## 2 Critical Analytical Parameters
### Ionization Mode for Detection of the Analytes  
Selection of the mass spectrometer ion source parameters is based on the chemical nature of the targeted NAFCs for any particular experiment. NAFCs and sulfonic acids in OSPW are typically analyzed with negative polarity due to the ready formation of anionic precursor ions, while many nitrogen-containing compounds can be preferentially ionized with positive polarity due to the basic nitrogen within the molecule. In some cases, more than half of the NAFCs extracted from OSPW at pH 1.5 are not classified as naphthenic acids [23]. Thus, multiple ionization techniques are needed for full NAFC characterization.  
Three common ionization sources are available to many analytical laboratories: (1) electrospray (ESI); (2) atmospheric pressure chemical ionization (APCI); and (3) atmospheric pressure photoionization (APPI). Polar compounds such as carboxylic acids and amines are typically ionized using ESI, where the compounds are ionized either in the mobile phase due to selective pH, or within the ion source, often in the presence of mobile phase additives to enhance the ionization process. Non-polar organics such as ketones and aldehydes can be analyzed by APCI [38], where a charge is induced on protic solvent molecules such as methanol, which then transfer the charge as a reagent gas to the analytes. APPI can be used for the analysis of non-polar aromatic compounds such as PAHs by using photoionization to directly ionize compounds when their ionization potential is lower than the energy of the APPI lamp [39; 40]. When the ionization potential of compounds is higher than the lamp, either the solvent or added dopant molecules (e.g., acetone or toluene) can be ionized, and this is followed by charge transfer to the target compounds. Both APCI and APPI can produce radicals as well as charged adducts [38].  
To demonstrate the need for additional ionization techniques and polarity, [PERSON] et al. compared both positive and negative polarities using ESI and APPI sources (Figure 6) [26]. The negative ion mode showed that most ions are in the 190-450 amu range, while the positive ion mode included ions up to 700 amu. The distribution within those ranges also changed along with the number of peaks. A greater number of peaks was observed in the positive-ion mode than negative-ion mode, and by APPI than ESI. The APPI positive-ion mode showed approximately 19,000 peaks.  
Some overlap and distinct differences exist in the ionization efficiencies of compounds using different ionization methods and polarities. [PERSON] et al. showed that compounds with the same empirical formula eluted with different chromatographic retention times in positive and negative mode ESI [27], indicating the presence of different compounds. Using ESI+, 162 and 85 compounds were identified in OSPW produced from surface mined and in situ extracted bitumen, respectively, that were not detected by ESI-. The O\({}_{2}\)+ species were determined to be dihydroxy, diketo, or ketohydroy groups based on MS\({}^{2}\) and MS\({}^{3}\) fragmentation and similar behaviour of model compounds. The authors noted that some sex hormones, such as testosterone, androstenedione, and 5-androstenedio, belong to these chemical classes and that OSPW has endocrine-disrupting effects. ESI does not ionize these functional groups in either positive or negative polarities. Thus, the analysis of OSPW for possible toxic compounds should include additional ionization techniques. Matrix effects, especially ion suppression, are a well-known phenomenon of ESI. Ion suppression with reverse-phase chromatography is most evident in samples containing high salts and other hydrophilic compounds. LC separations can help reduce salt-induced ion suppression compared to infusion by retaining organic compounds on the column while the non-retained salts are directed to waste. However, suppression effects are often evident due to other co-eluting compounds. APCI and APPI are less prone to ion suppression than ESI.