Anal. Chem.
2010, 82, 2463–2471
Development, Characterization, and Application of
Paper Spray Ionization
Jiangjiang Liu,†,‡ He Wang,‡ Nicholas E. Manicke,§ Jin-Ming Lin,*,† R. Graham Cooks,§,| and
Zheng Ouyang*,‡,|
The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua
University, Beijing 100084, China, Weldon School of Biomedical Engineering, Purdue University, West Lafayette,
Indiana 47907, Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, and Center for Analytical
Instrumentation Development, Purdue University, West Lafayette, Indiana 47907
Paper spray is developed as a direct sampling ionization
method for mass spectrometric analysis of complex
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mixtures. Ions of analyte are generated by applying a high
voltage to a paper triangle wetted with a small volume
(<10 µL) of solution. Samples can be preloaded onto the
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paper, added with the wetting solution, or transferred
from surfaces using the paper as a wipe. It is demon-
strated that paper spray is applicable to the analysis of a
wide variety of compounds, including small organic
compounds, peptides, and proteins. Procedures are
developed for analysis of dried biofluid spots and applied
to therapeutic drug monitoring with whole blood samples
and to illicit drug detection in raw urine samples. Limits
of detection of 50 ng/mL (or 20 pg absolute) are achieved
for atenolol in bovine blood. The combination of sample
collection from surfaces and paper spray ionization also
enables fast chemical screening at high sensitivity, for
example 100 pg of heroin distributed on a surface and
agrochemicals on fruit peels are detectable. Online de-
rivatization with a preloaded reagent is demonstrated for
analysis of cholesterol in human serum. The combination
of paper spray with miniature mass spectrometers offers
a powerful impetus to wide application of mass spectrom-
etry in nonlaboratory environments.
Mass spectrometry (MS) is one of the most versatile of
chemical analysis technologies. A strong trend is emerging in
which MS applications occur outside traditional analytical labora-
tory settings with major efforts going into two signature areas,
miniaturization of MS instruments1 and direct sampling ionization,
viz. ambient ionization MS.2 The aim in the former area is to
develop small highly portable and low cost devices for MS analysis,
while the successful development of the latter area is critical to
allow untrained personnel, such as clinical personnel or first
* To whom correspondence should be addressed. Phone: (86)10-62792343.
Fax: (86)10-62792343. E-mail: jmlin@mail.tsinghua.edu.cn (J.-M.L.). Phone: (765)
494-2214. Fax: (765) 496-1912. E-mail: ouyang@purdue.edu (Z.O.).
†
Tsinghua University.
‡
Weldon School of Biomedical Engineering, Purdue University.
§
Department of Chemistry, Purdue University.
|
Center for Analytical Instrumentation Development, Purdue University.
(1) Ouyang, Z.; Cooks, R. G. Annu. Rev. Anal. Chem. 2009, 2, 187–214.
(2) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311,
1566–1570.
10.1021/ac902854g  2010 American Chemical Society
Published on Web 02/16/2010
responders, to use MS equipment in situ and obtain immediate
results. An area of mass spectrometry developed since 2004,
ambient ionization, has emerged rapidly in the form of more than
20 separate methods2-4 all aiming at direct sampling of analytes
in untreated samples examined in their ambient state. The most
recent developments in this area are summarized in a recent
compilation of review and application papers.5
The capabilities of ambient ionization mass spectrometry have
been demonstrated with the direct analysis of pharmaceutical drug
compounds,6-14 monitoring of illicit chemicals (steroids,15
drugs16-18 and explosives8,16-21), characterization of biological
molecules,22-26 and sampling of complex matrices on sur-
(3) Harris, G. A.; Nyadong, L.; Fernandez, F. M. Analyst 2008, 133, 1297–
1301.
(4) Venter, T. i. B. S.; Nefliu, M.; Cooks, R. G. TrAC Trends Anal. Chem. 2008,
27, 284–290.
(5) Ouyang, Z.; Zhang, X. Analyst Special Issue: Ambient Mass Spectrometry;
2010.
(6) Takáts, Z.; Cotte-Rodriguez, I.; Talaty, N.; Chen, H.; Cooks, R. G. Chem.
Commun. 2005, 1950–1952.
(7) Wiseman, J. M.; Ifa, D. R.; Zhu, Y.; Kissinger, C. B.; Manicke, N. E.;
Kissinger, P. T.; Cooks, R. G. Proc. Natl. Acad. Sci. U. S. A. 2008, 105,
18120–18125.
(8) Takáts, Z.; Wiseman, J. M.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 1261–
1275.
(9) Weston, D. J.; Bateman, R.; Wilson, I. D.; Wood, T. R.; Creaser, C. S. Anal.
Chem. 2005, 77, 7572–7580.
(10) Hopfgartner, G.; Bourgogne, E. Mass Spectrom. Rev. 2003, 22, 195–214.
(11) Wang, H.; Liu, J.; Cooks, R. G.; Ouyang, Z. Angew. Chem., Int. Ed. 2010,
49, 877–880.
(12) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 7826–
7831.
(13) Williams, J. P.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2005, 19,
3643–3650.
(14) Williams, J. P.; Lock, R.; Patel, V. J.; Scrivens, J. H. Anal. Chem. 2006, 78,
7440–7445.
(15) Huang, G.; Chen, H.; Zhang, X.; Cooks, R. G.; Ouyang, Z. Anal. Chem.
2007, 79, 8327–8332.
(16) Harper, J. D.; Charipar, N. A.; Mulligan, C. C.; Zhang, X.; Cooks, R. G.;
Ouyang, Z. Anal. Chem. 2008, 80, 9097–9104.
(17) Ifa, D. R.; Jackson, A. U.; Paglia, G.; Cooks, R. G. Anal. Bioanal. Chem.
2009, 394, 1995–2008.
(18) Talaty, N.; Mulligan, C. C.; Justes, D. R.; Jackson, A. U.; Noll, R. J.; Cooks,
R. G. Analyst 2008, 133, 1532–1540.
(19) Cody, R. B.; Laramée, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 2297–
2302.
(20) Chen, H.; Venter, A.; Cooks, R. G. Chem. Commun. 2006, 2042–2044.
(21) Justes, D. R.; Talaty, N.; Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun.
2007, 2142–2144.
(22) Takáts, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306,
471–473.
Analytical Chemistry, Vol. 82, No. 6, March 15, 2010 2463
faces,6,11,17,18 food,27-29 urine,15,16,20,30-32 blood,8,11,32,33 and
tissue.7,25,34-38 Mass spectral information can be obtained instan-
taneously for analytes at trace-level concentrations without requir-
ing separation or preconcentration procedures. As new methods
with different sampling and ionization mechanisms are developed,
attention is also being given to the improvement of existing
methods for quantitative analysis, which is required for most
regulatory applications. In the demonstrated cases, the analytical
results are typically qualitative or semiquantitative when using
direct sampling, although significant improvements can be ob-
tained with the utilization of internal standards.39
It has been recognized previously that paper can be used for
electrospray.40 We recently reported a paper spray ionization
method,11 where a sample is loaded onto a piece of paper and
ions are generated directly for MS analysis by applying a high
voltage to the wetted paper. Paper is readily available at very low
cost and in different kinds and has great potential in developing
affordable diagnostics.41 Paper has well-established uses in chemi-
cal analysis, as in paper chromatography.42-45 Paper spray allows
direct MS analysis of the chemicals separated on paper after paper
chromatography.11 Paper has also played an important role in the
analysis of biological fluids, especially for blood and urine samples.
Paper-based test strips have been fully commercialized for early
pregnancy tests and the monitoring of blood glucose for
diabetics.46,47 Paper serves also as an excellent sample substrate
for dried blood spots.48 The direct analysis of therapeutic drug
(23) Shiea, J.; Huang, M.-Z.; Hsu, H.-J.; Lee, C.-Y.; Yuan, C.-H.; Beech, I.; Sunner,
J. Rapid Commun. Mass Spectrom. 2005, 19, 3701–3704.
(24) Nemes, P.; Vertes, A. Anal. Chem. 2007, 79, 8098–8106.
(25) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass.
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(26) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. Anal. Chem. 2008,
80, 6773–6778.
(27) Huang, G. M.; Zheng, O. Y.; Cooks, R. G. Chem. Commun. 2009, 556–
558.
(28) Garcia-Reyes, J. F.; Jackson, A. U.; Molina-Diaz, A.; Cooks, R. G. Anal. Chem.
2009, 81, 820–829.
(29) Garcia-Reyes, J. F.; Mazzoti, F.; Harper, J. D.; Charipar, N. A.; Oradu, S.;
Ouyang, Z.; Sindona, G.; Cooks, R. G. Rapid Commun. Mass Spectrom.
2009, 23, 3057–3062.
(30) Kauppila, T. J.; Talaty, N.; Kuuranne, T.; Kotiaho, T.; Kostiainen, R.; Cooks,
R. G. Analyst 2007, 868–875.
(31) Huang, G.; Chen, H.; Zhang, X.; Cooks, R. G.; Ouyang, Z. Anal. Chem.
2007, 79, 8327–8332.
(32) Hernández, F.; Sancho, J. V.; Pozo, O. J. Anal. Bioanal. Chem. 2005, 382,
934–946.
(33) Dethy, J.-M.; Ackermann, B. L.; Delatour, C.; Henion, J. D.; Schultz, G. A.
Anal. Chem. 2003, 75, 805–811.
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7, 493–496.
(35) Wiseman, J. M.; Ifa, D. R.; Venter, A.; Cooks, R. G. Nat. Protoc. 2008, 3,
517–524.
(36) Wiseman, J. M.; Puolitaival, S. M.; Takátsa, Z.; Cooks, R. G.; Caprioli, R. M.
Angew. Chem., Int. Ed. 2005, 44, 7094–7097.
(37) Wiseman, J. M.; Ifa, D. R.; Song, Q.; Cooks, R. G. Angew. Chem., Int. Ed.
2006, 45, 7188–7192.
(38) Talaty, N.; Takáts, Z.; Cooks, R. G. Analyst 2005, 130, 1624–1633.
(39) Manicke, N. E.; Kistler, T.; Ifa, D. R.; Cooks, R. G.; Ouyang, a. Z. J. Am.
Soc. Mass. Spectrom. 2009, 20, 321–325.
(40) Fenn, J. B. U.S. Patent 6,297,499, 2001.
(41) Arnaud, C. Chem. Eng. News 2009, 87, 50–53.
(42) Sherma, J.; Fried, B. Anal. Chem. 1982, 54, 45R–57R.
(43) Bush, I. E. Biochem. J. 1952, 50, 370–378.
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(46) Chin, C. D.; Linder, V.; Sia, S. K. Lab Chip 2007, 7, 41–57.
(47) Lode, P. v. Clin. Biochem. 2005, 38, 591–606.
(48) Spooner, N.; Lad, R.; Barfield, M. Anal. Chem. 2009, 81, 1557–1563.
2464 Analytical Chemistry, Vol. 82, No. 6, March 15, 2010
compounds in dried blood spots on paper was recently achieved
using paper spray, the case of imatinib in whole blood at
concentrations as low as 30 ppb being exemplary in detection limit
and linearity of response over its therapeutic range.11 These data
were obtained with the addition of deuterated imatinib as the
internal standard. With recent emphasis on using paper for dried
blood spot analysis in various pharmaceutical applications,48 paper
spray ionization represents a solution to fast quantitative analysis
at extremely low cost of consumables. The recent development
of paper-based microfluidic devices49-51 potentially allows sample
handling for in situ analysis to be developed using disposable
paper devices. Paper spray ionization should also allow direct
coupling of these sample handling devices with mass spectrom-
eters, especially miniature MS instruments,1 and this offers an
attractive direction of MS instrument development for in situ
chemical analysis applications such as point-of-care diagnosis.
In comparison with traditional ESI or nanospray ionization
sources, a distinct advantage of using paper for spray ionization
is the potential for making disposable sample cartridges for MS
analysis. Although some other porous materials potentially could
also be used for spray ionization,52 the commercial availability at
low cost and the ease of fabrication and chemical modification53-55
of paper make it a superior candidate for producing sample
cartridges that have multiple functions for simple sample treatment
and ionization. In this study, we report a relatively thorough
characterization of paper spray ionization and discuss its imple-
mentation in various applications involving the analysis of organic
and biological compounds.
EXPERIMENTAL SECTION
Six different papers used for paper spray were purchased from
Whatman (Whatman International Ltd., Maidstone, England): filter
papers with different pore size, grade 1 (11 µm), grade 2 (8 µm),
grade 595 (4-7 µm), grade 6 (3 µm), glass microfiber filter paper,
and chromatography paper. Bovine whole blood (with sodium
citrate as anticoagulant) was purchased from Innovative Research
(Novi, MI). All other reagents were purchased from Sigma-Aldrich
(Milwaukee, WI) and used without further purification. Methanol/
water solution (1:1, v/v) was used for paper spray unless otherwise
noted. Mass analysis was performed using a Thermo Fisher LTQ
mass spectrometer (Thermo Scientific Inc., San Jose, CA). The
temperature of the MS capillary inlet was typically set at 150 °C
unless otherwise noted. The tube lens voltage was set at 65 V.
Tandem mass spectra were recorded using collision-induced
dissociation (CID). The voltage used for paper spray ionization is
4.5 kV in positive mode and -3 kV in negative mode.
(49) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem.,
Int. Ed. 2007, 46, 1318–1320.
(50) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M. Proc. Natl. Acad. Sci.
U. S. A. 2008, 105, 19606–19611.
(51) Martinez, A. W.; Phillips, S. T.; Wiley, B. J.; Gupta, M.; Whitesides, G. M.
Lab Chip 2008, 8, 2146–2150.
(52) Tepper, G.; Kessick, R. Appl. Phys. Lett. 2009, 94, 084106.
(53) Carrilho, E.; Phillips, S. T.; Vella, S. J.; Martinez, A. W.; Whitesides, G. M.
Anal. Chem. 2009, 81, 5990–5998.
(54) Tan, I. H.; Silva, M. L. P. d.; Demarquette, N. R. J. Mater. Chem. 2001, 11,
1019–1025.
(55) Vesel, A.; Mozetic, M.; Hladnik, A.; Dolenc, J.; Zule, J.; Milosevic, S.;
Krstulovic, N.; Klanjsek-Gunde, M.; Hauptmann, N. J. Phys. D: Appl. Phys.
2007, 40, 3689–3696.
Figure 1. (a) Schematic illustration of paper spray for MS analysis. (b) MS spectrum and MS/MS spectra of a 10 µL methanol/water solution
containing heroin. (c) Measurement of the duration of the spray for cocaine solutions under various experimental conditions. Protonated cocaine
(m/z 304) was monitored, and each measurement was repeated three times.

RESULTS AND DISCUSSION
Figure 1a shows a schematic of a simple paper spray ionization
source. A piece of paper cut into a triangular shape, 10 mm long
and 5 mm wide at the base, is held by a copper clip with the apex
facing the inlet of the mass spectrometer and at a distance of 3
mm or more from it. A high voltage (3-5 kV) is applied to the
paper through the copper clip, and solutions containing analytes
are added onto the paper to generate ions for MS analysis. As
shown in Figure 1b and its inset, 10 µL methanol/water solutions
(1:1, v:v) containing 1 µg/mL and 1 ng/mL heroin were added to
the chromatography paper, and MS and MS/MS spectra were
recorded, respectively, using a positive high voltage of 4.5 kV.
The protonated heroin ion m/z 370 was detected, and its chemical
identity was confirmed with the MS/MS spectrum (Figure 1b
inset).
Paper, typically made from cellulose, is a hydrophilic porous
material that can hold a certain amount of liquid. Wet paper is
conductive, and the network of cellulose offers microchannels for
liquid (including dissolved analyte) transport. Presumably the high
electric potential applied between the paper triangle and MS inlet
generates an electric field that induces a charge that accumulates
at the apex of the paper triangle. Similar to electrospray, the
Analytical Chemistry, Vol. 82, No. 6, March 15, 2010 2465
Figure 2. Mass spectra of cocaine in methanol/water solution (10 µL, 200 ng/mL), sprayed from different papers. Whatman filter paper with
different pore sizes: (a) 3 µm, (b) 4-7 µm, (c) 8 µm, and (d) 11 µm, (e) glass fiber paper, and (f) chromatography paper. Spray voltage, 4.5 kV
for all tests.
Coulombic force breaks the liquid to form charged droplets, which
undergo subsequent desolvation processes to generate dry ions.
Paper spray ionization does not require sheath gas, and it works
well even when the MS inlet capillary temperature is set to room
temperature as in the case of heroin analysis (Figure 1b). When
a 10 µL methanol/water solution was applied to the paper triangle
as spray solvent, a Taylor cone was observed at the tip when
examined under a microscope. The mist of fine droplets formed
was clearly visible under strong illumination.11 The Taylor cone
and the spray typically disappear after about 1 min, presumably
because of the loss of solution via the spray as well as the
evaporation of solvent. The duration of paper spray was quanti-
tatively tested by applying methanol/water solutions in different
amounts and containing cocaine in different concentrations
(Figure 1c). For each solution, the peak intensity of the protonated
molecular ion of cocaine, m/z 304, was monitored to determine
the duration of the spray (inset of Figure 1c). In one set of tests,
three solutions of the same volume (10 µL) but different cocaine
concentrations were added in turn to paper; in another set of tests,
three solutions of different volumes but the same cocaine
concentration were added to paper; finally, a 10 µL methanol/
water solution at 1 ppm concentration was added to the paper
which was immediately covered with a piece of Teflon film to
minimize solvent evaporation. Three replicate measurements were
made for each type of test. Though slightly longer spray times
were achieved with solutions of higher analyte concentrations and
larger volumes, covering of the paper to minimize solvent
evaporation was effective in extending the spray ionization time.
For a paper triangle of described size (10 mm long and 5 mm
base), one addition of 5 µL to the paper allows for an analysis
time of more than 30 s, which is adequate for performing MS
and multiple tandem MS analyses for several analytes in an
2466 Analytical Chemistry, Vol. 82, No. 6, March 15, 2010
sample. It has been observed that the spray occurs simultaneously
at each of the three tips of the paper triangle, which indicates
that the macroscopic sharpness of the tips has an effect on spray
formation. The spray efficiency as a function of the tip sharpness
is being studied in a set of systematic experiments.
Six types of commercially available paper, including four filter
papers of different pore sizes, glass fiber paper, and chromatog-
raphy paper, were selected and tested as the substrate for paper
spray ionization. Filter papers and the chromatography paper were
made from cellulose, while the glass fiber paper was made from
glass microfiber. Paper triangles of the same dimensions were
cut from these materials, and a 10 µL methanol/water solution
containing cocaine (200 ng/mL) was added to each in order to
record the mass spectra. Different chemical backgrounds contain-
ing peaks of various intensities were observed for each type of
paper. The peak at m/z 151 is due to a chemical in methanol/
water solution and was also observed with nano ESI using the
same solution; other chemical noise peaks are most likely due to
the additives used in the paper industry, either as part of the
formulation or as process contaminants. For example, the peak
at m/z 301 was identified using MS/MS analysis as the sodium
adduct ion of dibutylphthalate, a commonly used industrial
plasticizer. The poorest performance for paper spray was observed
for glass fiber paper (Figure 2e), for which the total intensity was
2 orders of magnitude lower than any of the others and for which
the cocaine peak (m/z, 304) could not be identified. The highest
quality spectrum with the highest S/N value for cocaine was
obtained with chromatography paper, which was selected for the
subsequent experiments and applications reported. The thickness
of the chromatography paper is 0.18 mm. No deformation was
observed when wetted with 10 µL of solution. After the spray
solution has dried, the paper triangle can be reused for spray by
Figure 3. Characterization of the tolerance of paper spray ionization to position of the paper tip, using a cocaine solution (10 µL methanol/
water, 200 ng/mL) and chromatography paper. (a) Schematic illustration of the spatial movement of the paper triangle. (b) 2D contour plot
showing the relative intensity of m/z 304 as a function of the paper tip location on the x-y plane.
reloading the solution, and reproducible spectra can be obtained.
Rinsing the paper with the methanol/water solution or pure
methanol did not help to decrease the chemical noise significantly.
Immersing the paper for a long time (hours) in an appropriate
solution, dependent on the target chemicals to be removed, is
expected to be more effective; however, the physical and chemical
properties of the paper might also be changed.
As an important factor for judging the ease of use of an
ionization source, the tolerance to positioning was characterized.
A paper triangle was mounted on a 2D moving stage and was
moved 8 cm in the y-direction and 3 cm in the x-direction with a
2 mm increment for each step (Figure 3a). The cocaine solution
(1 µg/mL in methanol/water, 1:1 v/v) was continuously fed to
the paper from the bottom of the triangle to maintain the spray,
while a high voltage was applied and the peak intensity of the
protonated cocaine ion (m/z 304) was recorded as a function of
the position of the triangle tip. The peak intensities were first
normalized and then plotted as a contour map, as shown in Figure
3b. This shows that a stable high intensity can be obtained for
paper spray with the paper tip positioned anywhere in an area of
about 5 × 10 mm (x by y). Accurate positioning is not required
for implementation of paper spray.
To characterize the versatility of paper spray, sample solutions
(10 µL) containing a variety of chemicals, including amino acids,
peptides, proteins, herbicides, therapeutic drugs, and fatty acids,
were used (Figure 4). The spectra of serine, bradykinin 2-9,
cytochrome C, atrazine, and roxithromycin were recorded in the
positive ion mode, and methanol/water (1:1, v:v) was used as the
solvent for all five compounds except for cytochrome C, for which
acetic acid was added to the solution (1%) to improve the ionization
efficiency. Similar to ESI and nano-ESI, protonated molecules were
the main ionic species observed in the positive mode. Multiply
protonated ions were observed for peptides and proteins, and
charge state distributions typical of proteins were observed for
cytochrome c. Adduct ions [M + H + Na]2+ (m/z, 464) and [M
+ 2Na]2+ (m/z, 475) were also observed for bradykinin 2-9
(Figure 4b). For most of the analytes tested, good S/N for the
analyte peaks was obtained at concentration levels of about 1 µg/
mL, while the same or better S/N could be obtained in MS/MS
scans for concentrations 1 or 2 orders of magnitude lower. Limits
of detection (LOD) as low as 1 ng/mL were achievable for most
chemicals tested, especially for relatively small molecules such
as serine, atrazine, roxithromycin, heroin, cocaine, and methadone.
In the negative ionization mode of paper spray, it was found
that no spray plume or analyte ions could be observed when
methanol/water (1:1, v:v) was used as the solvent. Pure methanol
or solutions with high methanol concentrations therefore were
used to facilitate spray ionization. The deprotonated molecule was
observed for stearic acid (Figure 4f). With pure methanol, the
solvent surface tension is much lower, and charge accumulation
required for ionization is assumed to be much lowered. The
mechanisms responsible for this phenomenon are interesting and
being studied further.
As discussed above, a significant advantage of paper spray is
to generate ions for MS analysis directly from raw samples loaded
onto the paper substrate. Characterization of the sensitivity for
the analysis of pure analytes in methanol/water solutions does
not necessarily provide an accurate prediction for paper spray
performance in the analysis of real samples which involve complex
matrices. In this study, methods of analyzing dried spots of raw
samples have been developed with paper spray and tested for
various biological analytes. The monitoring of drugs in whole
blood is of critical importance in therapeutic drug development
and clinical disease treatment as well as in forensic applications.
Storing blood samples as dried blood spots on paper is the
standard means of sample storage for neonatal screening, and
recently it has also been explored for the analysis of therapeutic
drugs.48 Dried blood spots on paper can be stored for months
before analysis, without special storage conditions. The analysis
of whole blood using mass spectrometry typically requires the
removal of the blood cells and further separation processes to
clean the plasma. Using paper spray, direct MS analysis of the
therapeutic drugs in blood can be achieved easily by wetting the
paper with methanol/water solution without any sample prepara-
tion at all.
The detailed procedure for analysis of dried blood spots using
paper spray is as follows: 1) 0.4 µL of whole blood spiked with a
therapeutic drug was directly applied to the center of a triangle
of chromatography paper and was dried in air for about 1 min to
form a dried sample spot with a diameter of about 2 mm (Figure
5a); 10 µL of methanol/water (1:1, v/v) was then applied near
the base of the paper triangle with a 4.5 kV potential being applied,
while the MS spectra were recorded in the MS or MS/MS mode.
Analytical Chemistry, Vol. 82, No. 6, March 15, 2010 2467
Figure 4. Mass spectra of pure chemical solutions and corresponding tandem mass spectra. Spectra were obtained in the positive ion mode
for (a) serine, (b) bradykinin 2-9, (c) cytochrome C, (d) atrazine, (e) roxithromycin, and negative mode for (f) stearic acid.

As shown in Figure 5b, MS and MS/MS analyses were performed
for bovine blood samples spiked with atenolol, a β-blocker drug
used in cardiovascular disease. A S/N ratio of about 4 was
achieved with MS analysis of atenolol in dried blood spots at an
original concentration of 1 µg/mL (400 pg absolute analyte
amount), while a much better S/N could be achieved at much
lower concentrations with MS/MS analysis, for instance, at 50
ng/mL (20 pg absolute) as shown in Figure 5b, inset. As depicted
2468 Analytical Chemistry, Vol. 82, No. 6, March 15, 2010
in the photographs in Figure 5a, the dried blood spot was
dispersed after 1 min of spray time; presumably most of the blood
cells remained on the paper, while the drug and other molecules
dissolved in methanol/water were carried to the paper tip and
released in the spray. Adding an internal standard, such as a
deuterated drug compound, to the blood allowed good quantitation
(RSD < 5% for imatinib) of the drug concentrations in whole blood
samples to be achieved across a wide range of the concentration.11
Figure 5. Analysis of (a, b) dried blood spots for therapeutic drug and (c, d) a dried urine spot for an illicit drug; 0.4 µL samples used for each
dried sample spot, 10 µL of methanol/water (1:1, v:v) used for paper spray.
The concentration of the free drug could be much lower than that
for the total drug, dependent on how much of the drug is bound
to protein or contained in blood cells. Once the fractionation
between the free and bond drug is determined,56,57 during
experiments preparatory to therapeutic drug monitoring, the drug
concentration can be estimated based on the concentration
measured for the free drug in blood. Paper spray can be
implemented at low cost for a large number of measurements.
The same procedure was applied for the analysis of illicit drugs
in undiluted urine samples. Dried urine spots were prepared with
0.4 µL of spiked raw urine and were subsequently analyzed by
paper spray MS. Direct ESI or APCI MS analysis with raw urine
or even diluted urine samples typically does not provide good
sensitivity due to strong interference by the high concentration
of salts and other chemicals in the mixture. Using paper spray
with dried urine spots, the MS and MS/MS spectra were obtained
almost instantaneously when a 10 µL methanol/water solution was
applied to the paper bearing the dried spot, without any further
sample processing steps (Figure 5c,d). Heroin in raw urine at a
concentration of 125 ng/mL (50 pg in 0.4 µL) can be identified
easily with fragment ion peaks of good intensity visible in the MS/
MS spectrum. Development of the paper spray MS analysis of
dried sample spots provides a new means for direct characteriza-
tion of biofluids without sample preparation and consequently
without a need for human intervention during the analysis process.
This should have a significant impact on the implementation of
(56) Joachim, S.; Klaus, B.; Astrid, W.-L. J. Pharm. Sci. 2000, 89, 1008–1021.
(57) Michael, J. B.; Tracey, H. C.; John, A. W. J. Pharm. Sci. 2003, 92, 967–
974.
MS technologies for clinical analysis in laboratories as well as at
point-of-care facilities.
In addition to performing paper spray MS analysis with dried
spots of liquid samples, an attempt has also been made to do direct
paper spray with raw liquid samples that are not as viscous as
blood. In an experiment analyzing a cola drink, 10 µL of the liquid
was dropped on the paper with a positive or negative high voltage
applied for paper spray. In the positive mode, the peak due to
protonated caffeine (m/z 195) dominated the mass spectrum, due
to the high concentration of caffeine (100 µg/mL) in this drink.
In the negative mode, benzoate ion (m/z 121, from potassium
benzoate, a food preservative) and acesulfame anion (m/z 162,
from acesulfame potassium, an artificial sweetener) could be
identified easily with MS/MS analysis.
Another interesting application of paper spray which potentially
is very useful for fast chemical screening is the combination of
the “Swiffer” type sample collection procedure (as routinely done
at security check points) with paper spray to analyze the chemicals
on the surfaces of objects by MS. A piece of paper was used to
wipe the objects to be screened, and then paper spray MS was
used. For example, a piece of filter paper was used to wipe a
section of a desktop, and the paper could then be analyzed directly
after wetting the paper. In one experiment, 5 µL of methanol
containing a known amount of heroin was spotted on a 1 cm2 area
of a desktop, on which dust and traces of chemicals were also
present. This area was then wiped with a paper triangle,
prewetted with about 5 µL methanol/water, and used for
positive paper spray to analyze the samples collected. The mass
spectrum recorded for 50 ng of heroin on the desktop is shown
Analytical Chemistry, Vol. 82, No. 6, March 15, 2010 2469
Figure 6. Analysis of chemicals on surfaces using paper wiping and paper spray (a) and (b) heroin on a dirty desktop and (c) and (d)
agrochemicals on a lemon peel.
in Figure 6a; the protonated heroin molecule (m/z 370) was
observed. With MS/MS, the characteristic fragmentation pattern
of m/z 370 could be obtained to identify heroin on the desktop in
absolute amounts as low as 100 pg (as shown in Figure 6b).
Sample collection by paper wiping followed by analysis using
paper spray was also adapted for fast analysis of agrochemicals
on fruit. Chromatography paper (3 × 3 cm) wetted with methanol
was used to wipe a 10 cm2 area on the peel of a lemon purchased
from a grocery store. After the methanol had dried, a triangle
was cut from the center of the paper and used for paper spray
by applying a 10 µL methanol/water solution. The spectra
recorded (Figure 6c,d) show that a fungicide originally on the
lemon peel, thiabendazole (m/z 202 for protonated molecular ion
and m/z 224 for sodium adduct ion), had been collected onto the
paper and could be identified easily with MS and confirmed using
MS/MS analysis. Another fungicide imazalil (m/z 297) was also
observed to be present. In comparison with the in situ analysis of
the agrochemicals on fruits previously done using DESI28 and
LTP,58 the sample collection by paper wiping has the advantage
of collecting a relatively larger amount of analyte from a larger
surface area. High peak intensity (5.2 × 106) was obtained for
the protonated thiabendazole at m/z 202.
For analysis of target analytes which have relatively low
ionization efficiencies and relatively low concentrations in mix-
tures, derivatization is often necessary to provide adequate
sensitivity in the analysis. Online derivatization has been imple-
mented with desorption electrospray ionization by adding reagents
into the spray solution and significant improvements have been
achieved for analysis of steroids in urine31 and cholesterol in
(58) Wiley, J. S.; Garcı́a-Reyes, J. F.; Harper, J. D.; Charipar, N. A.; Ouyang, Z.;
Cooks, R. G. Analyst, in press. DOI: 10.1039/b919493.
2470 Analytical Chemistry, Vol. 82, No. 6, March 15, 2010
tissue.59 For paper spray, online derivatization can also be
implemented in a similar way by using methanol/water solutions
containing reagents appropriate for targeted analytes. If the
reagents to be used are stable on paper, they can also be added
onto the paper when the paper triangles and the sample cartridges
are fabricated, long before they are used to load and analyze the
samples. As a demonstration, 5 µL of methanol containing 500
ng of betaine aldehyde chloride was added onto a paper triangle
and allowed to dry to fabricate a sample substrate preloaded with
the derivatization reagent for the analysis of cholesterol in serum.
Online charge labeling with betaine aldehyde (BA) through its
reaction with hydroxyl groups has been demonstrated previously
to be very effective for the identification of cholesterol in tissue
using desorption electrospray ionization. When the paper triangle
was used for analysis, 2 µL of human serum was spotted onto the
paper to form a dried spot and then analyzed by using paper spray
ionization. A 10 µL ACN/CHCl3 (1:1 v:v) solution, instead of
methanol/water, was used for paper spray to avoid a reaction
between the betaine aldehyde and methanol. The comparison
between analysis using a blank and a reagent-preloaded paper
triangle is shown with Figure 7a,b. Without the derivatization
reagent, cholesterol-related peaks, such as the protonated ion
[Chol + H]+ (m/z 387), water loss [Chol + H - H2O]+ (m/z
369), and sodium adduction [Chol + Na]+ (m/z 409), could
not be observed (Figure 7a); with the derivatization reagent, the
ion [Chol + BA]+ was observed at m/z 488.6. MS/MS analysis
was performed for this ion, and a characteristic fragment ion59
m/z 369 was observed (Figure 7c). This method could be
implemented in the future for the development of paper-based
(59) Wu, C.; Ifa, D. R.; Manicke, N. E.; Cooks, R. G. Anal. Chem. 2009, 81,
7618–7624.
 sample cartridges, which have reagents preloaded for online
derivatization to improve the sensitivity and selectivity of the MS
analysis for targeted analytes in biofluid samples.

CONCLUSIONS
Paper spray ionization has been developed for direct MS
analysis of complex mixtures with minimum sample treatment.
Its capabilities have been characterized with the analysis of a
variety of compounds in a variety of physical forms and sample
matrices. While the mechanisms of the spray processes in the
positive and negative ion modes are being studied further, the
successful demonstrations of dried biofluid spot analysis and
the identification of particular chemicals from surfaces show the
potential of paper spray ionization for implementing MS analysis
for point-of-care diagnosis and other applications. The potential
of this methodology to combine with lab-on-a-chip processes
before the paper “ion source” while using miniature mass spec-
trometers for mass analysis opens many new avenues for ex-
ploration.

ACKNOWLEDGMENT
This study was supported by the Wallace H. Coulter Founda-
tion Early Career Award for Translation Research in Biomedical
Engineering, the U.S. National Science Foundation (Project CHE
0847205 and CHE 0848650), and the Natural Science Foundation
of China (Project No. 20728505).

Figure 7. Paper spray MS analysis of cholesterol in human serum

with (a) blank paper and (b, c) paper preloaded with betaine aldehyde Received for review December 15, 2009. Accepted
chloride (5 µL, 100 µg/mL). 2 µL of human serum spotted onto the January 27, 2010.
paper and 10 µL of ACN/CHCl3 (1:1 v:v) utilized as solvent for paper
spray. AC902854G

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010 2471