## 2 Critical Analytical Parameters
### Calibration Standards  
Quantitative analytical methods use authentic reference standards of high purity for each compound to be measured. For example, the Canadian Water Quality Guidelines list twelve PAHs that require monitoring for the protection of aquatic life ([PERSON], 2016). PAHs are well defined, limited in number, and commercially available. Many isotopically labelled analogs are also available that can be used as internal standard (ISTDs). The analysis is straightforward: a series of calibration standards is prepared at various concentrations, the response versus concentration is plotted for each compound, and sample concentrations are calculated from the generated calibration curves.  
Measuring the concentrations of NAs in OSPW is complex. Approximately 3000 elemental compositions have been found in OSPW samples from the oil sands in northern Alberta using LC with Orbitrap HRAM/MS, including O\({}_{1-6}\), NO\({}_{1-4}\), SO\({}_{1-4}\), NO\({}_{2}\)S, N, and S ([PERSON], 2016). Each chemical formula can further be expanded into a large number of combinatorial structural isomers ([PERSON], 2016) which do not separate well chromatographically. Commercial mixtures of calibration standards of this complexity are not available, and NA mixtures that are available are not representative of the wide variety of OSPWs. Model compounds representing various chemical classifications may provide a means for semi-quantitative analysis only.  
Figure 5: Effects on chromatography on fatty acid separation (C\({}_{8}\) to C\({}_{24}\)) resulting from reduced injection volume on HILIC LC column ([PERSON], 2016).  
The 'Naphthenic Acids Strategies Workshop' (Saskatoon, Canada, 24-25 November 2011) was held with the goal of reviewing the different routine methods used by laboratories for measuring, classifying, and quantitating NAFCs. Researchers from Canadian provincial and federal governments, universities (including one from the United Kingdom), and commercial laboratories were invited. The forum was also used to gain consensus on chemical classification terms for describing what was being measured, i.e., classical naphthenic acids (cNAs) and the naphthenic acid fraction compounds (NAFCs). Each lab gave a presentation outlining their acquisition approach and quantitation method and the findings were published [9]. Most of the review was concerned with the qualitative characterization of NAFCs as the first step in defining what was being measured.  
The following observation was made in the workshop review and is still true today, twelve years later: \"The need for the development of target analyses using authentic standards is evident as quantification of NAFC expressed as total NAs is currently at best, semi-quantitative\" [9]. No OSPW-derived reference standard has yet been produced for general availability. The consensus from the workshop was that the NARC fraction from OSPW was too complex for a single technique, and that a working group should be formed to develop protocols for standardized analyses, with interlaboratory studies designed to show conformance of results.  
In studying the impact of temperature, pH, and salinity changes on partitioning of NAs [22], 55 model NAs were selected based on their identification by other researchers [27; 38], including compounds from the following types: thiophene, indane, tetralin, cyclohexane, and adamantane. A comprehensive list of compounds such as this that spans a variety of elemental compositions may provide representative response factors that differ due to chemical structures. However, as noted earlier, OSPW from different sources contains different compounds. A selection of 39 model NAs showed up to 26-fold differences in response factors using LC/TOF [32], indicating that a one-size-fits-all approach to reference standards is problematic. The use of a commercially available Merichem NA mixture was recommended for semi-quantitation of classical NAs, due to the predominance of O\({}_{2}\) acids.