Unveiling a large fraction of hidden organosulfates in ambient organic aerosol

Domain: earth_science
Published: 2025-05-01
Editorial outcome: major_revision

Abstract
Organosulfates are key compounds driving the anthropogenic enhancement of ambient organic aerosol, however, total organosulfate quantification remains elusive due to their molecular diversity and the scarcity of authentic standards. Here, we present a solid-phase extraction method that isolates organosulfates from ambient aerosol samples and enables their identification and quantification using mass spectrometry and a charged aerosol detector, respectively. We investigate ambient aerosol samples from urban China and rural Germany and quantify ~130 and ~65 chromatographically resolved organosulfates, respectively, contributing less than ~2% to the total organic matter. We find a significantly larger organosulfate fraction appearing as a broad peak in the chromatograms from the charged aerosol detector. Confirming its origin from chromatographically non-resolved organosulfates, an all-ion fragmentation experiment reveals specific sulfate-related ions. Integrating this peak, we find the contribution of organosulfates to organic aerosol is 12-17% and ~21% in samples from urban China and rural Germany, respectively. These findings emphasise the potential of sulfur emission reduction for mitigating both sulfate-related and organic aerosol pollution.

Introduction
Atmospheric fine particulate matter (PM) has major implications for human health, Earth’s radiative balance and air quality1–3. Organic aerosol (OA) is a major component in ambient PM, typically contributing 30-50% of the fine aerosol mass in the lower troposphere, though this can reach up to 90% in pristine environments4–7. Therefore, understanding the chemical composition and atmospheric transformation of OA is vital for identifying their sources and developing effective mitigation strategies. Due to a large variety of natural and anthropogenic emissions, transformed by intricate multiphase chemical processes, the chemical characterization of secondary OA (SOA) is extremely challenging. Organosulfates (OSs) are an important subclass of OA8–13, comprising up to 30% of the OA12. They can form through different pathways such as heterogeneous reactions between OA and SO₂14–18, sulfuric acid-driven condensation reactions within aerosol particles16,19, or even through heterogeneous reactions on building surfaces20. The reactive uptake of semi-volatile organic compounds, particularly isoprene epoxydiols (IEPOX), onto acidic particles can result in the formation of low-volatile OSs and, consequently, add to the OA mass9,16,21–23. If the involved organics are of biogenic origin, SO emissions can effectively lead to an anthropogenic enhancement of biogenic SOA12,24,25. Further studies on OSs have demonstrated that they can change the hygroscopic growth of OA and even have the potential to promote heterogeneous ice nucleation26,27.

Several studies have analyzed OSs in different environments, e.g., in the urban OA of Chinese megacities, in which rapid formation of OSs during pollution events has been observed28–31. However, Brüggemann et al. reported significant sampling artifacts in the detection and quantification of monoterpene-derived OSs under atmospheric conditions32. Le Breton et al. used a Filter Inlet for Gases and Aerosols coupled to a Chemical Ionization Mass Spectrometer to measure OSs in near-real time, and therefore minimizing sampling artifacts, at a semi-rural site in Beijing. They quantified 17 single OSs, estimating their overall contribution to OA at 2%, suggesting that OSs are not merely artifacts of the sampling process33. Similarly, a study conducted in Beijing using high-performance liquid chromatography (HPLC) combined with high-resolution mass spectrometry (HRMS) reported a ~4% contribution of OSs to OA28.

In urban and rural OA, studies have shown significant seasonality in both biogenic and anthropogenic OSs at various sites, with biogenic OSs peaking in summer and anthropogenic OSs in winter34,35. In marine OA, in the Yellow and Bohai Seas, the contribution of biogenic OSs ranges from 0.04 to 6.9%36. In the central Amazon, Glasius et al. found ~0.1 and ~0.6 μg/m3 of isoprene-derived OSs downwind of Manaus during the wet and dry seasons, respectively37. OSs can be transported in the atmosphere over long distances, since relatively elevated OS concentrations (~20–40 ng/m3) during Arctic haze events at Svalbard have been reported, probably originating from northern Eurasia38. Even in Alpine ice core samples from the Fiescherhorn glacier, OSs from both biogenic and anthropogenic organics were detected in a sample that dates back to the year 198439.

Although the direct analysis of molecularly resolved OSs enables the determination of their contribution to total OA, a degree of uncertainty remains in using this approach since it relies on the individual detection of single molecules and the application of surrogate standards for quantification. If no authentic standard is available, Brüggemann et al. suggested camphor-10-sulfonic acid (C10H16O4S) as an appropriate surrogate standard40. Nevertheless, direct quantification can easily introduce a large uncertainty due to different ionization efficiencies or by missing low-abundant or chromatographically unresolved OSs that can result in the underestimation of the total content of OSs in ambient aerosol.

Besides the direct quantification of individual OSs, indirect methods are available that quantify the total content of OSs by determining the difference between total sulfur (e.g., determined by X-ray fluorescence) and inorganic sulfate (e.g., determined by ion chromatography). These indirect approaches have suggested that OSs contribute 6–14% and up to 20% of the total sulfate at a rural Hungarian site41 and in urban Shenzhen, China42, respectively, and 5–10% to the organic matter (OM) in the United States43. Aerosol mass spectrometry (AMS) has been used to quantify OSs in real time44–48. Farmer et al. reported ~12% of total sulfate being present as OSs in Riverside, California, by comparing the AMS measurements of total sulfate and the ion chromatography measurements of inorganic sulfate, while the same approach at a rural station in Germany indicated that OSs can account for up to 46% of OA during single pollution episodes47,48. Organosulfur abundance typically follows a seasonal cycle with the formation of isoprene-derived OSs during summer, providing direct evidence for the anthropogenic enhancement of biogenic SOA, although the majority of organosulfur species remain unattributed35,49. Thus, the larger OS concentrations consistently observed through indirect methods indicate that the direct quantification of individual OSs likely underestimates the total OS concentration in ambient OA.

To improve the detection of individual, low-abundant OSs in ambient aerosol samples, we developed a solid-phase extraction (SPE) method for their enrichment and fractionation. To overcome the need for authentic OS standards, we made use of HPLC coupled to a charged aerosol detector (CAD) alongside a HRMS for separation, quantification and identification, respectively. The CAD is favorable for the quantification of non-volatile species due to its universal response, regardless of the chemical structure50. Owing to the terminal sulfate groups (R-OSO3H), in particular, OSs exhibit a low saturation vapor pressure, thus making them well-suited for quantification using a CAD. In addition to the CAD measurements for quantification, we employed two different mass spectrometric experiments for unambiguous molecular identification: (1) full-scan measurements (full MS) as a basis for non-target analysis and unambiguous molecular formula attribution of chromatographically well-resolved compounds, and (2) all-ion fragmentation (AIF) experiments for the detection of OS-specific ion fragments across the whole chromatogram. Applied on ambient aerosol filters from different environments, we directly quantified a large fraction of unresolved OSs, indicating their significant presence in both urban and rural environments and, furthermore, highlighting that their contribution to the formation of the aerosol mass cannot be overlooked.

Results
Organosulfate isolation
In the field of atmospheric sciences, water or organic solvents are commonly used for liquid extraction of aerosol filter samples. The application of SPE for enrichment and fractionation of compounds is rarely practiced, but this sample preparation enables novel analytical approaches. Here, we used a mixed-mode anion-exchange (MAX) cartridge and reversed-phase sorbent that has a strong affinity to bind OSs, thus allowing the isolation of acidic compounds from the complex sample matrix of ambient aerosol filters39. A fundamental property of OSs, the terminal R-OSO3H group, leads to a higher acidity (pKa ~−2.4 to −4.6) for most of the (nitrooxy-) OSs compared to other acidic organic compounds in ambient air (e.g., nitro-phenols or organic acids), ensuring their almost complete dissociation under typical aerosol pH conditions (1–4)51.

We extracted PM2.5 filter samples from Handan, China, and the Taunus Observatory (TO), Germany, representing the North China Plain with severe air pollution, and a relatively clean, rural background station in central Europe, respectively. In the following, we first describe a representative sample from Handan with regard to our new enrichment and quantification approach, before discussing all analyzed samples from both field sites. We have compared the chemical fingerprint with retention time (RT) vs. the mass-to-charge ratio (m/z) of an aliquot of the native aerosol filter extract against the two isolated fractions and the flow through after SPE (Fig. 1). The two SPE fractions are diluted to the same concentration level as the native extract. This enabled a quantitative comparison and the evaluation of the absolute SPE-recovery of the single organic compounds detected by non-target analysis. Most of the identified organic compounds in the native extract were found to be smaller than 350 Da and eluted over the entire retention time range (Fig. 1a). CHNO compounds (containing carbon, hydrogen, nitrogen, and oxygen) were the predominant compound class, followed by CHO compounds. The sulfur-containing compounds (CHOS, CHNOS) contributed 37% (by intensity) to the overall detected compounds (Figs. 1a, 2). By using a weak acid dissolved in methanol for the first SPE elution, we removed the less acidic compounds, including (nitro-) aromatics and organic acids (Fig. 1b). Subsequently, we used diluted hydrochloric acid dissolved in methanol to elute the OSs from the cartridge. We did not observe any evidence for degradation of the OSs, confirming their chemical stability at low pH52. With this increased solvent acidity for the second SPE elution, we were able to elute the CHOS and CHNOS compounds and, hence, isolate the OSs in a specific fraction. The diluted aliquot of this fraction will now be referred to as the diluted OS fraction (Fig. 1c). The compounds in the flow through were found to be mainly neutral compounds that were not retained by the MAX cartridge (Fig. 1d).

Within the diluted OS fraction, the MS2 spectra revealed that over 99% of the CHOS and CHNOS compounds (by signal intensity) formed a fragment at m/z 96.9601 (HSO4−), indicating the existence of R-OSO3H groups53. Furthermore, 72% of the compounds in the OS fraction had an absolute recovery above 50% compared to the native extraction. Some OSs had a poorer recovery of between 10 and 50%, showing relatively small signal intensities (Fig. S1a). The low recovery of some compounds can be explained by their strong retention on the ion exchange cartridge. By extracting one sample via SPE in triplicate, we found that the intensity-weighted average of the relative standard deviation (RSD) of the SPE recovery was ~15% for OSs, with larger uncertainties for the low-intensity compounds (Fig. S1b). The recovered OSs spanned a large range regarding the polarity and mass-to-charge ratio, ranging from relatively small (~150 Da) and polar (short RT) to large (~350 Da) and non-polar compounds (long RT). However, we do note that the used SPE cartridge provides a low recovery of small and highly polar OSs, which may include certain isoprene-derived OSs, an important class of atmospheric OSs. To a lesser extent, reversed-phase LC (used in this study) is also less effective for separating these compounds, highlighting the complementary role of hydrophilic interaction LC methods in analyzing smaller OSs54.

With our SPE method, we obtained an overall mean OS (CHOS and CHNOS) recovery of 75% (with a standard deviation of 7%) for the six ambient aerosol samples (area-weighted mean, Fig. 2). We calculated the overall mean recovery by calculating the sum of all isolated OSs, instead of averaging the recovery of individual OSs. In particular, the mean overall recovery for CHOS-only OSs was found to be 89%. Distinguished by the sampling site, the recovery of all OSs (CHOS and CHNOS) for the Handan samples was 73%, with the CHOS-OS recovery being 91%. The OS recovery for the TO samples was 76%, with the recovery of CHOS-OSs also being 76%. The difference in CHOS-OS recovery for the two sites was likely caused by different OSs with different properties and total aerosol mass loadings. However, we note that larger, less polar OS compounds may exhibit a reduced extraction efficiency, which should be considered in future studies. Overall, the SPE isolation method was found to be capable of effectively isolating the OSs from complex aerosol filter extracts, thereby allowing the subsequent chemical analysis of the enriched fraction and the quantification of the OSs by the CAD.

Organosulfate enrichment and quantification
In the enriched OS fraction (second SPE elution after evaporation and reconsitution, see Fig. 2—workflow (C)), the OSs were enriched by a factor of ~240 compared to the native extraction, which thus enables their detection by the less sensitive CAD. This large enrichment factor also increased the number of detected OSs after the non-target analysis of HRMS data (diluted fraction vs. enriched fraction of sample “Handan 1”: 79 and 490 detected OSs, respectively; see Fig. 1c vs. Fig. 3a) due to the improved signal-to-noise ratio of low-abundant OSs. The molecular fingerprints of the enriched OS fraction of all samples are shown in Fig. S2. After SPE, the larger number of OSs revealed the specific molecular characteristics for both field sites, especially for the aliphatic OSs—likely to be of anthropogenic origin—that were clearly visible at the rural TO station during winter time22,55. For example, sample TO2 (Fig. 2), a PM2.5 sample obtained during relatively clean conditions, showed the largest increase in area-weighted CHOS compounds after enrichment. Note that the increase in relative abundance from TO2-A to TO2-C (Fig. 2) originates from the larger number of detected OSs following SPE-enrichment. The signal intensity of the enriched OS fraction was 2.8–3.4 times higher than the diluted OS fraction in the TO samples. For the Handan samples this factor ranged from 1.2 to 1.6 (Fig. 2). With our regular liquid extraction method (i.e., Fig. 1a, native extraction) many OSs remained below the HRMS detection limit, leading to a potential underestimation of the OS contribution to total OA, especially for rural stations.

Figure 3b shows the differences of the CAD chromatograms obtained for the native extract and the enriched OS fraction of the same sample (Handan 18.10.2018, daytime), and the corresponding aerosol filter blank that experienced the full SPE procedure. These chromatograms clearly demonstrate the necessity of the enrichment by SPE for quantification with CAD (Fig. 3b). Our regular liquid extraction of the ambient aerosol filters yielded no detectable peaks in the CAD, whereas strong signals appeared in the OS fractions following SPE. The two highest peaks in the CAD chromatograms (RT ~9.91 and ~16.66 min) appear in both the ambient aerosol samples and the blanks following SPE. The parallel detection by HRMS showed no signal of OSs, thus indicating background signals originated from either the SPE cartridge, HPLC column, or aerosol filter extraction procedures. Apart from these two peaks, the majority of the CAD peaks lay within a three seconds retention time tolerance within the summed extracted ion chromatograms (EICs) of the OSs, after correcting for the retention time offset between both detectors. Furthermore, the two largest CHNO-signals (m/z 182.01, RT ~11.01 and ~13.34 min; Fig. 3a) did not result in prominent CAD peaks (Fig. 3b), indicating that their ionization efficiency in HRMS was either very large, or that they were not detected by CAD due to volatilization. The two signals were tentatively identified as 3-nitrosalicylic acid (RT ~11.01 min) and 5-nitrosalicylic acid (RT ~13.34 min), which have been reported as markers for biomass burning56,57. From a chemical perspective, it is reasonable that these organic acids appear in the OS fraction, as the electron-withdrawing mesomeric effect (–M-effect) of the nitro group stabilizes the negative charge of the conjugated base of the carboxylic acid group (electron delocalization), thereby increasing the acidity of the nitrosalicylic acids.

For quantification of the OSs by CAD, we measured eight OS standards at five different concentrations evenly distributed across the mobile phase gradient. In contrast to (−)ESI-HRMS, the CAD has a universal response to the mass of non-volatile compounds, independent of their chemical structure and functionality (Fig. S3). However, by running an HPLC gradient, the CAD´s sensitivity increases with the increasing organic solvent fraction, which is why we calibrated the CAD with eight OS standards. The resulting calibration surface (Fig. S4) enables quantification of unknown signals across the whole chromatogram. It is worth mentioning that the sample matrix showed no signal suppression in the CAD measurements for all the standards used (Fig. S5), thus justifying the quantification obtained through external calibration. To quantify each individual OS, we subtracted a fitted baseline of the chromatographically unresolved peak from the CAD chromatogram (Fig. S6) and, subsequently, fitted the remaining peaks (Fig. S7b) by referencing the OS EICs (Fig. S7a). Following this approach, we quantified all the chromatographically resolved OSs based on the CAD chromatogram.

By comparing signals from the OSs in the HRMS against the CAD, we observed a strong discrepancy of the ionization efficiency for the different OSs. For instance, the signal of monoterpene-derived nitrooxy-OS C10H17NO7S (RT 18.1 min and m/z 294.0653; Fig. 3a and Fig. 3b) was ~20 times higher than the smaller nitrooxy-OS C6H11NO7S (RT 11.3 min and m/z 240.0183; Fig. 3a and Fig. 3b)10,16,28,58. However, their quantified concentrations obtained by CAD were 5.5 ng/m3 and 3.3 ng/m3 (Fig. 3b), respectively. Nevertheless, we found the monoterpene-derived nitrooxy-OS (C10H17NO7S) to be the most abundant OS in the representative Handan aerosol filter sample, thereby indicating the importance of the anthropogenic enhancement of biogenic emissions through OS formation. We identified the highest signal in HRMS (RT 21.7 min and m/z 209.1; Fig. 3a) as anthropogenic octyl sulfate (C8H18O4S) by comparing the retention time and fragmentation pattern with the authentic standard (Fig. S8), and quantified its concentration to 5.4 ng/m3. A recent study reported octyl sulfate as likely being formed through heterogeneous reactions of octyl hydroperoxide with sulfur dioxide or sulfuric acid59.

Overall, we were able to quantify ~130 individual OSs in each Handan sample and ~65 OSs in each Taunus Observatory sample (Tables S1–6). The total quantifiable OS concentrations by integration of chromatographically resolved OS peaks for the three Handan samples (18.10.2018, daytime; 21.10.2018, daytime; 21.10.2018, night-time) were 149 ng/m3, 157 ng/m3, and 156 ng/m3, respectively. The similar OS concentration of the three samples indicates relatively constant conditions during the two investigated winter days. Certainly, a full seasonal analysis would provide a more variable picture. In the rural samples at the TO, the OS concentrations ranged from 44–57 ng/m3. It is worth mentioning that the three TO aerosol filter samples used in this study were chosen from a large dataset of ~350 samples, collected from August 2021 to August 2022, due to their previously identified high occurrence of OSs. The difference in concentrations between the two sites indicated, as expected, a much stronger anthropogenic influence on OS formation in the North China Plain, although there was still a significant abundance of OSs at the European rural station.

Unresolved organosulfates
In five of the six investigated samples, we observed a chromatographically unresolved broad peak over the full range of the CAD chromatogram. We did not identify single OSs from the non-target analysis that can explain this broad chromatographic peak (Fig. 3). This is expected, as the non-target analysis requires sharp chromatographic peaks for their detection. A similar feature of the broad chromatographic peak appears in the CAD chromatograms of all three Handan samples (Fig. 3 and Figs. S10–S11). The CAD chromatograms for the TO samples were less elevated (Figs. S12–S14), but an unresolved broad peak was still significantly different from the field aerosol filter blank that experienced the whole sample preparation procedure. Our explanation for the broad peak in the CAD is that it was caused by chromatographically unresolved OSs that remained hidden after mass spectrometry-based non-target analysis. Possibly, a large number of low-abundant OSs contributed in sum to the CAD signal. To confirm the relationship between the broad peak in the CAD and the potentially hidden OSs, we performed an all-ion fragmentation (AIF) experiment. In this setup of the mass spectrometer, all formed ions are guided through the quadrupole into the collision cell where all ions are fragmented simultaneously. Specific ion fragments from OSs (HSO4−, HSO3−, SO4•− and SO3•−) are clearly visible in the Handan samples. We found that the sum of these four ions produced a similar broad peak over the whole AIF chromatogram compared to the CAD chromatogram (Fig. 4a, b). Therefore, we interpreted the elevated baseline in the AIF sample chromatogram to be caused by unresolved and low-abundant OSs in the sample. The blank measurement (with internal OS standards, numbers 1–8, Fig. 4b) did not result in an elevated baseline using the AIF mode. To investigate the correlation between the CAD and AIF measurements, we averaged the two chromatograms in three second intervals to plot the blank-subtracted scatter diagram (Fig. S15). In Fig. S15, the data points appear relatively widely scattered (R=0.45) with a low RT-dependence. After applying the calibration function on the integrated CAD area (see the polynomial function, Fig. S4), we found a clear RT-dependent gradient between the CAD concentration and the signal of OS fragment ions of the AIF mode (R=0.18). Division of the AIF chromatogram by the methanol content of the mobile phase at each consecutive time step resulted in an improved correlation between the OS concentration and AIF signal (Fig. 4d, R=0.90). This observation indicates that the ionization efficiency of the OSs is strongly dependent on the mobile phase composition, with a better ionization efficiency of OSs observed at a higher organic content. Therefore, we suggest that further studies can use this correlation between the quantitative OS measurements by CAD and the quotient of the OS AIF signal over the methanol mobile phase fraction, with the goal to quantify OSs via HRMS only. Besides this observation of a novel way to quantify the total content of resolved and unresolved OSs in ambient aerosol, the detection of OS-specific ions by AIF that correlate strongly with the CAD signal supports our hypothesis that the chromatographically unresolved broad peak in the CAD chromatograms was mainly induced by hidden OSs.

For total OS quantification, we fitted the chromatographically unresolved broad peak as one large peak (Fig. 5a). The resulting integration showed that ~2.3, ~2.8, and ~2.4 μg/m3 of OSs were chromatographically unresolved for the Handan 18.10.2018 daytime, 21.10.2018 daytime, and 21.10.2018 night-time samples, respectively. Figure 5b indicates that the chromatographically resolved OSs of the Handan samples contributed ~0.7-1% to the total organic matter (OM), while the overall area of the CAD chromatogram (representing the resolved and unresolved OSs) contributed 12-17% to the OM. Although the TO samples exhibited a lower total OM, we found an overall larger fractional contribution of OSs. The TO2 (14.01.2022, night-time) sample showed the lowest concentration of OSs, at ~0.04 μg/m3 that were chromatographically resolved. No chromatographically unresolved OSs were visible in this sample (Fig. S13). The contribution of the individually quantified OSs to the total OM was ~34%, although the uncertainty of this value remains large due to the low concentration of OSs (Fig. 5b). The homologous series of aliphatic OSs in this sample suggests an anthropogenic origin (Fig. S2e). Backward trajectories indicate a possible marine origin from the North Sea (Fig. S16) with aliphatic OSs being emitted by the shipping sector60. The concentration of total OSs for TO 11.12.2021 daytime and TO 28.02.2022 night-time was ~0.6 and ~0.8 μg/m3, hence, the total OSs contributed ~21% to the total OM for both samples (Fig. 5b). This large OS contribution to the total OM at TO is certainly an upper estimate since our sample selection for this field site was guided by a pre-evaluation of a full-year compositional analysis. Furthermore, we cannot rule out that a fraction of OSs is formed during aerosol filter sampling, although the SO2 concentrations at this rural background station are usually below quantifications limits for standard detectors. Hence, we infer that the majority of OSs observed at rural and remote stations forms during atmospheric transport. Analysis of the backward trajectories for TO3 indicate that the air masses originated from eastern Germany, southern Poland and the northern Czech Republic, which are Europe´s hotspots of coal combustion by power plants (Fig. S16)61. The time of atmospheric transport from this source area of around one day is twice as long as the actual aerosol filter sampling period, which speaks against significant OS artifact formation for the TO aerosol filter samples.

To evaluate the quantitative contribution of OSs to total OM, we compared the major quantification studies that followed either the direct or the indirect approaches (Fig. 5c and Table S7). The indirect methods determine a larger OS fraction contributing to the OM (on average ~15%), whereas the direct approaches reveal a smaller fraction of OSs in the OM (~2%)12,28,29,33,35,36,41,43,44,47,49,62–66. Our method presented here enables the direct quantification of the hidden OS fraction and, consequently, suggests that the direct quantification of molecularly resolved OSs generally underestimates the total OS content in ambient aerosol samples by roughly a factor of five to ten. Thus, the described quantification approach for estimating OSs using SPE and UHPLC-CAD enables the direct quantification with lower uncertainties compared to the approaches that employ two different methods. Further assumptions, for example, about the average molecular weight of OSs, are no longer necessary.

Our observation of the high contribution of OSs at both locations, in urban China and rural Europe, calls for further investigation of the variability and seasonality of OSs in atmospheric aerosol across the globe. The described observations are in agreement with the indirect quantification methods of organic sulfur species. Due to the enrichment of individual OSs that enable a more detailed OS fingerprint (Fig. S2), we are now able to evaluate the fractions of OSs that are purely anthropogenic (e.g., saturated aliphatic OSs) or whether they can be seen as anthropogenically enhanced OSs (e.g., sulfate from anthropogenic sources with a carbon backbone from biogenic sources). Especially, isoprene-derived OSs might still be underestimated using the described method, as we found the SPE-recovery for these polar compounds is around 30% (Fig. S17). Interestingly, some of the OSs at the rural Taunus Observatory indicate the long-range transport of anthropogenic pollution. The low abundance of terpene-derived OSs can be explained by very low biogenic emissions of the investigated samples from the winter season. Further analysis of year-long aerosol filter analyses should reveal the pure anthropogenic and mixed biogenic/anthropogenic OS quantities, thus enabling a better understanding of pollution over whole seasons at different locations.

Methods
Aerosol filter sampling, organic carbon analysis, and solid-phase extraction
Six 12 h integrated PM2.5 filter samples were collected at two sampling sites. The first sampling site was located at Hebei University of Engineering (36.57°N, 114.50°E), an urban site in Handan, China. We collected three Handan samples on preheated quartz fiber filters (203.2 mm × 254 mm, QM-A Quartz Microfiber Filters, Whatman™, China) with a high volume sampler (TH-1000C, Tianhong, China; sampling flow rate 1 m3/min) on 18.10.2018 (daytime, 08.00–19.30), 21.10.2018 (daytime, 08.00–19.30) and 21.10.2018 (night-time, 20.00–07.30). The second sampling site was located at the Taunus Observatory (TO; 50.22°N, 8.45°E, altitude 825 m), a mountain top site at the Kleiner Feldberg in Hesse, Germany. We collected three TO samples on preheated glass fiber filters (Ø 150 mm, MG 160, Ahlstrom-Munksjö, USA) by using a high volume sampler (DHA-80, Digitel, Switzerland, air volume 500 L/min) on 11.12.2021 (daytime, 07.00–19.00), 14.01.2022 (night-time, 19.00–07.00) and 28.02.2022 (night-time, 19.00–07.00).

We took 1.45 cm2 of each aerosol filter and performed the OC analysis by using a thermal/optical carbon analyzer (Model 4, Sunset Lab.). We deployed the NIOSH 870 protocol for Handan aerosol filters (quartz fiber filter) and the Semi-Continuous OC/EC Analyzer with a program featuring a lower temperature setting of up to 500 °C for TO aerosol filters (glass fiber filter). We used an OC-to-OM conversion factor of 1.6 and 1.8 for Handan and TO, with the assumption of 20% relative uncertainty, respectively12,67.

We sampled a 24.5 cm2 and a 13.0 cm2 filter punch from each Handan and TO ambient aerosol filter, respectively (we determined the area of the filter punch based on the amount of sampled air volume). We extracted the filter fragments by using an orbital shaker (Edmund Bühler GmbH, Germany) for 2 × 20 min, each time using 8 mL of a water/methanol mixture (98/2 (v/v); water: Milli-Q water, 18.2 MΩ·cm; Milli-Q® Reference A+ System with Millipak® Express 40 Filter (0.22 μm), Merck Millipore; methanol: UHPLC-MS grade, Thermo Scientific™). We combined the two consecutive native extractions, centrifuged at 4000 rpm for 40 min, and transferred ~14.5 mL of liquid supernatant to new vials. We saved an aliquot of the native extract for a direct measurement and used the remaining extract for the SPE separation.

After conditioning the SPE cartridge (Oasis MAX, 1 cc/10 mg bed weight, Ø 30 μm particles, Waters™, Milford, MA, USA) with 1 mL each of (1) methanol, (2) Milli-Q water, (3) 5% NH4OH in Milli-Q water, and (4) extraction solution (methanol/water mixture), we loaded the native aerosol filter extracts onto the cartridge and collected the flow through for further analysis. Omitting a drying step, we directly eluted the cartridge twice using (1) 6 mL of 2% formic acid in methanol, followed by (2) 3 mL of 5% formic acid and 0.5% hydrochloric acid in methanol (Fig. S18). The two elutions were collected separately. An aliquot of each elution was taken and diluted with the extraction solution to the same concentration level as the native extract.

We added 10 μL of dimethyl sulfoxide to the second elution as a preservative and to ensure the suspension of compounds before evaporating the sample under a gentle nitrogen flow to a volume of ~10 μL. After the evaporation, we used 50 μL methanol/water mixture (50/50 (v/v)) to re-dissolve the remaining sample for subsequent analysis.

UHPLC-CAD-HRMS analysis
We measured the native extract, the diluted first SPE elution, diluted second SPE elution and the flow-through via the Orbitrap HRMS (Q Exactive Focus™ Hybrid Quadrupol-Orbitrap Mass Spectrometer, Thermo Fisher Scientific™) equipped with a UHPLC system (Vanquish Flex UHPLC System, Thermo Fisher Scientific™). We operated a reversed-phase column (CORTECS T3, 120 Å, 2.7 µm, 3 mm × 150 mm, Waters) in the gradient mode (at 40 °C, still air) to achieve chromatographic separation. The eluents were Milli-Q water (eluent A) and methanol (eluent B), both containing 0.1% formic acid (v/v). The mobile phase gradient started with 1% eluent B (0–1 min) which was then increased linearly to 99% B (1–14 min), kept at 99% B (14–15.5 min), and then decreased to 1% B (15.5-16.5 min); the column remained at 1% B (16.5–20 min) for re-equilibration. The total method duration was 20 min with a flow rate of 0.4 mL/min and an injection volume of 4 μL. Ionization was conducted in the negative mode via a heated ESI (HESI-II Probe, Thermo Fisher Scientific™). Data acquisition for m/z was set from 75 to 750, with a resolving power of ~70,000 at m/z 200. The settings for the ion source were as follows: 8 psi auxiliary gas (nitrogen), 40 psi sheath gas (nitrogen), 3.5 kV spray voltage, and 350 °C gas temperature.

CAD has a sensitivity in the low-nanogram to high-microgram range (on column), which allows for the detection of trace levels of non-volatile compounds. Hence, we measured the concentrated second SPE elution using UHPLC-CAD/HRMS. The LC method was adjusted by increasing the column temperature to 50 °C and prolonging the total method duration to separate the detected compounds further. The mobile phase gradient started with 1% eluent B (0–1 min) increasing linearly to 99% B (1–34.5 min), and was kept at 99% B (34.5–36.5 min) and then decreased to 1% B (36.5–38 min), and the column remained at 1% B (38–40 min) to re-equilibrate the column. The total method duration was 40 min. We utilized a post-column flow splitter (Quicksplit 610, Analytical Scientific Instruments US) to direct 83.5% of the mobile phase flow (0.4 mL/min) into the CAD (0.334 mL/min) and the remaining flow into the MS (16.5%, 0.066 mL/min). Due to the split of the flow rate, the auxiliary and sheath gas pressures of the ion source were changed to 10 and 31.5 psi, respectively. The settings for the CAD were as follows: 25 °C evaporator temperature, 2 Hz data collection rate and 1.00 power function value. We subsequently measured the enriched second SPE elution in the all-ion fragmentation (AIF) mode via the UHPLC-HRMS with the same gradient as in the CAD measurement. AIF acquisitions were performed with a resolving power of ~70,000 at an m/z ratio of 200, using a stepped collisional energy of 30, 40, and 55 units, and an automatic gain control (AGC) target of 3 × 106.

Data analysis
The non-target analysis software Compound Discoverer (CD; version 3.3, Thermo Fisher Scientific™) was used to identify the chromatographic peaks. By comparing the exact mass, isotopic matching score and MS/MS fragmentation pattern with the database mzCloud™ (Thermo Fisher Scientific™), molecular formulae could be assigned to the detected peaks. The detailed settings of the CD-workflow for the UHPLC-HRMS and UHPLC-CAD/HRMS sequences are provided in Tables S8 and S9.

In brief, a threshold intensity of 1 × 104 and a peak rating of larger than five was applied to the two-dimensional coordinate system (UHPLC-HRMS sequence: y-axis RT 0–20 min, x-axis m/z 50–750; UHPLC-CAD/HRMS sequence: y-axis RT 0–40 min, x-axis m/z 50–750) for all measurements. Only chromatographic peaks exceeding these thresholds were analyzed. In addition, the maximum peak signal of a compound had to be three times higher than its signal in the blank sample to be considered in the ambient aerosol samples. Molecular formulae were calculated with the permitted elemental combinations of C n1 H n2 Br n3 Cl n4 N n5 O n6 P n7 S n8 (n1=1–90, n2=1–190, n3=0–3, n4=0–4, n5=0–4, n6=0–20, n7=0–1, and n8=0–3) and with a mass tolerance of 2 ppm. We set the minimum and maximum H to C ratios as 0.1 and 4.5, respectively, to filter out the chemically unrealistic compounds. We used the software Fityk to fit the individual CAD peaks based on the RT of the EIC of the MS data. CAD quantification of the total OSs was obtained using 95% confidence intervals of the fit. The obtained data from the non-target analysis were further processed and plotted by using self-written scripts using Matlab (Matlab R2023b, Mathworks).

Data availability
All data used to generate figures are available under https://zenodo.org/records/1460950268.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Ma et al. presents interesting, novel results regarding organosulfates in atmospheric aerosol particles. This group of compounds were discovered in aerosols less than two decades ago and their formation and occurrence have been studied in both laboratory and ambient settings. However there has been a discrepancy between the concentrations in ambient air inferred from unspecific total sulfur aerosol measurements and concentrations estimated based on chromatographically resolved individual compounds detected by mass spectrometry (MS). The present study bridges this gap in our understanding through development of another type of detection method for organosulfates by application of a so-called Charged Aerosol Detector (CAD) which enables quantification of the total mass in a chromatographic peak, without being affected by differences in ionization efficiency as with MS detectors. Furthermore the authors are able to detect a broad peak of unresolved organosulfates in the chromatograms. Together these observations enable the authors to conclude that their aerosol samples contain a much higher concentration of organosulfates than detected with standard chromatography-MS methods, which is closer to the observations from total sulfur measurements.\nThe study will be of broad interest to the scientific community, as it points out that organosulfates have a much larger prevalence in atmospheric aerosols than generally thought. I expect that the study will lead to many new investigations on formation and occurrence of organosulfates in the atmosphere.\nIn general the study is well presented. However the manuscript needs to be checked by a professional who is a native English speaker. One example of issues that needs to be corrected, is the wrong use of the word “exemplary” (e.g. Fig. 3 figure text). As there are several more instances of not grammatically correct English, the manuscript needs to be checked and corrected.\nSpecific comments\nLine 15: ambient samples -> ambient aerosol samples\nL17: I suggest to add “, respectively” after organosulfates\nL18-19: These sentences may not be clear to a more general reader.\nL25: Up to 90% OA in atmospheric aerosols seems very high. Maybe it is more relevant to state a more general value.\nL37: I was a bit surprised to see the paper by Le Breton et al. highlighted as the first paper presented in detail here, given that there are a number of earlier studies using UHPLC-MS as in the current work.\nL46-47: The use of camphor-10-sulfonic acid seems outdated, especially as the functional group is not a sulfate.\nL76-77: These first sentences are unclear. The general reader may not understand that the filter extraction procedure is similar, but sample preparation differs.\nL79-80: How does the sulfate group change acidity of nitrooxy-OSs?\nL83: Exemplary has a different meaning. Please correct. You probably mean that the sample is used as an example.\nL86-87: This sentence is not clear.\nL104: How large? Please be specific.\nL117: Can you state a standard deviation on the recovery?\nL123: Often it is necessary to add the word “aerosol” when you talk about filter extracts to make the sentence more precise.\nL140: What is the sensitivity of the CAD? Can this be described briefly, maybe in the experimental section?\nL152: It seems strange to refer to Figure 3b before 3a.\nL145: Are other data available to support the interpretation? Can TO really be described as remote (which is typically further away from anthropogenic sources)?\nL155: ambient filters -> suggest to change to: ambient aerosol filters - or similar.\nL164: What is the M-effect?\nL190: Two days can hardly be called a period.\nL192: It is very unclear to the reader what this means. How were the sample selected?\nL194: A verb seems to be missing here. It is important to point out that these were three selected samples during winter, where previous studies have observed relatively high OS concentrations.\nL249- and Fig. 5: For the calculation of OM from organosulfates, was the sulfate group omitted?\nL256: The North Sea is quite far away from TO compared to the closer densely populated and industrialized areas in Germany and Netherlands. The statement about shipping origin thus seems speculative.\nL259: Again the reader is wondering how these three samples were selected.\nL261: This has previously been observed by e.g. Glasius et al., Composition and sources of carbonaceous aerosols in Northern Europe during winter, Atmospheric Environment, 173, 2018, pages 127-141.\nL278: This seems like a very strong statement for apparently unpublished data.\nFigure 1: I suggest to make larger dots in the legend, so they are easier to see.\nFigure 2: In the description of the aerosol filter sample extraction in the figure, I suggest to list the composition of the extraction solvent.\nFigure 3: Is CHOP a common group of compounds in aerosols?", "reviewer_2": "Summary and Recommendation:\nThis manuscript examines the types and amounts of organosulfates (OSs) found in PM2.5 samples collected from Handan, China (urban) and Hesse, Germany (mountain top site that is remote) by using solid phase extraction (SPE) as a sample preparation step before RPLC-CAD-HRMS analysis. Although SPE has been rarely used in aerosol chemistry research, it helps in aiding the identification and quantification of OSs when using RPLC-CAD-HRMS. I should note that SPE was originally used in earlier work that analyzed PM2.5 samples collected from the SE USA (e.g., Gao et al., 2006, JGR-Atmospheres), but it was later neglected due to it removing small water-soluble OSs (such as the isoprene-derived OSs) in one of the washing steps. This latter recognition was so important to isoprene-derived SOA because using SPE caused these OSs to go initially unrecognized. With that said, to my knowledge, this study uses RPLC coupled with both CAD with ESI-HRMS detection. The CAD offers incredible potential to quantify OSs without the need for all of the authentic standards being available. In atmospheric chemistry, the lack of available authentic standards has been a huge hindrance, preventing models from being updated with the latest multiphase chemical processes that lead to low-volatility OSs. Overall, I find this manuscript exciting and mostly well-written, especially for atmospheric and aerosol chemists. Some readers not well-versed in analytical chemistry might find this paper somewhat difficult to read. However, I hope the Editor won't find this a reason to reject the paper. I do think the demonstration of a large un-resolved OS peak in the chromatogram is notable and appears to contribute a large OA mass to PM2.5 collected during the winter seasons in China and Germany. I have several major comments below that should be carefully addressed by the authors before publication in Nature Communications can be fully considered. Due to the nature of my comments below, I recommend the manuscript be reconsidered after major revisions.\nMajor Comments:\n1.) Filter collection artifacts:\nThere has been some important work to recently come out of Hartmut Hermann's lab (and I believe Marianne Glasius' lab) showing that filter sampling (especially with media like quartz fiber filters) is prone to positive sampling artifacts that lead to the production of certain OSs like m/z 294 (one of the nitrooxy OSs). Since high-volume filter samplers cannot use denuders (due to the high flowrates used) to remove gas-phase SO2 and NOx, it is possible that NOx and SO2 can absorb from the gas phase and react on these filter types yielding unwanted reactions that lead to OSs. Knowledgeable readers might wonder how much of the mass you quantify is related to these positive sampling artifacts?\n2.) Lines 29-31: The authors should cite the earliest papers here for OSs being discovered as an important subclass of OA, including the following:\nRomero and Oehme (2005, J. of Atmos. Chem.) - DOI: 10.1007/s10874-005-0594-y\nSurratt et al. (2007, ES&T) - DOI: 10.1021/es062081q\nIinuma et al. (2007, Atmos. Environ.) - https://doi.org/10.1016/j.atmosenv.2007.03.007\nIinuma et al. (2007, ES&T) - https://doi.org/10.1021/es070938t\nSurratt et al. (2008, JPCA) - https://doi.org/10.1021/jp802310p\nThe Surratt et al. (2008, JPCA) study estimated that upwards of 30% of particulate OM in Hungary could be OSs.\nAdditional work by Betsy Stone's group has also estimated OS contributions to OM, such as https://doi.org/10.5194/acp-19-3191-201. In that study, they estimated 16.5% of PM2.5 OC as OSs.\n3.) Lines 31-32: I'm a bit surprised the authors didn't cite any work on the reactive uptake of isoprene epoxydiols (IEPOX) yielding OSs in PM2.5. This is one of the major semi-volatile oxidation products out there that has helped clue us in as a research community on the role of reactive uptake (multiphase chemistry) onto wet acidic sulfate aerosols that yield OSs.\n4.) Lines 32-33: Similar to earlier lines in the introduction, simply citing review articles for statements like this doesn't seem appropriate. I think the authors should be citing some of the original studies that demonstrated the role of SO2 emissions in enhancing SOA from BVOC oxidations.\n5.) Lines 36-49: This paragraph is very European- and Asian-centric. I think this paragraph is fine, but a lot of prior OS work has also been done in the USA trying to quantify OSs in PM2.5 there. There has been a lot of work by the Surratt Group (UNC) and Stone Group (University of Iowa) on this topic using LC/MS methods and synthesizing authentic compounds. Furthermore, Jian Yu's Group (HKUST) in Hong Kong has also estimated OS contributions in PM2.5 and has synthesized authentic compounds as well.\n6.) Lines 92-93: How comfortable are the authors assuming that the majority of OSs are stable in the presence of a strong acid? Work from Matt Elrod's group (Oberlin College) has suggested that the stability of OSs depends on whether the OS is primary or tertiary. Did the authors consider testing their approach with different OS standards to confirm the stability of OS compounds?\n7.) Figure 1d:\nAs shown in Figure 1d, there are still some OS compounds that elute through what they call \"flow-through.\" Are these accounted for? One thing I want to note to the authors is that in Gao, Surratt, et al. (2006, JGR-Atmospheres), they employed an SPE separation to measure SOA. When they did this OSs were missed, especially the very water-soluble types (like the isoprene-derived OSs). Later Surratt et al. (2007, ES&T) found that by not using SPE, but rather a polar-end capped C18 column, they could measure the isoprene-derived OSs along with the larger monoterpene-derived OSs. Later, Betsy Stone's group (University of Iowa) and later Surratt's group (UNC) showed that HILIC separation is much more effective in resolving and quantifying isoprene-derived OSs and other water-soluble and smaller OSs. I'm curious, do the authors worry they are still missing water-soluble OSs that are not well retained on C18 columns? I noted the authors conducted reverse-phase liquid chromatography (RPLC) to separate and measure particulate OSs. However, it might be better for all of us in the community to use both HILIC and RPLC separations as orthogonal methods to better separate, characterize and quantify the OSs that range from water-soluble to less water-soluble. Do you agree? It might be worth mentioning the limitations of RPLC in past work and why HILIC methods have come about for the smaller OSs.\n8.) Line 117-118: How do the authors know they extracted all of the OS compounds and mass from their filters using 98% water and 2% methanol by volume solvent only? Were standards of OSs also examined through their extraction procedures? I think this would be helpful to include. Could larger OSs be missed if not soluble in water? Also, with water, do you have to worry about the hydrolysis of OSs?\n9.) Lines 121-122: I agree that the recoveries can be affected by what the authors state here. But what about also the matrix of the sample? Could that affect the recovery from the filters? For example, it is known that particulate PAHs can be hard to extract from certain filters depending on the matrix of the filter and the other components collected on the filter.\n10.) Lines 144-145: The authors note that there are OSs that are aliphatic and of anthropogenic origin. Are the authors aware of work by Riva et al. (2016, ACP)? This study demonstrated OSs form from the oxidation of alkanes in the presence of wet acidic sulfate aerosols. Riva et al. (2015, ES&T) also demonstrated OSs form from PAH oxidations in the presence of sulfate aerosols. Notably, in this latter study, they also observed sulfonates. Did you by chance observe any sulfonates in your PM2.5, possibly from the wintertime samples? What about other oxidation states of sulfur?\n11.) Minus the comment I made about the filter sampling artifacts above, I wonder if this method would show even higher OS fractions in fine particulate OM during the summer seasons when BVOC-derived oxidation products are known to yield substantial amounts of OS in the presence of sulfate? The fact the authors show fairly high amounts in winter, tells me the mass fractions could even be higher than previously thought in summer....\n12.) Some readers or reviewers might ask why the authors didn't try this RPLC-DAD-HRMS method on a well-characterized lab-generated SOA type like IEPOX SOA before analyzing field samples? The reason I raise this point is for IEPOX SOA our community has an analytical method (i.e., HILIC/ESI-HR-QTOFMS from both the Stone and Surratt Groups) and authentic standards (from the Surratt Group) to compare your quantitative method with. Furthermore, lab studies typically can denude unwanted gases (like SO2/NOx) before collecting onto Teflon filters (which are less prone to positive sampling artifacts like quartz)."}, "author_response": "Manuscript ID: NCOMMS-24-43312A-Z\nManuscript Type: Article\nCollection: Mass spectrometry method development\nResponse to reviewers\nReviewers’ Comments to the Authors:\nReviewer 1\nThe manuscript by Ma et al. presents interesting, novel results regarding organosulfates in atmospheric\naerosol particles. This group of compounds were discovered in aerosols less than two decades ago and\ntheir formation and occurrence have been studied in both laboratory and ambient settings. However\nthere has been a discrepancy between the concentrations in ambient air inferred from unspecific total\nsulfur aerosol measurements and concentrations estimated based on chromatographically resolved\nindividual compounds detected by mass spectrometry (MS). The present study bridges this gap in our\nunderstanding through development of another type of detection method for organosulfates by\napplication of a so-called Charged Aerosol Detector (CAD) which enables quantification of the total\nmass in a chromatographic peak, without being affected by differences in ionization efficiency as with\nMS detectors. Furthermore, the authors are able to detect a broad peak of unresolved organosulfates\nin the chromatograms. Together these observations enable the authors to conclude that their aerosol\nsamples contain a much higher concentration of organosulfates than detected with standard\nchromatography-MS methods, which is closer to the observations from total sulfur measurements.\nThe study will be of broad interest to the scientific community, as it points out that organosulfates have\na much larger prevalence in atmospheric aerosols than generally thought. I expect that the study will\nlead to many new investigations on formation and occurrence of organosulfates in the atmosphere.\nThank you very much for this positive feedback.\nIn general, the study is well presented. However, the manuscript needs to be checked by a professional\nwho is a native English speaker. One example of issues that needs to be corrected, is the wrong use of\nthe word “exemplary” (e.g., Fig. 3 figure text). As there are several more instances of not grammatically\ncorrect English, the manuscript needs to be checked and corrected.\nInitiated by this comment, we have completed a thorough language polishing by a professional\nservice and have made the necessary corrections. Specifically, we have replaced the word\n'exemplary' with 'representative' in the relevant sentences, including the figure caption of Fig. 3.\nLine 15: ambient samples -> ambient aerosol samples\nWe have revised 'ambient samples' to 'ambient aerosol samples' in line 16 (the track-changes\nversion) as recommended.\nL17: I suggest to add “, respectively” after organosulfates\nWe have added ', respectively' after 'organosulfates' in line 19 (the track-changes version) as\nrecommended.\"\nL18-19: These sentences may not be clear to a more general reader.\nWe changed the sentence in line 19-21 (the track-changes version) as follows:\n“We found a significantly larger organosulfate fraction appearing as a broad chromatographic peak\nin the charged aerosol detector. Confirming its origin from chromatographically non-resolved\norganosulfates, an all-ion-fragmentation experiment revealed specific sulfate-related ions.”\nWe agree that readers who have a more general background need to read through the manuscript\nto understand the idea behind the “all-ion-fragmentation experiment” – but the word limit of the\nabstract will not allow for a more detailed explanation of AIF experiments.\nL25: Up to 90% OA in atmospheric aerosols seems very high. Maybe it is more relevant to state a more\ngeneral value.\nWe agree that 90% OA in atmospheric aerosols may seem high. We have revised this to reflect a\nmore general and widely accepted value and added additional references to support the statement.\nThe new sentence was list in line 27-29 (the track-changes version) as follows:\n“Organic aerosols (OA) are a major component in ambient PM, typically contributing 30-50% of the\nfine aerosol mass in the lower troposphere, though this can reach up to 90% in pristine\nenvironments4–7. representing up to 90% of the fine aerosol mass in the lower troposphere4–7.”\nThe new references are:\n6. Zhang, Y., Cai, J., Wang, S., He, K. & Zheng, M. Review of receptor-based source apportionment\nresearch of fine particulate matter and its challenges in China. Sci. Total Environ. 586, 917–929\n(2017).\n7. Kanakidou, M. et al. Organic aerosol and global climate modelling: A review. Atmos. Chem. Phys.\n5, 1053–1123 (2005).\nL37: I was a bit surprised to see the paper by Le Breton et al. highlighted as the first paper presented in\ndetail here, given that there are a number of earlier studies using UHPLC-MS as in the current work.\nWe appreciate your observation regarding the choice of highlighted studies. To improve the logical\nflow of the text, in the revised version we first cite a paper by Brüggemann et al., which discusses\nOS-related sample artifacts. We then reference the work of LeBreton et al., whose near-real-time\nmethod minimizes the formation of OS artifacts, demonstrating that the observed OSs are not\npurely artifacts. Finally, we cite the study by Ma et al., who report similar results to LeBreton et al.,\nbut with filter collection and UHPLC-HRMS measurements, further supporting the validity of our\nfindings.\nWe changed the sentence in line 40-47 (the track-changes version) as follows:\n“Several studies have analysed OSs in different environments, e.g. in the urban OA of Chinese\nmegacities, in which rapid formation of OS during pollution events has been observed28–31. However,\nBrüggemann et al. reported significant sampling artifacts in the detection and quantification of\nmonoterpene-derived OSs under atmospheric conditions32. LeBreton et al. used a Filter Inlet for\nGases and Aerosols coupled to a Chemical Ionization Mass Spectrometer to measure OSs in near-\nreal time, and therefore minimizing sampling artifacts, at a semi-rural site in Beijing. They quantified\n17 single OSs, estimating their overall contribution to OA at 2%, suggesting that OSs are not merely\nartifacts of the sampling process33. Similarly, a study conducted in Beijing using high-performance\nliquid chromatography (HPLC) combined with high-resolution mass spectrometry (HRMS) reported\na ~4% contribution of OSs to OA28.\nThe new reference is:\n28. Ma, J. et al. Nontarget Screening Exhibits a Seasonal Cycle of PM2.5Organic Aerosol\nComposition in Beijing. Environ. Sci. Technol. 56, 7017–7028 (2022).\n32. Brüggemann, M. et al. Overestimation of Monoterpene Organosulfate Abundance in Aerosol\nParticles by Sampling in the Presence of SO2. Environ. Sci. Technol. Lett. 8, 206–211 (2020).\nL46-47: The use of camphor-10-sulfonic acid seems outdated, especially as the functional group is not\na sulfate.\nThank you for highlighting the importance of using correct surrogates for OS quantification. It is\nindeed true that ionization efficiencies can vary significantly between different OSs under\nelectrospray ionization. While compounds with the same functional group tend to exhibit more\nsimilar ionization efficiencies, the chemical structure of each compound also plays a crucial role. In\nthis case, camphor-10-sulfonic acid remains a suitable surrogate standard when authentic OSs are\nunavailable.\nBrüggemann et al. tested four authentic standards (2-hydroxy-α-pinene OS, 2-hydroxy-carene OS,\nlimonaketone OS, and 2-hydroxy-β-caryophyllene OS) along with two surrogates (octyl sulfate and\ncamphor-10-sulfonic acid), finding that calibration curves for these compounds could differ by up\nto two orders of magnitude (Fig. 1)1. Despite containing a sulfonic group, camphor-10-sulfonic acid\nexhibited a medium response compared to the other tested compounds (Fig. 1)1.\nFig. 1: Calibration curves of authentic terpene-derived OSs (solid lines) and commonly used surrogate\nstandards (dashed lines). The inset shows the calibration curves on a linear scale. Abbreviations:\napinOS (2-hydroxy-a-pinene OS), carenOS (2-hydroxy-carene OS), limketOS (limonaketone OS),\nbcaryOS (2-hydroxy-b-caryophyllene OS), cmphrsulfon (camphor-10-sulfonic acid) and MSA\n(methylsulfonic acid). Figure from Brüggemann et al.1\nAdditionally, we tested several authentic and surrogate OS standards under Orbitrap negative\nelectrospray ionization mode using an isocratic gradient. The results showed that while camphor-\n10-sulfonic acid displayed a slightly lower response than the average of these compounds, it\nremained within 20% (Fig. 2). Therefore, in the absence of other authentic OS standards, we believe\nit is justified to use camphor-10-sulfonic acid as a surrogate for quantifying ambient OSs.\nFig. 2: Peak area (mean ± SD) of ten OS standards (all at the same concentration levels of 2 ng/µL)\nmeasured with at an isocratic separation. The standards were prepared in one batch as an OS-mix. The\nmix contained: (1) Formaldehyde sodium bisulfite (CH SO Na), (2) Methyl sulfate sodium salt\n3 4\n(CH SO Na), (3) Sodium ethyl sulfate (C H SO Na), (4) Butyl sulfate (C H SO Na), (5) Camphor-10-\n3 4 2 5 4 4 10 4\nsulfonic acid (C H SO ), (6) 2-hydroxy-α-pinene OS (C H SO ), (7) Sodium octyl sulfate (C H SO Na),\n10 16 4 10 17 5 8 17 4\n(8) Sodium n-decyl sulfate (C H SO Na), (9) Sodium dodecyl sulfate (C H SO Na), and (10) Tetradecyl\n10 21 4 12 25 4\nsulfate sodium salt (C H SO Na). To test the stability of the instruments, we measured each standard\n14 29 4\nconcentration five times. The line (solid) is the average response of these compounds with a band\n(dash) of ± 20%.\nL76-77: These first sentences are unclear. The general reader may not understand that the filter\nextraction procedure is similar, but sample preparation differs.\nTo address this, we revised the sentence to explicitly state that while the filter extraction process\nis similar for all samples, the subsequent sample preparation is only for SPE.\nWe changed the sentence in line 90-93 (the track-changes version) as follows:\n“In the field of atmospheric sciences, water or organic solvents are commonly used for liquid\nextraction of aerosol filter samples. The application of SPE for enrichment and fractionation of\ncompounds is rarely practiced, but this sample preparation enables unique and novel analytical\napproaches. In the field of atmospheric sciences, where SPE techniques are not commonly applied,\nwe found unique analytical advantages over common filter extraction procedures. Here, Wwe\nused…”\nL79-80: How does the sulfate group change acidity of nitrooxy-OSs?\nThe sulfate group significantly lowers the pKa of OSs and nitrooxy-OSs to approximately -2.4 to -4.6.\nSince the atmospherically relevant pH of aerosols typically ranges from 1 to 4, much higher than\nthe pKa of OSs, all OSs and nitrooxy-OSs exist almost entirely in their dissociated (deprotonated)\nform under these conditions. As a result, the dissociation state of both OSs and nitrooxy-OSs is not\naffected by the specific pH within this range.\nTo improve clarity, we revised the sentence in lines 95–98 (tracked-changes version) as follows:\n“A fundamental property of OSs, the terminal R-OSO3H group, leads to a higher acidity (pKa ~ -2.4\nto -4.6) for most of the (nitrooxy-)OSs compared to other acidic organic compounds in ambient air\n(e.g. nitro-phenols or organic acids)., ensuring their almost complete dissociation under typical\naerosol pH conditions (1 – 4)51.”\nThe new reference is:\n51. Hyttinen, N., Elm, J., Malila, J., Calderón, S. M. & Prisle, N. L. Thermodynamic properties of\nisoprene-and monoterpene-derived organosulfates estimated with COSMOtherm. Atmos. Chem.\nPhys. 20, 5679–5696 (2020).\nL83: Exemplary has a different meaning. Please correct. You probably mean that the sample is used as\nan example.\nSee above, we have replaced the word 'exemplary' with 'representative' in the relevant sentences,\nthe new sentence in line 100-102 (the track-changes version) are as follows:\n“In the following, we first describe an representativeexemplary sample from Handan with regard\nto our new enrichment and quantification approach, before discussing all analysed samples from\nboth field sites.”\nL86-87: This sentence is not clear.\nWe rewrote the sentence in line 104-106 (in the track-changes version) as follows:\n“The two SPE fractions are diluted to the same concentration as the native extract. This enabled\nDilution of the SPE fractions to the same concentration level as the native extract enables a\nquantitative comparison and the evaluation of the absolute SPE-recovery of the single organic\ncompounds detected by non-target analysis.”\nL104: How large? Please be specific.\nThe molecular mass is up to ~350 Da, and the new sentence in line 124-126 (the track-changes\nversion) are as follows:\n“The recovered OSs span a large range regarding polarity and mass-to-charge ratio (m/z), ranging\nfrom relatively small (~150 Da) and polar (short RT) to large (~350 Da) and nonpolar compounds\n(long RT).”\nL117: Can you state a standard deviation on the recovery?\nThank you for your suggestion, the overall recovery for the sample Handan 1, Handan 2, Handan 3,\nTO1, TO2, and TO3 are 0.71, 0.84, 0.67, 0.70, 0.78, and 0.80, respectively. Therefore, the standard\ndeviation of the recovery is 6.6%, and we implement the SD in the manuscript. The new sentence\nin line 142-143 (the track-changes version) are as follows:\n“With our SPE method, we obtained an overall mean OS (CHOS and CHNOS) recovery of 75% (with\na standard deviation of 7%) for the six ambient aerosol samples (area-weighted mean, Fig. 2).”\nL123: Often it is necessary to add the word “aerosol” when you talk about filter extracts to make the\nsentence more precise.\nWe implement the comment in the manuscript. The new sentence in line 148-150 (the track-\nchanges version) is as follows:\n“Overall, the SPE isolation method is capable of effectively isolating the OSs from complex aerosol\nfilter extracts, thereby allowing the subsequent chemical analysis of the enriched fraction and the\nquantification of the OSs by the CAD.”\nL140: What is the sensitivity of the CAD? Can this be described briefly, maybe in the experimental\nsection?\nCAD is sensitive in the sub-nanogram range, and we put this information in the method part. We\nrevised the sentence in line 382-383 (the track-changes version) as follows:\n“CAD has a sensitivity in the low-nanogram to high-microgram range (on column), which allows for\nthe detection of trace levels of non-volatile compounds. Hence, wWe measured the concentrated\nsecond SPE elution with UHPLC-CAD/HRMS.”\nL152: It seems strange to refer to Figure 3b before 3a.\nThank you for your comment. Upon review, we can confirm that Figure 3a is referred to before\nFigure 3b in the manuscript. Specifically, Figure 3a is first mentioned in line 169, while Figure 3b is\nreferenced in line 180 (as seen in the track-changes version). We believe the current structure is\ncorrect, but we appreciate your attention.\nL145: Are other data available to support the interpretation? Can TO really be described as remote\n(which is typically further away from anthropogenic sources)?\nWe agree that the term “remote” is not entirely appropriate for describing the Taunus Observatory\n(TO). We changed the description to a “rural” site, given it located at 50.22∘ N, 8.44∘ E at 825 m\naltitude on top of Kleiner Feldberg in the Taunus Mountain range. In the North-West (the\ndominating wind direction) the area is dominated by forested and agricultural areas. Due to rural\nsetting, the observatory does have a European continental background character, allowing it to\ncapture long-range transport of pollutants in addition to local emissions.\nHowever, approx. 20 km south-east of the site is the city of Frankfurt (Main) in the center of the\nRhine–Main area with several industrial sources including chemical industry. This site also enables\nassess the emissions from this densely populated region.\nTherefore, while TO may not be truly remote, its location still allows for important observations of\nbroader regional and continental air quality phenomena. We carefully review the entire manuscript,\nreplacing \"remote TO\" and similar terms with \"rural TO\" and their equivalents.\nL155: ambient filters -> suggest to change to: ambient aerosol filters - or similar.\nWe implement the comment in the manuscript. The new sentence in line 182-184 (the track-\nchanges version) are as follows:\n“Our regular liquid extraction of the ambient aerosol filters yielded no detectable peaks in the CAD,\nwhereas strong signals appear in the OS fractions following SPE.”\nL164: What is the M-effect?\nIn organic chemistry, the “–M-effect” refers to the mesomeric effect. It describes how electron-\nwithdrawing or electron-donating groups attached to a conjugated system (like an aromatic ring)\ninfluence the distribution of electron density in the molecule through resonance.\nThe nitro group (–NO ) of nitrosalicylic acid is an electron-withdrawing group, which exerts a\n2\nnegative mesomeric effect (–M-effect). This effect pulls electron density away from the aromatic\nring through resonance.\nThis electron withdrawal increases the acidity of the carboxylic acid group on the same molecule\nbecause it stabilizes the negative charge on the conjugate base, making it easier for the carboxylic\nacid to lose a proton (H⁺).\nTo be clearer, we change the sentence in the line 192-196 (the track-changes version) as follows:\n“From a chemical perspective, it is reasonable that these organic acids appear in the OS fraction, as\nthe electron-withdrawing mesomeric effect (–M-effect) of the nitro group stabilizes the negative\ncharge of the conjugated base of the carboxylic acid group (electron delocalization), thereby\nincreasing the acidity of the nitrosalicylic acids. as the M-effect of the nitro group on the aromatic\nring increases the acidity of the carboxylic acid group of the nitrosalicylic acids.”\nL190: Two days can hardly be called a period.\nThank you for your comment. Instead of referring to it as a 'period,' we changed it as a 'duration of\ntwo investigated winter days'. The new sentence in line 220-221 (the track-changes version) are as\nfollows:\n“The similar OS concentrations of the three samples indicate relatively constant conditions during\nthe two investigated winter period days.”\nL192: It is very unclear to the reader what this means. How were the sample selected?\nWe provided additional details on how the samples are selected. The revised text in line 222-225\n(the track-changes version) are as follows:\n“It is worth mentioning that the three TO aerosol filter samples used in this study were chosen from\na large dataset of approximately 350 samples, collected from August 2021 to August 2022, due to\ntheir previously identified high occurrence of OSs. the three TO filter samples were selected for this\nstudy from an extensive time series of measurements based on their previously identified high OS\noccurrence”\nL194: A verb seems to be missing here. It is important to point out that these were three selected\nsamples during winter, where previous studies have observed relatively high OS concentrations.\nIn the revised version, we have mentioned that “the three selected samples from winter and have\nobserved relatively high OS concentrations” (see above), and we add the verb “a” in the text, The\nnew sentence in line 225-227 (the track-changes version) reads as follows:\n“The difference in concentrations between the two sites indicates, as expected, a much stronger\nanthropogenic influence on OS formation in the North China Plain, but still a significant abundance\nof OSs at a European rural station.”\nL249- and Fig. 5: For the calculation of OM from organosulfates, was the sulfate group omitted?\nNo, we did not omit the sulfate group. we measure the organic carbon (OC) on all the aerosol filters.\nTo estimate the organic matter (OM) content, we multiply the OC value for Handan aerosol filters\nby 1.62, and for TO aerosol filters, we multiply the OC value by 1.83.\nAs for the OSs quantification, we calibrate the CAD by using eight different standards at five\nconcentration levels. The 40 data points were fitted by using a second order polynomial function to\nget the calibration surface. Subsequently, we integrated the OS area detected in the ambient\nsample (which inherently includes the sulfate group) from the CAD chromatogram and input this\nvalue into the function to calculate its concentration. As a result, the OS fraction in OM represents\nan upper-limit estimation. The “other OM” (the legend of figure 5B) represent the OM value minus\nthe sum of the resolved and unresolved OS.\nL256: The North Sea is quite far away from TO compared to the closer densely populated and\nindustrialized areas in Germany and Netherlands. The statement about shipping origin thus seems\nspeculative.\nCertainly, the North Sea is quite distant from the sampling site (TO). However, the molecular\nfingerprint indicates that most of the detected compounds belong to a homologous series of long-\nchain aliphatic OSs, such as C H SO and C H SO (Fig. 3) and these compounds appear actually\nn 2n+2 4 n 2n 5\nat very low concentrations. Studies have suggested that ship emission are potential precursors of\nthese types of OSs4,5. At TO we also observe events of long-range transported pollution\n(unpublished work from our group) from South-Eastern Europe and Eastern Europe, hence, we\nbelieve that a significant source at the North Sea can be detected under the right transport\nconditions at TO. Even the “ultra-low sulfur” heavy fuel oil, which is the class of oil that is allowed\non the North Sea still contains up to 1000 ppm (0.1%) of sulfur. The back-trajectory analysis further\nsupports the hypothesis of a North Sea origin (Fig. 4).\nFig. 3: Molecular fingerprints (m/z-RT plot, Van Krevelen diagram, and Kroll diagram) of the enriched\nOS fractions (TO 2: 14.01.2022, night-time).\nFig. 4: Backward trajectories for samples TO-2.\nCertainly, there remain doubts about the exact origin of the OSs, therefore, we change the sentence\nin line 292-294 (the track-changes version) as follows:\n“The homologue series of aliphatic OSs in this sample indicatessuggests an anthropogenic origin\n(Fig. S2e), ). while Bbacktrajectories indicate a possible marine origin from the North Sea (Fig. S16)\nwith the aliphatic OS are likely being emitted by the shipping sector60.”\nL259: Again the reader is wondering how these three samples were selected.\nSee above (the answer for comments L192).\nL261: This has previously been observed by e.g. Glasius et al., Composition and sources of carbonaceous\naerosols in Northern Europe during winter, Atmospheric Environment, 173, 2018, pages 127-141.\nThank you for pointing out the study by Glasius et al. (2018), which we have now included as\nreference #61 in the line of 302 (the track-changes version).\n61. Glasius, M. et al. Composition and sources of carbonaceous aerosols in Northern Europe during\nwinter. Atmos. Environ. 173, 127–141 (2018).\nL278: This seems like a very strong statement for apparently unpublished data.\nWe believe that OSs with an aliphatic carbon backbone are certainly of anthropogenic origin, while\nOS of biogenic origin have been described by many other studies in the past. It can be expected\nthat a yearlong sampling that covers periods of high and low biogenic emissions (summer vs winter)\ncan enable identifying the OSs from biogenic origin. Therefore, we don’t think that this is such a\nstrong statement, but rather a realistic outlook what could be achieved with our described method\nin future work.\nFigure 1: I suggest to make larger dots in the legend, so they are easier to see.\nWe have updated Figure 1 accordingly (in line 131 of the track-changes version), with larger dots in\nthe legend to enhance clarity. New figure sees below:\nFigure 2: In the description of the aerosol filter sample extraction in the figure, I suggest to list the\ncomposition of the extraction solvent.\nWe have updated Figure 2 accordingly (in line 152 of the track-changes version), with the\ncomposition of the extraction solvent. New figure sees below:\nFigure 2. Schematic of the extraction and enrichment procedure. Relative abundance (%) of summed peak areas ((−)ESI)\nof the different compound classes in the native extraction (A), diluted OS fraction (B) and enriched OS fraction (C). The\ny-axis shows the relative abundance of the compound classes, normalised to the most intense sample (native extraction of the\nHandan 3 sample). For better clarity, the left y-axis refers to the Handan samples (black; 0-100%), and the right y-axis to the\nTaunus Observatory (TO) samples (pink; 0-20%). The samples used were Handan 1: 18.10.2018, daytime; Handan 2:\n21.10.2018, daytime; Handan 3: 21.10.2018, night-time; TO 1: 11.12.2021, daytime; TO 2: 14.01.2022, night-time, and TO 3:\n28.02.2022, night-time. In order to make the native extraction (A), the diluted OS fraction (B) and the enriched OS fraction (C)\ncomparable, we accounted for the enrichment factor by the SPE (~240) and the different experimental setup in (C): (A) and (B)\nwere acquired using HPLC-HRMS (100% of mobile phase flow into the MS) and (C) by using HPLC-HRMS/CAD (16.5% of\nmobile phase flow into the MS). The consistently larger relative abundance of OSs in fraction (C), therefore, solely originates\nfrom additionally detected OSs following SPE. Created in BioRender. Reininger, N. (2025) https://BioRender.com/n70h222.\nFigure 3: Is CHOP a common group of compounds in aerosols?\nYes, CHOP compounds, which refer to organic compounds containing carbon (C), hydrogen (H),\noxygen (O), and phosphorus (P), are common in aerosols, though they are less commonly discussed\ncompared to other groups like OSs or organonitrates. Due to their large anthropogenic sources,\nbeing classified as High Production Volume (HPV) chemicals, they are widely used as flame\nretardants or plasticizers. Generally, the primary emitted CHOP compounds (Fig. 5) don’t have\nacidic protons (e.g., TCEP, TCPP, TPhP, TBEP), however, their transformation products tend to have\nhydroxyl or carboxyl group (e.g., TCEP-1, TCEP-21, TCPP-38, TCPP-9, TPhP-6, TPhP-8)6, which\nenables the detection of these compounds in negative electrospray ionization mode (Fig. 6).\nFig. 5: some of the primary emitted CHOP compounds. Figure modified from Liu et al.6\nFig. 6: some of the transformation products (which contain acid protons) of primary CHOP compounds.\nFigure modified from Liu et al.6\nReviewer 2\nSummary and Recommendation:\nThis manuscript examines the types and amounts of organosulfates (OSs) found in PM2.5 samples\ncollected from Handan, China (urban) and Hesse, Germany (mountain top site that is remote) by using\nsolid phase extraction (SPE) as a sample preparation step before RPLC-CAD-HRMS analysis. Although\nSPE has been rarely used in aerosol chemistry research, it helps in aiding the identification and\nquantification of OSs when using RPLC-CAD-HRMS. I should note that SPE was originally used in earlier\nwork that analyzed PM2.5 samples collected from the SE USA (e.g., Gao et al., 2006, JGR-Atmospheres),\nbut it was later neglected due to it removing small water-soluble OSs (such as the isoprene-derived OSs)\nin one of the washing steps. This latter recognition was so important to isoprene-derived SOA because\nusing SPE caused these OSs to go initially unrecognized. With that said, to my knowledge, this study\nuses RPLC coupled with both CAD with ESI-HRMS detection. The CAD offers incredible potential to\nquantify OSs without the need for all of the authentic standards being available. In atmospheric\nchemistry, the lack of available authentic standards has been a huge hindrance, preventing models\nfrom being updated with the latest multiphase chemical processes that lead to low-volatility OSs.\nOverall, I find this manuscript exciting and mostly well-written, especially for atmospheric and aerosol\nchemists. Some readers not well-versed in analytical chemistry might find this paper somewhat difficult\nto read. However, I hope the Editor won't find this a reason to reject the paper. I do think the\ndemonstration of a large un-resolved OS peak in the chromatogram is notable and appears to\ncontribute a large OA mass to PM2.5 collected during the winter seasons in China and Germany. I have\nseveral major comments below that should be carefully addressed by the authors before publication in\nNature Communications can be fully considered. Due to the nature of my comments below, I\nrecommend the manuscript be reconsidered after major revisions.\nWe appreciate the recognition of the novelty and importance of our study.\nMajor Comments:\n1.) Filter collection artifacts:\nThere has been some important work to recently come out of Hartmut Hermann's lab (and I believe\nMarianne Glasius' lab) showing that filter sampling (especially with media like quartz fiber filters) is\nprone to positive sampling artifacts that lead to the production of certain OSs like m/z 294 (one of the\nnitrooxy OSs). Since high-volume filter samplers cannot use denuders (due to the high flowrates used)\nto remove gas-phase SO2 and NOx, it is possible that NOx and SO2 can absorb from the gas phase and\nreact on these filter types yielding unwanted reactions that lead to OSs. Knowledgeable readers might\nwonder how much of the mass you quantify is related to these positive sampling artifacts?\nThe work by Brüggemann et al. (from Prof. Hartmut Hermann's group) indeed highlights the\nformation of organosulfates (OSs) on quartz fiber filters during chamber experiments in the\npresence of SO₂.7 However, the conditions in their study differ significantly from those at our rural\nbackground site, where SO₂ concentrations are generally very low (e.g. the SO concentration of TO\n2\nduring our sampling day was lower than the detection limit of the SO detector). We infer that most\nanalysis for the TO2 (Fig.7a also in supplementary Information Fig. S16a) and TO3 (Fig. 7b also in\nsupplementary Information Fig. S16b) samples indicates distinct air mass origins. For TO2, the air\nmasses originated from the North Sea, suggesting a marine influence, with aliphatic OSs potentially\nemitted by the shipping sector (Fig. 3). In contrast, TO3 air masses originated in eastern Germany,\nsouthern Poland, and northern Czech Republic—key regions in Europe associated with coal\ncombustion by power plants. Particularly, the approximately one-day atmospheric transport time\nfrom the source region is twice the duration of the filter sampling period, suggesting minimal OS\nartifact formation in the TO filter samples.\nFig. 7: Backward trajectories for samples TO-2 (a) and TO-3 (b). Backward trajectories for Taunus\nObservatory (825 m.a.s.l.) starting every hour for the 12-hour filter sampling duration. Duration of\nbackward trajectories is 48 hours and was calculated based on the online web version of the NOAA\nHysplit Model.8\nWe also extracted the nitrooxy-OSs ion (m/z 294) from the LC-MS chromatograms of the solvent\nblank, ambient quartz filter blank, and ambient quartz filter sample (Fig. 8). The signal for all\nnitrooxy-OSs isomers is significantly higher in the ambient sample than in the solvent and filter\nblanks. Hence, this indicates the presence of OSs in the aerosol rather than their formation as filter\nartifacts.\nFig. 8: Comparison of extracted ion chromatograms (EICs) of nitrooxy-OSs (m/z 294.0653) in the\nsolvent blank, ambient quartz filter blank, and ambient quartz filter sample (Handan, October 21, 2018,\nnighttime), measured by HPLC-HRMS.\nWe also implemented several measures to minimize their impact. For each site, we collected\nmultiple filter blanks to subtract the background signal. These blanks were exposed for the same\nduration as the real filters, with the only difference being that the real filters operated with the\npump running for 11.5 hours. Additionally, during data processing with Compound Discoverer, we\napplied a stringent threshold to ensure data reliability, considering only signals in the ambient filters\nthat were at least ten times higher than those in the blanks as real signals.\nOverall, we believe that positive sampling artifacts have minimal influence on our quantified mass.\nHowever, we cannot entirely rule out the possibility of sampling artifacts, therefore, we change the\nsentence in the line 297-303 (the track-changes version) as follows:\n“Furthermore, we cannot rule out that a fraction of OSs is formed during filter sampling, although\nthe SO concentrations at this rural background station are usually below quantifications limits for\n2\nstandard detectors. Hence, we infer that the majority of OSs observed at rural and remote stations\nactually forms during atmospheric transport. Analysis of the Bbackward trajectories for TO 3\nindicate that the air masses originated from eastern Germany, southern Poland and the northern\nCzech Republic, which are Europe´s hotspots of coal combustion by power plants (Fig. S16)61. The\ntime of atmospheric transport from this source area of around one day is twice as long as the actual\nfilter sampling period, which speaks against significant OS artifact formation for the TO filter\nsamples.”\nAlso see answer to the first reviewer L37\nTo improve the logical flow of the introduction, we cite some new references in line 40-47 (the\ntrack-changes version):\nWe first cite a paper by Brüggemann et al., which discusses OS-related sample artifacts. We then\nreference the work of LeBreton et al., whose near-real-time method minimizes the formation of OS\nartifacts, demonstrating that the observed OSs are not purely artifacts. Finally, we cite the study by\nMa et al., who report similar results to LeBreton et al., but with filter collection and UHPLC-HRMS\nmeasurements, further supporting the validity of our findings.\nWe changed the sentence in line 40-47 (the track-changes version) as follows:\n“Several studies have analysed OSs in different environments, e.g. in the urban OA of Chinese\nmegacities, in which rapid formation of OS during pollution events has been observed28–31. However,\nBrüggemann et al. reported significant sampling artifacts in the detection and quantification of\nmonoterpene-derived OSs under atmospheric conditions32. LeBreton et al. used a Filter Inlet for\nGases and Aerosols coupled to a Chemical Ionization Mass Spectrometer to measure OSs in near-\nreal time, and therefore minimizing sampling artifacts, at a semi-rural site in Beijing. They quantified\n17 single OSs, estimating their overall contribution to OA at 2%, suggesting that OSs are not merely\nartifacts of the sampling process33. Similarly, a study conducted in Beijing using high-performance\nliquid chromatography (HPLC) combined with high-resolution mass spectrometry (HRMS) reported\na ~4% contribution of OSs to OA28.\nThe new references are:\n28. Ma, J. et al. Nontarget Screening Exhibits a Seasonal Cycle of PM2.5Organic Aerosol\nComposition in Beijing. Environ. Sci. Technol. 56, 7017–7028 (2022).\n32. Brüggemann, M. et al. Overestimation of Monoterpene Organosulfate Abundance in Aerosol\nParticles by Sampling in the Presence of SO2. Environ. Sci. Technol. Lett. 8, 206–211 (2020).\n2.) Lines 29-31: The authors should cite the earliest papers here for OSs being discovered as an\nimportant subclass of OA, including the following:\nRomero and Oehme (2005, J. of Atmos. Chem.) - DOI: 10.1007/s10874-005-0594-y\nSurratt et al. (2007, ES&T) - DOI: 10.1021/es062081q\nIinuma et al. (2007, Atmos. Environ.) - https://doi.org/10.1016/j.atmosenv.2007.03.007\nIinuma et al. (2007, ES&T) - https://doi.org/10.1021/es070938t\nSurratt et al. (2008, JPCA) - https://doi.org/10.1021/jp802310p\nThe Surratt et al. (2008, JPCA) study estimated that upwards of 30% of particulate OM in Hungary could\nbe OSs.\nAdditional work by Betsy Stone's group has also estimated OS contributions to OM, such as\nhttps://doi.org/10.5194/acp-19-3191-201. In that study, they estimated 16.5% of PM2.5 OC as OSs.\nThank you for pointing out these studies, we have included these studies as references #8-13 in the\nline of 32 (the track-changes version). The revised sentence are as follows:\n“Organosulfates (OSs) are an important subclass of OA8–13, comprising up to 30% of the OA12. They…”\nThe new references are:\n8. Romero, F. & Oehme, M. Organosulfates - A new component of humic-like substances in\natmospheric aerosols? J. Atmos. Chem. 52, 283–294 (2005).\n9. Surratt, J. D. et al. Evidence for organosulfates in secondary organic aerosol. Environ. Sci. Technol.\n41, 517–527 (2007).\n10. Iinuma, Y. et al. Evidence for the existence of organosulfates from β-pinene ozonolysis in\nambient secondary organic aerosol. 41, 6678–6683 (2007).\n11. Iinuma, Y., Müller, C., Böge, O., Gnauk, T. & Herrmann, H. The formation of organic sulfate esters\nin the limonene ozonolysis secondary organic aerosol (SOA) under acidic conditions. Atmos. Environ.\n41, 5571–5583 (2007).\n12. Surratt, J. D. et al. Organosulfate formation in biogenic secondary organic aerosol. J. Phys. Chem.\nA 112, 8345–8378 (2008).\n13. Hettiyadura, A. P. S., Al-Naiema, I. M., Hughes, D. D., Fang, T. & Stone, E. A. Organosulfates in\nAtlanta, Georgia: Anthropogenic influences on biogenic secondary organic aerosol formation.\nAtmos. Chem. Phys. 19, 3191–3206 (2019).\n3.) Lines 31-32: I'm a bit surprised the authors didn't cite any work on the reactive uptake of isoprene\nepoxydiols (IEPOX) yielding OSs in PM2.5. This is one of the major semi-volatile oxidation products out\nthere that has helped clue us in as a research community on the role of reactive uptake (multiphase\nchemistry) onto wet acidic sulfate aerosols that yield OSs.\nThank you for highlighting the relevance of the reactive uptake of isoprene epoxydiols (IEPOX) as a\nkey pathway for OS formation from isoprene. This mechanism indeed plays a crucial role in\nadvancing our understanding of multiphase chemistry on acidic sulfate aerosols, and being\nespecially relevant for high-isoprene emission regions such as the Amazon or the south-east US.\nWe have now included relevant references of the studies discussing the IEPOX yielding OSs. The\nsentence in the line of 34-36 (the track-changes version) now reads as follows:\n“The reactive uptake of semi-volatile organic compounds, particularly isoprene epoxydiols (IEPOX),\nonto acidic particles can result in the formation of low-volatile OSs and, consequently, add to the\nOA mass9,16,21–2316,21.”\nThe newly cited references:\n9. Surratt, J. D. et al. Evidence for organosulfates in secondary organic aerosol. Environ. Sci. Technol.\n41, 517–527 (2007).\n22. Riva, M. et al. Chemical Characterization of Secondary Organic Aerosol from Oxidation of\nIsoprene Hydroxyhydroperoxides. Environ. Sci. Technol. 50, 9889–9899 (2016).\n23. Surratt, J. D. et al. Chemical composition of secondary organic aerosol formed from the\nphotooxidation of isoprene. J. Phys. Chem. A 110, 9665–9690 (2006).\n4.) Lines 32-33: Similar to earlier lines in the introduction, simply citing review articles for statements\nlike this doesn't seem appropriate. I think the authors should be citing some of the original studies that\ndemonstrated the role of SO2 emissions in enhancing SOA from BVOC oxidations.\nWe agree that citing original studies demonstrating the role of SO₂ emissions in enhancing SOA\nformation from BVOC oxidations will strengthen the manuscript.\nIn response, we have included, in addition to the important review article by Brüggemann et al., a\nfew pivotal references in line of 37 (the track-changes version). Certainly, there are more\npublications on this topic, but we also have to consider that the journal has a certain limit on the\nnumber of references.\nThe newly cited references:\n12. Surratt, J. D. et al. Organosulfate formation in biogenic secondary organic aerosol. J. Phys. Chem.\nA 112, 8345–8378 (2008).\n25. Surratt, J. D. et al. Reactive intermediates revealed in secondary organic aerosol formation from\nisoprene. Proc. Natl. Acad. Sci. U. S. A. 107, 6640–6645 (2010).\n5.) Lines 36-49: This paragraph is very European- and Asian-centric. I think this paragraph is fine, but a\nlot of prior OS work has also been done in the USA trying to quantify OSs in PM2.5 there. There has\nbeen a lot of work by the Surratt Group (UNC) and Stone Group (University of Iowa) on this topic using\nLC/MS methods and synthesizing authentic compounds. Furthermore, Jian Yu's Group (HKUST) in Hong\nKong has also estimated OS contributions in PM2.5 and has synthesized authentic compounds as well.\nTo address this point, we have revised the paragraph in the line 48-52 (the track-changes version)\nto ensure a more comprehensive and balanced representation of global research efforts in this field.\nSimilar to the previous answer, there are more publications from America, but we need to consider\nthat the journal imposes a limit on the number of references.\nThe new text now as follows:\n“In urban and rural OA, studies have shown significant seasonality in both biogenic and\nanthropogenic OSs at various sites, with biogenic OSs peaking in summer and anthropogenic OSs in\nwinter34,35. LeBreton et al. quantified 17 single OSs and estimated their overall contribution as being\n2% to the OA at a semi-rural site in Beijing by using the Filter Inlet for Gases and Aerosol (FIGAERO)\ncoupled to a chemical ionisation mass spectrometer24. In marine OA, in the Yellow and Bohai Seas,\nthe contribution of biogenic OSs ranges from 0.04 to 6.9%36.”\nThe newly cited references:\n34. Meade, L. E. et al. Seasonal variations of fine particulate organosulfates derived from biogenic\nand anthropogenic hydrocarbons in the mid-Atlantic United States. Atmos. Environ. 145, 405–414\n(2016).\n35. Chen, Y. et al. Seasonal Contribution of Isoprene-Derived Organosulfates to Total Water-Soluble\nFine Particulate Organic Sulfur in the United States. ACS Earth Sp. Chem. 5, 2419–2432 (2021).\n6.) Lines 92-93: How comfortable are the authors assuming that the majority of OSs are stable in the\npresence of a strong acid? Work from Matt Elrod's group (Oberlin College) has suggested that the\nstability of OSs depends on whether the OS is primary or tertiary. Did the authors consider testing their\napproach with different OS standards to confirm the stability of OS compounds?\nTo confirm the stability of OSs under the acidic conditions for SPE elution used in our study, we\nconducted experiments with eight OS standards (one secondary OS (5) and one tertiary sulfonate\n(4)) with varying molecular structures. Figure 9 presents the extracted-ion chromatograms (EICs)\nfor individual standards before and after SPE, alongside their molecular structures and recoveries.\nWe observed that small, polar compounds (e.g., C1 and C2 compounds) either had lower recoveries\nor reacted under acidic conditions, consistent with observations noted in the manuscript (marked\nversion, lines 126–128). In contrast, the remaining compounds demonstrated high stability, with\nrecoveries close to 100% using our SPE method involving 0.5% HCl (Fig. 9). Additionally, no\ndifferences in recovery were observed between primary and secondary OS (tertiary sulfonate). The\nretention times for all standards remained within a 0.05-minute tolerance, indicating no chemical\ntransformation of these compounds.\nFig. 9: Comparison of extracted ion chromatograms (EICs) and molecular structures of the eight OS test\nstandards, measured by HPLC-HRMS, before and after SPE isolation (the post-SPE elution sample was\ndiluted by the theoretical SPE enrichment factor to enable the direct comparison with the pre-SPE\nsample).\nFurthermore, the nontarget analysis of ambient representative filter (Handan 1) shows 72% of the\ncompounds in the OS fraction had an absolute recovery greater than 50% compared to the native\nextraction. Some OSs exhibited poorer recoveries, ranging from 10% to 50%, with relatively low\nsignal intensities (Fig. 10a, in Supplementary Information Fig. S1a). By extracting this sample via SPE\nin triplicate, we determined that the intensity-weighted average relative standard deviation (RSD)\nof the SPE recovery for OSs was approximately 15%, with larger uncertainties for low-intensity\ncompounds (Fig. 10b, in Supplementary Information Fig. S1b).\nFig. 10: Evaluation of the SPE isolation recovery (a) and reproducibility (b) for individual compounds\nusing a PM2.5 filter extract from Handan (18.10.2018, daytime).\nThese results clearly demonstrate that the majority of OSs analyzed in this study remained stable\nunder the acidic SPE conditions employed. The high recoveries and consistent retention times of\nthe tested standards, combined with the highly reproducible nontarget screening results of\nambient OSs, show that our approach effectively captures most OS compounds with minimal\ndegradation or transformation.\nTo make the sentence more precise, we revised the sentence in lines 111–113 (tracked-changes\nversion) as follows:\n“Subsequently, we The majority of OSs are chemically stable which, thus, allowed the used of a\nstrong diluted hydrochloric acid dissolved in methanol to elute the OSs m OSs from the cartridge.\nWe did not observe any evidence for degradation of the OSs, confirming their chemical stability at\nlow pH52.”\n7.) Figure 1d:\nAs shown in Figure 1d, there are still some OS compounds that elute through what they call \"flow-\nthrough.\" Are these accounted for? One thing I want to note to the authors is that in Gao, Surratt, et\nal. (2006, JGR-Atmospheres), they employed an SPE separation to measure SOA. When they did this\nOSs were missed, especially the very water-soluble types (like the isoprene-derived OSs). Later Surratt\net al. (2007, ES&T) found that by not using SPE, but rather a polar-end capped C18 column, they could\nmeasure the isoprene-derived OSs along with the larger monoterpene-derived OSs. Later, Betsy Stone's\ngroup (University of Iowa) and later Surratt's group (UNC) showed that HILIC separation is much more\neffective in resolving and quantifying isoprene-derived OSs and other water-soluble and smaller OSs.\nI'm curious, do the authors worry they are still missing water-soluble OSs that are not well retained on\nC18 columns? I noted the authors conducted reverse-phase liquid chromatography (RPLC) to separate\nand measure particulate OSs. However, it might be better for all of us in the community to use both\nHILIC and RPLC separations as orthogonal methods to better separate, characterize and quantify the\nOSs that range from water-soluble to less water-soluble. Do you agree? It might be worth mentioning\nthe limitations of RPLC in past work and why HILIC methods have come about for the smaller OSs.\nWe appreciate that the reviewer mentions prior work on OS separation methods, particularly\nhighlighting the challenges of analyzing highly water-soluble OSs with RPLC and the advantages of\nalternative methods like HILIC. We agree that HILIC is the most favorable method for the separation\nof polar organic compounds.\nAccounting for flow-through OS compounds in Figure 1d:\nThe \"flow-through\" fraction observed in Figure 1d was not included in the recovery calculation.\nSince the solvent for the flow-through is 2% MeOH in H O, it is not possible to concentrate further,\n2\nand it cannot reach the detection limit of the CAD. Additionally, this fraction only contains a very\nsmall portion of the CHOS compounds. We have also highlighted this limitation of the method in\nthe manuscript (the track-changes version, lines 126–129).\nOS Retention on SPE MAX cartridge:\nIn this study, however, we use a mixed-mode anion-exchange (MAX) cartridge combined with a\nreversed-phase sorbent (Fig. 11). The quaternary ammonium cation (functional group ③) in the\ncartridge interacts with the highly acidic terminal R-OSO₃H group of OSs, ensuring strong retention\nof OSs.\nFig. 11: the polymer skeleton of the Mixed-mode Anion-eXchange cartridge with a reversed-phase\nsorbent.\nTo evaluate the SPE recovery of OSs from isoprene, we performed a new dedicated experiment.\nWe used an oxidation flow reactor to generate isoprene-derived OSs. Using a PAM-OFR, isoprene\nwas oxidized by O₃ (2 ppm) and photooxidation (OH was generated by UV light (λ = 254 nm) inside\nthe OFR). Additionally, we inject SO₂ (55 ppb) at relative humidity ~60%. The SOA exiting the OFR\npassed through two 50 cm denuders filled with charcoal to remove reactive gas-phase compounds.\nSOA particles were collected on glass fiber filters at a flow rate of 3 L min⁻¹ over 120 minutes.\nWe extracted the filters and applied the SPE method described in the paper, using this time a HILIC\ncolumn for the separation of isoprene OSs. A comparison of compound intensities across different\nfractions, based on non-target analysis, revealed that the second SPE elution recovered only ~30%\nof the OSs present in the native elution. However, the missing OSs were not detected in the flow-\nthrough fraction (Fig. 12). We infer that these OSs were either strongly retained on the anion-\nexchange cartridge or they are more reactive compared to longer chain OS.\nFig. 12: Summed peak areas of different compound classes in the isoprene-derived OS sample across\nthe native extraction, first elution, second elution, and flow-through, analyzed using the HILIC column\n(both first and second elutions were diluted by the theoretical SPE enrichment factor to enable the\ndirect comparison of areas).\nWe are currently working to isolate isoprene-derived OSs by testing different elution strategies for\nthe SPE, in case the isoprene-OS are retained very strongly to the solid phase material. However,\nsuch detailed method development is beyond the scope of this manuscript. Instead, we focus on\nthe scientific novelty of our findings, specifically the discovery of a significant fraction of\nchromatographically unresolved OSs in ambient aerosols, which appear throughout the\nchromatogram of the RPLC-column.\nDiscussion of the limitations of RPLC:\nFor this study, we focused on RPLC because it allowed for effective separation and quantification\nof the majority of OSs detected in ambient aerosol samples. However, we agree that using HILIC as\nan orthogonal method alongside RPLC would provide a more comprehensive characterization of\nOSs. Therefore, revisions were made to the manuscript in the line of 126-129 (the track-changes\nversion):\n“However, we did note that the used SPE cartridge provides a low recovery of weakly retained some\nsmall and highly polar OSs (e.g. C -C ), which may include certain isoprene-derived OSs, an\n2 3\nimportant class of atmospheric OSs. To a lesser extent, reversed-phase LC (used in this study) is also\nless effective for separating these compounds, highlighting the complementary role of hydrophilic\ninteraction LC methods in analyzing smaller OSs54.”\nWe have added a new reference #54 in the line 129 (the track-changes version) to support our\ndiscussion:\n54. Cui, T. et al. Development of a hydrophilic interaction liquid chromatography (HILIC) method\nfor the chemical characterization of water-soluble isoprene epoxydiolydiol (IEPOX)-derived secondary\norganic aerosol. Environ. Sci. Process. Impacts 20, 1524–1536 (2018).\nWe added a sentence to the manuscript in the line of 317-318 (the track-changes version) and\nincluded the Fig.12 in the supplementary Information as Figure S17:\n“Especially, isoprene-derived OSs might still be underestimated using the described method, as we\nfound the SPE-recovery for these polar compounds is around 30% (Fig. S17).”\n8.) Line 117-118: How do the authors know they extracted all of the OS compounds and mass from their\nfilters using 98% water and 2% methanol by volume solvent only? Were standards of OSs also examined\nthrough their extraction procedures? I think this would be helpful to include. Could larger OSs be missed\nif not soluble in water? Also, with water, do you have to worry about the hydrolysis of OSs?\nWe agree it is important to evaluate extraction efficiencies of the used solvent in this study. Since\nOSs are highly water-soluble, we are confident that a water-based extraction solvent is effective for\nextracting the majority of OSs. Of course, the extraction efficiencies may be lower for non-polar\nOSs, which is why we also tested the method with additional experiment by using authentic\nstandards, as described below:\nWe prepared a solution of eight OS standards with varying molecular structures (see Fig. 9 question\n6) and spiked the same amount of the standard mix onto the following filters: 1) Handan ambient\nblank (quartz fiber), 2) Handan ambient sample (quartz fiber), 3) TO ambient blank (glass fiber), and\n4) TO ambient sample (glass fiber). These filters were then extracted twice with a water-based\nsolvent (2% MeOH in H O), and the extracts were combined. The extracts were analyzed by LC-\nfilter materials. The extracted ion chromatogram of the eight standards from the five\nmeasurements (LC-HRMS chromatogram) are displayed in Fig. 13. We calculated the extraction\nefficiency of the standard mix for the four filter extracts based on the pure standards measurement\n(Fig. 14).\nUsing our extraction procedure, we found that the recovery of the standards spiked onto different\nfilter materials was consistently above 80%, except for dodecyl sulfate (C H SO ⁻) on the Handan\n12 25 4\nambient filter, which was slightly below 80% (Fig. 14).\nAdditionally, we considered the possibility of hydrolysis during extraction but observed no\nsignificant degradation of OSs under the conditions used, as also verified through the stability of\nstandards (Fig. 9). These results support the reliability of our extraction method in capturing the\nmajority of OS compounds without significant loss or reaction.\nFig. 13: Comparison of extracted ion chromatograms for the eight OS standards across different filter\nmaterials.\nFig. 14 Comparison of the responses of the eight OS standards across different filter materials. The\nblack line is normalized to the pure standard measurement.\n9.) Lines 121-122: I agree that the recoveries can be affected by what the authors state here. But what\nabout also the matrix of the sample? Could that affect the recovery from the filters? For example, it is\nknown that particulate PAHs can be hard to extract from certain filters depending on the matrix of the\nfilter and the other components collected on the filter.\nWe also tested our extraction procedure across different filters and filter materials and found that\nthe recovery of almost all the standards spiked onto these filters was consistently above 80%.\nSee the answer to question 8.\n10.) Lines 144-145: The authors note that there are OSs that are aliphatic and of anthropogenic origin.\nAre the authors aware of work by Riva et al. (2016, ACP)? This study demonstrated OSs form from the\noxidation of alkanes in the presence of wet acidic sulfate aerosols. Riva et al. (2015, ES&T) also\ndemonstrated OSs form from PAH oxidations in the presence of sulfate aerosols. Notably, in this latter\nstudy, they also observed sulfonates. Did you by chance observe any sulfonates in your PM2.5, possibly\nfrom the wintertime samples? What about other oxidation states of sulfur?\nThank you for highlighting the studies by Riva et al. (2016, ACP) and Riva et al. (2015, ES&T). We are\naware of these publications, which strongly support our hypothesis that some OSs are aliphatic and\nof anthropogenic origin. Accordingly, we have cited these studies in the revised manuscript (the\ntrack-changes version, line 173). The new references #22 and #55:\n22. Riva, M. et al. Chemical Characterization of Secondary Organic Aerosol from Oxidation of\nIsoprene Hydroxyhydroperoxides. Environ. Sci. Technol. 50, 9889–9899 (2016).\n55. Riva, M. et al. Chemical characterization of organosulfates in secondary organic aerosol\nderived from the photooxidation of alkanes. Atmos. Chem. Phys. 16, 11001–11018 (2016).\nWe observed some sulfonates in our winter samples, such as C₅H₁₂SO₃ and C₈H₁₈SO₃ (Fig. 15,\ndaytime sample from Handan on 18-10-2018). However, their intensities were lower compared to\nthose of OSs. All other sulfur-containing compounds identified in our samples were OSs, with sulfur\nin an oxidation state of +6.\nFig. 15: Extracted ion chromatograms for C₅H₁₂SO₃ and C₈H₁₈SO₃.\n11.) Minus the comment I made about the filter sampling artifacts above, I wonder if this method would\nshow even higher OS fractions in fine particulate OM during the summer seasons when BVOC-derived\noxidation products are known to yield substantial amounts of OS in the presence of sulfate? The fact\nthe authors show fairly high amounts in winter, tells me the mass fractions could even be higher than\npreviously thought in summer....\nTo explore this hypothesis, we conducted a new experiment involving solid-phase extraction (SPE)\nand HPLC-CAD-HRMS analysis using a summer season sample collected on July 2, 2019, from\nHandan, China. Our results revealed a larger fraction of unresolved OS in this sample (Fig. 16). This\nfinding supports the idea that BVOC-derived oxidation products significantly contribute to OS\nformation in the presence of sulfate under summer (haze) conditions.\nUnfortunately, our group currently lacks the instrumentation required to measure organic carbon\n(OC) on the filters, preventing us from calculating the OS fractions in organic matter. As part of our\nfuture work, we plan to analyze yearlong filter samples with this method. This will help distinguish\npurely anthropogenic from mixed biogenic/anthropogenic OS contributions, providing a more\ncomprehensive understanding of seasonal variations across different locations.\nFig.16: Comparison of the chromatograms of the enriched OS fraction of the Handan filter (July 2, 2019,\nblack) and blank (red).\n12.) Some readers or reviewers might ask why the authors didn't try this RPLC-DAD-HRMS method on\na well-characterized lab-generated SOA type like IEPOX SOA before analyzing field samples? The reason\nI raise this point is for IEPOX SOA our community has an analytical method (i.e., HILIC/ESI-HR-QTOFMS\nfrom both the Stone and Surratt Groups) and authentic standards (from the Surratt Group) to compare\nyour quantitative method with. Furthermore, lab studies typically can denude unwanted gases (like\nSO2/NOx) before collecting onto Teflon filters (which are less prone to positive sampling artifacts like\nquartz).\nSee answer 7. We generated an isoprene SOA filter, but encountered difficulties eluting the OSs\nfrom the MAX SPE cartridge. We are currently optimizing the method, such as adjusting the acid\nused to elute the cartridge. However, we believe that a detailed method development will be better\nsuited for a separate publication.\nThat said, we recognize the potential of HILIC for more effectively separating isoprene-derived OSs\ncompared to RPLC (Fig. 17), as HILIC offers a complementary approach for polar compound analysis.\nWe are actively working on integrating our SPE method with HILIC-CAD-HRMS to enhance the\nseparation and detection of isoprene-derived OSs.\nFig. 17 Comparison of the effects of separation of C H SO using the RPLC and HILIC.\n5 12 7\nReferences:\n1. Brüggemann, M. et al. Quantification of known and unknown terpenoid organosulfates in\nPM10 using untargeted LC–HRMS/MS: contrasting summertime rural Germany and the North\nChina Plain. Environ. Chem. 16, 333–346 (2019).\n2. Wang, Y. et al. Comparative Study of Particulate Organosulfates in Contrasting Atmospheric\nEnvironments: Field Evidence for the Significant Influence of Anthropogenic Sulfate and NOx.\nEnviron. Sci. Technol. Lett. 7, 787–794 (2020).\n3. D. Surratt, J. et al. Organosulfate formation in biogenic secondary organic aerosol. J. Phys.\nChem. A 112, 8345–8378 (2008).\n4. Qi, L. et al. Molecular characterization of atmospheric particulate organosulfates in a port\nenvironment using ultrahigh resolution mass spectrometry: Identification of traffic emissions.\nJ. Hazard. Mater. 419, 126431 (2021).\n5. Huang, L. et al. Biogenic and Anthropogenic Contributions to Atmospheric Organosulfates in a\nTypical Megacity in Eastern China. J. Geophys. Res. Atmos. 128, e2023JD038848 (2023).\n6. Liu, Q. et al. Uncovering global-scale risks from commercial chemicals in air. Nat. 2021\n6007889 600, 456–461 (2021).\n7. Brüggemann, M. et al. Overestimation of Monoterpene Organosulfate Abundance in Aerosol\nParticles by Sampling in the Presence of SO2. Environ. Sci. Technol. Lett. 8, 206–211 (2020).\n8. Stein, A. F. et al. NOAA’s HYSPLIT Atmospheric Transport and Dispersion Modeling System.\nBull. Am. Meteorol. Soc. 96, 2059–2077 (2015)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a very good job addressing the comments from the reviewers. However, I have a few additional comments to the response.\nLine 18-19\n“We found a significantly larger organosulfate fraction appearing as a broad chromatographic peak in the charged aerosol detector.” You do not find peaks inside the charged aerosol detector, but maybe in the chromatograms from it. Please use correct wording.\nRegarding the investigation of responses of different OS standards, it can be discussed whether the first compound formaldehyde sodium bisulfite has a sufficient response or it should be excluded from the calculation of average response and variation, when evaluating the suitability of camphor-sulphonic acid as reference standard.\n“The application of SPE for enrichment and fractionation of compounds is rarely practiced, but this sample preparation enables unique and novel analytical approaches.” As the other reviewer pointed out, SPE was already used by Gao et al., 2006, JGR-Atmospheres. Furthermore SPE is used to extract HULIS in aerosol samples for quantification and characterization (e.g. Baduel, C., Voisin, D., and Jaffrezo, J. L.: Comparison of analytical methods for Humic Like Substances (HULIS) measurements in atmospheric particles, Atmos. Chem. Phys., 9, 5949–5962, https://doi.org/10.5194/acp-9-5949-2009, 2009.). Based on these considerations, I suggest removing “unique” here.\nOS/OM. It is important to clarify in the text, that here the OS mass fraction includes the sulphate group. Meanwhile some of the previous studies listed in Table S7 may have either included or excluded the mass of the sulfate group in the OS fraction. Did the authors check this?"}, "author_response": "Manuscript ID: NCOMMS-24-43312B\nManuscript Type: Article\nCollection: Mass spectrometry method development\nResponse to reviewer\nReviewers’ Comments to the Authors:\nReviewer 1\nThe authors have done a very good job addressing the comments from the reviewers. However, I have\na few additional comments to the response.\nThank you for your positive assessment and for taking the time to review our responses.\nLine 18-19\n“We found a significantly larger organosulfate fraction appearing as a broad chromatographic peak\nin the charged aerosol detector.” You do not find peaks inside the charged aerosol detector, but\nmaybe in the chromatograms from it. Please use correct wording.\nWe changed the sentence in line 19-20 (the track-changes version) as follows:\n“We found a significantly larger organosulfate fraction appearing as a broad chromatographic\npeak in the chromatograms from the charged aerosol detector.”\nRegarding the investigation of responses of different OS standards, it can be discussed whether the\nfirst compound formaldehyde sodium bisulfite has a sufficient response or it should be excluded from\nthe calculation of average response and variation, when evaluating the suitability of camphor-\nsulphonic acid as reference standard.\nThank you for pointing out the low response of formaldehyde sodium bisulfite. However, we\nbelieve its lower response in ESI is real, and we cannot rule out that other organosulfates exhibit\ndeviating response from the average. Hence, including this compound accounts for the overall\nlarger uncertainty with ESI quantification.\nCompared to the other tested standards, it has the shortest retention time under reversed-phase\nliquid chromatography, indicating higher polarity. This suggests it is well-suited for ionization using\nan electrospray ionization source. Additionally, simultaneous injection of all standards into a\ncharged aerosol detector showed that formaldehyde sodium bisulfite had a similar response to the\nother standards, with deviations within 20% (Fig. 1). This indicates that it fully elutes from the\nchromatographic column, supporting its inclusion in the evaluation of camphor-sulfonic acid as a\nreference standard.\nFigure 1. Comparison of the CAD (blue, upper figure) and HRMS (orange, lower figure) response\n(mean ± SD) of ten OS standards at an isocratic gradient. The standards were prepared in one batch\nas an OS-mix. The mix contained: (1) Formaldehyde sodium bisulfite (CH3SO4Na), (2) Methyl sulfate\nsodium salt (CH3SO4Na), (3) Sodium ethyl sulfate (C2H5SO4Na), (4) Butyl sulfate sodium salt\n(C4H10SO4Na), (5) Camphor-10-sulfonic acid (C10H16SO4), (6) 2-hydroxy-α-pinene OS (C10H18SO5),\n(7) Sodium octyl sulfate (C8H17SO4Na), (8) Sodium n-decyl sulfate (C10H21SO4Na), (9) Sodium\ndodecyl sulfate (C12H25SO4Na), and (10) Tetradecyl sulfate sodium salt (C14H29SO4Na). Each OS\nstandard had a concentration of 2 ng/µL. To test the stability of the instruments, we measured each\nstandard concentration five times. The HPLC flowrate was set to 0.4 mL/min, with 83.5% being\ndirected into the CAD and 16.5% being directed into the MS. The line (solid) is the average response\nof these compounds with a band (dash) of ± 20%.\n“The application of SPE for enrichment and fractionation of compounds is rarely practiced, but this\nsample preparation enables unique and novel analytical approaches.” As the other reviewer pointed\nout, SPE was already used by Gao et al., 2006, JGR-Atmospheres. Furthermore SPE is used to extract\nHULIS in aerosol samples for quantification and characterization (e.g. Baduel, C., Voisin, D., and\nJaffrezo, J. L.: Comparison of analytical methods for Humic Like Substances (HULIS) measurements in\natmospheric particles, Atmos. Chem. Phys., 9, 5949–5962, https://doi.org/10.5194/acp-9-5949-2009,\n2009.). Based on these considerations, I suggest removing “unique” here.\nWe changed the sentence in line 88-89 (the track-changes version) as follows:\n“The application of SPE for enrichment and fractionation of compounds is rarely practiced, but this\nsample preparation enables unique and novel analytical approaches.”\nOS/OM. It is important to clarify in the text, that here the OS mass fraction includes the sulphate\ngroup. Meanwhile some of the previous studies listed in Table S7 may have either included or\nexcluded the mass of the sulfate group in the OS fraction. Did the authors check this?\nTo clarify the fact that we include sulphate group for the OSs mass calculation, we added a sentence\nin line 630 (the track-changes version, in the figure 5’s caption) as follows:\n“We note that the OSs mass fraction includes the sulphate group.”\nWe also checked the references listed in Table S7, and found that the majority of studies include\nthe sulphate group in the OS/OM calculation. As not all papers provide all the necessary information\n(e.g. we had to assume an OC to OM conversion factor), therefore, we do not change the table in\nthe SI.\nDirect quantification\nlocation Particle size OS fraction in OM (%) Sulphate group Instruments Reference\nBeijing, China PM2.5 0.31 Include HPLC/MS Wang et al., 2018 2\nShanghai, China PM2.5 0.62 Include UPLC/ToF-MS Wang et al., 2021 3\nBeijing (Winter), China PM2.5 0.7 Include UHPLC/Orbitrap-MS Ma et al., 2022 4\nXi’an (Winter), China PM2.5 1.4 Include UHPLC/ToF-MS Glasius et al., 2022 5\n4 sites, Asia PM2.5 1.4 Include UPLC/ ToF-MS Stone et al., 2012 6\nBeijing, China PM1 2 Include? FIGAERO ToF-CIMS Le Breton et al., 2018 7\n19 sites, USA PM2.5 3.22 Include UPLC/ToF-MS Chen et al., 2021 8\nBohai and Yellow Sea, China PM2.5 3.47 Include UHPLC/Orbitrap-MS Wang et al., 2023 9\nBeijing (Summer), China PM2.5 4 Include UHPLC/Orbitrap-MS Ma et al., 2022 4\nXi’an (Summer), China PM2.5 7 Include UHPLC/ToF-MS Glasius et al., 2022 5\nIndirect quantification\nFairbanks, USA PM2.5 1.29 Exclude? XRF, IC Shakya et al., 2013 10\nHongkong, China PM1 5 (Minimum estimation) Include HR-ToF-AMS Huang et al., 2015 11\n334 sites, USA PM2.5 7.5 Exclude? XRF, IC Shakya et al., 2015 12\n12 sites, USA PM2.5 8.12 Include XRF, IC Tolocka et al., 2012 13\nRiverside, USA PM1.5 12 Include AMS, IC Farmer et al., 2010 14\n19 sites, USA PM2.5 14.51 ? ICP−OES, IC Chen et al., 2021 8\nBeijing, China PM2.5 25.05 Include? XRF, IC He et al., 2001 15\nK-puszta, Hungary PM2.5 29 Include XRF, IC Lukács et al., 2009 16\nK-puszta, Hungary PM2.5 30 Include XRF, IC Surratt et al., 2008 17\nReviewer 1's comments regarding response to Reviewer 2\nI have now carefully read the responses to point 7 and onwards from the other reviewer.\nOverall, I think the authors have addressed the comments well, and this has led to a better\nunderstanding of the limitations of the method, as well as suggestions for future work.\n7: The authors have thoroughly addressed the issue by performing a new study of isoprene-derived OS\nand they have added additional text to the manuscript to describe the associated uncertainty. This\nissue however does not diminish the novelty and overall importance of the study.\n8: The authors have correctly studied extraction efficiency and degradation. A recovery below 80% is\nobserved for the largest standard compound. As some of the OS compounds observed in ambient\nsamples are even larger (and thus probably less polar), the authors could mention in the text, that a\nslightly lower recovery was observed for dodecylsulfate and this may also be relevant for larger OS in\ngeneral.\n9, 10, 11 : The authors have adequately addressed the questions.\n12: I agree with the reviewer that it would have been interesting to analyze a well-characterized\nlaboratory-generated OS samples using authentic standards, however the best characterized system\nis isoprene, which the authors now investigated, but also observed issues with recovery of the OS\nproducts. Overall, there is not a standard, reproducible method to generate and verify OS analysis, so\nthe authors have done a good job in addressing the question here. Future work will hopefully lead to\nan optimized method to study isoprene-derived OS.\nThank you for your thoughtful assessment and for recognizing our efforts in addressing the\ncomments. We thank for your positive feedback on the improvements made to the manuscript,\nincluding the clarification of limitations and future research directions.\nRegarding point 8, add one sentence in line 142-143 (the track-changes version)\n“However, we note that larger, less polar OS compounds may exhibit a reduced extraction\nefficiency, which should be considered in future studies.”\nOnce again, we appreciate your time and effort in reviewing our work and providing valuable\ninsights that have helped strengthen the study."}]

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