LC/MS/MS Method for Analysis of Naphthenic Acids from Aqueous Samples

Transcription

1 LC/MS/MS Method for Analysis of Naphthenic Acids from Aqueous Samples By: Million Woudneh 1, Coreen Hamilton 1, Guanghui Wang 1, Preston McEachern 2 1 Axys Analytical Services Ltd., Sidney, BC 2 Alberta Environment, Edmonton, AB

2 1. Introduction Naphthenic Acids (NA) Definition 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 bitumens Extracted from oil sands during the caustic hot water production process Project purpose Develop a sensitive method that is: able to sufficiently characterize NAs for fingerprinting is useful for comparative data evaluation Overcome the false positive effects associated with GC MS

3 Model compounds Model compounds were used for initial method development Refined Merichem Abietic acid Model Compounds Cyclohexanecarboxylic acid Cyclohexanebutyric acid Decanoic acid 1 pyrenebutyric acid (PYB) 5ß cholanic acid 2. Experimental Approach Refined Merichem (from Merichem chemicals) was used to validate method Table 1. Internal Standards n Decanoic acid d19 n Dodecanoic acid d23 n Tetradecanoic acid d27 n hexadecanoic acid d31 n octadecanoic acid d35 n Eicosanoic acid d39 Instrumental method ( ) ESI vs (+) ESI LC/MS/MS ( ESI) NAs produce good parent signal but poor product ions (+ESI) derivatization with EDC, EDC= 1 Ethyl 3 [3 dimethylaopropyl]carbodiimide Hydrochloride

4 2.1 (+ESI) Parent Ions Selection WG29394 WG29394 L (1.173) Scan ES+ 2.29e Decanoic acid derivative m/z In both cases no signal in ( ESI) WG ul/, unit mass res. WG29568 L (2.374) Cm (18:149) Scan ES+ 1.66e Decanoic acid d19 derivative m/z

5 WG29394 WG29394 L (1.173) Scan ES+ 2.29e m/z (+ESI) Parent and Daughter Ions Selection Table 2. Analyte Decanoic acid Decanoic acid d19 Parent ions Selected MRM Daughter ions In both cases no signal in (-ESI) WG ul/, unit mass res. WG29568 L (2.374) Cm (18:149) Scan ES+ 1.66e m/z

6 2.3 Target Naphthenic Acids Target naphthenic acids were selected based on: prevalence in surface water samples presence in refined Merichem Table 3. n = not applicable peak area for various naphthenic acids in a surface water sample z= z= z= z= z= z= z= Total

7 2.4 Analyte Separation Separation by z number Sample Chromatograms for isomer group n=17, Z= 12 to for a solution of purified Merichem (154 ug/ml) NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM C17H22O2 (NA Z -12) F1:MRM of 32 channels,es+ 414.> e+5 NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM C17H24O2 (NA Z -1); F1:MRM of 32 channels,es+ 416.> e+5 NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM C17H26O2 (NA Z -8) F1:MRM of 32 channels,es+ 418.> e+5 NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM 1 NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM 1 NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM 1 F2:MRM of 32 channels,es+ C17H28O2 (NA Z -6) 42.> e+6 F2:MRM of 32 channels,es+ C17H3O2 (NA Z -4) 422.> e+6 F2:MRM of 32 channels,es+ C17H32O2 (NA Z -2) 424.> e+6 NA9J_238S5 Sm ooth(sg,2x1) 1,WG31245,1/1ul WG ,,SPM 1 F2:MRM of 32 channels,es+ C17H34O2 (NA Z -) 426.> e

8 2.4 Analyte Separation Separation by n number Sample Chromatograms for isomer group n=12 to n19 for Z= 6 for a solution of purified Merichem (154 ug/ml) NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA 98-2 C12H18O2 (NA Z -6);12.55; ; * 9.66 NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA C13H2O2 (NA Z -6);16.13; ; NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA C14H22O2 (NA Z -6);19.16; ;743422* C15H24O2 (NA Z -6);22.33; ; * F1:MRM of 32 channels,es > e+6 F1:MRM of 32 channels,es > e+6 F1:MRM of 32 channels,es+ 378.> e+6 F1:MRM of 32 channels,es+ 392.> e+6 NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA 98-2 F1:MRM of 32 channels,es+ 46.>129 C16H26O2 (NA Z -6);24.72; ;521842* 5.298e+6 NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA 98-2 NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA 98-2 F2:MRM of 32 channels,es+ 42.>129 C17H28O2 (NA Z -6);26.48; ;337946* 3.431e+6 F2:MRM of 32 channels,es+ 434.>129 C18H3O2 (NA Z -6);27.88; ; e+6 NAP_15S11 Smooth(SG,2x1) 1,WG34185,1/1ul WG ,i,SAR MC NA 98-2 F2:MRM of 32 channels,es+ 448.>129 C19H32O2 (NA Z -6);29.1; ;712858* 7.238e

9 2.5 Standard characterization and Analyte Quantification Analyte Quantification Two quantification standards were selected: n Decanoic acid d19 and n hexadecanoic acid d31 Atrazine 13C3 was used as injection (recovery) standard Response factor from 1 pyrenebutyric acid was used for target analyte quantification Merichem Characterization 32.8 ug of purified Merichem was detered to be equivalent to 53.2 ug of NA as pyrenebutyric acid (PYB) equivalents (detered from six replicates) The concentration of each NA isomer group was characterized using PYB

10 3. Method Summary Analysis Flowchart Preparation of POCIS samples Extraction of POCIS samples + Column elution with 6 ml of MeOH Extract Derivatization +1 EDC at 6 o C Instrumental Analysis (+ESI) LC/MS/MS Data Quantification and Reporting Data reported as PYB

11 4. Method Performance Data Table 4. Demonstration of Precision and Accuracy Isomer group C12 Z1 C12 Z8 C12 Z6 C12 Z4 C12 Z2 C12 Z C12 Z= to 1 Average ( Rec.) n= RSD C13 Z= to 1 C14 Z= to 12 C15 Z= to 12 C16 Z= to 12 C17 Z= to 12 C18 Z= to 12 C19 Z= to 12 C2 Z=2 to 12 C21 Z=2 to

12 4.1 Field sample data Percentage Peak Intensities for NA in Purified Merichem 9 8 PeakArea Z= Z=-4 Z=-8 Z=-12 n values Z=-12 Z=-1 Z=-8 Z=-6 Z=-4 Z=-2 Z= Percentage Peak Intensities for NA in Surface Water Sample from Northern Alberta 8 7 Peak Area z= z=-4 z=-8 z=-12 n values z=-12 z=-1 z=-8 z=-6 z=-4 z=-2 z=

13 Field sample data cotd Concentrations of NA in surface water samples from Northern Alberta as PYB equivalents n=12, z=-6 n=12, z= n=13, z=-2 n=14, z=-6 n=14, z= n=15, z=-8 n=15, z=-2 n=16, z=-1 n=16, z=-4 n=17, z=-12 n=17, z=-6 n=17, z= n=18, z=-8 n=18, z=-2 n=19, z=-1 n=19, z=-4 n=2, z=-1 n=2, z=-4 n=21, z=-12 n=21, z=-6 NA isomer group ng/sample

14 Summary #1. #2. #3. #4. #5. First quantitative LC/MS/MS method for analysis of Naphthenic acids The method is based on derivatization of NA compounds with 1 EDC Naphthenic acids are separated based on carbon number and extent of cyclization 1 Pyrenebutyric acid simplifies quantification and data interpretation Suitability of the method is demonstrated by analysis of surface water samples from North Alberta

15 Supplemental information

16 Demonstration of Method Linearity Example Linear Regression 4 45 PYB equivalents of n= 12 (ng) R 2 =.9989 R 2 =.9976 R 2 =.9997 R 2 = Amount of NA (ug) Linear (n=12, z=-12 (right) Linear (n=12, z= (right) Linear (n=21, z=12 (left) Linear (n=21, z= (left) 3 15 PYB equivalents of n=21 (ng)

17 WG29394 WG29394 L (1.173) Scan ES+ 2.29e m/z WG ul/, unit mass res. WG29568 L (2.374) Cm (18:149) Scan ES+ 1.66e NA +EDC product m/z (+ESI) Product and Daughter Ions Selection Naphthenic acid Table 2. Analyte Decanoic acid Decanoic acid d19 Parent ions Selected MRM Daughter ions In both cases no signal in (-ESI) Proposed Fragmentation Pattern EDC R1 = Alkyl from NA R2 = CH 3 CH 2 -

18 Analyte Separation, A note on Z= analytes NA9J_238S1 Smooth(SG,2x1) 1,WG31245,1/1ul WG ,, BLK 1 Laboratory blank F1:MRM of 32 channels,es+ 356.>129 C12H24O2 (NA Z ) 2.612e NA9J_238S5 Smooth(SG,2x1) 1,WG31245,1/1ul WG ,,SPM 1 Field sample C12H24O2 (NA Z ) F1:MRM of 32 channels,es+ 356.> e+7 Laboratory background due to fatty acids is a challenge in NA analysis This can be corrected by consistently taking out the sharp peaks corresponding to the straight chain isomer for Z= and sometimes for Z=

19 Chromatographic conditions Table Time () LC-Gradient Mobile phase composition A =7 B = 3 A = 5 B = 5 A = 45 B = 55 A = 2 B = 8 A = B =1 LC flow (ml/) A = B = 1 A = 7 B =3 A =7 B = 3 Column = Xtera C18, 1 cm, 2.1 mm i.d., 3.5 µm Solvent A =.1 HCOOH &.1 HCOONH4 in HPLC H2O Solvent B = Methanol.2.2.2

20 Chromatographic conditions Analysis batch compositions Lab Blank uses 5 mg HLB Spiked Matrix uses 5 mg HLB spiked SAR (surrogate, authentic and recover mix) Calibration linear calibration to demonstrate linearity Model compound calibration (PYB) bracketing Run order Linearity model compound linearity Bracketing Cal Model compound solvent SAR Bracketing cal model compound processed sample SAR (surrogate, authentic and recover mix) Solvent SAR - containing Merichem NA Solvent SAR containing model compound NA Spiked sample processed NA spiked SAR Solvent blank Method blank