lc-ms/ms analysis of naphthenic acids in environmental waters coreen hamilton, million b. woudneh...

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



4.354e+006C17H32O2 (NA Z -2)

28.10

min

%

0

100



5.115e+006C17H30O2 (NA Z -4)

27.25

min

%

0

100



1.806e+006C17H28O2 (NA Z -6)

26.26

min

%

0

100



4.263e+005C17H26O2 (NA Z -8)

24.77

5.658.91

min

%

0

100



2.231e+005C17H24O2 (NA Z -10);22.37

5.9313.58

min

%

0

100



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


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



1.604e+006C18H30O2 (NA Z -6);27.88;3318019.75;1598737

min%-2

98



3.431e+006C17H28O2 (NA Z -6);26.48;6575263.50;3379046*

min%-2

98



5.298e+006C16H26O2 (NA Z -6);24.72;11846904.00;5218042*

min

%-2

98



6.960e+006C15H24O2 (NA Z -6);22.33;17806836.00;6854148*

min%-2

98



7.985e+006C14H22O2 (NA Z -6);19.16;20282282.00;7434202*

min%-2

98



6.695e+006C13H20O2 (NA Z -6);16.13;17679876.00;6593902

min%-2

98



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


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



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

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)

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