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IMPROVED UHPLC-MS/MS METHODS FOR ANALYSIS OF ISOPRENE-DERIVED ORGANOSULFATES Grzegorz Spólnik, Paulina Wach, Krzysztof J. Rudzinski, Krzysztof Skotak, Witold Danikiewicz, and Rafal Szmigielski Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05060 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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Analytical Chemistry

IMPROVED UHPLC-MS/MS METHODS FOR ANALYSIS OF ISOPRENE-DERIVED ORGANOSULFATES GRZEGORZ SPOLNIK*†, PAULINA WACH‡, KRZYSZTOF J. RUDZINSKI‡, KRZYSZTOF SKOTAK&, WITOLD DANIKIEWICZ† AND RAFAL SZMIGIELSKI*‡ †

Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, 01-224, Poland Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, 01-224, Poland & Institute of Environmental Protection, National Research Institute, Warsaw, 00-548, Poland Corresponding Authors: Rafal Szmigielski Grzegorz Spolnik Phone: +48 22 343 3402 Phone: +48 22 343 22 11 Fax: +48 22 3433333 Fax: +48 22 632 66 81 E-mail: [email protected] E-mail: [email protected] ‡

ABSTRACT: The secondary organic aerosol (SOA) is an important yet not fully characterized constituent of atmospheric particulate matter. A number of different techniques and chromatographic methods are currently used for the analysis of SOA, so the comparison of results from different laboratories poses a challenge. So far, tentative structures have been suggested for many organosulfur compounds that have been identified as markers of the formation of SOA, including isoprene-derived organosulfates. Despite the effectiveness and robustness of LC-MS/MS analyses, the structural profiling of positional isomers of recently discovered organosulfates with molecular weights (MWs) of 214 and 212 from isoprene was entirely unsuccessful. Here, we developed a UHPLC combined with high-resolution tandem mass spectrometric method that significantly improves the separation efficiency and detection sensitivity of these compounds in aerosol matrices. We discovered that selection of the proper solvent for SOA extracts was a key factor in improving the separation parameters. Later, we took advantage of the enhanced sensitivity, combined with a short scan time window, to perform detailed structural mass-spectrometric studies. For the first time, we elucidate a number of isomers of the MW 214 and the MW 212 organosulfates and provide strong evidence for their molecular structures. The structure of trihydroxyketone sulfate MW 214 that we propose has not been previously reported. The methods we designed can be easily applied in other laboratories to foster an easy comparison of related qualitative and quantitative data obtained throughout the world.

Atmospheric aerosols are a major pollutant of the Earth’s atmosphere. They consist of fine particulate matter with diameters below 100 µm that are suspended in the air. A large proportion of aerosols originate in the atmosphere through the chemical transformation of volatile organic compounds (VOCs) followed by gas-to-particle partitioning and/or transfer, followed by further reactions therein1-3. These aerosols are known as secondary organic aerosols (SOA). Consequently, SOA particles are complex mixtures of organic and inorganic compounds that have a negative impact on human health, influence the biosphere and take part in climate change4,5.

methyltetrol sulfates12 have been systematically discovered as relevant markers of SOA formation from α-pinene and isoprene, respectively. The compounds were quantified in ambient aerosol samples from many sites13-16. The studies revealed the presence of other monoalkyl sulfate esters (organosulfates, OS) with low17-21 and high molecular weights (MW)22, nitroorganic compounds23 and carboxylated derivatives thereof. Most of these compounds are strongly hydrophilic and occur as minute quantities in samples. Thus, they are difficult to separate or analyze with conventional techniques and resist detailed identification of molecular structure, including differentiation of positional and/or stereo isomers.

The organic fraction of atmospheric aerosols is one of the most important subjects of recent atmospheric studies6. The molecular identification and quantification of organic aerosol components, as well as revealing their precursors and formation routes, have been strongly emphasized as crucial challenges7. The intensive research on SOA formation and composition has resulted in numerous species that have been identified as markers of their natural precursors4,7-10. For instance, 3methyl-1,2,3-butanetricarboxylic acid (MBTCA)11 and 2-

Prior to 2005, organosulfates in aerosol samples were largely overlooked because GC-MS techniques failed to measure them due to their low volatilities and chemical instability to derivatization24. Therefore, several hyphenated methods have been used to analyze organosulfates in aerosol samples, including ion mobility spectrometry-MS25, capillary electrophoresis-MS26,27, LC-MS19,20,28-30 or UHPLC-MS31-35 techniques in the negative ion mode. The main drawbacks of these methods

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for organosulfate speciation arise from a close similarity of their isomeric forms, which impedes efficient chromatographic separation, as well as the lack of authentic standards for full validation of quantitative methods7. A number of compounds could not be readily identified due to the lack of dedicated mass spectral libraries and authentic standards. Among these compounds were the isoprene-derived C5 organosulfates that have been detected in abundant amounts in recent field studies32,36 and smog-chamber experiments37,38. Their structures have been tentatively proposed36,39 (Chart 1: sulfate ester of 2methyltetrol, OS 216; cyclic methyltrihydroxyaldehyde hemiacetal, OS 214 and methyldihydroxylactone, OS 212) but without differentiation between their regio- and stereoisomers. The chromatographic separation methods have failed to provide baseline-separated peaks (e.g., OS 154 and OS 216)40. For some other SOA isoprene organosulfates (e.g., OS 212 and OS 214), only groups of peaks were reported with no clear indication of the number of isomers present17,41. Under these conditions, the capabilities of mass spectrometric structural analysis were insufficient to provide satisfactory results. Therefore, the quantification of SOA components of complex isomeric profiles remains a challenge for analytical atmospheric chemists.

Chart 1. Postulated structures of the C5 isoprene derived organosulfates. The observable positional isomers differ in the positions of methyl and sulfate ester moieties.

The goal of this study was to optimise and improve commonly used UHPLC methods and create a tool for the qualitative comparison for SOA compositions obtained in different environmental laboratories. Naturally, the tool is intended to provide reproducible results for SOA samples of different origins. Thus, it should accommodate differences in matrix composition and be sensitive.. We analyzed, in detail, the chromatographic behavior of the most polar components of SOA under conditions of different buffers, pH values, column temperatures and flow rates. We optimized the MS parameters to increase the method sensitivity. Finally, we revised the sample preparation procedure by checking the influence of sample solvents on the results. Another goal of this work was to apply the improved method to the structural analysis of the main isoprene-derived C5 organosulfates, which is of great importance for the recognition of atmospheric SOA.

EXPERIMENTAL SECTION Sample collection and extraction. Samples of ambient PM2.5 aerosol (particulate matter smaller than 2.5 µm) were collected on 15-cm quartz filters using a high-volume aerosol sampler (Digitel DHA-80, Switzerland) at the Diabla Góra field station, located in the Puszcza Borecka forest in northeastern Poland (54°07’29.52″N and 22°02’17.08″E) from July 26-31, 2014. Before sampling, the filters were pre-baked in a labora-

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tory oven at ca. 500 °C for 24 h to remove organic traces. Located far from cities, industrial facilities and heavy-traffic roads, the Diabla Gora forest is considered to be a regional background station for monitoring clean air masses. The site is densely covered by broad-leaf forests, which provide an important local source for isoprene release. The filters were exposed for 24 hours in two-week series and were then wrapped in paper and stored at -20 °C before further analysis. Two 1-cm2 punches from each filter were extracted three times with 10 ml of methanol using an Orbital shaker (Biosan, Poland). The extracts were combined and evaporated to ca. 1 ml, filtered through a 0.2 µm PTFE membrane syringe filter and dried under a gentle stream of nitrogen. Dry samples were dissolved in 140 µl of 1:1 v/v methanol-water mixture and were analyzed. To verify the influence of the sample solvents, the samples were evaporated under a gentle stream of nitrogen and were re-dissolved in pure water and/or pure methanol. Chromatographic methods. In our approach, we used an Acquity I-Class liquid chromatograph (Waters) coupled with a Synapt G2-S Q-TOF mass spectrometer (Waters). All of the variable experimental conditions are described in detail in the following section. The optimized RP-C18 chromatographic conditions were: Acquity HSS T3 1.8 µm column (2.1 x 100 mm) (Waters) with temperature set to 40 °C and the gradient elution started from 100 % of mobile phase A (10 mM ammonium acetate) with a 0.35 ml/min flow rate for 1.7 minutes, then changed to 100 % of mobile phase B (methanol) with a flow rate 0.25 ml/min in 4.7 minutes and maintained constant for 1 minute. Since our analytical target was the most polar fraction of the SOA that eluted in the first two minutes, a steep gradient slope was set to save time. The optimized HILIC conditions were the Acquity BEH Amide 1.7 µm column (2.1 x 100 mm) (Waters) with the temperature set to 45 °C and the gradient elution started from 100 % of mobile phase B (acetonitrile : water 95:5 (v/v) with 10 mmol ammonium acetate adjusted to pH 9 using NH3 solution) for 3 minutes then changed to 40 % of mobile phase A (water with 10 mmol ammonium acetate adjusted to pH 9 using NH3 solution) for 3 minutes and maintained constant for 1 minute, then brought back to initial conditions that were maintained for 5 minutes to equilibrate the column. The flow rate was 0.5 ml/min. Mass spectrometer parameters. We used a Synapt G2-S mass spectrometer that was equipped with an electrospray ion source to perform all experiments. The instrument worked in the negative ion mode in the sensitivity mode with the resolving power of the TOF analyzer 20000 FWHM. An external calibration on sodium formate was used with a lock spray correction on the leucine-enkephalin spectrum that was generated by the lock spray source. The exact mass measurements for all peaks were performed within 3 mDa mass error. A 0.1 s scan time was set for the full scan experiments and a 0.2 s scan time was set for the MS/MS experiments. The optimized source parameters were: capillary voltage -0.3 kV, cone voltage -15 V, desolvation gas flow 800 L/h with the temperature 350 °C, nebulizer gas pressure 5.5 bar, source temperature 120 °C. All presented MS/MS experiments data was recorded using collision energy 20 eV. The EIC window was +/-0.0015. The instrument was controlled and the data analysis was performed with the MassLynx 4.1 software.


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RESULTS AND DISCUSSION The reversed phase chromatography on C18 column was our first method of choice. We performed the mobile phase and column temperature optimization process to improve the separation efficiency. The description of this process was presented in Supplementary Information The matrix effect and the sample solvent. A commonly applied procedure for the preparation of filter SOA samples 7,20 consists of the extraction, filtration, evaporation and reconstitution of aerosol extracts in a methanol : water solution13,32,37,40. Some of the chromatograms that were obtained in this manner, especially with higher injection volumes, resulted in either peak-broadening or the appearance of ghost peaks (Figure S5). This phenomenon can be explained in two ways, either by the injection matrix effect or by an incorrect selection of the sample solvent. The injection matrix effect can be easily verified by a sample-to-sample comparison, in which the different samples are prepared in exactly the same procedure. Our longterm experience with SOA sample analysis showed that the ambient aerosol samples that were collected during different field campaigns and prepared using the same procedure could result in varying quality of the chromatographic separation despite of the use of the exact same chromatographic parameters. For example, PM2.5 aerosol samples collected from Kpuszta in Hungary (details are given elsewhere20) revealed significantly better isomeric separation for the MW 212 organosulfate than those collected from the Diabla Góra forest in Poland, despite the fact that the former extract was far more concentrated in terms of the analyte concentration (Figure 1). The MW 212 organosulfate is another key biogenic SOA component and has recently been reported from field36 and laboratory studies31. The matrix effect that occurs in the aerosol filter sample analyses is difficult to avoid owing to an inability to remove an inorganic matrix from the SOA samples without risking an uncontrolled loss of polar analytes31.

Figure 1. Comparison of chromatographic responses acquired for ambient fine aerosol extracts collected at two different sampling sites, K-puszta (top) and Diabla Gora (bottom). EICs are given for the m/z 210.991 ion corresponding to regioisomers of lactonebased isoprene organosulfate. The analyses were performed using non-optimized conditions and are presented to show the unmanaged matrix effect.

The common reconstitution of the dried aerosol filter extract using a methanol : water mixture before the analysis was used to ensure good solubility of all known and possible components of the SOA samples. However, the composition of the sample solvent should be different when the analysis starts, with the majority of the aqueous mobile phase at the injection point. When the injected sample contains greater amounts of the stronger eluent (in this case, less polar - methanol), it forms in the zone at the front of the LC column, where an excess of the stronger eluent is present. Providing that the analyte suffers from weak retention in the column, it is very sensitive for such a small change in the characteristic of the mobile phase, and thus it takes a lot of time for it to escape from the zone during the retention process. A large volume of analyte can travel through the column in the strong eluent excess zone, forming an additional peak (ghost peak), or needs some time to escape, which causes a broad peak as shown in Figure S5.



To the best of our knowledge, the only examples of organosulfate LC-MS analysis using water as the only solvent were presented by Gómez-González and co-workers42,43. However, for the isocratic flow, the authors did not use 100 % water in the onset of a gradient elution, and thus, their chromatography did not show a significant improvement of the separation, which is in stark contrast to our results. We modified the sample solvent composition in combination with the optimized method and verified how it affected the retention factor of the polar SOA organosulfates. First, we added the buffer (mobile phase A) to the sample in different amounts and observed that, together with an increasing buffer-to-sample ratio, the peak shapes and the resolution were markedly improved (Figure 2). The best result was achieved for the sample that was evaporated and re-dissolved in a pure buffer solution. No ghost peaks were observed under these conditions. Since the buffer addition to the sample could be treated as an irreversible interference in the sample composition and in its stability, the reconstitution of the sample in pure water proved to be a good alternative (Figure S6). Moreover, the separation quality of the SOA samples that were re-dissolved in buffer or pure water was far less dependent on the injection volume. As a result, this approach - significantly increased method sensitivity. Even with an injection volume of 10 µl, peak resolution was retained. Hence, the proposed sample preparation is a crucial step in the chemical analysis of the SOA samples that contain several isomeric organosulfates and could be applied in the structural identification of these species (details in the section on the structural differentiation of isoprene-derived compounds).

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much better. For the compounds OS 212 and OS 214, we obtained much more effective separation. The latter is another key SOA component and recently has been reported in data from both field36 and laboratory31 studies. We obtained several major peaks that were fully resolved (OS 214) or qualitatively separated (OS 212) (Figure 3). The HILIC method is less dependent on sample solvent, although a pure less-polar solvent worked slightly better. The drawback of the HILIC method was its low versatility with respect to the compounds that had very different polarity and poor retention of less polar compounds, for example, nitroxyorganosulfates of α-pinene (Figure S7). The more difficult to obtain reproducibility in the HILIC method was the second reason for regarding it only as a supporting method. It must be stressed, however, that in the case of the isoprene-derived organosulfates that are discussed in the following part of this paper, the HILIC method results in a more effective chromatographic separation. For this reason, it is recommended to select the methodology according to the purpose of analysis, with respect to its advantages and disadvantages. Mass spectrometer parameters. The main advantage of our mass spectrometric approach was using a 0.1 s scan time. It produced a much denser set of points on the chromatogram and empowered us to differentiate the peaks that are separated in much less than 0.01 min. It was very important for the analysis of the samples that contained hundreds of nearly inseparable compounds. It was also important to determine whether or not the less-abundant ion was derived from source fragmentation of the co-eluting ion and to confirm that it was a separate compound. Furthermore, we optimized the Synapt spectrometer parameters, showing that only -0.3 kV of capillary voltage is the optimal setting regarding the sensitivity. Moreover, the optimization of the parameters that affected sensitivity allowed us to apply a 0.2 s scan time to the MS/MS experiments, which was a crucial procedure for determining the coeluting isomeric MW 212 organosulfates.

Figure 2. Differences in the separation quality of regioisomers of the MW 212 isoprene organosulfate with respect to various solvent compositions. The appearance of ghost peaks (RT = 0.85 min) and peak-broadening in the range 0.90 – 1.35 min. is clearly visible.

HILIC chromatography. As the second method of choice for the analysis of organosulfates, we used HILIC chromatography separation with a BEH Amide column with the same elution conditions that were suggested by Hettiyadura and coworkers36. In contrast to their report, the results obtained in our analyses were substantially better than those they presented. However, for the OS 216, we obtained very similar extracted ion current (EIC) chromatograms, but the peak resolution was

Figure 3. EICs for MWs 216, 214 and 212 isoprene-derived organosulfates with elemental formulas C5H11O7S, C5H9O7S and C5H7O7S, respectively, recorded in a HILIC mode.

Structural differentiation of organosulfates. Using the optimized methods, we analyzed the group of isoprene derived organosulfates (OS 216, OS 214 and OS 212) in detail. Structures for these compounds were tentatively proposed else-


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where as sulfate esters of 2-methyltetrols (OS 216), cyclic hemiacetals (OS 214) and lactones (OS 212), respectively (Chart 1)24,36, but no isomeric assignment was performed. We aimed to use the improved chromatographic methods to gain insights into the structural profile of their isomers on the basis of detailed interpretation of fragment ion mass spectra.

OS 212. In the EIC for the m/z 210.991 ion that was recorded in a RP-C18 mode, we observed four well-separated peaks and one additional shoulder peak. Peaks at 0.63 and 0.85 min

Figure 4. Chromatographic traces recorded for the two m/z 211/211 and m/z 211/97 ion pairs for the Diabla Gora fine aerosol extract using C18 (top panel) and HILIC (bottom panel) chromatography. A detailed analysis of the MS/MS profiles for the m/z 211 organosulfate allows for in-depth structural differentiation of seven isomers thereof.

correspond to compounds with the same molecular formula but different linear structures and were identified as carboxylic acids. Accidentally, the peak with RT = 0.85 min (top panel in Figure 2) overlaps with the ghost peak that is shown in the bottom panel. However, the HILIC method showed the presence of seven not fully baseline-separated peaks on the EIC plot for the same exact mass (Figure 3). To rationalize this incoherence, we performed a detailed analysis of the MS/MS experimental data that were recorded with the 0.2 s scan time. EIC traces for the parent ion m/z 211 and fragment ion m/z 97 that were obtained with a C18 column revealed that the first two peaks on the chromatogram were composed of two pairs of co-eluting different compounds. This increased the number of peaks to seven, exactly the same number as in the chromatogram that was obtained with the HILIC method (Figure 4). As shown, the 211→211 trace gives a peak when the m/z 211 precursor ion is nearly intact upon fragmentation at a given collision energy and the 211→97 gives a peak when the precursor ion m/z 211 is easily fragmented, affording the HSO4ion (m/z 97). When the peaks in different traces are shifted as in the C18 analysis for peaks 1 and 2 or 3 and 4, we can deduce that there are two isomeric compounds that differ slightly in retention time. This results in the merging of two peaks into one in the single ion trace (top panel in Figure 2) and explains a missing number of isomers. When we precisely collected the fragmentation spectra for the parts of the peaks that did not overlap with others and compared the spectra from the C18 and HILIC methods, we found seven pairs of identical MS/MS spectra (Figures S8-S14). We could ascertain that the OS212 compound was a mixture of seven structurally similar isomers and assigned every peak on the chromatograms to a unique compound. Further attempts to assign structures of OS 212 single isomers using their distinct fragmentation behaviors were based on the tendency to detach the bisulfate (HSO4-) anion. It is restricted

by the accessibility of the vicinal hydrogen atom in a cis configuration relative to the -OSO3- group, which results from the mechanism of this particular fragmentation reaction44. In only two spectra that correspond to peaks 2 and 7, the abundance of the m/z 97 ion was close to zero (for all product ion spectra, see Figures S8-S14). Instead, the m/z 193 peaks were observed, indicating the loss of water. These results clearly show that 2 and 7 are two diastereoisomers of lactone, bearing both methyl and free hydroxyl groups in position 4 (compounds 1a and 1b, Chart 2). In these molecules, there are no hydrogen atoms vicinal to the –OSO3- group, but there are hydrogen atoms vicinal to the OH group, which disables the elimination of HSO4- and enables the elimination of water. The structures of the other five hydrogen sulfates cannot be established unequivocally; however, some reasonable proposals can be formulated.

Chart 2. Tentative structures of the isomers of OS 212. O 1

2

3

O 5

O OSO 3H

4

OH CH3

1a,b peaks 2 and 7

O OH

O

O OSO3H CH3

OSO 3 H CH3 OH 3a, b

2a, b peaks 1, 3 and 5

O

OH CH3

O

OSO3H 4a, b peaks 4 and 6

In the MS/MS spectra of the m/z 211 ions that correspond to chromatographic peaks 1, 3 and 5 in Figure 4, the m/z 97 fragment ion is the only one, or is at least the most abundant. In contrast, in the case of chromatographic peaks 4 and 6, the relative intensities of the parent ion (m/z 211) and the fragment



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ion peak (m/z 97) are comparable, indicating that elimination of the HSO4- ion is still possible; however, the fragmentation reaction is hampered. Comparing the structures of likely isomers (Chart 2), it is apparent that in organosulfates 4a and 4b, there is only one vicinal hydrogen atom cis to the -OSO3residue, so there is only one fragmentation channel leading to the elimination of the HSO4- ion. In the case of compounds 2 and 3, there are at least three hydrogen atoms in positions that enable the cis-elimination reaction, so the m/z 97 ion should be more abundant. Consequently, it is very likely that chromatographic peaks 1, 3 and 5 correspond to three to four possible structures: 2a, b and 3a, b. OS 214. Five well-separated peaks were revealed using the HILIC method for the EIC of the m/z 213.007 (C5H9O7S) ion, which was far more informative than the RP-C18 analysis in which we obtained an unseparated, broad group of peaks (Figure 5). We performed a detailed analysis of high-resolution fragmentation spectra for all five compounds that were separated in HILIC mode analysis (Figure 6). The spectrum for the peak with a retention time of 1.07 min revealed the presence of the m/z 183 fragment ion with the composition C4H7O6S, which was formed by the loss of a formaldehyde molecule. This is a fingerprint for a terminal hydroxymethyl group neighboring the double bond in the α position. The same fragmentation reaction was observed for organosulfates that were obtained from in-cloud laboratory processing of methyl vinyl ketone17 and/or isoprene19. A subsequent loss of water, giving rise to the m/z 165 fragment ion, is observed when sulfate residue is situated at the tertiary carbon atom. Taking this into account, molecular structures other than those of trihydroxyketone sulfate that are presented in Scheme 1 are very unlikely for this compound. This structure has not been reported previously. Four other chromatographic peaks represented two pairs of isomeric compounds with very similar MS/MS spectra (Figure 6) that undergo two kinds of main fragmentation reactions. First is the loss of the C2H4O2 (60 Da) neutral fragment. This kind of fragmentation reaction is linked to the presence of αhydroxy formyl residue in the structure. To confirm this hypothesis, the fragmentation reaction of a commercially available compound that contained this moiety was performed. In the fragmentation spectrum of the D-glucose-6-phosphate anion (Figure S15), the same loss of a C2H4O2 fragment occurred, which supported the presence of a α-hydroxy formyl group in the investigated OS structures.

Figure 5. The chromatographic profiles for the MW 214 organosulfate (C5H9O7S) recorded in the C18 and HILIC modes. The group of peaks in the range of 0.5 – 0.9 min. in the HILIC trace is related to different isomeric forms, which could be linked to different molecular structures.

Additionally, according to the NMR spectrum of D-glucose-6phosphate, it was present in the water solution in a cyclic form, which indicated that the hemiacetal structure did not affect this type of fragmentation. By analogy, we proposed the presence of a α-hydroxymethyl formyl group as a part of the structures that were responsible for the loss of 74 Da (C3H6O2) fragmentation reaction (Scheme 1). We concluded that the four compounds represented two pairs of diastereoisomers of organosulfates of five-membered ring hemiacetals. To form such a structure, the terminal carbon atoms must bear formyl and hydroxyl functional groups. The loss of C2H4O2 and C3H6O2 fragments confirms that both ends of an isoprene skeleton can bear either the hydroxyl or formyl residues. Consequently, only two pairs of diastereoisomeric structures are likely to occur (Scheme 1). The absence of the compounds where the sulfate group is situated next to the formyl group can be explained by the substituent effect of the sulfate group on the formyl group, which makes it more sensitive for the oxidation of the corresponding acid. In the case of OS 212 compounds (γ-lactones described in previous paragraph) that can be formed spontaneously from the 4-hydroxy acids, we do observe compounds with a sulfate group at the α-position to the carbonyl group. They are probably formed by the stepwise oxidation of the 2methyltetrols sulfates (OS 216), which could be more effective in such a configuration of functional groups. OS 216. In the EIC for the m/z 215.023 that was obtained with the RP-C18 method, two very well-separated peaks were present, which correspond with previous reports17,40,42. The chromatographic profile that was obtained with the HILIC method showed four baseline-separated peaks, which is also consistent with data reported elsewhere36,41 (Figure 7). However, the final verification of the proposed isomeric structures for the MW 216 organosulfate would require the synthesis of authentic standards, which remains a challenging research task.

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Analytical Chemistry were reported by other research groups36,41, and we also observed them in our analysis) confirmed that the three most possible ones formed through the IEPOX ring opening mechanism37,45,46. The structures are presented in Chart 3.

Figure 6. The negative ion fragment spectra (CE = 20 eV) that were acquired for all baseline-resolved isomers of the MW 214 isoprene organosulfate in HILIC chromatography.

Scheme 1. The proposed structures and main fragmentation pathways of deprotonated MW214 organosulfates. H 3C OH O OSO 3O

OH O

H

5 (RT = 1.07 min)

OSO 3-

O m/z 183, C4 H7 O6 S

OSO 3 CH 3 6a, b

(RT = 1.43 and 1.93 min)

O

O

OSO 3CH3

m/z 153, C3H5O 5S

Chart 3. Molecular structures of three possible positional isomers of the organosulfates with the MW 216. H 3C

OH

H3 C

OH OSO 3-

HO

CH3 OSO 3-

OSO 3 OH

HO

OH

OH

8a, b

9a, b

H3C

OH OH

HO OSO 310a, b

7a, b (RT = 1.53 and 1.70 min)

- C2 H4O2

- CH2 O

H 3C OH

HO

HO

Figure 7. The EIC chromatograms for the m/z 215/97 ion pair (A) and 215/215 ion pair (B) related to the MW 216 organosulfate. The chromatograms obtained in the C18 mode and HILIC mode were overlain on one graph and normalized to compare their relative intensities. The vertical dashed line separates the chromatograms that were obtained with different methods.

- C3 H6O2

O OSO3

-

m/z 139, C2H3O 5S

Nevertheless, the negative ion fragment mass spectra could provide the structural features of these isomers. The relative intensities of the m/z 215 and m/z 97 ions (Figure S16) seem to be similar for the first and last two peaks on the HILIC chromatogram (Figure 7). This observation allows us to hypothesize that these isomeric pairs are diastereoisomeric to each other. The same regularity was observed in the fragmentation spectra that were recorded for two peaks separated on a C18 column. It clearly indicates that stereoisomeric forms of OS 216 could not be separated using this method and only positional isomers are distinguishable. According to the mass spectra, the position of the sulfate group in the C5 skeleton could not be predicted, but the occurrence of three likely pairs of diastereoisomers (two minor peaks that were eluted later

CONCLUSIONS The chemical analysis of organosulfates – which are important markers of atmospheric secondary organic aerosols – was greatly improved by careful tuning of the sample preparation and chromatographic separation, followed by meticulous tandem mass spectral analysis. We developed a simple method that allowed for the improved separation of early eluting isoprene-related organosulfates, followed by a detailed structural analysis. We propose that our method be applied in other laboratories to ensure characterization of organosulfates in SOA samples from various sites around the world, and can be extended to the quantification thereof. The detailed understanding of the isomeric profiles of SOA organosulfates provides clues for the analysis of their formation and processes in the atmosphere that naturally contribute to the formation and aging of SOA. We postulated, for the first time, the actual number of observable isomers of the isoprene-derived MW 212 and MW 214 organosulfates. We suggest that the lactones (MW 212) with adjacent sulfate and carbonyl groups originated from the corresponding hemiacetals (MW 214) with adja-



cent groups that we did not observe in the samples. The most likely reason was that the adjacent groups in hemiacetals accelerated the oxidation of formyl groups to carboxyl groups, followed by subsequent lactonization. Accordingly, we cannot exclude that the abovementioned isoprene derivatives are closely related compounds, and it is very likely that MW 212 and MW 214 organosulfates can be formed in the oxidation process of the sulfated 2-methyltetrols. These relationships provide us the possibility to investigate the SOA aging process through the quantification of the relationships of the compounds.

ASSOCIATED CONTENT Supporting Information Description of the process of the mobile phase and column temperature optimization is provided. Graphs are presenting the separation dependence on a buffer type, pH values, column temperatures and the sample solvent. Given are also the EIC for nitroxyorganosulfates of alpha-pinene as well as MS/MS spectra for MW 212 and MW 216 organosulfates and for D-glucose-6phosphate. Likely structures of MW 216 organosulfates isomers are proposed.

AUTHOR INFORMATION Corresponding Authors * Rafal Szmigielski. Email: [email protected] Phone: +48 22 343 3402 * Grzegorz Spolnik. Email: [email protected] Phone: +48 22 343 2211

Author Contributions GS designed experiments, conducted LC-MS analyses, performed data interpretation and wrote the manuscript. PW prepared the ambient filter samples and participated in LC-MS analyses and data interpretation. KJR consulted and corrected the manuscript. KS collected the ambient filter samples. RS and WD consulted the data interpretation, assisted in writing and correcting the manuscript. All the authors read and approved the final manuscript.

ACKNOWLEDGMENTS The research was partially supported by funds from the Polish National Science Centre Grant (Nr OPUS82014/15/B/ST10/04276). The authors are grateful to Prof. Willy Maenhout and Prof. Magda Claeys for providing the K-puszta ambient aerosol samples collected in Hungary and for the fruitful scientific discussion.

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