1. LC-MS/MS Analysis of Naphthenic Acids in Environmental Waters Coreen Hamilton, Million B. Woudneh & Guanghui Wang Presented at Workshop on Analytical Strategies for Naphthenic Acids NHRC, Saskatoon Nov. 24 -25, 2011

2. OUTLINE Background Some Starting Considerations Method Results Questions

3. BACKGROUND: AXYS Involvement in Naphthenic Acids (NA) Spring 2008: Alberta Environment Passive Sampling of Athabasca Watershed –POCIS – Naphthenic Acids –SPMDs – PAHs and Alkylated PAHs –Monthly sampling for 2 years Spring 2011: Addition of Water Analysis –Extension of Method to water analysis –Applied to Tailings water, Surface water, Groundwater and Process water

4. Starting Considerations in Method Development Chemical Standards –Consideration of literature - “Merichem acids”, model compounds Scope of NA species measured Target Detection Limits –Low ng per isomer group per sample –Small sample size for higher [NA] in process streams Reporting Conventions –Z=0 to include or not to include? Compound Specific Analysis –Individual compound analysis not pursued – applicability, technical, time & cost considerations

5. Current Method: Potential Applications Concentration of individual isomer groups by ‘n’ and ‘z’ number NA concentration and composition variability over space & time; –Fingerprinting sources (e.g. watersheds) –Ecotoxicology applications – focusing research effort on identifiable isomer groups –Process waters for identification of significant isomer groups

6. Aligning Methods – Key Points Standardized quantification basis Standardized reporting conventions – n, z Wish list – availability of native and labelled standards Benchmarking performance of methods Define methods by “fitness for purpose”

7. What the method measures and how are they selected? Table 1. % contribution of various NA to total NA in surface water sample nz=-12z=-10z=-8z=-6z=-4z=-2z=-0Total 9n/a 0 0.13 10 n/a 0000 11 n/a 0.410.10 00.51 12 n/a 0.621.500.2802.40 13 n/a 2.133.330.530.026.01 14 n/a 0.685.255.070.770.0411.8 15 1.281.091.867.595.480.900.0518.3 16 2.581.542.707.474.450.680.0619.5 17 3.521.702.604.922.770.460.0516.0 18 3.431.591.802.681.470.320.0211.3 19 2.321.101.151.420.810.15 06.95 20 1.850.710.540.600.350.06 04.11 21 0.970.390.240.290.150.02 02.06 22 0.430.140.080.050.02 000.72 23 0.170.030.020.01 0000.23 n/a = not applicable Target NAs were selected based on prevalence in Northern Alberta surface water

8. What the method measures and how are they selected? Table 1. % contribution of various NA to total NA in surface water sample nz=-12z=-10z=-8z=-6z=-4z=-2z=-0Total 9n/a 0 0.13 10 n/a 0000 11 n/a 0.410.10 00.51 12 n/a 0.621.500.2802.40 13 n/a 2.133.330.530.026.01 14 n/a 0.685.255.070.770.0411.8 15 1.281.091.867.595.480.900.0518.3 16 2.581.542.707.474.450.680.0619.5 17 3.521.702.604.922.770.460.0516.0 18 3.431.591.802.681.470.320.0211.3 19 2.321.101.151.420.810.15 06.95 20 1.850.710.540.600.350.06 04.11 21 0.970.390.240.290.150.02 02.06 22 0.430.140.080.050.02 000.72 23 0.170.030.020.01 0000.23 n/a = not applicable by structure Target NAs were selected based on prevalence in Northern Alberta surface water

9. Analytical report for NAs from a POCIS in surface water sample

10. Analytical report for NAs from a POCIS in surface water sample continued …

11. Method Scope – Matrices Analyzed Aqueous samples collected on POCIS samplers Process waters Surface waters Ground waters

12. Method Summary – Environmental Waters and POCIS Samples Analysis Flowchart Spike labeled standards HLB Extraction POCIS or grab water samples Solvent Reduction Extract Derivatization +1-EDC at 60 o C Instrumental Analysis (+ESI) LC/MS/MS Data Quantification and Reporting Data reported as PYB

13. Extract Derivatization R1 = Alkyl from NA R2 = CH 3 CH 2 - Naphthenic acid EDC NA +EDC product Derivatization & Proposed Fragmentation Pattern Produces strong daughter ion in MS/MS ESI+ monitoring provides a thousand times sensitivity improvement Common daughter ion for all analytes provides uniform response factor and simplifies quantitation

14. LC MS/MS Instrumental Analysis Reverse phase LC with C18 column Mobile phases: HCOOH/HCOONH4 + & CH3OH ESI+ using MRM monitoring Run time = 45 min

15. Quantification Approach: All NAs quantified as PYB equivalents Initial instrument calibration –PYB-based –IS: D19-Decanoic & D31-Hexadecanoic acids Demonstration of linearity for NAs Daily instrument calibration check Sample quantification Inter-batch QC using Merichem acids

16. Instrument linearity is demonstrated for each NA isomer group using Merichem standard of varying concentration

17. Analyte Separation (fixed N; varying Z) Sample Chromatograms for isomer group n=17, Z=-12 to 0 for a solution of purified Merichem (154 ug/mL)

18. Analyte Separation (fixed Z; varying N) Sample Chromatograms for isomer group n=12 to n19 for Z=-6 for a solution of purified Merichem (154 ug/mL)

19. Key Elements of Method QC Initial demonstration of accuracy and precision Internal standard quanitification for all NAs with IS recovery specifications Blank <100ng per sample per isomer group Inter batch QC using extracted Merichem acids

20. Method Performance in POCIS and Aqueous Samples Table 3.Demonstration of Precision and Accuracy POCIS samplesAqueous samples High-levelLow-levelHigh-level Isomer group (% Rec.) n=5 %RSD (% Rec.) n=5 %RSD (% Rec.) n=5 %RSD C12-Z692.78.967.54.272.64.9 C12-Z494.29.172.54.378.33.9 C12-Z295.57.676.32.784.94.1 C12-Z092.04.070.74.078.44.2 ∑C12 Z=0 to-1093.971.878.5 ∑C13 Z=0 to-1095.370.579.7 ∑C14 Z=0 to-1210365.574.7 ∑C15 Z=0 to-1210279.792.0 ∑C16 Z=0 to-1210679.392.2 ∑C17 Z=0 to-1211277.7101 ∑C18 Z=0 to-1212098.6105 ∑C19 Z=0 to-1211396.697.9 ∑C20 Z=2 to-1296.296.597.9 ∑C21 Z=2 to-1210389.292.9

21. Laboratory Blanks Laboratory background due to fatty acids is a consideration in NA analysis This can be corrected by consistently excluding the peak area corresponding to the straight chain isomer for Z=0 and sometimes for Z=-2 Laboratory blank Sample

22. Laboratory Blanks

24. Spatial NA Concentration Patterns in Waters of Northern Alberta Total Conc. (ug): Site 1 = 68 Site 2 = 191 Site 3 = 2890

25. Temporal NA Concentration Patterns in Waters of Northern Alberta Total Conc. (ug): Site 1 = Low Site 2 = Medium Site 3 = High

26. Normalized Relative Response & Analyte Patterns: Surface Water & Standard Merichem Surface water

27. Normalized Analyte Patterns in Various Water Matrices Process Water Tailings pond Ground water Surface water

28. Analyte Patterns in Water Matrices and Standard Process Water Merichem Surface water Tailings pond water Ground water

29. Method Features Simplified quantitation and data interpretation via: –Single compound equivalence (PYB) –Internal standard use Derivatization, LC and tandem MS combine to provide a high degree of specificity for NAs Method adaptable to: –Expanded target analytes e.g. aromatics, dicarboxylic acids –Alternate quantification references beyond PYB

30. Summary Currently applied, validated, quantitative LC MS/MS method for analysis of NA isomer groups Demonstrated method suitability for: –Surface, ground & process water samples –Wide NA concentration range Data reported as total NA concentrations and as Isomer Group concentrations

31. Acknowledgements Alberta Environment Phil Fedorak (University of Alberta) Erik Krogh & Chris Gill (Vancouver Island University)