Million B. Woudneh, M. Coreen Hamilton, Jonathan P. Benskin, Guanghui Wang, John R. Cosgrove SETAC, Nashville November 2013

Transcription

1 Analysis of Naphthenic Acids (NAs) in Tissue: A derivatization based LC-MS/MS approach Million B. Woudneh, M. Coreen Hamilton, Jonathan P. Benskin, Guanghui Wang, John R. Cosgrove SETAC, Nashville November 213

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3 Overview: Introduction Method scope Method summary Challenges in NA analysis Method validation data Questions

4 Introduction: Naphthenic acids: A complex mixture of organic acids that occur in crude oil and oil sand bitumen. Extracted from oil sands during the caustic hot water or Steam Assisted Gravity Drainages (SAGD) production process. Produced as waste product in aqueous phase of upgrading and refining processes. Chemical raw material produced through refining process

5 Definition Naphthenic Acids Intro and Analyte Selection - A complex mixture of saturated, cyclic and non-cyclic carboxylic acids described by the formula CnH2n+zO2, Where, n=# of C atoms, z, represents hydrogen deficiency Occurrence - Naturally occurring in crude oils and oil sand bitumen - Extracted from oil sands during the caustic hot water production process - Z =, Z = -2 have contributions (fatty acids) that are naturally occurring important compositional information for assessing changes in Naphthenic acid concentrations. Project purpose - Develop a sensitive method that is: - able to sufficiently characterize NAs enabling source characterization and fingerprinting in field samples, including differentiation of Z= and Z=2 from NA totals - is useful for comparative data evaluation for spatial and temporal changes in occurrence - Overcome the false positive effects associated with GC-MS and FTIR

6 Naphthenic Acids - Isomer Groups What to Measure? Naphthenic Acids Over 2, possible compounds, not practical to measure all compounds routinely Prevalence and toxicity of individual NA compounds not well developed Very limited specific compound standards and surrogates, technical Merichem standards used Use of Isomer Groups Provide wide variety of information on NA isomer groups to monitor spatial and temporal concentrations - occurrence and change in space and over time Identify prevalent and important isomer groups for further study Application to Tissue May demonstrate change in NA concentration / pattern over space and time Specific biota may have unique isomer groups for use as biomarkers

7 Naphthenic Acid Isomer Group Selection (Highlighted) AXYS Aqueous Method MLA-77 / Solid Method MLA 91 peak area for various naphthenic acid isomer groups in a surface water* sample n z=-12 z=-1 z=-8 z=-6 z=-4 z=-2 z=- Total 9 n/a n/a n/a n/a n/a n/a n/a n/a n/a 11 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a = not applicable. * Surface water suspected of contaation from Oil sands.

8 Naphthenic Acid Isomer Group Selection Specific to Tissue Type Analyzed AXYS Tissue Method MLA- 92 Table 3. peak area for various naphthenic acid isomer groups in a surface water sample, isomer groups quantified in tissue (fish, frogs) highlighted n z=-12 z=-1 z=-8 z=-6 z=-4 z=-2 z=- Total 9 n/a n/a n/a n/a n/a n/a n/a n/a n/a 11 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a = not applicable

9 Tissue method scope: Marker NA isomers n=12,z=-6 n=12,z=-4 Target analytes C n H 2n+z O 2, where, n = # of C atoms and Z = hydrogen deficiency n=12,z=-2 n=12,z= n=13,z=-6 n=13,z=-4 n=13,z=-2 n=13,z= n=14,z=-8 n=14,z=-6 n=14, Z=-4 n=14, Z=-2 n=14, Z= n=15, Z=-12 n=15, Z=-1 n=15, Z=-8 Matrices: - Tissues Applicability: - For spatial and temporal trends as well as fingerprinting applications Quantification: - Pyrenebutyric acid equivalents Method detection limits ng/g, wet, for a 1 g tissue. n=15, Z=-6 n=15, Z=-4 n=15, Z=-2

10 Method summary: Derivatization and fragmentation Extraction and cleanup - Shaker table extraction with CHCl 3 :MeOH:H 3 O + - Extract cleanup uses SAX (2 g, 4µm) Ref: Young et al. 27. Chemosphere 68:

11 MERICHEM STANDARD C12H18O2 (Z -6) C13H2O2 (Z -6) C14H22O2 (Z -6) Chromatographic separation C15H24O2 (Z -6) LC Column : Xterra C18, 1. cm, 2.1 mm i.d., 3.5 μm C16H26O2 (Z -6) Mobile Phase:.1 formate buffer and methanol C17H28O2 (Z -6) C18H3O2 (Z -6) C19H32O2 (Z -6) C2H34O2 (Z -6) Retention time ()

12 Laboratory background : Fatty acids 1 A. Lab Blank C18H36O2 (Z=) B. Standard 3:42 C18H36O2 (Z= ) Retention time () Retention time () By convention, straight chain Z= NA isomer is excluded.

13 Intrinsic interferences : Fatty acids, Z= A. Un-spiked tissue extract B. Merichem standard C12H24O2 (Z= ) C12H24O2 (Z= ) C. Spiked tissue extract D. Spiked and extracted C12H24O2 (Z= ) C12H24O2 (Z= )

14 Intrinsic interferences : Fatty acids, Z=-2 A. Un-spiked tissue extract B. Merichem standard C. Spiked tissue extract C15H28O2 (Z =-2) C15H28O2 (Z=-2) By convention, straight chain Z-2 NA isomer is excluded.

15 Intrinsic interferences : Fatty acids, Z<-2 A. Merichem standard B. gamma-linolenic acid C. Mixed Merichem and linoleic acid 1 C18H3O2 (Z=-6) C18H3O2 (Z=-6) C18H3O2 (Z=-6) Fatty acid isomers with Z<-2 cannot be separated from NAs with the same formula.

16 Intrinsic interferences : Resin acids A. Pimaric acid standard B. Merichem standard C. Merichem + Pimaric acid standard C2H3O2 (Z=-1) C2H3O2 (Z -1) C2H3O2 (Z =-1) Resin acids present interference for n=2, Z=-1 and -8 NA isomer groups.

17 Isomer groups n=12,z=-6 n=12,z=-4 n=12,z=-2 n=12,z= n=13,z=-6 n=13,z=-4 n=13,z=-2 Potential marker NA isomer groups: n=13,z= n=14,z=-8 n=14,z=-6 n=14, Z=-4 n=14, Z=-2 n=14, Z= n=15, Z=-12 n=15, Z=-1 n=15, Z=-8 n=15, Z=-6 n=15, Z=-4 n=15, Z=-2

18 Method selectivity: 1. Dicarboxylic acids 2. Hetero atomic acids 3. Hydroxy acids

19 Study design considerations: Control samples: - Samples that are known to NOT be impacted by a potential NA source A sample of suspected NA source: - e.g. Oil Sands Process Water (OSPW) Target / suspected impacted sample: - Samples of tissue from the target organism in the potentially affected area Statistical considerations - Good experimental design and number of replicates

20 Method applicability: Special and temporal change measurement For fingerprinting

21 Method applicability: Special and temporal change measurement For fingerprinting Surface water Scores for PC1 (49.9 ) versus PC2 (38.6 ) 8 6 PC2 Score Well water Process water Tailings pond water "Refined Merichem" PC1 Score

22 Method validation data: Isomer group rec. n=5, (RSD) n=12,z=-6 96 (8) n=12,z=-4 98 (8) n=12,z=-2 94 (8) n=12,z= 81 (13) n=13,z=-6 14 (7) n=13,z=-4 18 (7) n=13,z=-2 18 (8) n=13,z= 9 (9) n=14,z= (12) n=14,z=-6 19 (7) n=14, Z= (6) n=14, Z=-2 95 (4) n=14, Z= 87 (3) n=15, Z= (27) n=15, Z= (11) n=15, Z=-8 126(5) n=15, Z= (6) n=15, Z= (5) n=15, Z=-2 89 (3)

23 Quality control: Surrogates rec. n=5 D15-Adamentane Carboxylic Acid 49.8 D9-Anthracene Carboxylic Acid 6.2 D4-Lithochlolic Acid C4 Mono-n-octyl phthalate 51.7 Derivatization controls D23-Dodecanoic Acid D35-Octadecanoic Acid 78.3 N/A Recovery standards 13C3-Atrazine 12

24 Summary: A versatile derivatization and (+ESI) MS/MS fragmentation method was developed for monitoring NAs in tissue. - Enabled MRM, increased specificity, sensitivity and lowered detection limits. Tissue samples present intrinsic interference to analysis of fatty acids. - Chromatographic separation was employed to imize the impact of endogenous fatty acids on NA data. Fingerprinting and source characterization have been shown to be useful as a fingerprinting tool. - Multivariate methods such as principal components analysis and discriant analysis have been shown to group samples based on NA isomer group covariance (source).