## 2 Critical Analytical Parameters
### On-Line Chromatography vs. Injection or Infusion  
Injection or infusion of samples directly into the source of an LC/MS is a fast and convenient method for qualitative characterization of OSPW. Injection or infusion is used more frequently with FT-ICR MS compared to other instrumental methods for two main reasons. First, the ultrahigh-resolution accurate mass feature allows for mass separation from interfering compounds. Second, the acquisition rate of some instruments may be too slow to produce high-resolution spectra when using LC separations [29]. A downside to infusion can be the presence of high salt levels that result in ion suppression and the formation of salt-bridged dimers, both of which can be reduced by using on-line chromatography (LC/MS).  
NAs are classified as weak acids, with pKa values calculated to be 3.5, 4.8, and 6.8 for O\({}_{2}\), O\({}_{3}\), and O\({}_{4}\) species, respectively [25]. Under acidic conditions (pH \(<\) 1.5), the acids will be present in their neutral form and become more hydrophobic. Condensed phase-membrane introduction mass spectrometry (CP-MIMS) used this property of NAs to calculate the pKa of many compounds of environmental concern, including pharmaceuticals, algal toxin biomarkers, and naphthalene acids [30]. The technique uses a semipermeable polydimethylsiloxane membrane probe immersed into pH-controlled solution. Neutral hydrophobic compounds permeate the membrane and infuse into the MS source with a methanol carrier, leaving salts and other ionic interferences behind. This approach works for both individual compounds in solution at parts-per-billion concentrations as well as complex mixtures such as OSPW. The CP-MIMS technique was extended to monitor the compositional changes in OSPW in a constructed wetland treatment system (CWTS), using unit mass quadrupole MS. The analysis took-15 min per sample and ion suppression effects were corrected through the use of lauric acid-d\({}_{2}\) ISTD infused into the methanol stream [31]. Because LC separation is not required, the technique is proposed as an approach to implement in situ monitoring of treatment systems with relatively inexpensive MS systems.  
An ISO/IEC 17025-validated method for cNAs using reversed-phase LC separation and TOF analysis showed excellent linearity (R\({}^{2}\) = 0.9996) based on average response factors for all detected O\({}_{2}\) acids [32]. Many researchers have used reversed-phase chromatography to provide additional cleanup for samples, separation from salts that are concentrated in OSPW, and to increase resolution by compound separation over time [9; 28; 33; 34]. When using reversed phase LC, the mobile phase is typically acidic to neutralize the NAs, allowing retention on the LC column. Negative polarity ionization takes place in the source of the MS. Mobile phase modifiers such as ammonium acetate or ammonium hydroxide can lead to ionization in solution, enhancing ESI signals, although column retention can decrease as a result. Hydrophobic interaction liquid chromatography (HILIC) can be used as an alternative separation technique, where anionic NAs in a high organic mobile phase are selectively retained, reversing elution order, and providing the benefits of ion formation before entering the source, enhancing the response ([PERSON] et al., unpublished). HILIC requires longer equilibration times between injections and is sensitive to method parameters such as solvent strength in the vial and injection volume (Figure 5).