lc-ms-ms analysis of pah’s their derivativesms analysis ......2011/08/16 · lc-ms-ms analysis of...

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


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)

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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.

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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


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.

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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


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.

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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


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


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


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


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


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


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”


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)


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


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


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

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

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

lc-ms-ms analysis of pah’s their derivativesms analysis ......2011/08/16 · lc-ms-ms analysis of...

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