Optimization and Application of Paper-based Spray Ionization Mass

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Optimization and Application of Paper-based Spray Ionization Mass Spectrometry for Analysis of Natural Organic Matter Donghwi Kim, Joonhee Lee, Byungjoo Kim, and Sunghwan Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02668 • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 21, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Optimization and Application of Paper-based Spray Ionization Mass Spectrometry for Analysis of Natural Organic Matter Donghwi Kim[a], Joonhee Lee[b], Byungjoo Kim[b], *, and Sunghwan Kim[a], [c],* [a]

Department of Chemistry, Kyungpook National University, Daegu 41566, Republic of Korea Center for Analytical Chemistry, Division of Chemical & Medical Metrology, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea [c] Green-Nano Materials Research Center, Daegu 41566, Republic of Korea [b]

ABSTRACT: In this study, paper-based ionization techniques—paper spray ionization (PSI) and paper spray chemical ionization (PSCI)—were evaluated and applied for high-resolution mass spectrometry (MS)-based analysis of natural organic matter (NOM). Methanol:isopropyl alcohol (50:50, v/v) and ethanol emerged as good spray solvents for PSI and hexane:dichloromethane (50:50, v/v) was a good spray solvent for PSCI. PSI-MS spectra could be obtained with NOM samples in the microgram scale, which is a critical advantage over conventional electrospray ionization (ESI)-MS when the amount of available sample is limited. In addition, PSI is more tolerant to salt contamination than ESI for NOM analysis. PSCI preferentially ionized less polar compounds, which may not be ionized well using ESI. Therefore, PSCI can be used as a complimentary method to ESI or PSI. Comparison of the numbers of peaks obtained with ESI-, PSI-, and PSCI-MS showed that employing PSI and PSCI can increase the number of compounds that can be detected by high-resolution MS. In conclusion, the data presented in this study showed that PSI and PSCI are suitable ionization techniques for NOM analysis. To the best of our knowledge, this is the first study evaluating and applying PSI and PSCI for NOM analysis.

Natural organic matter (NOM) plays an important role in the global carbon cycle, for soil fertility, and for plant nutrition;1-3 hence, understanding its chemical composition is important. Despite its vital environmental roles, NOM has not yet been fully characterized at the molecular level because it is one of the most complex and heterogeneous mixtures in the environment, mainly composed of carbon, hydrogen, oxygen, and other minor heteroatoms like nitrogen and sulfur.4-6 High-resolution mass spectrometry (HRMS) techniques, such as Orbitrap and Fourier-transform ion cyclotron resonance (FT-ICR) MS, have been used for better understanding of NOM.7-15 Most of the reported studies on NOM have been conducted by using electrospray ionization (ESI) combined with HRMS due to its high efficiency in detecting polar compounds.12,16 The well-known problems associated with ESI are adduct formation and degradation of the spectral signal-tonoise (S/N) ratio caused by salt contamination in the samples.17-22 Salts can interfere with the ESI process resulting in loss of ion transfer efficiency or desolvation efficiency,18,21,23 and can limit the dynamic range of analysis and cause clogging of the ESI tip.24-27 NOM samples are often extracted from water or soil with considerable amounts of salts. Therefore, an appropriate desalting process, such as solid-phase extraction (SPE)23,28-30 and size exclusion chromatography,19,31-34 is required before MS analysis. However, these desalting processes are time-consuming and results in loss of analytes. Paper spray ionization (PSI) is a convenient and sensitive ionization technique that can be used for analyzing polar compounds in a complex mixture.35-38 PSI has increased tolerance

to salt contamination and is inherently free from the clogging issue.38-40 Paper spray chemical ionization (PSCI) is another paper-based ionization technique that can efficiently ionize less- or non-polar analytes in complex mixtures.41 Therefore, PSI and PSCI can be effective for NOM analysis. Despite these potentials, PSI- and PSCI-MS have not yet been applied for NOM analyses, to the best of our knowledge. In this study, PSI/PSCI-MS processes were optimized and evaluated to characterize NOM at the molecular level.

EXPERIMENTAL Sample Preparation Suwannee River fluvic acid (SRFA) was purchased from the International Humic Substances Society (IHSS). For PSI/PSCI-MS, 10 mg/mL SRFA solution in methanol (SigmaAldrich GmbH, Hamburg, Germany) was prepared. Methanol, methanol:water (J.T. Baker, Phillipsburg, NJ, USA) solution (50:50, v/v), 2-butanol (Sigma-Aldrich GmbH, Hamburg, Germany), isopropyl alcohol (J.T. Baker, Deventer, Holland), methanol:isopropyl alcohol solution (50:50, v/v), and ethanol (Fluka, Germany) were used as spray solvents to optimize PSI-MS analysis of SRFA. n-Hexane (Honeywell Burdick & Jackson, Ulsan, Korea) and n-hexane:dichloromethane (Honeywell Burdick & Jackson, Ulsan, Korea) solution (50:50, v/v) were used as spray solvents for PSCI-MS. All the solvents used were HPLC grade. For ESI-MS, SRFA was dissolved in methanol:water (50:50, v/v) to obtain a final concentration of 1 mg/mL. Instrumentation

ACS Paragon Plus Environment

Analytical Chemistry Analyses were performed on a Q-Exactive quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA). The instrument setup for PSI and PSCI is provided in the supporting information (Figure S1). Chromatography paper (Whatman 1 Chr, U.K.) was cut into a triangle of approximately 7 mm base width × 14 mm height. Paper tips were pre-cleaned by sequential 5 min sonication in methanol, followed by dichloromethane and dried at 65 °C for 4 hr. For PSI-MS analysis, 1 µL of sample solution was spotted onto the paper tip using a 10 µL Hamilton syringe (Hamilton, Reno, NV) and was dried at room temperature. The sample-loaded paper was placed at a distance of 5 mm in front of the MS inlet. Each spray solvent was fed to the paper tip by a syringe pump (Fusion 100T, Chemyx, Stafford, TX, USA) at a flow rate of 8–10 µL/min. Spray voltage of 2-5 kV was applied to the paper and other parameters were as follows: mass resolution 140,000 at m/z 200; transfer temperature 300 °C; S-Lens level 50 V. For PSCI-MS, 5 µL of sample solution was loaded onto the paper tip and each spray solvent was fed at a flow rate of 40 µL/min. A spray voltage of 6 kV was applied to the paper tip and other experimental conditions were the same as those used for PSI. For ESI-MS, 1 mg/mL of SRFA solution dissolved in methanol:water solution (50:50, v/v) was infused at a flow rate of 10 µL/min. The spray voltage was set at 3 kV and the sheath gas flow rate was set at 7 (arbitrary units), while the other parameters were the same as those used for PSI. Data Processing Spectral interpretation was performed using in-house developed software with an auto-mated peak-picking algorithm for more reliable and faster results.42,43 Peaks were assigned to chemical formulae within a mass error of 1.5 ppm and typical conditions for humic substance analysis were considered (CcHhNnOoSs; c unlimited, h unlimited, 0 ≤ n ≤ 3, 0 ≤ o ≤ 30, 0 ≤ s ≤ 3).

RESULTS AND DISCUSSION Optimization and Evaluation of PSI & PSCI for SRFA Analysis *

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Figure 2. (a) Comparison of ionization efficiencies obtained at different magnitudes of applied voltage, and (b) spray jet images taken at each voltage using ethanol as a spray solvent.

To optimize the experimental conditions of PSI-MS for NOM analysis, SRFA was analyzed. Various solvent compositions including methanol:water (5:5, v/v), methanol (MeOH), isopropyl alcohol (IPA), 2-butanol (BuOH), methanol:isopropyl alcohol (5:5, v/v), and ethanol (EtOH) were tested to examine the ionization efficiency in (−) PSI-MS. Mass spectra obtained using various solvents are presented in Figure 1. Background peaks generated by solvents and/or paper substrates were typically observed around m/z 300 and the intensity of each background peak varied depending on the applied solvents. The details of observed background peaks are listed in Table S-1. Isopropyl alcohol, methanol:isopropyl alcohol solution (5:5, v/v), and ethanol gave the best MS signal in terms of peak intensity and signal duration. For example, MS signals last more than 10 min, consuming only 10 µg of fluvic acid when ethanol was used as the spray solvent (Figure S2). Interestingly, PSI-MS for fluvic acid did not give a good response in water:methanol solution (5:5, v/v), which is the most commonly used solvent for ESI-MS analysis of NOM. Ryan D. Espy and co-workers determined that spraying of water/organic mixtures with more than 50% water are difficult in PSI as the solvent composition is made more viscous by increasing the water content.44 To test the effect of applied voltage on the ionization efficiency of PSI-MS, various magnitudes of voltage were applied to the paper tip and the respective spectra were obtained. The summed abundances of peaks are presented in Figure 2-a. The MS signals show greater abundance at an applied voltage of 3.0 kV when either MeOH:IPA solution (5:5, v/v) or IPA was used as the spray solvent. Significant decrease in MS signals was observed when voltages larger than 3.5 kV was applied. When ethanol was used as the spray solvent, signal intensity was highest at 2.5 kV; however, signals of good intensities were observed over a wider range of applied voltage (2.2–2.8 kV). The averaged signal to noise ratios (S/N) of top 1000 peaks in the spectra shown in Figure 2 were 122.43 with EtOH, 119.56 with MeOH/IPA, and 110.39 with IPA. Therefore, based on the data presented in Figures 1 and 2, ethanol is considered the ideal solution for NOM analysis using PSI-MS.

Figure 1. Full mass spectra of SRFA obtained by negative-ionmode PSI with various solvents. Marked peaks are background peaks generated by solvents and/or paper substrates.


Page 3 of 12 (a)

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velocities at voltages above 3.5 kV decreased the observed signal intensities. Figure 3 shows the effect of salt concentration on the MS signal of SRFA in negative ESI and negative PSI modes. In negative ESI mode, the addition of NaCl to the sample solution shifts the peak distribution toward lower m/z and reduces the signal intensities corresponding to the analytes. To identify the peak shift and MS signal reduction, averaged mass values for each spectrum were calculated using the following equation: ∑ ⁄ = ∑ where An and (⁄ )n designate the abundance of the nth peak and its m/z value observed in the MS spectra, respectively. Only the peaks having S/N ratio higher than 3 including 13C peaks were considered in this calculation. The calculated average m/z values are provided in Table S2. Spectrum shift is observed in both ESI and PSI modes with increasing salt concentration. The average m/z values calculated from the ESIMS and PSI-MS spectra were reduced by 13% and 5%, respectively, when 0.05% NaCl was added. When NaCl was added, abundance of (-) ESI-MS signal was significantly reduced, especially for the peaks above m/z 500, while no significant signal reduction was not observed in the () PSI mode (insets of Figure 3 and Table S3). These results show that PSI has greater tolerance to salt contamination than ESI. PSCI works with non-polar solvents and spray voltages as high as 6 kV and can ionize non- or less-polar molecules.41 To optimize the experimental conditions of PSCI-MS for SRFA study, hexane (Hex) and Hex:DCM solution (5:5, v/v) were

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Figure 3. Broadband and expanded spectra of SRFA samples containing 0%, 0.005%, and 0.05% NaCl, obtained by negativeion-mode (a) ESI and (b) PSI. To clarify the effect of the added salt on MS spectrum, the Y-axis scales in broadband spectra are fixed at 3 × 106 and 4 × 106 for ESI and PSI, respectively.

Paper spray jet images at different voltages were obtained using a digital camera in a dark room with LED light illumination (Figure 2-b). Under these conditions, spray jets similar to Taylor cone-jet were observed at the paper tip.44,45 The diameter of the spray plume increased continuously as the applied spray voltage was increased from 2 to 4 kV. However, no visible spray could be observed at the paper tip at voltages below 2 kV. The spray mechanism is well documented in a previous report in terms of droplet sizes and velocity of droplets.44 For the SFRA experiment, wider angles of spray plume and faster

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Figure 4. Comparison of SRFA data obtained by negative-ion-mode ESI, PSI, and PSCI; (a) Broadband spectra, (b) Expanded spectra at the 2 different nominal masses: 341 Da and 471 Da, and (c) major chemical class distribution.


Analytical Chemistry tested in the negative-ion mode with an applied voltage of 6 kV. DCM can be used as a PSCI solvent, but using only DCM produces ice crystals at the edge of the paper tip without adding non-volatile solutes like acids, bases, or other solvents.40,41 Therefore, pure DCM was not considered in this study. (-) PSCI-MS spectra of SRFA obtained using Hex and Hex:DCM (5:5, v/v) solvents are presented in Figure S3, in which 2309 peaks above S/N > 3 were observed with Hex and 3276 peaks were observed with Hex:DCM (5:5, v/v). In addition, higher abundance of peaks was obtained when Hex:DCM (5:5, v/v) was used as the solvent. Therefore, Hex:DCM (5:5, v/v) was determined to be a suitable solvent for (-) PSCI-MS analysis of SRFA. MS/MS analysis was performed for both PSI and PSCI using higher-energy collisional dissociation (HCD). Precursor (or parent) ions were selected using an isolation window of 1 m/z, which were then fragmented using HCD with a normalized collision energy (NCE) of 30%. The MS/MS spectra of each precursor ion are presented in Figure S4. The fragmentation resulted in losses of m/z 18 and 44, corresponding to the losses of neutral species H2O and CO2 from singly charged ions (Figure S5). It is well documented that humic substrate ions tend to lose these fragments.46-48 Therefore, the data presented in Figure S5 shows that the peaks detected by PSI- and PSCI-MS have the characteristics of SFRA that agree with the ones observed with ESI-MS. Comparison of ESI, PSI, and PSCI-MS spectra Figures 4a and b show the full mass spectra and expanded spectra of SRFA obtained by (-) ESI, PSI, and PSCI-MS, obtained using the optimized conditions described in the previous section. The observed heteroatom class distributions are shown in Figure 4c. The data presented in Figure 4 show that (-) PSI-MS and (-) ESI-MS provide similar results in terms of peak distribution and major chemical class distribution. For example, the overall m/z distribution in Figure 4a and class distribution in Figure 4c are similar for the results obtained from (-) PSI-MS and (-) ESI-MS. However, it is important to note the difference between ESI and PSI in terms of the amount of sample consumed and that (a)

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Van Krevelen Diagram - Multi Level

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1.01.0 0.9

0.750

0.8

0.7

0.75

0.7

0.6

0.6

0.50

0.50.5

0.50

0.50.5

0.500

0.4

0.500

0.4

0.25

0.3

0.25

0.3

0.250

0.2

0.250

0.2

0.1

00.00.0 0

PSI

1.8

1.3

0.1

0.00

00.00.0 0

0.00

0.2

0.2

0.4

0.4

0.6 O/C Atomic Ratio

0.6

0.8

0.8

1.0

1.0

1.2

1.2

0.00

0.00

0.2

0.2

0.4

0.4

O/C Ratio

(c)

0.6

1.0

1.0

1.2

1.2

2.0 Lipid-like Protein-like

1.7 1.6

1.51.4

Carbohydrates-like

1.5

1.5

1.2 1.1

1.000

1.00

1.01.0 0.9

0.750

0.8

0.75

H/C Ratio

1.3 H/C Atomic Ratio

0.8

0.8

(d) PSCI

1.8


O/C Ratio


2.02.0 1.9

H/C Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Unsaturated hydrocarbon Lignin-like

1.0

Tannin-like

0.7 0.6

0.50

0.50.5

0.500

0.5

Condensed aromatic structure

0.4

0.25

0.3

0.250

0.2 0.1

00.00.0 0

0.00

0.00

0.2

0.2

0.4

0.4


0.6

0.8

0.8

O/C Ratio

1.0

1.0

1.2

1.2

0 0

0.2

0.4

0.6

0.8

O/C Ratio

1.0

1.2

Page 4 of 12

ESI 158 53 571 1075

PSCI

880

526 83

PSI

Figure 6. Venn diagram showing the number of identified peaks in negative-ion-mode ESI, PSI, and PSCI.

required for analysis. In this study, about 10 of SRFA sample was used to obtain (-) ESI-MS data for 1 min. However, practically, at least 50 of SRFA sample is required for a single (-) ESI-MS analysis because a syringe and line have to be filled for injection. In case of (-) PSI-MS, data collected over 10 min could be obtained with 10 of sample (Figure S2). For PSI, no line has to be filled and hence the amount of sample consumed is the same as that required for the analysis. The small amounts of sample consumed and required for analysis provide a critical advantage to PSI when the amount of sample is limited. (-) PSCI-MS analysis gives different results from those obtained with ESI and PSI in terms of peak distribution and major chemical composition (Figure 4). In Figures 4b and c, it is shown that (-) PSCI is more effective in ionizing molecules with less number of oxygen atoms than PSI and ESI. For example, O6 class compounds are the most abundant in (-) PSCIMS spectra but O9 and O10 class compounds the most abundant are in (-) PSI-MS and (-) ESI-MS. To further compare the molecules that can be identified by the different ionization techniques, van Krevelen diagrams, a useful visualization method to interpret MS data obtained from NOM, were generated and presented in Figure 5.11,49,50 The circles in the Figure 5d are used as broad indicators representing the major biogeochemical classes of commonly identified compounds in NOM.8,12,15,51 All the spectra show that lignin and tannin-type molecules are dominant in the SRFA and this agrees with previous reports.52,53 The most abundant peaks in PSCI spectra were shown between 0.3 < O/C < 0.5 and 1.2 < H/C < 1.4. In contrast, the most abundant peaks observed by ESI and PSI were present between 0.5 < O/C < 0.8 and 0.6 < H/C < 1.0. This show that PSCI is more sensitive toward less polar lignin type molecules. The number of assigned peaks in the spectra obtained with (-) ESI, PSI, and PSCI-MS are summarized as a Venn diagram in Figure 6. The lists of compounds used to generate the Venn diagrams are provided in the supporting information (Table S4). A total of 3346 unique compounds were observed from the SRFA by using (-) ESI, PSI, and PSCI-MS. Among them, 1075 peaks were detected by all three techniques and 880 peaks were uniquely found in (-) PSCI-MS data. The peaks uniquely observed by PSI were mainly attributed to oxygenrich compounds such as O18, O19, O20, and O21 class com-

Figure 5. Van Krevelen diagrams for identifying oxygencontaining molecules in SRFA observed by (a) (-) ESI, (b) (-) PSI, PSI, and (c) (-) PSCI. Relative abundances of peaks adjusted to maximum 1 were shown as color codes. (d) Regions associated with major biogeochemical classes of compounds that are commonly detected in NOM. ACS Paragon Plus Environment

Page 5 of 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pounds at higher masses with m/z > 500. The compounds uniquely observed by PSCI-MS mainly included O3, O4, and O5 class compounds. Therefore, the data presented in Figure 6 and Table S4 show that application of PSI and PSCI-MS along with conventional ESI can significantly improve the number of compounds that can be identified in NOM analysis.

Sunghwan Kim: 0000-0002-3364-7367 Byungjoo Kim: 0000-0002-6557-5919

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

CONCLUSIONS

ACKNOWLEDGMENT

In this study, PSI and PSCI ionization techniques were optimized and applied to characterize NOM at the molecular level. The data presented herein show the advantages of using PSI and PSCI for NOM analysis. Compared to ESI-MS, PSIMS requires less amount of sample, and has increased salt tolerance. PSCI can provide data complementary to either ESI or PSI because it can ionize less-polar compounds. Oxygenrich compounds are abundantly detected using PSI and ESI, but low-oxygen-content compounds are preferentially ionized by PSCI. In summary, paper-based spray ionization can provide more comprehensive molecular information on NOM. Therefore, we propose that PSI and PSCI should be included as effective ionization methods for mass spectroscopic studies of NOM.

The authors acknowledge support for this work by the Korea Research Institute of Standards and Science under the project ‘Establishing Measurement Standards for Food and Clinical Nutrients’, grant no. 14011049.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The supporting information file contains the following information: Figure S-1. Procedures for paper spray ionization (PSI) and paper spray chemical ionization (PSCI). Figure S-2. (a) Total ion current chromatogram of SRFA in negative-ion-mode PSI, (b) broadband and (c) expanded mass spectra of SRFA averaged for three different time domains. Figure S-3. Full mass spectra of SRFA obtained by negative-ionmode PSCI with Hex and Hex/DCM solution (50:50, v/v). Figure S-4. Negative-ion-mode PSI and PSCI tandem mass spectra of three different precursor ions detected in SRFA: MS/MS spectra of (a) m/z 325, (b) m/z 369, and (c) m/z 381. Figure S-5. Broadband and expanded MS/MS spectra of SRFA obtained by negative-ion-mode PSI and PSCI. Precursor ion at m/z 381 was selected using an isolation window of 1 m/z. Table S-1. The details of observed background peaks. Table S-2. The averaged m/z values obtained by negative-ionmode ESI and PSI-MS at different salt concentrations. Table S-3. The averaged peak abundance of SRFA obtained by negative-ion-mode ESI and PSI-MS at different salt concentrations. Table S-4. The peak lists of identified compounds in each ESI-, PSI-, and PSCI-MS. (XLSX)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Phone: +82-53-950-5333 * E-mail: [email protected]; Phone: +82-42-868-5367

ORCID

REFERENCES (1) Kononova, M. M. Soil organic matter: its nature, its role in soil formation and in soil fertility; Pergamon Press: Oxford, 1966. (2) Brown, S.; Lugo, A. E. Biotropica 1982, 14, 161-187. (3) Falkowski, P.; Scholes, R. J.; Boyle, E.; Canadell, J.; Canfield, D.; Elser, J.; Gruber, N.; Hibbard, K.; Högberg, P.; Linder, S.; Mackenzie, F. T.; Moore III, B.; Pedersen, T.; Rosenthal, Y.; Seitzinger, S.; Smetacek, V.; Steffen, W. Science 2000, 290, 291-296. (4) Kuwatsuka, S.; Tsutsuki, K.; Kumada, K. Soil Sci. Plant Nutr. 1978, 24, 337-347. (5) Christensen, J. B.; Jensen, D. L.; GrØN, C.; Filip, Z.; Christensen, T. H. Water Res. 1998, 32, 125-135. (6) Li, Y.; Harir, M.; Lucio, M.; Gonsior, M.; Koch, B. P.; SchmittKopplin, P.; Hertkorn, N. Water Res. 2016, 106, 477-487. (7) Wang, C.-F.; Fan, X.; Zhang, F.; Wang, S.-Z.; Zhao, Y.-P.; Zhao, X.Y.; Zhao, W.; Zhu, T.-G.; Lu, J.-L.; Wei, X.-Y. RSC Adv. 2017, 7, 2067720684. (8) Reemtsma, T. J. Chromatogr. A 2009, 1216, 3687-3701. (9) Hawkes, J. A.; Dittmar, T.; Patriarca, C.; Tranvik, L.; Bergquist, J. Anal. Chem. 2016, 88, 7698-7704. (10) Phungsai, P.; Kurisu, F.; Kasuga, I.; Furumai, H. Water Res. 2016, 100, 526-536. (11) Hertkorn, N.; Frommberger, M.; Witt, M.; Koch, B. P.; SchmittKopplin, P.; Perdue, E. M. Anal. Chem. 2008, 80, 8908-8919. (12) Sleighter, R. L.; Hatcher, P. G. J. Mass Spectrom. 2007, 42, 559-574. (13) Cao, D.; Huang, H.; Hu, M.; Cui, L.; Geng, F.; Rao, Z.; Niu, H.; Cai, Y.; Kang, Y. Anal. Chim. Acta 2015, 866, 48-58. (14) Blackburn, J. W. T.; Kew, W.; Graham, M. C.; Uhrín, D. Anal. Chem. 2017, 89, 4382-4386. (15) Patriarca, C.; Bergquist, J.; Sjöberg, P. J. R.; Tranvik, L.; Hawkes, J. A. Environ. Sci. Technol. 2018, 52, 2091-2099. (16) Lu, Y.; Li, X.; Mesfioui, R.; Bauer, J. E.; Chambers, R. M.; Canuel, E. A.; Hatcher, P. G. PLoS One 2016, 10, e0145639. (17) Cech, N. B.; Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362-387. (18) Constantopoulos, T. L.; Jackson, G. S.; Enke, C. G. J. Am. Soc. Mass Spectrom. 1999, 10, 625-634. (19) Hoque, E.; Wolf, M.; Teichmann, G.; Peller, E.; Schimmack, W.; Buckau, G. J. Chromatogr. A 2003, 1017, 97-105. (20) Kruve, A.; Kaupmees, K.; Liigand, J.; Oss, M.; Leito, I. J. Mass Spectrom. 2013, 48, 695-702. (21) Metwally, H.; McAllister, R. G.; Konermann, L. Anal. Chem. 2015, 87, 2434-2442. (22) Pan, P.; McLuckey, S. A. Anal. Chem. 2003, 75, 5468-5474. (23) Fountain, K. J.; Gilar, M.; Gebler, J. C. Rapid Commun. Mass Spectrom. 2004, 18, 1295-1302. (24) Annesley, T. M. Clin. Chem. 2003, 49, 1041-1044. (25) Furey, A.; Moriarty, M.; Bane, V.; Kinsella, B.; Lehane, M. Talanta 2013, 115, 104-122. (26) Belov, M. E.; Anderson, G. A.; Angell, N. H.; Shen, Y.; Tolic, N.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2001, 73, 5052-5060. (27) Wu, L.; Han, D. K. Expert Rev. Proteomics 2006, 3, 611-619. (28) Gilar, M.; Belenky, A.; Wang, B. H. J. Chromatogr. A 2001, 921, 313. (29) Dittmar, T.; Koch, B.; Hertkorn, N.; Kattner, G. Limnol. Oceanogr.: Methods 2008, 6, 230-235. (30) Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W. Glycoconjugate J. 1998, 15, 737-747.


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Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for TOC graphic specifications.

Insert Table of Contents artwork here (a)

(b)

ESI


2.0 2.0 1.9

1.9 1.8 1.7

1.6

1.6

1.5 1.5

1.5 1.5

1.000

1.00

0.9 0.750

0.8

0.75

H/C Atomic Ratio

1.3

1.1

H/C Ratio

H/C Atomic Ratio

H/C Ratio

PSI

1.4

1.3 1.2

1.0 1.0

1.2 1.1

0.7

0.50

0.750

0.8

0.6

0.500

1.00

0.9

0.7

0.5 0.5

1.000

1.0 1.0

0.6

0.75 0.50

0.500

0.5 0.5

0.4

0.4

0.25

0.3

0.2 0.1

0.00

0.0 0 0.0 0

0.00

0.2

0.2

0.4

0.4


0.6

0.8

0.8

1.0

1.0

0.25

0.3

0.250

0.2 0.1 0.0 0 0.0 0

PSI


2.0 2.0

ESI

1.8 1.7

1.4

1.2

1.2

0.250

0.00

0.00

0.2

0.4

0.2

0.4

O/C Ratio

(c)

(d)

PSCI

1.9


0.6

0.8

0.8

1.0

1.2

1.0

1.2

O/C Ratio

Van Krevelen Diagram - Multi Level 2.0

2.0

ESI

PSCI

158

1.8 1.7 1.6 1.5

1.5

1.4

53

H/C Atomic Ratio

1.3

H/C Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 12

1.2 1.1

571

1.000

1.00

1.0 1.0

1075

0.9 0.750

0.8

0.75

0.7 0.6

0.50

0.500

0.5 0.5 0.4

0.25

0.3

0.250

0.2 0.1 0.0 0 0.0 0

PSCI

880

526 83

0.00

0.00

0.2

0.2

0.4

0.4


0.6

0.8

0.8

1.0

1.0

1.2

1.2

O/C Ratio


PSI

PSI_N_1CHR_IHSS_Fulvic_5000ppm_2ul_3kv_MeOHDW #80-206 RT: 0.70-1.80 AV: 127 NL: 9.45E6 T: FTMS - p NSI Full ms [111.0000-1200.0000]

Page 7 of 12

PSI_N_1CHR_IHSS_fulvic_5000ppm_2ul_3kv_IPA_FR10 #204-324 RT: 1.78-2.83 AV: 121 NL: 8.61E6 T: FTMS - p NSI Full ms [111.0000-1200.0000] 100

*

100

100

95

Analytical Chemistry 100 95

75

65

*

*

60

60 55 50 45

40

40 35 30 25

20

20


80

85

H2O/MeOH Relative Abundance


Relative Abundance

80

70

*

*

85

0 100

300

400

300

500

500

600

700

700

800

m/z

*

100

100

95

900

900

1000

1100

1100

1200

m/z

45

40

40 35 30 25

20

20

200

300

300

400

500

500

600

700

700

800

m/z

*

95

900

900

1000

1100

1100

1200

m/z

90

75 70

*

65

60

60 55 50 45

40

40 35 30 25

20

20

*

0 100


85

2-BuOH

80

80

Relative Abundance


50

100

85

75

*

70 65

60

60 55 50 45

40

40 35 30 25

20

20

10

0 100 5

0

0

200

300

400

300

500

500

600

700

700

800

m/z

PSI_N_1CHR_IHSS_Fulvic_5000ppm_2ul_3.9kv_MeOH #90-215 RT: 0.78-1.88 AV: 126 NL: 2.70E7 T: FTMS - p NSI Full ms [111.0000-1200.0000] 100

*

900

900

1000

1100

1100

1200

200

m/z

95 90

* *

75 70 65

60

Relative Abundance

60 55 50 45

40

40 35

*

30 25

20

500

500

600

700

700

800

m/z

900

900

1000

1100

1100

1200

20

1100

1200

m/z

*

85

MeOH


85

80

400

*

100

80

300

300

PSI_N_1CHR_IHSS_Fulvic_5000ppm_2ul_3kv_EtOH_FR10 #115-228 RT: 1.00-1.99 AV: 114 NL: 5.05E6 T: FTMS - p NSI Full ms [111.0000-1200.0000] 100

90

80

80

EtOH

75 70 65

60

60 55 50 45

40

40

*

35 30 25

20

20 15

15

ACS Paragon Plus 0Environment 10

10

0 100

MeOH/IPA

80

80

15

5


55

PSI_N_1CHR_IHSS_Fulvic_10000ppm_1ul_3kv_MeOHIPA_180115201920 #230-351 RT: 2.01-3.06 AV: 122 NL: 8.38E6 T: FTMS - p NSI Full ms [111.0000-1200.0000] 100

90

Relative Abundance

*

60

0

200

PSI_N_1CHR_IHSS_Fulvic_5000ppm_2ul_3kv_BuOH_FR10 #344-461 RT: 3.00-4.02 AV: 118 NL: 2.07E7 T: FTMS - p NSI Full ms [111.0000-1200.0000]

95

65

60

5

0

100

70

0 100

5

10

75

10

10

15

IPA

80

80

15

15

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

*

90

90

5

5

0

0

200

300

300

400

500

500

600

700

700

m/z

m/z

800

900

900

1000

1100

1100

1200

100

200

300

300

400

500

500

600

700

700

m/z

m/z

800

900

900

1000

1100

(a) 88

(b)


Page 8 of 12

2.0kV

2.5kV

1 cm

1 cm

3.0kV

3.5kV

1 cm

1 cm

Summed abundance

8.0E+08 7.0E+08

88 6.0x10 6.0E+08 6.0x10 5.0E+08 4.0E+08

88 3.0x10 3.0E+08 3.0x10 2.0E+08

1.0E+08

0

0.0E+00 0

2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.6 3.8 3.8 4.0 4.0 2.4 2.6 2.8 3.0 3.2 3.4

SprayVoltage voltage (kV) Spray (kV)

Spray solvent : MeOH/IPA Spray solvent : MeOH/IPA

1.8E+09

Summed abundance

Summed abundance

9 2.0E+09 2.0x10 1.6E+09

9 1.5x10 1.4E+09 1.2E+09

9 1.0E+09 1.0x10 8.0E+08 6.0E+08

9 0.5x10 4.0E+08 2.0E+08

0 2.4 2.4 2.6 2.6 2.8 2.8 3.0 3.0 3.2 3.2 3.4 3.4 3.6 3.6 3.8 3.8 4.0 4.0

0.0E+00

Spray (kV) SprayVoltage voltage (kV)

99 2.0x10 2.0E+09 2.0x10

Spay solvent : EtOH: EtOH Spray solvent

4.0kV

1.8E+09

Summed abundance

Summed abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Summed abundance

9.0x10 9.0E+08 9.0x10

Spray solvent : IPA Spray solvent : IPA

1.6E+09

1.5x109 1.5x10 1.4E+09 1.2E+09

9 1.0x10 1.0E+09 1.0x10 8.0E+08

6.0E+08 0.5x10 0.5x1099

4.0E+08 2.0E+08

ACS Paragon Plus Environment 0 2.0 2.0 2.2 2.2 2.4 2.4 2.6 2.6 2.8 2.8 3.0 3.0 3.2 3.2 3.4 3.4 3.6 3.6 3.8 3.8 4.0 4.0

0.0E+00 0

Spray (kV) SprayVoltage voltage (kV)

1 cm

Page 9 of 12

(b)

3.0x106

4.0x106

4000000

2900000

3800000

2800000

psi_n_ihss_fulvic_10000ppm_1ul_3kv_etoh_180121195511 #109-230 RT: 0.95-2.01 AV: 122 NL: 5.00E5 T: FTMS - p NSI Full ms [110.0000-1200.0000]

ESI_N_IHSS_fulvic_1000ppm_2.7kv_DWMeOH #1-100 RT: 0.01-0.87 AV: 100 NL: 4.01E5 T: FTMS - p ESI Full ms [100.0000-1200.0000] 400000

2700000

3600000

2600000

280000 260000


240000

2200000

2.0x106

2100000

220000 200000 180000 160000

2000000

140000 120000

1900000

100000 80000

1800000

60000 40000

1700000

20000 0 600

1600000

605

610

600

1500000 1400000

615

620

625

630

620

635

640

645

640

650 m/z

655

660

665

670

660

675

680

685

690

680

695

700

700

m/z

1300000 1200000

1.0x106

1100000 1000000 900000 800000 700000 600000 500000 400000

380000

320000

2800000

300000 280000 260000 240000 220000 200000 180000 160000

2600000

140000 120000 100000

2400000

80000 60000 40000 20000

2200000

0 600

2.0x106

300

300

400

500

500

600

700

800

700

900

1000

900

m/z

1100

1100

625

630

635

640

645

640

650 m/z

655

660

665

670

660

675

680

685

690

695

700

680

700

1100

1200

m/z

1400000 1200000

1.0x106

1000000 800000 600000

200

300

300

400

500

500

600

700

800

700

900

1000

900

m/z

psi_n_ihss_fulvic_10000ppm_1ul_0.005pnacl_3kv_etoh_180121201834 #66-188 RT: 0.58-1.64 AV: 123 NL: 4.00E6 T: FTMS - p NSI Full ms [110.0000-1200.0000]

1100

m/z

4000000 3800000

psi_n_ihss_fulvic_10000ppm_1ul_0.005pnacl_3kv_etoh_180121201834 #67-185 RT: 0.58-1.61 AV: 119 NL: 5.00E5 T: FTMS - p NSI Full ms [110.0000-1200.0000]

esi_n_ihss_fulvic_1000ppm_2.7kv_0.005pnacl_dwmeoh_sg7 #1-100 RT: 0.01-0.87 AV: 100 NL: 4.00E5 T: FTMS - p ESI Full ms [100.0000-1200.0000] 400000

3600000

380000

320000 300000

2400000

280000 260000

2300000

240000

Relative Abundance

2200000

2.0x106

2100000

220000 200000 180000 160000

2000000

140000 120000

1900000

100000 80000

1800000

60000 40000

1700000

20000 0 600

1600000

605

610

600

1500000 1400000

500000 480000 460000

340000

615

620

625

630

620

635

640

645

640

650 m/z

655

660

665

670

660

675

680

685

690

680

695

700

700

m/z

1300000 1200000

1.0x106

1100000 1000000 900000 800000 700000 600000 500000

440000

3400000

420000 400000 380000

3200000

360000

3.0x106

340000 320000 300000

Relative Abundance

2500000

Relative Abundance

360000

Absolute abundance

2600000

400000

3000000 2800000

280000 260000 240000 220000 200000 180000 160000

2600000

140000 120000 100000

2400000

80000 60000 40000 20000

2200000

0 600

2.0x106

605

610

600

2000000

615

620

625

630

620

635

640

645

640

650 m/z

655

660

665

670

660

675

680

685

690

695

700

680

700

1100

1200

m/z

1800000 1600000 1400000 1200000

1.0x106

1000000 800000 600000

300000

400000

200000

200000

0 100 6 4.0x10

100000

0

200

300

400

300

500

600

700

500

800

900

700

2900000

1000

900

m/z


1100

1200

1100

200

300

300

400

500

500

600

700

m/z

800

700

900

1000

900

m/z

psi_n_ihss_fulvic_10000ppm_1ul_0.05pnacl_3kv_etoh #159-278 RT: 1.39-2.42 AV: 120 NL: 4.00E6 T: FTMS - p NSI Full ms [110.0000-1200.0000]

1100

m/z

4000000 3800000

2800000

psi_n_ihss_fulvic_10000ppm_1ul_0.05pnacl_3kv_etoh #159-278 RT: 1.39-2.42 AV: 120 NL: 5.00E5 T: FTMS - p NSI Full ms [110.0000-1200.0000]


2700000

3600000

500000 480000

380000

460000

340000

2500000

320000

2400000

280000

300000

260000


240000

2200000

2.0x106

2100000

220000 200000 180000 160000

2000000

140000 120000

1900000

100000 80000

1800000

60000 40000

1700000

20000 0 600

1600000

605

600

1500000 1400000

610

615

620

625

620

630

635

640

645

640

650 m/z

655

660

665

660

670

675

680

685

680

690

695

700

m/z

1300000

700

1200000

1.0x106

1100000 1000000 900000 800000 700000 600000 500000 400000

440000

3400000

420000 400000 380000

3200000

360000

3.0x106

340000 320000

3000000

300000

Relative Abundance

360000

Relative Abundance

2600000

Absolute abundance

Relative Abundance

620

620

1600000

0 100 6 4.0x10

1200

m/z

2700000

Absolute abundance

615

1800000

2800000

2800000

280000 260000 240000 220000 200000 180000 160000

2600000

140000 120000 100000

2400000

80000 60000 40000 20000

2200000

0 600

2.0x106

605

600

2000000

610

615

620

625

620

630

635

640

645

640

650 m/z

655

660

665

660

670

675

680

685

690

695

700

1100

1200

1600000 1400000 1200000

1.0x106

1000000 800000 600000 400000

200000

200000

100000

200

300

300

400

500

500

600

700

700

m/z

m/z

800

900

900

1000

ACS Paragon Plus Environment 1100

1100

1200

0 100 0

200

300

300

400

500

500

600

700

700

m/z

m/z

800

900

900

1000

700

680

m/z

1800000

300000

0 100

610

0

200

2900000

0 100

605

600

2000000

200000


Relative Abundance

340000

3000000

400000

100000

Absolute abundance

360000

3.0x106

200000

0.005 % NaCl

420000 400000

3200000

300000

0 100

440000

3400000

Relative Abundance

300000

Relative Abundance

320000

2400000

0 100 6 3.0x10

480000 460000

2500000

0 100

500000

360000 340000

Absolute abundance

Relative Abundance

Absolute abundance

Non-NaCl

380000

0 100 6 3.0x10

Negative PSI psi_n_ihss_fulvic_10000ppm_1ul_3kv_etoh_180121195511 #108-226 RT: 0.94-1.97 AV: 119 NL: 4.00E6 T: FTMS - p NSI Full ms [110.0000-1200.0000]

ESI_N_IHSS_fulvic_1000ppm_2.7kv_DWMeOH #1-100 RT: 0.01-0.87 AV: 100 NL: 3.00E6 T: FTMS - p ESI Full ms [100.0000-1200.0000] 3000000

0.05 % NaCl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55


Negative ESI

(a)

1100


(a) RT: 0.01-0.87

AV: 100

NL: 4.27E6

100

[C15H17O9]-

100

95

[C14H13O10]-

90 85 80 75

(H2O/MeOH)

60

60

40

40

20

20

0 100

0 100

60

Relative Abundance

[C16H21O8]-

80

65

55

50

[C22H17O12][C23H21O11][C H O13][C24H25O10]NL: 3.55E6 esi_n_ihss_fulvic_1000ppm_3kv _dwmeoh#1-100 RT: 0.01-0.87 21 AV: 100 T: FTMS - p ESI Ful13 l ms [100.0000-1200.0000]

100

Negative ESI

70

80

45

NL: 7.47E5 esi_n_ihss_fulvic_1000ppm_3kv _dwmeoh#1-100 RT: 0.01-0.87 AV: 100 T: FTMS - p ESI Full ms [100.0000-1200.0000]

[C25H29O9]-

50

40 35 30 25

15 10

0 100

5

0 100 200 300 400 PSI_N_1CHR_IHSS_fulvic_5000ppm_2ul_3kv_EtOH_Chro #75-539 T: FTMS - p NSI Full ms [100.0000-1200.0000] 100

500 RT: 0.65-4.70 AV: 465

600 NL: 7.80E6

700

800

900

1000

1100

1200

m/z

NL: 8.03E6 PSI_N_1CHR_IHSS_fulvic_5000 ppm_2ul_3kv_EtOH_Chro#116306 RT: 1.01-2.67 AV: 191 T: FTMS - p NSI Full ms [100.0000-1200.0000]

95 90

80 75 70

(EtOH)

65 60

80

55

50

50 45 40 35 30 25

[C60 13H9O11]40

20

80

Relative Abundance

Negative PSI

85

Relative Abundance


Relative Abundance

20

NL: 2.80E6 PSI_N_1CHR_IHSS_fulvic_5000 ppm_2ul_3kv_EtOH_Chro#116306 RT: 1.01-2.67 AV: 191 T: FTMS - p NSI Full ms [100.0000-1200.0000]

[C19H21O14]-

60 40

20

20

0 100

0 100

15 10

0 100

5

0 100 200 300 400 PSCI_N_1CHR_IHSS_SRFA_10000ppm_5ul_HexDCM_1PFA #33-183 T: FTMS - p NSI Full ms [100.0000-1200.0000] 100

500 RT: 0.29-1.60

AV: 151

600 NL: 7.37E6

700

800

900

1000

1100

1200

m/z

95

[C17H25O7]80 -

90 85 80 75

NL: 3.29E6 psci_n_1chr_ihss_srfa_10000pp m_5ul_hexdcm_1pfa#38-114 RT: 0.33-0.99 AV: 77 T: FTMS p NSI Full ms [100.0000-1200.-0000]

Negative PSCI

80

(Hex/DCM)

60

[C18H60 29O6]

40

40

20

20

0

0

70 65 60

[C26H33O8]-

50

50 45 40 35 30 25 20 15 10

0 100 5

0 100

(c)

200

300

400

300

500

600

700

800

m/z

500

700

900

1000

900

1100

1200

1100

340.95 341.00 341.05 341.10 341.15 341.20 341.25 472.90 340.95 341.05 341.25 m/341.15 z

m/z

17

472.95

473.00 473.05 473.10 473.15 473.20 473.25 473.30 473.35 473.00 473.10 473.20 473.30 m/z

m/z

m/z

16 16 15

ESI PSI PSCI

14 14 13

% Relative Abundance

12 12 11

10 10 9

88 7

66 5

44 3

22 1

00

Paragon Plus Environment Class O1 O2 O3 O4 O5 O6 O7 O8 O9 OACS 10 O 11 O12 O13 O14 O15 O16 O17 O18 O19 O20 O21 O22 O23 O24 O25 O1

O2

O3

O4

O5

O6

O7

O8

O9

O10

O11

O12

O13

O14

Class

O15

O16

O17

O18

O19

O20

NL: 3.95E5 psci_n_1chr_ihss_srfa_10000pp m_5ul_hexdcm_1pfa#38-114 RT: 0.33-0.99 AV: 77 T: FTMS p NSI Full ms [100.0000-1200.0000]

[C27H37O7]-

55


Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

(b) ESI_N_IHSS_fulvic_1000ppm_3kv_DWMeOH #1-100 T: FTMS - p ESI Full ms [100.0000-1200.0000] 100

Page 10 of 12

O21

O22

O23

O24

O25

Page 11 of 12

(a)

(b)


2.0


2.0

2.0

2.0

ESI

1.9 1.8

1.8

1.7

1.7

1.6

1.6

1.5

1.5

1.5 1.4

1.4

1.3

1.1

1.000

1.00

1.0

1.0 0.9

0.750

0.8

0.75

H/C Atomic Ratio

1.2

H/C Ratio

H/C Atomic Ratio

H/C Ratio

1.3

1.1

1.000

1.00

1.0 0.9

0.750

0.8

0.75

0.7

0.6

0.6

0.50

0.5

0.500

0.5

0.50

0.500

0.5

0.5 0.4

0.4

0.25 0.250

0.3

0.25

0.3

0.250

0.2

0.2 0.1 0.0

1.2

1.0

0.7

0

PSI

1.9

1.5

0.1

0.00

0

0.00 0.0

0

0.2

0.2

0.4

0.4


0.6

0.8

0.8

1.0

1.0

0.0

1.2

1.2

0.00

0.00 0.0

0

0.2

0.2

0.4

0.4

O/C Ratio

(c)

1.0

1.2

1.2

Lipid-like Protein-like

1.7 1.6

1.5

Carbohydrates-like

1.5

1.5 1.4

1.2 1.1

1.000

1.00

1.0

1.0 0.9

0.750

0.8

0.75

H/C Ratio

1.3

H/C Atomic Ratio

0.8

1.0

2.0

PSCI

1.8

0.6

0.8

(d)

2.0 1.9


O/C Ratio


2.0

H/C Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55


Unsaturated hydrocarbon Lignin-like

1.0

Tannin-like

0.7 0.6

0.50

0.5

0.500

0.5

0.5

Condensed aromatic structure

0.4

0.25

0.3

0.250

0.2 0.1

0

0.0

0.00

0.00 0.0

0

0.2

0.2

0.4

0.4


0.6

0.8

0.8

1.0

1.0

1.2

1.2

0

O/C Ratio

0

0.2

0.4

0.6

0.8

O/C Ratio ACS Paragon Plus Environment

1.0

1.2

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 12 of 12

ESI 158

53 571

1075

PSCI

880

526 83


PSI

Optimization and Application of Paper-based Spray Ionization Mass

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