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Silver-ion Solid Phase Extraction Separation of Classical, Aromatic, Oxidized, and Heteroatomic Naphthenic Acids from Oil Sands Process-Affected Water Rongfu Huang, Yuan Chen, and Mohamed Gamal El-Din Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01350 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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Environmental Science & Technology

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Silver-ion Solid Phase Extraction Separation of Classical, Aromatic, Oxidized, and

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Heteroatomic Naphthenic Acids from Oil Sands Process-Affected Water

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Rongfu Huang, Yuan Chen, Mohamed Gamal El-Din*

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Department of Civil and Environmental Engineering, University of Alberta, Edmonton,

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Alberta, T6G 1H9, Canada

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Word count: 7308. Text: 4308 (abstract and main text); Big figure (4): 2400; Big table

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(1): 600

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* Corresponding Author:

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Mohamed Gamal El-Din, Ph.D., P.Eng.

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7-285 Donadeo Innovation Centre for Engineering

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Department of Civil and Environmental Engineering

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University of Alberta, Edmonton, Alberta, Canada, T6G 1H9

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Tel.: +1-780-492-5124

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Fax: +1-780-492-0249

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E-mail address: [email protected]

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1

ACS Paragon Plus Environment


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Abstract

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The separation of classical, aromatic, oxidized, and heteroatomic (sulfur-containing)

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naphthenic acids (NAs) species from unprocessed and ozone-treated oil sands process-

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affected water (OSPW) was performed using silver-ion (Ag-ion) solid phase extraction

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(SPE) without requirement of pre-methylation for NAs. OSPW samples before SPE and

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SPE fractions were characterized using ultra performance liquid chromatography ion

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mobility time-of-flight mass spectrometry (UPLC-IM-TOFMS) to corroborate the

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separation of distinct NA species. The mass spectrum identification applied a mass

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tolerance of ±1.5 mDa due to the mass errors of NAs were measured within this range,

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allowing the identification of O2S–NAs from O2–NAs. Moreover, separated NA species

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facilitated the tandem mass spectrometry (MS/MS) characterization of NA compounds

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due to the removal of matrix and simplified composition. MS/MS results showed that

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classical, aromatic, oxidized, and sulfur-containing NA compounds were eluted into

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individual SPE fractions. Overall results indicated that the separation of NA species using

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Ag-ion SPE is a valuable method to extract individual NA species that are of great

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interest for environmental toxicology and wastewater treatment research, to conduct

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species-specific studies. Furthermore, the separated NA species in mg level could be

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widely used as the standard materials for environmental monitoring of NAs from various

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contamination sites.

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INTRODUCTION

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Naphthenic acids (NAs) have been reported as the toxic polar constituents of

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process water from various petroleum production processes, including conventional and

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unconventional petroleum reservations, such as oil sands.1 In recent decades, the rapid

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development of Alberta (Canada) oil sands industry has produced huge amounts of oil

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sands process-affected water (OSPW), which are of increasing concern due to the

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presence of persistent toxic compounds, such as NAs, which are abundantly present,

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adversely affecting the environmental and public health.1-5 Both acute and chronic

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toxicities of NAs species have been assessed towards different organisms including

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goldfish, larval zebrafish, Pimephales promelas, Vibrio fischeri, and the mammalian

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immune system.6-9 NAs have the empirical formulae as CnH(2n+Z)Ox and CnH(2n+Z)OyS,

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where “n” is the carbon number (7≤n≤26), “Z” is zero or a negative even integer (0≤-

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Z≤18) that specifies the hydrogen deficiency resulting from ring or unsaturated bond

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formation, and “x” and “y” represent the number of oxygen atoms (2≤x≤5, 2≤y≤4).

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Classical NAs have x = 2 and oxidized NAs have x > 2.

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Liquid-liquid extraction (LLE) and/or solid phase extraction (SPE) were commonly

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used as sample preparation steps in various analytical and toxicological studies to extract

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NAs from OSPW.6, 8, 10-12 For example, fractionation using pH-dependent LLE has been

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performed to study the dissociate constants for NA species; however, LLE at different pH

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conditions was not able to separate distinct NA species.12 Due to NAs are a complex

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mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, the separation of

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distinct NA species would simplify their quantification and facilitate related analytical,

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toxicological, and engineering investigation of NAs. A number of solid phase materials

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for SPE, including activated carbon, cellulose, iron oxides (magnetite and goethite),

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polyaniline, and three types of biochar derived from biomass, were examined to remove

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NAs from OSPW and the results showed that activated carbon had the highest removal

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efficiency (95%) for NAs but none of these solid phase materials were found to separate

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classical, oxidized, or sulfur-containing NA species.13

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The silver-ion (Ag-ion) solid phase extraction was initially applied to fractionate

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methylated fatty acids via multiple-step elution based on saturation degree or cis/trans

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molecular structure.14 Pre-methylation for fatty acids was necessary to modify the

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molecular polarity, facilitating Ag-ion SPE separation, though other structure-related

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properties (e.g. molecular toxicity) also changed after methylation. Scarlett and Reinardy

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et al. applied similar methylation to NAs, and based on GC×GC-MS determination,

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found fractionation (hexane/ethyl ether mixture as eluent) of aromatic and nonaromatic

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(or acyclic) NAs using Ag-ion SPE.6, 11 Though the toxicity of separated aromatic and

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nonaromatic NAs was assessed towards larvae zebrafish after de-methylation to

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methylated NAs, the methylation and de-methylation reagents and reactions reduced the

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accuracy of the toxicity assessment for NA species from OSPW.6 Apparently, the

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requirement for methylation in this method was a limitation that not only slowed the

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entire process but also restrained the application of fractionated products, which were

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only roughly separated as aromatic and nonaromatic species.

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In this work, we found that, Ag-ion SPE could be used to separate classical,

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aromatic, oxidized, and sulfur-containing NAs without requirement of pre-methylation

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for NAs, using hexane/acetone mixture as the eluent solvent. This finding is crucially

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important because the method is able to separate classical, aromatic, oxidized, and sulfur-

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containing NA species, including but not limit to aromatic and nonaromatic NAs. By

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using the products generated from this method, future environmental toxicology or

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engineering studies could be conducted to specific NA species without being interfered

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by unnecessary methylation and de-methylation steps. The ultra-performance liquid

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chromatography ion mobility time-of-flight mass spectrometry (UPLC-IM-TOFMS),

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which was previously applied in a number of studies for the characterization of OSPW

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samples,15, 16 was used to characterize the unprocessed and ozonated OSPWs before SPE

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and SPE fractions for comparison (e.g., ozonation could remove largely the aromatic

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NAs15 that are to be separated via Ag-ion SPE), in order to corroborate the fractionation

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process for the separation of NA species. The identification of compounds from spectra

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using accurate mass matching with a mass tolerance of ±1.5 mDa, based on estimated

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mass errors between measured and calculated masses for NAs, allowed the identification

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of sulfur-containing NAs from O2–NAs. The separated NA species facilitated the

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subsequent tandem mass spectrometry (MS/MS) determination of structural information

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for different NAs species due to the removal of matrix and simplified composition

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compared to unprocessed OSPW. This work aims to achieve the separation of classical,

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aromatic, oxidized, and sulfur-containing NAs from OSPW using Ag-ion SPE and to

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verify the composition of fractions using UPLC-IM-TOFMS and MS/MS analysis,

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contributing to future analytical, toxicological, and engineering studies as well as

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providing the NA standard material for future comprehensive environmental monitoring

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of NA contamination from various sources.

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EXPERIMENTAL

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Reagents and materials

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Fresh OSPW was collected in December 2014 from a tailings pond located in Fort

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McMurray, Alberta, Canada and stored at 4 ºC prior to use. H2SO4 (Sigma-Aldrich, ON)

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was used to adjust the solution pH. Dichloromethane (DCM), hexane, and acetone

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(Fisher Scientific, ON) were used in the liquid-liquid extraction and/or solid phase

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extraction processes. Discovery Ag-ion SPE tube (Sigma-Aldrich, ON) was used for

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solid phase extraction. Optima-grade water, methanol, and acetonitrile (Fisher Scientific,

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ON) were used in UPLC-IM-TOFMS analysis.

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Ag-ion solid phase extraction

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The unprocessed and ozonated OSPWs were extracted using Ag-ion SPE tubes for

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comparative characterization. The ozonated OSPW was prepared from unprocessed

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OSPW with an utilized ozone dose of 80 mg/L, which was used in previous study17, by

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sparging with an ozone generator (WEDECO, GSO-40, Germany). Detailed explanation

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of the ozonation experiments can be found in the Supplementary Information (SI). The

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pre-extracted sample fraction in hexane was required prior to the Ag-ion SPE process.11

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However, liquid-liquid extraction directly using hexane could change the composition of

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NA species from OSPW.18 Thus, the unprocessed and ozonated OSPWs were firstly

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extracted using DCM. OSPW was mixed uniformly using a motor driven paddle mixer

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before dividing into 500 mL working aliquots, which were then centrifuged at 10,000

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rpm for 10 minutes to remove the suspended particles. H2SO4 solution (1.8 M) was added

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dropwise to adjust the pH of OSPW supernatant (pH 9.4) to 2.0 prior to extraction. In

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each extraction, a 500 mL sample was extracted with 250 mL DCM (90 mL, 80 mL, and 6


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80 mL sequentially with sample: total solvent = 2:1). The organic layers were separated,

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combined (~250 mL), and air-dried completely under a fume hood at room temperature.

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Each fraction was re-dissolved into 10 mL hexane and stored at 4 ºC prior to use.

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For Ag-ion solid phase extraction, the Ag-ion SPE tube was pre-conditioned using

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5 mL acetone for three times and then pre-equilibrated using 5 mL hexane for three times.

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The OSPW extract (5 mL) was loaded onto the cartridge and then rinsed using 5 mL

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hexane for three times. SPE fractions were achieved via varying the composition and

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polarity of eluent solvent mixture, and the elution process was optimized as follows:

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fraction 1− fraction 7 (F1−F7) using 5 mL 97/3 (v/v) hexane/acetone for eluting each

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fraction, F8−F13 using 5 mL 93/7 (v/v) hexane/acetone for each, F14−F16 using 5 mL

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88/12 (v/v) hexane/acetone for each, F17−F19 using 5 mL 82/18 (v/v) hexane/acetone for

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each, and F20−F22 using 5 mL acetone for each. 100% acetone for F20−F22 was applied

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to elute out remaining NAs from the SPE cartridge. The SPE fractions were collected in

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test tubes, which were weighed precisely before use, and air-dried completely in the fume

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hood at room temperature. The test tubes with dried fractions were weighed again to give

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the mass of each fraction (Table S1). Dried fractions were re-dissolved using 1 mL 50/50

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acetonitrile/water, with the addition of 1 mg/L myristic acid-1-13C as the internal standard

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to surrogate the instrument fluctuation in analysis, and stored at 4 ºC prior to analysis.

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Instrument method

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An ultra-performance liquid chromatography ion mobility time-of-flight mass

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spectrometry (UPLC-IM-TOFMS) was applied to achieve two-dimensional (2D)

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separation (drift vs. retention time) with integrated travel wave ion mobility mass

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spectrometry (TWIMS) (Synapt G2, Waters Canada). The drift time of the TWIMS

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provided an extra dimension for the separation of molecules based on relative molecule

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sizes. The chromatographic separations were performed using a Waters UPLC Phenyl-

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BEH column (1.7 µm, 150 mm × 1 mm) with a prefilter (0.2 µm). The mobile phases

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were 10 mM ammonium acetate in water (A) and 10 mM ammonium acetate in 50/50

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methanol/acetonitrile (B). The elution gradient was 0−2 min, 1%B; 3 min, 60%B; 7 min,

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70%B; 13 min, 95%B; 14 min, back to 1%B until 20 min to equilibrate the column with a

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flow rate of 100 µL/min. The column temperature was 50 °C while the sample

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temperature was 10 °C. The UPLC method was developed and verified previously.12, 19

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The samples were analyzed with the electrospray ionization (ESI) source, operating

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in negative ion mode, TWIMS in Mobility TOF mode, and the TOF analyzer in high-

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resolution mode (mass resolution of ~40000 at m/z 1431). The gas control was set as 0

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mL/min Source, 2.0 mL/min Trap, 180 mL/min Helium Cell, and 90 mL/min for IMS.

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The data acquisition process was controlled using MassLynx software and the peak

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detection from 2D separation spectra was performed using DriftScope software. The peak

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detection parameters were: chromatographic mode; minimum chromatographic peak

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width, 2.0 min; drift peak width range, 8–42 bins (bin is the drift time unit), and

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minimum intensity threshold (MIT) set as 150 counts for unprocessed and ozonated

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OSPWs and 300 counts for SPE fractions. The peak detection provided the accurate

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masses for each peak, which were then assigned to each of NA species based on exact

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mass matching with a tolerance of ±1.5 mDa. To investigate the structural information of

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the separated species, an MS/MS method was applied with the UPLC method above and

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the ion mobility function deactivated to allow acquiring sufficient signal of product ions

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from complicated samples. The quadrupole was used as a mass filter to choose the parent

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ions. Typically, the small and large product ions are observed separately with low and

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high collision energy (CE) conditions, respectively. In this method, CE was set with a

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range as 25-50 eV to observe the small and large product ions simultaneously, which

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helped to reveal a thorough demonstration of the structural information for the target

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compounds.

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RESULTS AND DISCUSSION

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In this study, the identification of OyS–NAs species from Ox–NAs was achieved

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using the UPLC-IM-TOFMS method based on accurate mass matching with a mass

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tolerance of ±1.5 mDa, due to the mass errors of NAs were measured within this range.

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To separate a mass difference of 3.4 mDa from O2S–NAs to O2–NAs, a mass resolution

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of ~120,000 is typically required for mass spectrometry alone in theory.20 We previous

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reported TOFMS measurements with a mass resolution of ~40,000 and a mass tolerance

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of ±3.0 mDa for peak assignments, and under these conditions, the separation of a mass

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difference of 3.4 mDa could not be achieved.15 As improvement, the present work

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showed that, for current UPLC-IM-TOFMS method, UPLC combined with TWIMS was

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able to separate OyS–NAs from Ox–NAs before these analytes entered the TOFMS mass

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analyzer, obtaining extraordinary mass accuracy for the method. Therefore, the

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identification of OyS–NAs species from OxS–NAs was achieved with a mass tolerance of

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±1.5 mDa for peak assignment. To elucidate this improvement, mass errors between

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measured and theoretical masses for NAs were investigated (Fig. S1 in the SI) by

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comparing O2–NAs and O2S–NAs species in the unprocessed OSPW (Figs. S1a,b), O2–

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NAs and O2S–NAs species in SPE fraction F15 from unprocessed OSPW (Figs. S1c,d),

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and O4–NAs and O4S–NAs species in SPE fraction F15 from ozonated OSPW (Figs.

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S1e,f). The intensity distributions of these samples and fractions, regarding carbon and –

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Z numbers, are shown in Figs. S2-S4, respectively. Fig. S1a showed that the mass errors

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of O2–NAs were grouped into two clusters, one cluster centered at 0 mDa that was

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identified as O2–NAs and another cluster centered at around -3.0 mDa that was actually

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not O2–NAs and subsequently identified as O2S–NAs in Fig. S1b, with a mass tolerance

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of ±1.5 mDa as boundary. It is worth noting that a portion of O2–NAs (upper cluster in

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Fig. S1a) was not present in the mass-overlapping region of O2S–NAs15, thus this portion

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of O2–NAs was not shown in the upper cluster of Fig. S1b. As further validation using

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separated fractions with reduced complexity for composition, compared to OSPW, Figs.

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S1c-f showed separated clusters of two NA species (O2–NAs v.s. O2S–NAs and O4–NAs

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v.s. O4S–NAs) in 2D separation maps with clear boundaries of ±1.5 mDa. Therefore, the

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peak assignment with a mass tolerance of ±1.5 mDa was validated for the identification

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of OyS–NAs species from Ox–NAs and was applied to the UPLC-IM-TOFMS

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characterization and determination of the compositions of OSPWs and SPE fractions to

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corroborate the Ag-ion SPE separation of distinct NA species.

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The unprocessed and ozonated OSPW samples were at first determined by two-

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dimensional (2D) drift time v.s. retention time separation (Figs. 1a,b) using UPLC-IM-

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TOFMS, and Ox–NAs (2≤x≤5) and OyS–NAs (2≤y≤4) species were identified using

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accurate mass matching (Figs. 1c,d). The intensity distributions of NA species, in terms

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of carbon and –Z numbers, are shown in Figs. S2,S5 for the unprocessed and ozonated

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OSPWs, respectively, and the comparison of intensity for distinct NA species is shown in

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Fig. S6. Fig. 1c,d showed that O2–NAs were observed with two separated clusters and the

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lower (in position) cluster was previously verified as saturated acyclic O2–NAs using

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Merichem NA standard.15 The upper cluster of O2–NAs was aromatic O2–NAs based on

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the facts that this cluster was clearly separated from the cluster of saturated acyclic O2–

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NAs and was greatly consumed during the ozonation process (Fig. 1d) as was previously

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reported.15, 16 The results shown in Fig. 1c,d indicated that, in general, the polarity of NA

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compounds follows the order as classical O2–NAs < aromatic O2–NAs < O2S–NAs < O3–

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NAs < O3S–NAs ~ O4S–NAs < O4–NAs < O5–NAs based on the fact that high polarity of

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compounds reduce the retention time on a reverse-phase column21. In addition, few NAs

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from distinct NA species with similar retention time were actually separated in the drift

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time dimension. The similar retention time of few NAs from distinct NA species resulted

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from similar molecular polarity. Increased abundances were found for O3–NAs and O5–

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NAs (Fig. S6), indicating that the ozonation process oxidized NAs, producing NAs with

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more oxygen atoms (e.g. O2–NAs to O3–NAs). Fig. S6 also showed that OyS–NAs were

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greatly consumed during the ozonation process. The results from the unprocessed and

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ozonated OSPWs were compared with subsequent characterization results from SPE

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fractions of the unprocessed and ozonated OSPWs as effective validation method.

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Ag-ion SPE separations have been performed without pre-methylation steps for

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unprocessed and ozonated OSPWs. The eluent solvent for SPE was a mixture of hexane

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(polarity index 0.1) and acetone (polarity index 5.1).21 The percentage of acetone in the

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eluent solvent was increased gradually (3% for fraction 1- fraction 7 (F1-F7), 7% for F8-

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F13, 12% for F14-F16, 18% for F17-F19, and 100% for F20−F22) to elevate the solvent

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polarity.22 Weights of dried fractions (Table S1) showed that the organic matters were

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eluted out with fraction masses within 0.96-1.88 mg for unprocessed/ozonated OSPWs,

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except for the spikes at F20 (3.91/4.22 mg), which was resulted from a sharp increase of

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eluent solvent polarity from 18% to 100% acetone. The sum of fraction masses in Table

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S1 showed that, after ozonation, the total mass of extracted organic matter was reduced

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by 10.3% (30.05 mg to 26.94 mg). This is because ozonation consumed a portion of

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organic compounds and/or some ozonation products were not eluted out from the SPE

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tube. The results of fraction masses also indicated that the gradual increase of eluent

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polarity for multiple elution processes fractionated the organic matters from OSPWs,

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facilitating the subsequent characterization and determination of NA species in separated

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fractions.

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The SPE fractions for unprocessed and ozonated OSPWs were analyzed using

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UPLC-IM-TOFMS to produce 2D separation maps as shown in Fig. S7 (F1-F22 for

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unprocessed OSPW) and Fig. S8 (F1-F22 for ozonated OSPW). Ox–NAs (2≤x≤5) and

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OyS–NAs (2≤y≤4) species were identified and shown in Fig. 2 (F2-F20 from unprocessed

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OSPW) and Fig. 3 (F2-F20 from ozonated OSPW) for comparison. NAs were not

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detected in F1, F2, F21, F22 from unprocessed OSPW (Fig. S7) and F1, F21, and F22

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from ozonated OSPW (Fig. S8) due to NAs were either not readily to be eluted out or

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already completely eluted.

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The intensity distributions of Ox–NAs and OyS–NAs, in terms of carbon and –Z

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numbers, are shown in DATASET1 for all 44 SPE fractions from unprocessed and

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ozonated OSPWs and Fig. 4 compares the intensity (total peak area) of all NAs species in

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SPE fractions. Figs. 2,S7 show that NAs species were eluted out gradually and separated

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clearly into SPE fractions, e.g. classical (saturated acyclic) O2–NAs were separated in F3-

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F5 and aromatic O2–NAs in F7-F11 (F7-F11 also contained O3–NAs) based on the

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separated position of two clusters in accord with Fig. 1c, indicating the physical

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separation of classical and aromatic O2–NAs. Similar separation was observed in Figs.

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3,S8, showing that classical O2–NAs were separated in F2-F5 and the aromatic O2–NAs

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in F8-F10 (F8-F10 also contained O3–NAs). The disruption of intensity for O2–NAs (Fig.

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4a) further confirmed the separation of classical and aromatic O2–NAs species. It was

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also found that the consumption of aromatic O2–NAs in the ozonation process was

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greater than that of classical O2–NAs. This is aligned with the observation reached from

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the ozonated OSPW before SPE.

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O2S–NAs were eluted into F11-F16 (Figs. 2,4e), though some other species were

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also detected in these fractions, e.g. aromatic O2–NAs in F11-F13, O3–NAs in F11-F16,

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and O4–NAs in F16. O2S–NAs were completely consumed during the ozonation process

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(Figs. 3,4e). The consumption of O2S–NAs is consistent with previous results of

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ozonated OSPW before SPE (Fig. S6). O3–NAs were eluted into F8-F16 (Figs. 2,3,4b)

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and O4–NAs were eluted into F16-F20 (Figs. 2,3,4c). A slight amount of O5–NAs was

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eluted into F20 of unprocessed OSPW (Figs. 2,4d), though the ozonated OSPW before

283

SPE actually had higher abundance of O5–NAs than unprocessed OSPW (Fig. S6). This

284

is because more oxygen atoms in NAs actually required increasing the eluent solvent

285

polarity (Figs. 4a-d). The affinity of O5–NAs to solid phase was too strong to be eluted in

286

current solvent polarity conditions. Slight amounts of O3S–NAs in F16-F20 (Figs. 2,4f)

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and O4S–NAs in F14-F20 (Figs. 2,3,4g) were observed but not clearly separated. This

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should be attributed to their strong affinity to solid phase as well as their low initial

289

concentration, prohibiting further investigation. The use of stronger solvent polarity

290

(higher than that of acetone) could help elute the O5–, O3S–, and O4S–NAs. Overall

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results indicated that the elution of distinct NAs species into individual SPE fractions

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(e.g. classical O2–NAs in F3-F5, aromatic O2–NAs in F7-11, O3–NAs in F8-16, O4–NAs

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in F16-F20, O2S–NAs in F11-F16 for SPE fractions from OSPW) was achieved using

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Ag-ion SPE based on the NA compound polarity (primary) and the molecular size for a

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few NAs from distinct species but with similar retention time (polarity). Ag-ion SPE

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separation of OSPW NAs is an important advancement for NA species-specific studies in

297

wastewater treatment and toxicological research. Furthermore, the separated NA species

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in mg level using this method could be widely used in environmental monitoring

299

programs as standard materials, given commercial NA standard comprised of only

300

classical O2–NAs, e.g. Merichem NA standard (Merichem Co.) or Fluka NA standard

301

(Sigma Aldrich), and no NA mixture standard has been produced from synthetic

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chemistry as reported in the published literature.

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Fractions separated using Ag-ion SPE reduced the complexity of composition and

304

matrix compared to unprocessed OSPW samples, facilitating the MS/MS determination

305

and characterization of NA species with reduced interference. Fig. S9 showed the

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MS/MS spectra of NA compounds in SPE fractions from unprocessed OSPW and

307

detailed information is summarized in Table 1. Mass errors were listed for all peaks with

308

the average value of -1.8 mDa. The –Z numbers were calculated for all ions, assuming

309

that the addition of one proton as negative electrospray ionization removed one proton

310

from each molecule, to rule out predicted chemical structure with impossible Z number

311

(e.g. –Z was much bigger for product ions than precursor ions).

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Two abundant compounds for each of O2–NAs, O3–NAs, O4–NAs, and O2S–NAs

313

were chosen for MS/MS analysis, except for O5–NAs, O3S–NAs, and O4S–NAs due to

14


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314

their low intensity. Parent ions of acyclic O2–NAs were not broken down to generate

315

product ions, indicating low reactivity of acyclic O2–NAs.23 Parent ions of aromatic O2–

316

NAs were found to lose –COOH group because of the strong C–C bonding in phenyl

317

rings (bond lengths are 1.381 Å for C–C in phenyl rings and 1.514 Å for regular C–C).24

318

For O3–NAs and O4–NAs, the product ion was found to contain at least one oxygen atom

319

that could be the single hydroxyl group in the molecules when losing –COOH group.12

320

Product ions of O2S–NAs usually contained the sulfur atom while losing –COOH, due to

321

the bonding energy of C–S is higher than C–C (713.3 ± 1.2 KJ/mol for C–S and 618.3 ±

322

15.4 KJ/mol for C–C)24, making C–S harder to break. Some product ions could be

323

predicted as either O2–NAs or O2S–NAs based on the accurate masses, but actually only

324

one structure is reasonable. As illustration, 133.0090 Da (Table 1) could be identified as

325

C8H5S- (-2.2 mDa error) or C11H1- (1.2 Da error), latter of which was, however, not a

326

reasonable chemical structure with –Z = 20 much bigger than the precursor ion (–Z = 14).

327

Because of the intramolecular rearrangement reaction occurring to the hydroxyl group

328

during electrospray ionization, an increase of –Z by 2 for product ions was expected.25

329

MS/MS characterized distinct NA species in separated SPE fractions and the results

330

validated Ag-ion SPE separation and UPLC-IM-TOFMS identification of Ox–NAs and

331

OyS–NAs species.

332

333

Environmental Significance

334

NAs are present in process water of crude oil and unconventional oil, e.g., oil sands,

335

causing public concern to environmental health.1 Previous toxicological studies have

336

compared the toxicity in aromatic and nonaromatic species, and similar assessment has 15



337

not been performed widely on distinct NA species (e.g., oxidized or heteroatomic NAs

338

species) because they were not readily separated.11 NAs are also the primary reason for

339

corrosion damage to oil refinery equipment, with sulfur-containing compounds playing

340

an important role.26 It is expected that distinct NAs species can be separated, paving the

341

way to other related studies. In this work, Ag-ion SPE was used to fractionate Ox–NAs

342

and OyS–NAs from unprocessed and ozonated OSPWs and the determination and

343

characterization of fractions were performed using UPLC-IM-TOFMS and MS/MS

344

analyses. This method is important to the research community due to the method is able

345

to separate classical, aromatic, oxidized, and sulfur-containing NA species, including but

346

not limit to aromatic and nonaromatic NAs. Future water treatment and toxicological

347

studies could benefit from separated NA species without being interfered from

348

unnecessary methylation and de-methylation. Moreover, the proposed method separated

349

NA species that could be widely used as the standard material in the environmental

350

monitoring and be an advancement compared with current Fluka or Merichem NA

351

standards that are comprised of only classical NAs.

352

353

ASSOCIATED CONTENTS

354

Supporting Information

355

Additional information as noted in the text, including description of the ozonation

356

process, figures/tables for ion mobility spectra, SPE fraction masses, NAs peak areas for

357

OSPWs and two SPE fractions, as well as MS/MS spectra. Another additional file

358

provided an integrated dataset for NAs peak areas of all SPE fractions in terms of carbon

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359

and Z numbers. The materials are available free of charge via the Internet at

360

http://pubs.acs.org.

361

362

ACKNOWLEDGEMENTS

363

This research was supported by research grants from the Helmholtz-Alberta

364

Initiative (HAI) and a Natural Sciences and Engineering Research Council of Canada

365

(NSERC) Senior Industrial Research Chair in Oil Sands Tailings Water Treatment

366

through the support by Syncrude Canada Ltd., Suncor Energy Inc., Shell Canada,

367

Canadian Natural Resources Ltd., Total E&P Canada Ltd., EPCOR Water Services,

368

IOWC Technologies Inc., Alberta Innovates - Energy and Environment Solution, and

369

Alberta Environment and Parks. The authors also thank Dr. Md. Shahinoor Islam for

370

conducing the ozonation experiments.

371 372

REFERENCES

373 374 375 376 377 378 379 380 381 382 383 384 385 386

1. Headley, J. V.; Peru, K. M.; Barrow, M. P., Advances in mass spectrometric characterization of naphthenic acids fraction compounds in oil sands environmental samples and crude oil-A review. Mass Spectrom. Rev. 2016, 35, (2), 311-28. 2. Hudson, P. V., History of environmental contamination by oil sands extraction. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, (5), 1569-1570. 3. Kurek, J.; Kirk, J. L.; Muir, D. C. G.; Wang, X.; Evans, M. S.; Smol, J. P., Legacy of a half century of Athabasca oil sands development recorded by lake ecosystems. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, (5), 1761-1765. 4. Swigert, J. P.; Lee, C.; Wong, D. C. L.; White, R.; Scarlett, A. G.; West, C. E.; Rowland, S. J., Aquatic hazard assessment of a commercial sample of naphthenic acids. Chemosphere 2015, 124, 1-9. 5. Kim, E.-S.; Liu, Y.; Gamal El-Din, M., Evaluation of membrane fouling for in-line filtration of oil sands process-affected water: the effects of pretreatment conditions. Environ. Sci. Technol. 2012, 46, (5), 2877-2884.

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6. Scarlett, A. G.; Reinardy, H. C.; Henry, T. B.; West, C. E.; Frank, R. A.; Hewitt, L. M.; Rowland, S. J., Acute toxicity of aromatic and non-aromatic fractions of naphthenic acids extracted from oil sands process-affected water to larval zebrafish. Chemosphere 2013, 93, (2), 415-420. 7. He, Y.; Patterson, S.; Wang, N.; Hecker, M.; Martin, J. W.; El-Din, M. G.; Giesy, J. P.; Wiseman, S. B., Toxicity of untreated and ozone-treated oil sands process-affected water (OSPW) to early life stages of the fathead minnow (Pimephales promelas). Water Res. 2012, 46, (19), 6359-6368. 8. Wang, N.; Chelme-Ayala, P.; Perez-Estrada, L.; Garcia-Garcia, E.; Pun, J.; Martin, J. W.; Belosevic, M.; Gamal El-Din, M., Impact of Ozonation on Naphthenic Acids Speciation and Toxicity of Oil Sands Process-Affected Water to Vibrio fischeri and Mammalian Immune System. Environ. Sci. Technol. 2013, 47, (12), 6518-6526. 9. Tollefsen, K. E.; Petersen, K.; Rowland, S. J., Toxicity of Synthetic Naphthenic Acids and Mixtures of These to Fish Liver Cells. Environ. Sci. Technol. 2012, 46, (9), 5143-5150. 10. Garcia-Garcia, E.; Pun, J.; Hodgkinson, J.; Perez-Estrada, L. A.; El-Din, M. G.; Smith, D. W.; Martin, J. W.; Belosevic, M., Commercial naphthenic acids and the organic fraction of oil sands process water induce different effects on proinflammatory gene expression and macrophage phagocytosis in mice. J. Appl. Toxicol. 2012, 32, (12), 968-79. 11. Reinardy, H. C.; Scarlett, A. G.; Henry, T. B.; West, C. E.; Hewitt, L. M.; Frank, R. A.; Rowland, S. J., Aromatic naphthenic acids in oil sands process-affected water, resolved by GCxGC-MS, only weakly induce the gene for vitellogenin production in zebrafish (Danio rerio) larvae. Environ. Sci. Technol. 2013, 47, (12), 6614-6620. 12. Huang, R.; Sun, N.; Chelme-Ayala, P.; McPhedran, K. N.; Changalov, M.; Gamal ElDin, M., Fractionation of oil sands-process affected water using pH-dependent extractions: A study of dissociation constants for naphthenic acids species. Chemosphere 2015, 127, 291-296. 13. Mohamed, M. H.; Wilson, L. D.; Shah, J. R.; Bailey, J.; Peru, K. M.; Headley, J. V., A novel solid-state fractionation of naphthenic acid fraction components from oil sands process-affected water. Chemosphere 2015, 136, 252-8. 14. Nuernberg, K.; Dannenberger, D.; Ender, K.; Nuernberg, G., Comparison of different methylation methods for the analysis of conjugated linoleic acid isomers by silver ion HPLC in beef lipids. J. Agric. Food Chem. 2007, 55, (3), 598-602. 15. Huang, R.; McPhedran, K. N.; Gamal El-Din, M., Ultra Performance Liquid Chromatography Ion Mobility Time-of-Flight Mass Spectrometry Characterization of Naphthenic Acids Species from Oil Sands Process-Affected Water. Environ. Sci. Technol. 2015, 49, (19), 11737-45. 16. Sun, N.; Chelme-Ayala, P.; Klamerth, N.; McPhedran, K. N.; Islam, M. S.; PerezEstrada, L.; Drzewicz, P.; Blunt, B. J.; Reichert, M.; Hagen, M.; Tierney, K. B.; Belosevic, M.; Gamal El-Din, M., Advanced analytical mass spectrometric techniques and bioassays to characterize untreated and ozonated oil sands processaffected water. Environ. Sci. Technol. 2014, 48, (19), 11090-11099. 17. Huang, R.; McPhedran, K. N.; Yang, L.; El-Din, M. G., Characterization and distribution of metal and nonmetal elements in the Alberta oil sands region of Canada. Chemosphere 2016, 147, 218-229.

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18. Huang, R.; McPhedran, K. N.; Sun, N.; Chelme-Ayala, P.; Gamal El-Din, M., Investigation of the impact of organic solvent type and solution pH on the extraction efficiency of naphthenic acids from oil sands process-affected water. Chemosphere 2016, 146, 472-477. 19. Hwang, G.; Dong, T.; Islam, M. S.; Sheng, Z.; Perez-Estrada, L. A.; Liu, Y.; Gamal El-Din, M., The impacts of ozonation on oil sands process-affected water biodegradability and biofilm formation characteristics in bioreactors. Bioresour. Technol. 2013, 130, 269-277. 20. Pereira, A. S.; Bhattacharjee, S.; Martin, J. W., Characterization of Oil Sands Process-Affected Waters by Liquid Chromatography Orbitrap Mass Spectrometry. Environ. Sci. Technol. 2013, 47, (10), 5504-5513. 21. Snyder, L. R.; Kirkland, J. J.; Dolan, J. W., Introduction to Modern Liquid Chromatography, 3rd Edition. Wiley: New Jersey, 2010. 22. Jouyban, A.; Soltanpour, S.; Chan, H.-K., A simple relationship between dielectric constant of mixed solvents with solvent composition and temperature. Int. J. Pharm. 2004, 269, (2), 353-360. 23. Headley, J. V.; Peru, K. M.; Armstrong, S. A.; Han, X.; Martin, J. W.; Mapolelo, M. M.; Smith, D. F.; Rogers, R. P.; Marshall, A. G., Aquatic plant-derived changes in oil sands naphthenic acid signatures determined by low-, high- and ultrahigh-resolution mass spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, (4), 515-522. 24. Lide, D. R., CRC Handbook of Chemistry and Physics, 90th Edition. CRC: Boca Raton, 2010. 25. Robinson, J. W.; Frame, E. M. S.; Frame II, G. M., Undergraduate Instrumental Analysis, 6th Ed. Marcel Dekker: New York, 2005. 26. Mejia-Miranda, C.; Laverde, D.; Molina V, D., Correlation for Predicting Corrosivity of Crude Oils Using Proton Nuclear Magnetic Resonance and Chemometric Methods. Energ. Fuel. 2015, 29, (11), 7595-7600.

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Table 1. Summary of parent and product ions for NA species from MS/MS spectra of SPE fractions from unprocessed OSPW. NA Measured Error Chemical Retention -Z Note Fraction Species Mass (Da) (mDa) Formulae time (min) acyclic 209.1530 -1.2 C13H21O24 Parent ion 5.16 F4 O2−NA acyclic 237.1845 -1.0 C15H25O24 Parent ion 6.08 F4 O2−NA aromatic 243.1385 -2.7 C16H19O212 Parent ion 5.07 F8 O2−NA 225.1261 -1.8 C16H17O14 Product ion 199.1503 1.6 C15H1910 Product ion 197.1331 0.1 C15H17 12 Product ion 183.1172 -0.2 C14H15 12 Product ion 143.0812 -4.9 C11H11 10 Product ion aromatic 257.1512 -3.0 C17H21O212 Parent ion 5.32 F8 O2−NA 239.1432 -0.4 C17H19O14 Product ion 213.1653 1.0 C16H21 10 Product ion 171.1179 0.5 C13H15 10 Product ion 157.1009 -0.8 C12H13 10 Product ion O3−NA 239.1635 -1.2 C14H23O34 Parent ion 4.74 F9 222.1566 -5.4 C14H22O2 5 Product ion 195.1750 0.1 C13H23O 2 Product ion 193.1569 -2.3 C13H21O 4 Product ion 139.1138 1.5 C9H15O2 Product ion O3−NA 249.1505 1.4 C15H21O3 8 Parent ion 4.71 F11 221.1549 0.7 C14H21O2 6 Product ion 205.1600 0.8 C14H21O 6 Product ion 203.1438 0.2 C14H19O8 Product ion 163.1126 0.3 C11H15O 6 Product ion 148.0916 2.8 C10H12O 7 Product ion O4−NA 309.2036 -3.0 C18H29O4 6 Parent ion 4.71 F17 291.1934 -2.6 C18H27O38 Product ion 265.2144 -2.4 C17H29O24 Product ion 247.2027 -3.5 C17H27O 6 Product ion O4−NA 323.2181 -4.1 C19H31O4 6 Parent ion 4.88 F17 305.2102 -1.5 C19H29O38 Product ion 279.2300 -2.4 C18H31O24 Product ion 261.2202 -1.6 C18H29O 6 Product ion O2S−NA 259.0710 -8.3 C15H15O2S 14 Parent ion 4.94 F15 213.0697 -4.1 C14H13S14 Product ion 201.0723 -1.5 C13H13S12 Product ion



O2S−NA

148.0305 133.0078 123.0231 287.1037 243.1154 201.0723 161.0379 133.0090

-4.2 -2.2 -3.7 -6.9 -5.3 -1.5 -4.6 -2.2

C9H8SC8H5SC7H7SC17H19O2SC16H19SC13H13SC10H9SC8H5S-

9 10 6 14 12 12 10 10


Product ion Product ion Product ion Parent ion Product ion Product ion Product ion Product ion

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5.49

F15

Page 23 of 26


Figure 1. 2D separation maps for unprocessed (a) and ozonated (b) OSPW. Colors on maps indicate the relative intensity, with light-yellow colored cluster illustrating the areas of most abundant peaks. The horizontal strip at 2.5 ms is an artifact of the samples matrix. The spectrum peaks were acquired using DriftScope (Waters Canada) and indicated as black markers. The Ox−NAs (2≤x≤5) and OyS−NAs (2≤y≤4) species were identified based on the match of accurate masses for unprocessed (c) and ozonated (d) OSPW.



Figure 2. The Ox−NAs (2≤x≤5) and OyS−NAs (2≤y≤4) species were identified based on the match of accurate masses for Ag-ion SPE fractions F2-F20 from unprocessed OSPW. No NAs was detected in the Ag-ion SPE fractions F1, F2, F21, and F22.


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Figure 3. The Ox−NAs (2≤x≤5) and OyS−NAs (2≤y≤4) species were identified based on the match of accurate masses for Ag-ion SPE fractions F2-F20 from ozonated OSPW. No NAs was detected in the Ag-ion SPE fractions F1, F21, and F22.



Figure 4. Comparison of intensity (peak area) for O2−NAs (a), O3−NAs (b), O4−NAs (c), O5−NAs (d), O2S−NAs (e), O3S−NAs (f), and O4S−NAs (g) in Ag-ion SPE fractions of unprocessed and ozonated OSPWs.


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