International Journal of Analytical ChemistryVolume 2012 (2012), Article ID 382021, 6 pagesdoi:10.1155/2012/382021

Research Article

Ultra High-Mass Resolution Paper Spray by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Kevin D. Quinn, Charmion I. Cruickshank, and Troy D. Wood

Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA

Received 1 September 2011; Revised 3 February 2012; Accepted 3 February 2012

Academic Editor: Charles L. Wilkins

Copyright © 2012 Kevin D. Quinn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Paper Spray Ionization is an atmospheric pressure ionization technique that utilizes an offline electro-osmotic flow to generate ions off a paper medium. This technique can be performed on a Bruker SolariX Fourier transform ion cyclotron resonance mass spectrometer by modifying the existing nanospray source. High-resolution paper spray spectra were obtained for both organic and biological samples to demonstrate the benefit of linking the technique with a high-resolution mass analyzer. Error values in the range 0.23 to 2.14 ppm were obtained for calf lung surfactant extract with broadband mass resolving power (m/ ) above 60,000 utilizing an external calibration standard.

1. Introduction

Since the first practical implementation of electrospray ionization (ESI) with mass spectrometry reported by Yamashita and Fenn in 1984, a number of different atmospheric pressure ionization (API) techniques have been developed [3]. Many API techniques require extensive sample preparation as well as costly consumables to obtain mass spectra. One of the most cost effective and widely available media for chemical separation and analysis is paper. It has been shown that an electro-osmotic flow similar to that of static electrospray can be utilized to interface a triangle of wetted paper to a mass spectrometer (MS); this form of analysis is called paper spray (PS) [4–9].

The use of a chromatographic medium such as paper allows for the analysis of complex mixtures since separations will occur as the analyte is being driven along the paper medium [7]. Separating analytes on a paper medium provides the benefits of reduced probability of ion suppression and rapid analysis. The paper medium allows for analysis of complex samples such as biological tissue and dried blood spots which are not suitable for traditional infusion techniques due to their complex matrices [4, 7].

One limitation to PS, like many MS techniques that utilize incomplete chromatographic separations is the detector being utilized to perform the analysis. When dealing with complex samples or unknown samples, there is a need for high-quality data in order to determine what is being analyzed. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers are powerful tools for analysis of complex mixtures due to their unsurpassed capability to obtain broadband high-resolution mass spectra with high mass accuracy. Utilizing PS with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer as a detector allows for rapid analysis of both unknowns and/or complex mixtures.

In this present work, we show the suitability of a 12 Tesla FT-ICR MS to obtain high-resolution broadband mass spectra utilizing a coffee filter paper spray medium. High-resolution mass spectra were first obtained for polypropylene glycol (PPG) and polyethylene glycol (PEG) standards to test the source. Second, urine and calf lung surfactant extract (CLSE) were analyzed to test the PS source for analysis of complex mixtures not suitable for direct infusion.

2. Materials, Methods, and Instrumentation

2.1. Chemicals

All solvents used were HPLC grade obtained from Sigma Aldrich (St. Louis, MO, USA). PPG was obtained in the form of a tuning standard from Applied Biosystems (Bedford, MA, USA). PEG standard was made by dissolving 1 mg of PEG 2000 obtained from Sigma Aldrich (St. Louis, MO, USA) in 1 mL of HPLC grade water. Coffee filter, Wegmans brand (Rochester, NY, USA), was cut by hand into an isosceles triangle with dimensions of approximately 5 mm (base) by 10 mm (height).

2.2. PPG and PEG

Samples for PPG and PEG PS analysis were prepared by wetting the coffee filter triangle with 10 μL of the standard solutions. The wet filter paper samples were allowed to dry under ambient conditions before PS analysis was performed. Mass spectra were then obtained by wetting the dried paper triangle with 10 μL of 50 : 50 methanol : water and using the method described in Section 2.5.

2.3. CLSE

CLSE was obtained from ONY Inc. (Amherst, NY, USA). Prior to analysis, the sample was stored in chloroform in the refrigerator. Samples for CLSE analysis were produced by wetting a coffee paper triangle with 2 μL of CLSE and allowing the sample to dry under ambient conditions before PS analysis was performed. Mass spectra were then obtained by wetting the paper with 10 μL of 50 : 50 methanol : water using the instrument method described in Section 2.5.

2.4. Urine

Urine samples were collected from two healthy adult males into sterile polypropylene centrifuge tubes (VWR Scientific) and immediately placed in the freezer. The samples were collected in accordance with the Health Sciences Institutional Review Board at the University at Buffalo (HSIRB no. CHE0050411E). Prior to analysis, the samples were thawed to room temperature and 1 mL was pipetted into 2 mL sample vials (Agilent, Santa Clara, CA, USA) for use. Ten μL was injected directly onto a triangular coffee filter and allowed to dry under ambient conditions. Once dry, the filter paper was attached to the mass spectrometer via a copper clip and rewetted using 50 : 50 methanol : water for PS analysis using the instrument method described in Section 2.5.

2.5. Paper Spray FT-ICR Method

High-resolution spectra were obtained on a 12T Bruker SolariX FT-ICR MS (Bruker Daltonics Inc., Billerica, MA, USA) utilizing a modified Bruker nanoelectrospray source. The positive potential was obtained by grounding the paper triangle using a copper clip and applying a negative voltage at the inlet to the mass spectrometer (Figure 1 shows an image of the source). Mass spectra were obtained at various mass to charge ranges with a pre-FT-ICR-trap hexapole accumulation time of 0.400 seconds and an FT-ICR trap accumulation time of 0.025 seconds. All mass spectra were obtained in the positive mode at a spray voltage of 2 kV. Mass resolving power (m/∆m50%) was defined in accordance with the literature [10].

(a)

(b)

Figure 1: PS source utilizing a modified Bruker nano-ESI source, a copper clip, and cable to ground the paper triangle to the chasse of the instrument.

3. Results and Discussion

3.1. Polymer Standards

Polymeric standards were utilized to determine the suitability of PS with high-resolution FT-ICR MS. In FT-ICR MS the resolution of the mass spectra is relative to m/z as well as the number of data points that can be obtained during the detection process. High-resolution spectra require a time domain signal stable for longer than several hundred milliseconds. Polyethylene glycol and polypropylene glycol produce a broad m/z range of ions ideal for testing the suitability of PS with high-resolution mass spectrometry. High-resolution mass spectra of PEG and PPG are displayed in Figures 2 and 3, respectively. Identifies of the oligomer ion detected and their associated errors is given in Tables 1 and 2, respectively.

Table 1: PEG standards identified using paper spray mass spectrometry. The PEG mass spectrum was obtained with both an excitation and detection frequency range of 1.5 MHz.

PEG
Experimental m/z
Theoretical m/z
Mass measurement error (Δppm)
Mass resolving power (M/ΔM50%)
[H(OCH₂CH₂)nOH+Na]⁺

1009.577639
1009.57651
−0.12
46309

1053.60274
1053.60273
0.01
42870

1097.62886
1097.62894
−0.07
40306

1141.65527
1141.65515
0.02
38791

1185.68128
1185.68137
−0.08
37199

1229.70779
1229.70758
0.17
35953

1273.73391
1273.7338
0.09
34776

1317.76035
1317.76001
0.26
33645

1361.78640
1361.78623
0.12
32579

1405.81232
1405.81244
−0.09
31288

1449.83868
1449.83866
0.01
30101

1493.86539
1493.86487
0.35
29219

1537.89172
1537.89109
0.41
28219

1581.91742
1581.9173
0.08
27254

1625.94358
1625.94352
0.04
26391

1669.96968
1669.96973
−0.03
25567

1713.99520
1713.99595
−0.44
24691

Table 2: PPG standards identified using paper spray mass spectrometry. The PPG mass spectrum was obtained with excitation and detection frequency range of 1.5 MHz.

PPG
Experimental m/z
Theoretical m/z
Mass measurement error (Δppm)
Mass resolving power (M/ΔM50%)
[C₃O₂H₇(C₃H₆O)nC₆H₁₃O₂+Na]⁺

1666.17197
1666.17200
−0.02
22686

1724.21224
1724.21386
−0.94
23629

1782.25781
1782.25573
1.17
23817

1840.29829
1840.29760
0.37
23808

1898.34113
1898.33946
0.88
23139

1956.37992
1956.38133
−0.72
22968

2014.41937
2014.42319
−1.90
22638

2072.46849
2072.46505
1.66
21991

2130.50088
2130.50692
−2.84
21503

2188.55370
2188.54878
2.25
20679

2246.59219
2246.59065
0.69
19685

2304.63182
2304.63254
−0.31
17868

2362.67370
2362.67438
−0.29
16365

Figure 2: Polyethylene glycol paper spray mass spectrum; identification of oligomers as given in the literature [1].

Figure 3: Polypropylene glycol paper spray mass spectrum; identifications of oligomers as given in the literature [2].

Signal-to-noise obtained for PEG and PPG after tuning spectra was in excess of 100/1 with absolute intensities that varied shot-to-shot by less than 20%. This allowed ample time for adjustment of the filter paper towards the inlet of the mass spectrometer so as to optimize instrument setup and analysis. Utilizing polymers of known values (provided by the manufacturers; polydispersity not given) such as PEG 2000 and PPG 2000 provides a detection window several hundred m/z wide for broad mass accuracy calibrations for FT-ICR MS.

3.2. Calf Lung Surfactant Extract

Calf lung surfactant extract (CLSE), which is commercially available in drug form as Infasurf, is used to treat respiratory distress syndrome [11]. CLSE is obtained by doing a total lipid extraction on the lavage fluid from a slaughtered calf lung [11, 12]. Phosphatidylcholine (PC) makes 79% of the molar distribution of lipids and consists of a choline head group attached to the phospholipid with varying degrees of unsaturation in the fatty acid chains [2]. Based on the optimized setup determined from previously analyzing the standards, CLSE was then analyzed. PS mass spectra of CLSE are given in Figures 4 and 5, and the identities with associated error provided in Table 3.

Table 3: CLSE compounds identified using paper spray mass spectrometry. The phospholipids detected are denoted using the lipid numbers nomenclature to indicate degrees of unsaturation, and PC refers to phosphatidylcholine. The CLSE mass spectrum was obtained with excitation and detection frequency range of 1.5 MHz.

Experimental m/z
Theoretical m/z
Mass measurement error (Δppm)
Mass resolving power (M/ΔM50%)
Compound

728.52151
728.52008
1.96
61637
16 : 0/14 : 0 PC [M+Na]⁺

732.55505
732.55378
1.73
60967
16/16 PC [M+H]⁺ 1 degree of unsaturation

734.57100
734.56943
2.14
62820
16 : 0/16 : 0 PC [M+H]⁺

754.53660
754.53572
1.17
57074
16/16 PC [M+Na]⁺ 1 degree of unsaturation

756.55257
756.55137
1.59
57597
16 : 0/16 : 0 PC [M+Na]⁺

760.58591
760.58508
1.09
55950
16/18 PC [M+H]⁺ 1 degree of unsaturation

782.56728
782.56702
0.33
52802
16/18 PC [M+Na]⁺ 1 degree of unsaturation

798.54114
798.54096
0.23
48188
16/18 PC [M+K]⁺ 1 degree of unsaturation

808.58322
808.58267
0.68
48764
18/18 PC [M+Na]⁺ 2 degrees of unsaturation

Figure 4: Broadband PS mass spectrum of 2 μL CLSE, the spectrum was obtained by signal averaging 48 scans (arbitrary).

Figure 5: Inset of broadband PS mass spectrum of CLSE over m/z 776–791.

3.3. Urine

Due to the complex nature of urine, many small molecules and metabolites were detected at varying intensities using PS with FT-ICR. PS mass spectra, showing numerous lipids and other metabolites obtained from the analyzed urine specimens of two healthy adult male subjects, are shown in Figure 6; an inset of one of these PS mass spectra is shown in Figure 7 while the other is shown in Figure 8. A few of the metabolites identified in the 27-year-old volunteer using the exact mass capability of FT-ICR are provided in Table 4. Metabolites identified in the 44-year-old volunteer using the exact mass capability of FT-ICR are given in Table 5. Of note, the 44-year-old volunteer, who was taking prescribed vitamin D supplements, showed indications of the vitamin D metabolite 25-Hydroxyvitamin D2-25-glucuronide in the urine. Interestingly, both adult male subjects had indications of one or more glucuronides in urine. This may signal a particularly strong propensity for glucuronides to ionize via the PS ionization mechanism; this capability of PS should be further explored.

Table 4: Compounds identified using paper spray mass spectrometry on urine sample from 27-year-old volunteer. The urine mass spectrum was obtained with both an excitation and detection frequency range of 1.5 MHz.

Compound
Species
Formula
Experimental m/z
Theoretical m/z
Mass measurement error (Δppm)

3-Hexaprenyl-4,5-Dihydroxybenzoic acid
[M + Na]
C₃₇H₅₄O₄Na
585.39288
585.39143
2.5

3′-hydroxy-e,e-caroten-3-one
[M + CH₃OH + H]
C₄₁H₅₉O₃
599.44790
599.44587
3.4

Vitamin D2 3-glucuronide
[M + CH₃OH + H]
C₃₅H₅₇O₈
605.40328
605.40480
2.5

Coenzyme Q10
[M + K]
C₅₉H₉₀O₄K
901.64549
901.64707
1.8

Table 5: Compounds identified using paper spray mass spectrometry on urine sample from 44-year-old volunteer.  The urine mass spectrum was obtained with excitation and detection frequency range of 1.5 MHz.

Compound
Species
Formula
Experimental m/z
Theoretical m/z
Mass measurement error (Δppm)

Vitamin K1
[M + K]
C₃₁H₄₆O₂K
489.31176
489.31294
2.4

16-alpha, 17-beta-estriol 17-beta-D-glucuronide
[M + CH₃OH + H]
C₂₅H₃₇O₁₀
497.23617
497.23812
3.9

Tetrahydroaldosterone-3-glucuronide
[M + H]
C₂₇H₄₁O₁₁
541.26279
541.26434
2.9

25-Hydroxyvitamin D2-25-glucuronide
[M + CH₃OH + H]
C₃₅H₅₇O₉
621.39946
621.39971
0.4

(a)

(b)

Figure 6: PS mass spectra of urine specimens from two healthy male individuals: 27 year-old (a) and 44 year-old (b).

Figure 7: Inset of PS mass spectrum for urine specimen from 27-year-old volunteer over m/z 582–608.

Figure 8: Inset of PS mass spectrum for urine specimen from 44-year-old volunteer being treated with prescription vitamin D supplement over m/z 480–560.

4. Conclusions

We developed a PS source for Bruker FT-ICR instrumentation that can be utilized for a broad range of chemical species. The PS source was tested on a range of samples and determined the source was suitable for both polymer and biological samples. The PS source provided an API medium for the analysis of complex mixtures while mitigating the possibility of cross contamination due to the disposable nature of coffee filter paper.

Nomenclature

ESI: | Electrospray ionization
API: | Atmospheric pressure ionization
MS: | Mass spectrometer
PS: | Paper spray
FT-ICR: | Fourier transform ion cyclotron resonance
PPG: | Polypropylene glycol
PEG: | Polyethylene glycol
CLSE: | Calf lung surfactant extract
PC: | Phosphatidylcholine
HSIRB: | Health Sciences Institutional Review Board.

Acknowledgments

Donation of the CLSE sample from ONY, Inc. and funding from the National Institutes of Health for the 12-tesla FT-ICR (S10RR029517 to T.D.W.) is gratefully acknowledged. This paper is dedicated in remembrance of Dr. Bruce A. Holm.

References

G. Montaudo, M. S. Montaudo, C. Puglisi, and F. Samperi, “Characterization of polymers by matrix-assisted laser desorption ionization—time of flight mass spectrometry. End group determination and molecular weight estimates in poly(ethylene glycols),” Macromolecules, vol. 28, no. 13, pp. 4562–4569, 1995.

A. T. Jackson, S. E. Slade, K. Thalassinos, and J. H. Scrivens, “End-group characterisation of poly(propylene glycol)s by means of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS),” Analytical and Bioanalytical Chemistry, vol. 392, no. 4, pp. 643–650, 2008.

M. Yamashita and J. B. Fenn, “Electrospray ion source. Another variation on the free-jet theme,” Journal of Physical Chemistry, vol. 88, no. 20, pp. 4451–4459, 1984.

H. Wang, N. E. Manicke, Q. Yang et al., “Direct analysis of biological tissue by paper spray mass spectrometry,” Analytical Chemistry, vol. 83, no. 4, pp. 1197–1201, 2011.

L. Anyin, H. Wang, Z. Ouyang, and R. G. Cooks, “Paper spray ionization of polar analytes using non-polar solvents,” Chemical Communications, vol. 47, no. 10, pp. 2811–2813, 2011.

J. Liu, H. Wang, N. E. Manicke, J. M. Lin, R. G. Cooks, and Z. Ouyang, “Development, characterization, and application of paper spray ionization,” Analytical Chemistry, vol. 82, no. 6, pp. 2463–2471, 2010.

H. Wang, J. Liu, R. Graham Cooks, and Z. Ouyang, “Paper spray for direct analysis of complex mixtures using mass spectrometry,” Angewandte Chemie, vol. 49, no. 5, pp. 877–880, 2010.

R. G. Cooks, N. E. Manicke, A. L. Dill et al., “New ionization methods and miniature mass spectrometers for biomedicine: DESI imaging for cancer diagnostics and paper spray ionization for therapeutic drug monitoring,” Faraday Discussions, vol. 149, pp. 247–267, 2011.

R. Haddad, H. M. S. Milagre, R. R. Catharino, and M. N. Eberlin, “Easy ambient sonic-spray ionization mass spectrometry combined with thin-layer chromatography,” Analytical Chemistry, vol. 80, no. 8, pp. 2744–2750, 2008.

A. G. Marshall, C. L. Hendrickson, and G. S. Jackson, “Fourier transform ion cyclotron resonance mass spectrometry: a primer,” Mass Spectrometry Reviews, vol. 17, no. 1, pp. 1–35, 1998.

R. H. Notter, E. A. Egan, and M. S. Kwong, “Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age,” Pediatric Research, vol. 19, no. 6, pp. 569–577, 1985.

E. G. Blugh and W. J. Dyer, “A rapid method of total lipid extraction and purification,” Canadian Journal of Biochemistry and Physiology, vol. 37, no. 8, pp. 911–917, 1959.