Capillary HPLC/QTOF-MS for Characterizing Complex Naphthenic Acid Mixtures and Their Microbial Transformation. M. Bataineh, † A. C. Scott, ‡ P. M. Fedorak, ‡ and J. W. Martin † * Department of Laboratory Medicine and Pathology, 10-102 Clinical Sciences Building, University of Alberta, Edmonton, AB T6G 2G3, Canada, and Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada A rapidly expanding oil sands industry in Canada pro- duces and indefinitely stores large volumes of toxic aqueous tailings containing high concentrations of naph- thenic acids (NAs), a complex mixture of naturally occur- ring aliphatic or alicyclic carboxylic acids. Although there is an acknowledged need to reduce the environmental risks posed by NAs, little is understood about their environmental fate due to a lack of appropriate analytical methods. A dilute-and-shoot reversed-phase capillary HPLC/QTOF-MS method was developed that combines high specificity and sensitivity, quantitative capabilities, the ability to detect novel transformation products, and new structural information within each NA isomer class. HPLC separated NAs, based on carbon number, degree of cyclization, and the extent of alkyl branching, and in so doing increased analytical sensitivity up to 350-fold while providing additional specificity compared to infusion techniques. For tailings water, an interlaboratory study revealed many differences in isomer class profiles com- pared to an established GC/MS method, much of which was attributed to the misclassification of oxidized NAs (i.e., NA + O) by low-resolution GC/MS. HPLC/QTOF- MS enabled the detection of oxidized products in the same chromatographic run, and Van Krevelen diagrams were adapted to visualize the complex data. A marked decrease of retention times was evident in Syncrude tailings water compared to a commercial mixture, suggesting that tail- ings water is dominated by highly persistent alkyl- substituted isomers. A biodegradation study revealed that tailings water microorganisms preferentially deplete the least alkyl-substituted fraction and may be responsible for the NA profile in aged tailings water. Oil sands regions of northern Alberta, Canada, contain an estimated 1.7 trillion barrels of oil in the form of bitumen, 1 representing the second largest deposit of crude oil in the world. A rapidly expanding industry extracts surface-mined bitumen from the sand using alkaline hot water, resulting in large volumes of aqueous tailings that must be contained on site due to toxicity and a no-discharge policy. The toxicity of these tailings has largely been attributed to naphthenic acids (NAs), 2 a complex mixture of naturally occurring aliphatic and (poly-)alicyclic carboxylic acids having the general formula C n H 2n+Z O 2 , where n is the carbon number and Z is zero or a negative even number defining the hydrogen deficiency due to cyclization (e.g., 0, -2, -4, etc.). The structure of the many isomers within each isomer class (i.e., for each n and Z combination) is poorly understood. NAs are regarded as persistent, 3 and total concentrations in tailings ponds can range from 20 to 120 mg L -1 . 4-6 Storage of tailings water represents a temporary solution but is a substantial cost to the industry, and the risk of large spills or of NAs leaching into surrounding aquatic environments (previously detected in groundwater 2,5 ) grows with the size of the industry. A more sustainable solution would be to reduce the toxicity of these aqueous tailings; thus, there is an urgent need to understand the fate of NAs under a variety of engineered scenarios. NA fate studies are currently hampered by a lack of adequate analytical methods 5 that can be rapidly applied to accurately and simultaneously determine NA isomer class profiles, quantify degradation rates, and identify transformation products in complex environmental samples. To date, methods based on MS have provided the most useful data for characterizing NA mixtures in water samples, including GC/MS or GC/GC/MS of volatile NA derivatives, 4,7-10 and direct liquid infusion analysis using atmo- * To whom correspondence should be addressed. Phone: (780)492-1190. Fax: (780)492-7800. E-mail: jon.martin@ualberta.ca. † Department of Laboratory Medicine and Pathology. ‡ Department of Biological Sciences. (1) MacLean, D. Syncrude Facts; Fort MacMurray, AB: Syncrude Canada Ltd. 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Chem. 2006, 78, 8354-8361 8354 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006 10.1021/ac061562p CCC: $33.50 © 2006 American Chemical Society Published on Web 11/18/2006