## 2 Critical Analytical Parameters  
### Choice of Extraction Solvent  
The solvent used for NAFC extraction from water should be based on the chemical properties of target compounds. For example, in the case of relatively non-polar PAHs, lipophilic solvents that are immiscible with water are used. Cyclohexane and dichloromethane are common choices for neutral compounds and are directly amenable to GC analysis following a drying step to remove residual water, typically by passing the solvent through a column of anhydrous sodium sulfate. NAs have surfactant properties, with hydrophobic ring structures and carbon side chains, as well as hydrophilic carboxylic acid groups. Extraction and concentration of NAFCs from OSPW is carried out to lower detection limits, much like that performed for PAH analysis but with additional consideration of aqueous pH that takes advantage of the ionic and neutral forms of the acids. Use of high pH (between 10 and 12) during partitioning will ionize NAs for their retention in the water phase, while non-polar compounds are extracted into the organic solvent and discarded. Lowering of pH to ~2 will then neutralize NAFCs, so they partition into the organic phase and are dried, and concentrated. The choice of extraction solvent can also align with the solvent used to prepare the sample for MS analysis. For example, a procedure was reported where an NAFC extract concentrate was solvent-adjusted to prepare a series of fractions for nanospray infusion into the ESI source (Figure 3) [18]. The NAFC solution was acidified to pH 2.5, then the neutral NAFCs were extracted into dichloromethane (DCM), concentrated, and the solvent  
Figure 2: O\({}_{2}\) DBE plots from ESI- Orbitrap (**top**) and FT-ICR MS (**bottom**) data using acidic (brown) and basic (green) conditions [17]. was exchanged into acetonitrile. The acetonitrile extract was then amended with five solvents used separately or in various combinations: Milli-Q water (9.0 polarity index, PI)/acetonitrile (ACN; 5.8 PI); ACN; methanol (MeOH; 5.1 PI)/ACN; DCM (3.1 PI)/ACN; and 1-octanol (<3.9 PI)/ACN. Ammonia solution (35% \(v/v\) in water) was added to each solution to improve deprotonation of NAs in the MS source. Of the five solvents, water/ACN, ACN, and MeOH/ACN were similar in their respective spectra. The DCM/ACN and 1-octanol/ACN solvents, however, showed marked differences in intensity and molecular weight distribution compared to the other three, particularly when plotted as carbon number vs. z-values and broken out as monocarboxylate O\({}_{2}\) vs. dicarboxylate O\({}_{4}\) species (Figure 4, ACN and MeOH/ACN not shown). The authors attributed the difference in NA characterization as an effect of greater solubility of higher-molecular-weight acids in the lipophilic solvents DCM and 1-octanol. Researchers using a more polar solvent system would, therefore, reach a very different conclusion about the NAFC characterization of the same OSPW sample. More recent work has shown the effects of the apparent pH of the transfer solvent (mobile phase), where identical OSPW extracts were analyzed by Orbitrap and FT-ICR-MS in different laboratories using both acidic and basic mobile phases on each instrument [17]. In this particular application, neither the Orbitrap (flow injection analysis) nor the FT-ICR-MS (infusion) used chromatographic separation before ionization in the source of the mass spectrometer. A flow injection experiment would result in a mixture of the mobile phase and the solvent in the sample vial when the sample reaches the source within seconds, but infusion contains the vial solvent alone. These solvent differences would result in a different apparent pH in the source and therefore different spectra. On-line chromatography will diffuse the sample vial solvent to a greater extent by the time the sample arrives at the source, with the apparent pH being that of the mobile phase alone. Figure 3: Summary of liquid–liquid extraction of NAFCs from OPSW.