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LC MS MS Analysis of PAH’s Their DerivativesLC-MS-MS Analysis of PAH’s, Their Derivatives and 3 Oil Dispersants in Sea Water Rolf Kern Applications ChemistRolf Kern, Applications ChemistFoster City Mass Spectrometry Lab
IntroductionIntroduction
Some well known, and many less well known maritime based oil spillsspills.
Exxon Valdez, Prince William Sound, Alaska March of 1989– 10 million gallonsg
BP Deepwater Horizon, April, of 2010– 4.9 million barrels or 205 million gallons
June 2011, Conoco-Phillips rig off North Eastern China –Unverifiable amount.
Unfortunately common events Unfortunately common events.
2 © 2011 AB SCIEX
IntroductionIntroduction
PAH’s make up 0.2% to 7% of crude oil
Some of these are toxic (carcinogenic, mutagenic), with the most well known being Benzo(a)pyrene.
Alkylated PAH’s are less well studied and in some cases may Alkylated PAH s are less well studied and in some cases may be more toxic.
Oxidation products (OPAH’s) are also not well studied, but are more water soluble, and possibly more bioavailable.
32 PAH’s are classified as Priority Pollutants by the EPA, with 16 being commonly monitored (Method 8270 1625)16 being commonly monitored (Method 8270, 1625)
3 © 2011 AB SCIEX
IntroductionIntroduction
Dispersants often used to “clean up” spills.
Mixtures of surfactants to break up oil slicks, prevent them form washing onshore.
Some controversy around use Some controversy around use.
Components can be toxic.
Two products that were used during Deepwater Horizon made Two products that were used during Deepwater Horizon made by Nalco –
– Corexit EC9500A (Propylene Glycol, light petroleum distillates, DOSS)C it EC9527A (2 B t th l P l Gl l DOSS)– Corexit EC9527A (2-Butoxyethanol, Propylene Glycol, DOSS)
By some estimates, as much as 1,000,000 gallons of these two products were used.
4 © 2011 AB SCIEX
IntroductionIntroduction
Explored methods for analysis of PAH’s and common dispersant componentsdispersant components.
Included analysis of common alkylated PAH’s and some oxidized metabolites.
Based methods on direct injection of sea water
Compared sensitivity for various ionization techniques.p y q
Addressed common problems with analysis.
5 © 2011 AB SCIEX
PAH AnalysisPAH Analysis
C10H8 C11H10 CH C12H10PAH’s and some naphthalene azulene
C10H8(MW: 128.063)
C11H10(MW: 142.078) 1-methylnaphthalene
CH3
CH3
2-methylnaphthalene
C12H10(MW: 154.078)
acenaphthene biphenyl
triphenylene chrysene benzo(a)anthracene benzo(b)anthracene
C18H12(MW: 228.094)anthracene phenanthrene
C14H10(MW: 178.078)
common methyl derivatives.
Shaded in red are
fluoranthene pyrene
C16H10(MW: 202.078)
C20H12
indeno(1,2,3-cd)pyrene benzo(ghi)perylene
C22H12(MW: 276.094)
dibenzo(a,h)anthracene pentacene
C22H14(MW: 278.110)
considered toxic.
benzo(j)fluoranthene benzo(k)fluoranthene benzo(b)fluoranthene benzo(e)pyrene benzo(a)pyrene perylene
C20H12(MW: 252.084)
AcenaphthyleneC12H8
2,6-dimethylnaphthaleneC12H12
CH3
CH3
FluoreneC13H10
2,3,5-trimethylnaphthaleneC13H14
CH3
CH3
CH3
C12H8(MW: 152.062)
C12H12(MW: 156.094)
C13H10(MW: 166.078)
C13H14(MW: 170.110)
DibenzothiopheneC12H8S
S
1-methylphenathreneC15H12
CH3
7,12-dimethylbenzo(a)anthraceneC20H16
CH3
CH3
CoroneneC24H12
RubreneC42H28
(MW: 532.219)
6 © 2011 AB SCIEX
(MW: 184.035)(MW: 192.084) (MW: 256.125) (MW: 300.094)
PAH Analysis
OH
O
OO
C10H8O C10H6O2
PAH Analysis
Oxidative PAH Metabolites 1-Naphthol
OH
2-Naphthol 1,2-NaphthoquinoneO
1,4-Naphthoquinone
C10H8O(MW: 144.058)
C10H6O2(MW: 158.037)
OH
1-Anthracenol
OH
9-Anthracenol
C14H10O(MW: 194.073)
OH
2-HydroxyfluoreneOH
9-Hydroxyfluorene
C13H10O(MW: 182.073)
OH9-Phenanthrol
described in Literature.
Difficult to impossible to find some of these as analytical
C18H12O(MW: 244.089) OH
2-Hydroxychrysene
OH
3-Hydroxychrysene
C20H12O(MW: 244.089)
OH
2-Hydroxybenzo(a)pyrene
OH
6-Hydroxybenzo(a)pyrene
OH O
OH
O OO O
standards.
Toxicity unknown for many of these.
1-AcenaphthenolC12H10O
(MW: 170.073)
2-Hydroxy-9-fluorenoneC13H8O2
(MW: 196.052)
9-FluorenoneC13H8O
(MW: 180.058)
AcenaphthenequinoneC12H6O2
(MW: 182.037)
OAnthraquinoneC14H10O2
(MW: 210.068)
OH
O
OH
OH
O
1-HydroxypyreneC16H10O(MW: 218.073)
OH
Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxideC20H14O3
(MW: 302.094)
9,10-Dihydrobenzo(a)pyrene-7(8H)-oneC20H14O
(MW: 270.104)
7 © 2011 AB SCIEX
PAH AnalysisPAH Analysis
Commonly analyzed by GC and GC-MS or LC-Fluorescence.
For analysis in water:– Wanted to develop an LC-MS-MS technique to minimize (or eliminate)
sample preparation.– Shorter analytical run time.– Greater specificity than fluorescence.
Potentially problematic because they have no obvious readily Potentially problematic because they have no obvious, readily ionizable functional group.
8 © 2011 AB SCIEX
PAH Analysis - IonizationPAH Analysis - Ionization Ionization efficiencies investigated using benzo(a)pyrene
[M]+ radical was observed as charged species for all ionization[M] radical was observed as charged species for all ionization modes.
APPI was tested using: no dopant, toluene, anisole chloroben ene best 0 3 ml/minchlorobenzene best, 0.3 ml/min
APCI was chosen for best combination of sensitivity and robustness
Table 1: Comparison of ionization methods and detector types. MRM m/z = 252.09 → 224.06 monitored4000QTRAP TM: 5-μL injections in triplicate per concentration (1 pg/mL to 1,000 ng/mL); unit - unit resolutionLinear regression with "1/x" weighting was used to obtain slope sensitivity, intercept, S/N, etc.; retention time = 5.40 min
ionization/ Slope Intercept Lowest conc. S/N Linearity range Notedetector cps/(ng/mL) cps detected(ng/mL) at LD
APCI 98.4 -21.5 1.00 8.9 1 - 1,000 robustESI 2,890.0 68.2 0.10 12.5 0.1 - 100 non-linear above 100 ng/mL
APPI 400.7 40.7 10.00 11.3 10 - 1,000 noisy backgroundUV (254 nm) 13.8 152.0 10.00 2.9 10 - 1,000 high background
9 © 2011 AB SCIEX
( ) g gFL(EX=260; EM=460 nm) 841.0 15.2 0.10 15.2 0.1 - 1,000 high carry-over
PAH AnalysisPAH Analysis
MS/MS of benzo(a)pyrene – from LC-MS-MS runMS/MS of benzo(a)pyrene from LC MS MS run
MS2 (QQQ Based Product Ion Scan)QTRAP scans much faster than QQQ MS2 (QQQ Based Product Ion Scan)resulting in higher quality spectra for ID purposes.
EPI (QTRAP based Product Ion Scan)
Retro Diels-Alders fragments (loss of H2and CH2) are observed EPI (QTRAP based Product Ion Scan)for most PAH’s.
10 © 2011 AB SCIEX
PAH Analysis - ChromatographyPAH Analysis - ChromatographyTime(min) Module Events Parameter
0.01 Pumps Pump B Conc. 40.007 00 Pumps Pump B Conc 60 00
MPA: H2O7.00 Pumps Pump B Conc. 60.00
14.00 Det. A Emission wavelength 352.0014.01 Det. A Emission wavelength 440.0016.00 Pumps Pump B Conc. 100.0018.90 Pumps Pump B Conc. 100.0018.90 Pumps Total Flow 0.5019 00 Pumps Total Flow 1 00
MPB: CH3CN
GL Sciences Inertsil ODS-P HP 3m 2 1x250mm 19.00 Pumps Total Flow 1.00
23.00 Det. A Emission wavelength 440.0023.10 Det. A Emission wavelength 420.0025.00 Pumps Pump B Conc. 100.0025.00 Pumps Total Flow 1.0025.10 Pumps Total Flow 0.5025 11 Pumps Pump B Conc 40 00
HP 3m, 2.1x250mm
Shimadzu Nexera UHPLC system with RF-20Axs
25.11 Pumps Pump B Conc. 40.0029.00 Det. A Emission wavelength 420.0029.10 Det. A Emission wavelength 352.0030.00 System Controller Stop
Excitation Wavelength=260 nm; Lamp=D2; Gains x4; Sensitivity LowResponse 1 5 sec
yfluorescence detector.
Quantitative work done on API 5000 mass spectrometer Response=1.5 secAPI 5000 mass spectrometer
11 © 2011 AB SCIEX
PAH Analysis - ChromatographyPAH Analysis - Chromatography
XIC of +MRM (66 pairs): 128.070/102.060 Da ID: azulene_102 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-uL.wiff... Max. 4.1e5 cps.
8.45
XIC of +MRM (66 pairs): 128.070/102.060 Da ID: azulene_102 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-uL.wiff... Max. 4.1e5 cps.
8.45
XIC of +MRM (66 pairs): 142.060/115.050 Da ID: 1-methylnaphthalene_115 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P A... Max. 2.3e5 cps.
10.40
XIC of +MRM (66 pairs): 142.060/115.050 Da ID: 1-methylnaphthalene_115 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P A... Max. 2.3e5 cps.
10.40
XIC of +MRM (66 pairs): 154.070/153.070 Da ID: acenaphthene_153 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-... Max. 4.0e5 cps.
11.20
XIC of +MRM (66 pairs): 154.070/153.070 Da ID: acenaphthene_153 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-... Max. 4.0e5 cps.
11.20
XIC of +MRM (66 pairs): 178.090/176.080 Da ID: phenanthrene_176 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-... Max. 7.2e4 cps.
7 2e4 13.69
XIC of +MRM (66 pairs): 178.090/176.080 Da ID: phenanthrene_176 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-... Max. 7.2e4 cps.
7 2e4 13.69
C8H10 C11H10 C12H10 C14H10XIC of +MRM (66 pairs): 228.070/226.070 Da ID: tripheneylene_226 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-u... Max. 3.0e5 cps.
x 40.018 45
XIC of +MRM (66 pairs): 228.070/226.070 Da ID: tripheneylene_226 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-u... Max. 3.0e5 cps.
x 40.018 45
C18H12
1 4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
Inte
nsity
, cps
Azulene
1 4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
Inte
nsity
, cps
Azulene
8 0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
2.3e5In
tens
ity, c
ps10.40
10.77
1-methylnaphthalne
2-methylnaphthalene
8 0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
2.3e5In
tens
ity, c
ps10.40
10.77
1-methylnaphthalne
2-methylnaphthalene
1 4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
Inte
nsity
, cps
10.22
Biphenyl Acenaphthene
1 4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
Inte
nsity
, cps
10.22
Biphenyl Acenaphthene
2 5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e47.2e4
Inte
nsity
, cps
12.55
PhenanthreneAnthracene
2 5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e47.2e4
Inte
nsity
, cps
12.55
PhenanthreneAnthracene
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
Inte
nsity
, cps
18.45
16.99
19.25
Triphenylene Benzo(a)anthracene
Chrysene
Benzo(b)anthracene1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
Inte
nsity
, cps
18.45
16.99
19.25
Triphenylene Benzo(a)anthracene
Chrysene
Benzo(b)anthracene
6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
Naphthalene
6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
Naphthalene
9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
20.8719.91
24.31
21.93 23.00
15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
20.8719.91
24.31
21.93 23.00
mrm TIC XIC of +MRM (66 pairs): 276.080/274.080 Da ID: indeno(1,2,3-cd)perylene_274 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-... Max. 2.5e5 cps.
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
2.3e5
2.4e5
2.5e530.42
Dibenzo(a,h)anthracene
cascadingBenzo(ghi)perylene
Indeno(1,2,3-cd)pyrene
XIC of +MRM (66 pairs): 252.080/250.080 Da ID: benzo(e)pyrene_250 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-1... Max. 1.5e6 cps.
1 0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e61.5e6 20.85
21.91
Benzo(j)fluoranthene
Perylene
Benzo(e)pyrene
XIC of +MRM (66 pairs): 252.080/250.080 Da ID: benzo(e)pyrene_250 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-1... Max. 1.5e6 cps.
1 0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e61.5e6 20.85
21.91
Benzo(j)fluoranthene
Perylene
Benzo(e)pyrene
C20H10 C22H10
Fluorescence0 0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
Inte
nsity
, cps
26.45
28.66
0 0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
Inte
nsity
, cps
24.31
22.97
Benzo(a)pyrene
Benzo(k)fluorantheneBbF
0 0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
Inte
nsity
, cps
24.31
22.97
Benzo(a)pyrene
Benzo(k)fluorantheneBbF
12 © 2011 AB SCIEX
23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5Time, min
0.020.6 20.8 21.0 21.2 21.4 21.6 21.8 22.0 22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0 24.2 24.4 24.6 24.8 25.0
Time, min
0.020.6 20.8 21.0 21.2 21.4 21.6 21.8 22.0 22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0 24.2 24.4 24.6 24.8 25.0
Time, min
0.0
PAH AnalysisPAH Analysis
Optimized compounds, developed chromatography, how else can we increase sensitivity without resorting to sample prep?can we increase sensitivity without resorting to sample prep?
For APCI good correlation with injection volume & s/n
Measurements were in triplicates. S/N, retention time, peak width, theoretical plate are averages of 3 measurements5‐µL Injections Slope linear correlation Detection S/N at Retention time peak width Theoreticalm/z or UV sensitivity coefficient limit (ng/mL) detection limit (min) base(min) plate
m/z = 252.09 → 224.06 100.0 0.9981 1 8.5 5.64 0.139 479m/z = 252.09 → 250.09 397.0 0.9986 1 13.8 5.48 0.234 481
UV @ 254 nm 14.5 0.9983 10 7.2 7.19 0.290 446
10‐µL Injections Slope linear correlation Detection S/N at Retention time peak width Theoretical
BaP 5-ul injection.rdb (252 - 224): "Linear" Regression ("1 / x" weighting): y = 100 x + -2.33 (r = 0.9983)
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
1.05e5
Area, cou
nts
5-µL injection
y = 100 X – 2.33
m/z or UV sensitivity coefficient limit(ng/mL) detection limit (min) base(min) platem/z = 252.09 → 224.06 221.0 0.9829 1 17.9 5.32 0.169 453m/z = 252.09 → 250.09 863.0 0.9830 1 26.6 5.33 0.269 454
UV @ 254 nm 28.6 0.9856 10 21.5 5.28 0.217 447
20‐µL Injections Slope linear correlation Detection S/N at Retention time peak width Theoreticalm/z or UV sensitivity coefficient limit(ng/mL) detection limit (min) base(min) plate
m/z = 252.09 → 224.06 519.0 0.9952 1 33.1 5.43 0.230 472m/z = 252.09 → 250.09 1980.0 0.9961 0.1 5.8 5.41 0.157 469
UV@ 254 nm 53 8 0 6848 10 15 5 5 25 0 229 442
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000Concentration, ng/mL
0.00
5000.00
1.00e4
1.50e4
BaP 40-ul injection.rdb (252 - 224): "Linear" Regression ("1 / x" weighting): y = 1.22e+003 x + -28.3 (r = 0.9939)
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
9.0e5
9.5e5
1.0e6
1.1e6
1.1e6
1.2e6
1.2e6
1.3e6
1.3e6
coun
ts
40-µL injection
y = 1.22e3 X -10.1
UV @ 254 nm 53.8 0.6848 10 15.5 5.25 0.229 442
40‐µL Injections Slope linear correlation Detection S/N at Retention time Peak width Theoreticalm/z or UV sensitivity coefficient limit(ng/mL) detection limit (min) base(min) plate
m/z = 252.09 → 224.06 1220.0 0.9939 0.1 6.5 5.36 0.103 461m/z = 252.09 → 250.09 4480.0 0.9955 0.1 5.1 5.37 0.171 461
UV @ 254 nm 107.0 0.9996 1 6.2 5.32 0.230 453 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000Concentration, ng/mL
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
Are
a,
13 © 2011 AB SCIEX
PAH AnalysisPAH Analysis
Oyster extract prepared using slightly modified NOAA methodXIC of +MRM (76 pairs): 128.060/78.050 amu Expected RT: 9.6 ID: naphthalene_78 from Sample 68 (OYS 0-3 + 500 uL ACN) of PAH ca... Max. 3.5e4 cps.
6000.0
8000.0
1.0e4
1.2e4
1.4e4
nten
sity
, cps
Scheduled MRM
XIC of +MRM (76 pairs): 128.060/78.050 amu Expected RT: 9.6 ID: naphthalene_78 from Sample 68 (OYS 0-3 + 500 uL ACN) of PAH ca... Max. 3.5e4 cps.
6000.0
8000.0
1.0e4
1.2e4
1.4e4
nten
sity
, cps
Scheduled MRM
9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0Time, min
0.0
2000.0
4000.0
In
9.72 9.85
9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0Time, min
0.0
2000.0
4000.0
In
9.72 9.85
XIC of +MRM (76 pairs): 128.060/78.050 amu Expected RT: 9.6 ID: naphthalene_78 from Sample 3 (Air sample) of Environment Canada.... Max. 4.7e4 cps.
4.5e5
5.0e5
5.5e5
Solvent extracted air sampling disks
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
Intensity, cps
9.72
14 © 2011 AB SCIEX
7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0Time, min
0.0
9.919.369.21
PAH Analysis - MetabolitesPAH Analysis - Metabolites
Oxidative metabolism of benzo(a)pyrene is well studied.
CYP450 substrate
+MS2 (267.10) CE (55): Period 3, 20.522 to 20.848 min from Sample 5 (Benzo(a)pyrene incubate) of pos Q 3 incubation study_MSMS.wi... Max. 6.4e5 cps.
4.5e5
5.0e5
5.5e5
6.0e5
6.4e5
x 25.0239.1
+14 DaRLM incubation yielded expoxide
2.0e5
2.5e5
3.0e5
3.5e5
4.0e599.0
15 © 2011 AB SCIEX
80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z , Da
0.0
5.0e4
1.0e5
1.5e5
238.3
267.1
-28 Da
H2C=CH2
PAH AnalysisPAH Analysis
16 © 2011 AB SCIEX
PAH AnalysisPAH Analysis
Developed a method for:26 PAH’– 26 PAH’s
– 6 Alkylated (methyl) species– 11 Oxidized degradants
5 l b l d i t l t d d– 5 labeled internal standards
Chromatographically separates isobaric compounds
S b b d t ti li it i t th t i ill b b d Sub ppb detection limit in water – other matrices will be based on sample prep techniques.
17 © 2011 AB SCIEX
Dispersant AnalysisDispersant Analysis
Developed methods for:DOSS (di t l lf i i id)– DOSS (dioctylsulfosuccinic acid)
– 2-Butoxyethanol– Di(propylene glycol) tert-butyl ether
2-butoxyethanol is present in Corexit EC9527A
OOH
dioctylsulfosuccinic acid sodium (DOSS) MW: 444.21, 423.24
2-butoxyethanolMW: 118.10
Di(propylene glycol) tert-butyl MW: 190.16
O
18 © 2011 AB SCIEX
Dispersant AnalysisDispersant Analysis
2-Butoxyethanol & D(ipropylene glycol) tert-butyl ether, no strong ionizable groups Do associate well with NH4
+ in ESIgroups. Do associate well with NH4 in ESI.
+Q3: 0.000 to 0.769 min from Sample 1 (2-butoxyethanol 1 ng_uL) of 2-butoxyethanol pos Q3 MS.wiff (Turbo Spray) Max. 9.6e5 cps.
7 0e5
8.0e5
9.0e59.6e5 141.0
+Q3: 0.100 to 5.710 min from Sample 2 (dipropylene glycol monobutyl ether 100 pg_uL) of dipropylene glycol monobutyl ether posQ3.wif... Max. 1.8e6 cps.
1.4e6
1.6e6
1.8e6191.1
MNH4+
MH+
MNa+MH+
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
Inte
nsity
, cps
119.1 136.1
104.9
129.0
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
Inte
nsity
, cps
179.1
213.1208.1
192.1193.1
4
MNH4+ MNa+
100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134 136 138 140 142 144 146 148 150m/z, Da
+MS2 (119.10) CE (30): 0.050 to 2.304 min from Sample 1 (2-butoxyethanol 1 ng_uL) of 2-butoxyethanol pos DS=119.wiff (Turbo Spray)... Max. 5.1e5 cps.
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
cps
62.9
150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250m/z, Da
0.0
+MS2 (191.16) CE (10): 0.000 to 0.551 min from Sample 1 (dipropylene glycol monobutyl ether 10 pg_uL) of dipropylene glycol monobut... Max. 6.9e5 cps.
4.0e5
5.0e5
6.0e5
6.9e5cp
s
115.0
58.9
191 1
MS/MS of MH+MS/MS of MH+
15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130m/z, Da
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
Inte
nsity
,
119.0
45.0
57.0
41.0 101.0
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200m/z, Da
0.0
1.0e5
2.0e5
3.0e5
Inte
nsity
, 191.1
117.0 173.156.9 101.097.1
19 © 2011 AB SCIEX
2-butoxyethanol Dipropylene glycol t-butyl ether
Dispersant AnalysisDispersant Analysis
DOSS ionizes in both negative mode and positive mode. Positive mode also works best as an NH + adduct
-MS2 (421.23) CE (-30): 0.050 to 2.104 min from Sample 1 (docusate 1 ng_uL) of docusate neg DS=421 CEM=1500.wiff (Turbo Spray), ... Max. 7.0e5 cps.
6.0e57.0e5
..x 20.0
421.080.9
works best as an NH4+ adduct.
Negative mode
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z, Da
0.0
2.0e5
4.0e5
Inte
n..
227.0290.997.0 160.9 183.3
+MS2 (423.24) CE (30): 0.000 to 1.052 min from Sample 1 (docusate 1 ng_uL) of Docusate pos DS=423.wiff (Turbo Spray), Smoothed Max. 8946.7 cps.
8947 71.1
ega e odeMS/MS [M-H]-
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420m/z Da
0
2000
4000
6000
80008947
Inte
n...
57.0 199.1113.1 414.9
423.444.9 89.1 406.4133.0
Positive mode MS/MS [M+H]+
m/z, Da +MS2 (440.27) CE (15): 0.000 to 1.553 min from Sample 1 (docusate 1 ng_uL) of Docusate pos DS=440.wiff (Turbo Spray), Smoothed Max. 9.2e4 cps.
2 0 4
4.0e4
6.0e4
8.0e49.2e4
Inte
n...
113.1
199.0
71.0 311.2
423.357.0 440.4
Positive mode MS/MS [M+NH4]+
20 © 2011 AB SCIEX
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z, Da
0.0
2.0e4217.1
181.298.9
[ 4]
Dispersant AnalysisDispersant AnalysisStandard compound optimization techniques. Work done on an API 5000 triple quadrupole mass spectrometer.p q p p
Negative ion mode MRM TableM1 M3 Dwell Time ID DP EP CE CXP
421.24 80.90 50 dioctylsulfosuccinate_81 -210 -10 -45 -34.0421.24 183.10 50 dioctylsulfosuccinate_183 -210 -10 -45 -24.0421.24 227.10 50 dioctylsulfosuccinate_227 -210 -10 -34 -28.0421.24 290.90 50 dioctylsulfosuccinate_291 -210 -10 -33 -37.0
Positive ion mode MRM TableM1 M3 Dwell Time ID DP EP CE CXP
119 11 45 05 50 2-butoxyethanol 119 45 52 10 15 6 7119.11 45.05 50 2-butoxyethanol 119_45 52 10 15 6.7119.11 63.08 50 2-butoxyethanol 119_63 52 10 9 9.8136.11 45.05 50 2-butoxyethanol 136_45 7 10 21 5.5136.11 63.08 50 2-butoxyethanol 136_63 7 10 13 6.5191.16 59.07 50 di(propylene glycol) t -butyl ether_191_59 72 10 17 8.8191.16 115.09 50 di(propylene glycol) t -butyl ether 191 115 72 10 10 12.3191.16 115.09 50 di(propylene glycol) t butyl ether_191_115 72 10 10 12.3208.19 59.07 50 di(propylene glycol) t -butyl ether_208_59 6.5 10 23 8.9208.19 115.09 50 di(propylene glycol) t -butyl ether_208_115 6.5 10 16 13.0423.24 113.10 50 dioctylsulfosuccinic acid_423_113 197 10 13.4 13.0423.24 199.10 50 dioctylsulfosuccinic acid_423_199 197 10 15 15.7440.27 113.10 50 dioctylsulfosuccinic acid_440_113 152 10 18 18.3
21 © 2011 AB SCIEX
440.27 199.10 50 dioctylsulfosuccinic acid_440_199 152 10 20 20.5
Dispersant AnalysisDispersant Analysis
MPA: H2O + 5 mM ammonium acetate
MPB: CH3CN + 5 mM ammonium acetate
Total gradient flow of 0.5 ml/min
Z b RRHD SB C18 l 50 X 2 1 id Zorbax RRHD SB-C18 column, 50 mm X 2.1 mm id, 1.8 μm HPLC column
A
123 6
A
123 6
Column
waste
MS
Column
MSwaste
A
123 6
A
123 6
A
123 6
123 6
A
123 6
A
123 6
A
123 6
123 6
Column
waste
MS
Column
MSwaste3
4 563
4 56 waste
Pump C Pump C
waste34 5
634 5
634 5
634 5
634 5
634 5
634 5
634 5
6 waste
Pump C Pump C
waste
22 © 2011 AB SCIEX
Valve Position “0” Valve Position “1”Valve Position “0” Valve Position “1”
Dispersant AnalysisDispersant Analysis
These compounds – particularly DOSS - are very “sticky”, and thus highly tprone to carryover.
Used the Shimadzu NEXERA’s autosampler rinse program to dramatically reduce injection to injection carryover.
Fig. 13. Internal/External Rinse steps usedR0 = reagent alcohol; R1 = acetonitrile; R2 = 50% water + 50% acetonitrile
23 © 2011 AB SCIEX
Dispersant AnalysisDispersant AnalysisBecause DOSS is more sensitive in negative mode and the compounds are chromatographically resolved, a 2 period experiment was used.
TIC: from Sample 1 (1 ng_uL mix) of pos-neg non s-MRM.wiff (Turbo Spray) Max. 3.7e6 cps.
0 0
1.0e6
2.0e6
3.0e63.7e6
In...
4.57
+e -e
TIC: from Sample 1 (1 ng_uL mix) of pos-neg non s-MRM.wiff (Turbo Spray) Max. 3.7e6 cps.
0 0
1.0e6
2.0e6
3.0e63.7e6
In...
4.57
+e -e
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min
0.0
XIC of +MRM (12 pairs): Period 1, 119.110/45.050 Da ID: 2-butoxyethanol 119_45 from Sample 1 (1 ng_uL mix) of pos-neg non s-MRM.... Max. 1.0e4 cps.
5000.00
1.00e4
In...
3.83
2-butoxyethanol (RT 3.83min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min
0.0
XIC of +MRM (12 pairs): Period 1, 119.110/45.050 Da ID: 2-butoxyethanol 119_45 from Sample 1 (1 ng_uL mix) of pos-neg non s-MRM.... Max. 1.0e4 cps.
5000.00
1.00e4
In...
3.83
2-butoxyethanol (RT 3.83min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.00
XIC of +MRM (12 pairs): Period 1, 191.160/59.070 Da ID: dipropylene glycol butyl ether_191_59 from Sample 1 (1 ng_uL mix) of pos-ne... Max. 1.9e6 cps.
1.0e6
1.5e61.9e6
In...
4.57
Di(propylene glycol) t-butyl ether (4.57 min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.00
XIC of +MRM (12 pairs): Period 1, 191.160/59.070 Da ID: dipropylene glycol butyl ether_191_59 from Sample 1 (1 ng_uL mix) of pos-ne... Max. 1.9e6 cps.
1.0e6
1.5e61.9e6
In...
4.57
Di(propylene glycol) t-butyl ether (4.57 min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.0
5.0e5
XIC of -MRM (4 pairs): Period 2, 421.240/80.900 Da ID: dioctylsulfosuccinate_81 from Sample 1 (1 ng_uL mix) of pos-neg non s-MRM.... Max. 1.5e5 cps.
1.0e5
1.5e5
.
5.47
Dioctylsulfosuccinate (5 47 min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.0
5.0e5
XIC of -MRM (4 pairs): Period 2, 421.240/80.900 Da ID: dioctylsulfosuccinate_81 from Sample 1 (1 ng_uL mix) of pos-neg non s-MRM.... Max. 1.5e5 cps.
1.0e5
1.5e5
.
5.47
Dioctylsulfosuccinate (5 47 min)
24 © 2011 AB SCIEX
5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0Time, min
0.0
5.0e4Int.. Dioctylsulfosuccinate (5.47 min)
5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0Time, min
0.0
5.0e4Int.. Dioctylsulfosuccinate (5.47 min)
Dispersant AnalysisDispersant Analysis
2-butoxyethanol 136.163.1 LOD 18ng/ml
Di l l l t b t lDipropylene glycol t-butyl ether 19159 LOD 0.5 ng/ml
25 © 2011 AB SCIEX
Dispersant AnalysisDispersant Analysis
DOSS Pos modeDOSS Pos mode 440199.1, 0.9 ng/mL
DOSS Neg mode 421.280.9, 0.8 ng/mLg
Neg mode is actually ~13x more sensitive than Pos
26 © 2011 AB SCIEX
more sensitive than Pos mode – carryover still a problem!
Conclusions & Further WorkConclusions & Further Work
Developed reliable, sensitive methods that should be fairly easy f t l b t i l tfor most labs to implement.
– Sub ppb LLOQ’s for PAH’s & related compounds– Low to sub ppb LOD’s for dispersant compounds
Relatively short run times compared to traditional, GC based approaches.
N l i d f t l No sample prep required for water samples.
27 © 2011 AB SCIEX
Conclusions & Further WorkConclusions & Further Work
Challenges for PAH analysis– Lack of standards for alkylated and oxidized PAH’s– Lack of action limits/toxicity data for oxidized PAH’s– Need to investigate whether additional PAH metabolites/degradants are
t i th iti ill i tpresent in the maritime spill environment.
Challenges for dispersant analysis– Even though reduced, carryover is still a problem.Even though reduced, carryover is still a problem.
28 © 2011 AB SCIEX
Conclusions & Further WorkConclusions & Further Work
Q ti ?Questions?
29 © 2011 AB SCIEX
With Thanks to:With Thanks to:
Takeo Sakuma Rebecca Wittrig
ABSCIEX71 Four Valley Drive, Concord, ON, L4K
Stacy TremintinTimothy L. HoffmanYunYun ZouCarmai Seto Rebecca Wittrig Scott Kragerud
4V8
Scott Kragerud Robert I. EllisDeolinda Fernandes Fouad Khalaf Christopher D. Borton
C ti C b ll Shi d S i tifi I t tCurtis CampbellMasatoshi Takahashi
Shimadzu Scientific Instruments 7102 Riverwood Drive, Columbia, MD
Kein’ichi Suzuki GL Sciences, Inc. 22-1 Nishishinjuku 6-chome, Shinjuku-ku, Tokyo, 163-1130, JAPAN
Jack Cochran Restek Corporation,110 Benner Circle, Bellefonte, PA 16823
30 © 2011 AB SCIEX
Questions and Answers
31 © 2011 AB SCIEX
Some ReferencesSome References
OSU Superfund PAH & OPAH Monitoring http://oregonstate.edu/superfund/oilspill
BaP Metabolism BaP Metabolism http://herkules.oulu.fi/isbn9514270398/html/x203.html
32 © 2011 AB SCIEX