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LC-MS/MS Analysis of Naphthenic Acids in Environmental Waters
Coreen Hamilton, Million B. Woudneh & Guanghui WangPresented at
Workshop on Analytical Strategies for Naphthenic AcidsNHRC, SaskatoonNov. 24 -25, 2011
OUTLINE
• Background
• Some Starting Considerations
• Method
• Results
• Questions
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
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
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
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”
What the method measures and how are they selected?
Table 1. % contribution of various NA to total NA in surface water samplen z=-12 z=-10 z=-8 z=-6 z=-4 z=-2 z=-0 Total9 n/a n/a n/a n/a n/a 0 0.13 0.13
10 n/a n/a n/a n/a 0 0 0 0
11 n/a n/a n/a n/a 0.41 0.10 0 0.51
12 n/a n/a n/a 0.62 1.50 0.28 0 2.40
13 n/a n/a n/a 2.13 3.33 0.53 0.02 6.01
14 n/a n/a 0.68 5.25 5.07 0.77 0.04 11.8
15 1.28 1.09 1.86 7.59 5.48 0.90 0.05 18.316 2.58 1.54 2.70 7.47 4.45 0.68 0.06 19.517 3.52 1.70 2.60 4.92 2.77 0.46 0.05 16.018 3.43 1.59 1.80 2.68 1.47 0.32 0.02 11.319 2.32 1.10 1.15 1.42 0.81 0.15 0 6.9520 1.85 0.71 0.54 0.60 0.35 0.06 0 4.1121 0.97 0.39 0.24 0.29 0.15 0.02 0 2.0622 0.43 0.14 0.08 0.05 0.02 0 0 0.7223 0.17 0.03 0.02 0.01 0 0 0 0.23
n/a = not applicable
Target NAs were selected based on prevalence in Northern Alberta surface water
What the method measures and how are they selected?
Table 1. % contribution of various NA to total NA in surface water samplen z=-12 z=-10 z=-8 z=-6 z=-4 z=-2 z=-0 Total9 n/a n/a n/a n/a n/a 0 0.13 0.13
10 n/a n/a n/a n/a 0 0 0 0
11 n/a n/a n/a n/a 0.41 0.10 0 0.51
12 n/a n/a n/a 0.62 1.50 0.28 0 2.40
13 n/a n/a n/a 2.13 3.33 0.53 0.02 6.01
14 n/a n/a 0.68 5.25 5.07 0.77 0.04 11.8
15 1.28 1.09 1.86 7.59 5.48 0.90 0.05 18.316 2.58 1.54 2.70 7.47 4.45 0.68 0.06 19.517 3.52 1.70 2.60 4.92 2.77 0.46 0.05 16.018 3.43 1.59 1.80 2.68 1.47 0.32 0.02 11.319 2.32 1.10 1.15 1.42 0.81 0.15 0 6.9520 1.85 0.71 0.54 0.60 0.35 0.06 0 4.1121 0.97 0.39 0.24 0.29 0.15 0.02 0 2.0622 0.43 0.14 0.08 0.05 0.02 0 0 0.7223 0.17 0.03 0.02 0.01 0 0 0 0.23
n/a = not applicable by structure
Target NAs were selected based on prevalence in Northern Alberta surface water
Analytical report for NAs from a POCIS in surface water sample
Analytical report for NAs from a POCIS in surface water sample continued …
Method Scope – Matrices Analyzed
• Aqueous samples collected on POCIS samplers
• Process waters
• Surface waters
• Ground waters
Method Summary – Environmental Waters and POCIS Samples
Analysis Flowchart
Spike labeled standardsHLB Extraction
POCIS or grab water samples
Solvent Reduction
Extract Derivatization
+1-EDC at 60oC
Instrumental Analysis
(+ESI) LC/MS/MS
Data Quantification and Reporting
Data reported as PYB
Extract Derivatization
R1 C
O
OH+
N C N N H+ Cl-R2
R1 C
O
N
R2
C
O
HN N
R1 = Alkyl from NAR2 = CH3CH2
-
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
LC MS/MS Instrumental Analysis
• Reverse phase LC with C18 column
• Mobile phases: HCOOH/HCOONH4+ & CH3OH
• ESI+ using MRM monitoring
• Run time = 45 min
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
Instrument linearity is demonstrated for each NA isomer group using Merichem standard of varying concentration
R2 = 0.9997
R2 = 0.9989
R2 = 0.9971
R2 = 0.9976
0
10000
20000
30000
40000
0 10 20 30 40 50 60 70 80 90
Amount of NA (ug)
PYB
equi
vale
nts
of n
= 12
(ng)
0
150
300
450
PYB
equi
vale
nts
of n
=21
(ng)
Linear (n=12, z=-12 (right) Linear (n=12, z=0 (right)Linear (n=21, z=12 (left) Linear (n=21, z=0 (left)
Analyte Separation (fixed N; varying Z)
min10.0 15.0 20.0 25.0 30.0 35.0
%
0
100
F2:MRM of 32 channels,ES+426.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
1.555e+006C17H34O2 (NA Z -0)
28.8131.57
min
%
0
100
F2:MRM of 32 channels,ES+424.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
4.354e+006C17H32O2 (NA Z -2)
28.10
min
%
0
100
F2:MRM of 32 channels,ES+422.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
5.115e+006C17H30O2 (NA Z -4)
27.25
min
%
0
100
F2:MRM of 32 channels,ES+420.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
1.806e+006C17H28O2 (NA Z -6)
26.26
min
%
0
100
F1:MRM of 32 channels,ES+418.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
4.263e+005C17H26O2 (NA Z -8)
24.77
5.658.91
min
%
0
100
F1:MRM of 32 channels,ES+416.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
2.231e+005C17H24O2 (NA Z -10);22.37
5.9313.58
min
%
0
100
F1:MRM of 32 channels,ES+414.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
4.357e+00528.03
C17H22O2 (NA Z -12)20.385.58
Sample Chromatograms for isomer group n=17, Z=-12 to 0 for a solution of purified Merichem (154 ug/mL)
Analyte Separation (fixed Z; varying N)
min6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0
%-2
98
F2:MRM of 32 channels,ES+448.0>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
7.238e+005C19H32O2 (NA Z -6);29.01;1357979.25;712858*
min
%-2
98
F2:MRM of 32 channels,ES+434.0>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
1.604e+006C18H30O2 (NA Z -6);27.88;3318019.75;1598737
min%-2
98
F2:MRM of 32 channels,ES+420.0>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
3.431e+006C17H28O2 (NA Z -6);26.48;6575263.50;3379046*
min%-2
98
F1:MRM of 32 channels,ES+406.0>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
5.298e+006C16H26O2 (NA Z -6);24.72;11846904.00;5218042*
min
%-2
98
F1:MRM of 32 channels,ES+392.0>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
6.960e+006C15H24O2 (NA Z -6);22.33;17806836.00;6854148*
min%-2
98
F1:MRM of 32 channels,ES+378.0>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
7.985e+006C14H22O2 (NA Z -6);19.16;20282282.00;7434202*
min%-2
98
F1:MRM of 32 channels,ES+363.9>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
6.695e+006C13H20O2 (NA Z -6);16.13;17679876.00;6593902
min%-2
98
F1:MRM of 32 channels,ES+349.90>129
NA0P_150S11 Smooth(SG,2x1)1,WG34185,10/1000ul WG34185-104,i,SAR MC NA
4.202e+006C12H18O2 (NA Z -6);12.55;7782365.00;4138734*
9.66
Sample Chromatograms for isomer group n=12 to n19 for Z=-6 for a solution of purified Merichem (154 ug/mL)
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
Method Performance in POCIS and Aqueous Samples
Table 3. Demonstration of Precision and Accuracy
POCIS samples Aqueous samples
High-level Low-level High-level
Isomer group (% Rec.) n=5
%RSD (% Rec.) n=5
%RSD (% Rec.) n=5
%RSD
C12-Z6 92.7 8.9 67.5 4.2 72.6 4.9
C12-Z4 94.2 9.1 72.5 4.3 78.3 3.9
C12-Z2 95.5 7.6 76.3 2.7 84.9 4.1
C12-Z0 92.0 4.0 70.7 4.0 78.4 4.2
∑C12 Z=0 to-10 93.9 71.8 78.5
∑C13 Z=0 to-10 95.3 70.5 79.7
∑C14 Z=0 to-12 103 65.5 74.7
∑C15 Z=0 to-12 102 79.7 92.0
∑C16 Z=0 to-12 106 79.3 92.2
∑C17 Z=0 to-12 112 77.7 101
∑C18 Z=0 to-12 120 98.6 105
∑C19 Z=0 to-12 113 96.6 97.9
∑C20 Z=2 to-12 96.2 96.5 97.9
∑C21 Z=2 to-12 103 89.2 92.9
Laboratory Blanks
min6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0
%
0
100
F1:MRM of 32 channels,ES+356.0>129
NA9J_238S10 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-101,, BLK
2.612e+006C12H24O2 (NA Z 0)24.35
1381249.252593763
15.14
min6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0
%
0
100
F1:MRM of 32 channels,ES+356.0>129
NA9J_238S05 Smooth(SG,2x1)1,WG31245,10/1000ul WG31245-102,,SPM
2.318e+00724.35
C12H24O2 (NA Z 0)22.79
30512178.008980136
•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
Laboratory Blanks
Laboratory Blanks
Spatial NA Concentration Patterns in Waters of Northern Alberta
Samples Collected in July 2010
0
2
4
6
8
10
12
14
Con
cent
ratio
n (u
g)
0
50
100
150
200
250
300
Site 1 (left) Site 2 (left) Site 3 (right)
Total Conc. (ug): Site 1 = 68 Site 2 = 191Site 3 = 2890
Temporal NA Concentration Patterns in Waters of Northern Alberta
Seasonal Variation of NA
0
1000
2000
3000
4000
5000
6000
Mar-09 Apr-09 May-09 Jun-09 Jul-09 Aug-09 Sep-09 Oct-09 Nov-09 Dec-09 Jan-10 Feb-10 Mar-10 Apr-10 May-10 Jun-10 Jul-10
Total
NA
Conc
. (ug
)
0
10
20
30
40
50
60
70
80
Site 2 (left) Site 3 (left) Site 1 (right)
Total Conc. (ug): Site 1 = LowSite 2 = MediumSite 3 = High
Normalized Relative Response & Analyte Patterns: Surface Water & Standard
0.00
0.20
0.40
0.60
0.80
1.00
1.20
MC SAR
Merichem
0.00
0.20
0.40
0.60
0.80
1.00
1.20
L1655-3 L1655-4
Surface water
Normalized Analyte Patterns in Various Water Matrices
0.00
0.20
0.40
0.60
0.80
1.00
1.20
L1655-7 L1655-8
0.00
0.20
0.40
0.60
0.80
1.00
1.20
L1655-5 L1655-6
Process Water
Tailings pond
0.00
0.20
0.40
0.60
0.80
1.00
1.20
n=12
, z=-6
n=12
, z=0
n=13
, z=-2
n=14
, z=-6
n=14
, z=0
n=15
, z=-8
n=15
, z=-2
n=16
, z=-1
0
n=16
, z=-4
n=17
, z=-1
2
n=17
, z=-6
n=17
, z=0
n=18
, z=-8
n=18
, z=-2
n=19
, z=-1
0
n=19
, z=-4
n=20
, z=-1
2
n=20
, z=-6
n=21
, z=-1
2
n=21
, z=-6
L1655-1 L1655-2
Ground water
0.00
0.20
0.40
0.60
0.80
1.00
1.20
L1655-3 L1655-4
Surface water
12 13 14 15 16 17 18 19 20 21
Z=-12
Z=-8
Z=-4
Z=0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Rel
ativ
e p
eak
inte
nsi
ties
Carbon number
12 13 1415 16 17
18 1920
21
Z=-12
Z=-10
Z=-8
Z=-6
Z=-4Z=-2
Z=0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Rel
ativ
e p
eak
inte
nsi
ties
Carbon number
12 13 14 15 16 17 18 19 20 21
Z=-12
Z=-8
Z=-4
Z=0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Rel
ativ
e p
eak
inte
nsi
ties
Carbon number
Analyte Patterns in Water Matrices and Standard
Process Water
Merichem
Surface water
Tailings pond waterGround water
12 13 14 15 1617 18 19 20
21
Z=-12
Z=-8
Z=-4
Z=0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Rel
ativ
e p
eak
inte
nsi
ties
Carbon number
12 13 14 15 16 17 18 19 20 21
Z=-12
Z=-8
Z=-4
Z=0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Rel
ativ
e p
eak
inte
nsi
ties
Carbon number
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
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
Acknowledgements
• Alberta Environment
• Phil Fedorak (University of Alberta)
• Erik Krogh & Chris Gill (Vancouver Island University)