM)
analysis (transitions m/z 150 to 91 and m/z 158 to 94 for the
analyte and internal standard, respectively; Figure 2 inset).
Figure 2 shows a plot of the measured analyte-to-internal
standard ratios (A/IS) as a function of the original analyte
concentration in blood. A good linearity was obtained, which
is governed by the partitioning process (see derivation in the
Supporting Information). Relative standard deviations
(RSDs) better than 10% were obtained for samples with
concentrations higher than 10 ngmLꢀ1.
Chemical derivatization is an effective way of altering the
properties of the target analytes to improve the efficiency of
the separation or ionization for MS analysis.[7] For example,
the steroids in urine or blood samples are expected to be
extracted well into an organic phase using the SFME;
however, the efficiency of the subsequent ionization by
nanoESI would be low due to the low proton affinity of the
steroid molecules. The reaction with hydroxylamine has
hydroxylamine
hydroxylamine
urine
urine
hydroxylamine
hydroxylamine
5
5
0.2
0.8
[a] NA=not applied.
equilibrium if the blood samples were diluted to reduce the
viscosity. The distribution of the analyte between the sample
and extraction phase can be relatively estimated by the
partitioning coefficient (logP, see Equation S1 and derivation
in the Supporting Information). The concentration of meth-
amphetamine (logP value of 2.1) in the organic extraction
solvent can be 100 times higher than in the urine sample after
SFME, which certainly explains the good LOD of
0.03 ngmLꢀ1 achieved with urine samples (Table 1). The
logP value for benzoylecgonine is ꢀ0.6, which means it has
higher solubility in urine than in organic solvents and the
extraction into ethyl acetate was a dilution process; however,
an LOD of 0.08 ngmLꢀ1 was nevertheless achieved. This
indicates that the limiting factor in the detection of the
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previously been proved to be effective in improving the
ionization efficiency of the steroids,[8] and thereby was used in
this study as an example. An additional liquid plug of 5 mL
water containing 50 mm hydroxylamine was injected between
the 5 mL ethyl acetate and 5 mL urine sample spiked with
200 ngmLꢀ1 epitestosterone (Figure 3). With five SFME
Figure 4. a) The reaction scheme of the enzymatic conversion of
acetylthiocholine (ATCh) into thiocholine (TCh) catalyzed by cholines-
terase (ChE). b) Progression curve of ATCh digestion determined by
SFME-nanoESI. The incubation was for 30 min and catalyzed by blood
cholinesterase (ChE) at room temperature. Acetylthiocholine iodide
was added as the enzyme substrate into human whole blood at a final
concentration of 1.8 mgmLꢀ1 before incubation. The intensity ratios of
product ions from thiocholine (m/z 102!61) and enzyme substrate
(m/z 162!103) were monitored using MRM; c) Evaluation of blood
ChE with different levels of enzyme inhibition. The ChE activity in the
blood sample was determined by SFME-nanoESI after 5 min incuba-
tion.
thiocholine (TCh, Figure 4a). The blood sample was diluted
10 times to slow down the reaction rate as well as to facilitate
the slug flows for SFME. The substrate acetylthiocholine
iodine was added into the diluted blood sample at a concen-
tration of 1.8 mgmLꢀ1, and then a 5 mL sample was taken
immediately and injected into the capillary with a 5 mL
extraction phase. The capillary with the sample and the
extraction solvent was left at room temperature (258C) for
incubation. The SFME-nanoESI could be performed repeat-
edly on the same sample and the ratio of the substrate ATCh
and the reaction product TCh (see Figure S7 in the Support-
ing Information) could be monitored as a function of time to
characterize the enzymatic activity of the ChE. A potential
problem in this approach would be the organic solvent
damaging the enzyme function. The impact of the organic
extraction phase was investigated for ethyl acetate and other
solvents such as chloroform with 5 min incubation. It was
found that the reduction of ChE activity through contact with
ethyl acetate was minimal, but much more severe (more than
60% decrease) with chloroform (see the Supporting Infor-
mation). A weakly polar solvent such as ethyl acetate can
better preserve the enzyme structures.[9]
The SFME-nanoESI technique was performed repeatedly
over 30 min using ethyl acetate as the extraction solvent, with
5 cycles for SFME and 5 s nanoESI at 1500 V for each
analysis. The TCh/ACTh ratio is plotted as a function of time
in Figure 4b, which is characteristic of the enzymatic activity
of the ChE. An enzyme inihibition study was then carried out
as a validation of this method. Two ChE inhibitors, donepezil
(a therapeutic drug for Alzhimerꢀs disease) and ethion (a
neuron toxicant), were spiked seperately into blood samples,
Figure 3. a) Reactive SFME-nanoESI with a reagent plug injected
between the biofluid sample and the extraction solvent. MS/MS
spectra from b) direct and c) reactive SFME-nanoESI analysis of
200 ngmLꢀ1 epitestosterone in synthetic urine. 5 mL water containing
50 mm hydroxylamine was used as the liquid reagent plug.
cycles, the hydroxylamine solution mixed well with the
urine sample. The MS/MS analysis of the reaction product
m/z 304 produced spectra of significantly improved signal-to-
noise ratios (S/N, Figure 3b,c). The reactive SFME-nanoESI
technique was applied for analysis of a series of anabolic
steroids in 5 mL urine samples, including epitestosterone, 6-
dehydrocholestenone, 5a-androstan-3b,17b-diol-16-one, and
stigmastadienone, with LODs of 0.7, 0.6, 0.2, and 0.8 ngmLꢀ1
obtained, respectively (Table 1).
The use of the liquid–liquid extraction process with SFME
allows the analysis to now be performed directly with wet
blood samples. This provides an opportunity for probing the
chemical and biological properties that only exist with the
original liquid samples. For example, the enzymatic functions
of the proteins are typically quenched in the dried blood spots
or after the traditional laboratory procedure for sample
extraction. In this study, we applied the SFME-nanoESI
technique for monitoring the enzymatic activity of cholines-
terase (ChE) in whole blood samples. The ChE facilitates the
enzymatic conversion of acetylthiocholine (ATCh) into
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Inv. (Novi, MI, USA). The human pooled blood for the enzymatic
thereby simulating enzyme inhibition to different degrees.
The compromised enzyme activities were then determined
uisng the SFME-nanoESI method with 5 min incubation. In
comparion with the blood samples without adding the
inhibitors, the defficiencies measured are resported in Fig-
ure 4c for blood samples treated with donpezil at 25 ngmLꢀ1
and 5 mgmLꢀ1, and with ethion at 10 mgmLꢀ1. The percent
decreases observed are consistent with the findings reported
for previouse studies.[10]
reaction study was purchased from BioreclamationIVT (Baltimore,
MD, USA). The synthetic urine was purchased from CST Technol-
ogies (Great Neck, NY, USA). The steroids were purchased from
Steraloids Inc. (Newport, RI, USA). All other chemicals were
purchased from Sigma–Aldrich (St. Louis, MO, USA).
Received: August 19, 2014
Published online: October 3, 2014
In summary, the combination of slug-flow microextraction
with nanoESI enabled a highly sensitive direct analysis of
organic compounds in biofluids. Multiple types of processes
for sample treatments, which traditionally require complex
setups in the laboratory, can now be incorporated into a one-
step analysis with an extremely simplified operation proce-
dure. Since the biofluid samples are directly analyzed without
being made into dried spots, an efficient liquid–liquid
extraction can be designed based on the partitioning process.
The chemical and biological properties of wet biofluids can
also be retained and characterized thereby. The extraction
process can be turned on and off by controlling the move-
ments of the sample and extraction plugs. This allows an on-
line monitoring of the chemical and biological reactions in
a biofluid sample of only 5 mL. With the increasing interest in
the translation of MS technologies to clinical applications, this
development has a profound implication on designing dis-
posable sample cartridges with adequate function for direct
analysis. This could ultimately lead to an elimination of the
traditional laboratory procedures that require complex setups
and expertize. Its implementation with miniature mass
spectrometers[11] would produce a powerful solution for
POC diagnosis.
Keywords: analytical methods · mass spectrometry ·
microextraction · online reaction
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[1] a) R. G. Cooks, Z. Ouyang, Z. Takats, J. M. Wiseman, Science
2006, 311, 1566 – 1570; b) M. E. Monge, G. A. Harris, P. Dwivedi,
F. M. Fernꢁndez, Chem. Rev. 2013, 113, 2269 – 2308.
[2] H. Wang, J. J. Liu, R. G. Cooks, Z. Ouyang, Angew. Chem. Int.
Ed. 2010, 49, 877 – 880; Angew. Chem. 2010, 122, 889 – 892.
[3] a) J. R. Burns, C. Ramshaw, Lab on a Chip 2001, 1, 10 – 15;
b) M. N. Kashid, Y. M. Harshe, D. W. Agar, Ind. Eng. Chem. Res.
2007, 46, 8420 – 8430.
[4] a) R. H. Silber, C. C. Porter, J. Biol. Chem. 1954, 210, 923 – 932;
b) C. Casari, A. R. J. Andrews, Forensic Sci. Int. 2001, 120, 165 –
171.
[5] D. H. Chace, Chem. Rev. 2001, 101, 445 – 478.
[6] J. Liu, R. G. Cooks, Z. Ouyang, Anal. Chem. 2013, 85, 5632 –
5636.
[7] J. R. Yates III, Nat. Methods 2011, 8, 633 – 637.
[8] G. Huang, H. Chen, X. Zhang, R. G. Cooks, Z. Ouyang, Anal.
Chem. 2007, 79, 8327 – 8332.
[9] a) K. Nakanishi, T. Kamikubo, R. Matsuno, Bio/Technology
1985, 3, 459 – 464; b) K. F. Ilett, W. D. Reid, I. G. Sipes, G.
Krishna, Exp. Mol. Pathol. 1973, 19, 215 – 229.
[10] a) S. L. Rogers, L. T. Friedhoff, J. T. Apter, R. W. Richter, J. T.
Hartford, T. M. Walshe, B. Baumel, R. D. Linden, F. C. Kinney,
R. S. Doody, R. L. Borison, G. L. Ahem, Dementia 1996, 7, 293 –
303; b) A. Dewan, A. B. Patel, R. R. Pal, U. J. Jani, V. C. Singel,
M. D. Panchal, Clin. Toxicol. 2008, 46, 85 – 88.
Experimental Section
All the experiments were carried out with a TSQ Quantum Access
Max mass spectrometer (Thermo Fisher Scientific, San Jose, CA,
USA). The bovine blood was purchased from Innovative Research
[11] L. Li, T.-C. Chen, Y. Ren, P. I. Hendricks, R. G. Cooks, Z.
Ouyang, Anal. Chem. 2014, 86, 2909 – 2916.
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