LC/MS/MS Analysis of PAH's, Their Derivatives, and 3 Oil Dispersants

LC-MS-MS Analysis of PAH’s , Their Derivatives and 3 Oil Dispersants in Sea Water Rolf Kern, Applications Chemist Foster City Mass Spectrometry Lab Introduction

 Some well known, and many less well known maritime based oil spills.  Exxon Valdez, Prince William Sound, Alaska March of 1989 – 10 million gallons  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 .

 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 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)

 Dispersants often used to “clean up” spills.  Mixtures of surfactants to break up oil slicks, prevent them form washing onshore.  Some controversy around use.  Components can be toxic.  Two products that were used during Deepwater Horizon made by Nalco – – Corexit EC9500A (Propylene Glycol, light petroleum distillates, DOSS) – Corex it EC9527A (2-Bu toxyeth anol , P ropyl ene Gl ycol , DOSS)  By some estimates, as much as 1,000,000 gallons of these two products were used.

 Explored methods for analysis of PAH’s and common dispersant components.  Included analysis of common alkylated PAH’s and some oxidized metabolites.  Based methods on direct injection of sea water  Comppyared sensitivity for various ionization techni ques.  Addressed common problems with analysis.

PAH’s and some C10H8 C11H10 H3C C12H10 (MW: (MW: (MW: 154.078) naphthalene azulene CH3 common methyl 128.063) 142.078) 1-methylnaphthalene 2-methylnaphthalene acenaphthene biphenyl derivatives. C14H10 C18H12 (MW: (MW: 178.078) anthracene phenanthrene 228.094) Shaded in red are triphenylene chrysene benzo(a)anthracene benzo(b)anthracene

considered toxic. C16H10 C22H12 C22H14 (MW: (MW: (MW: 202.078) 276.094) 278.110) fluoranthene pyrene indeno(1,2,3-cd)pyrene benzo(ghi)perylene dibenzo(a,h)anthracene pentacene

C20H12 (MW: 252.084) benzo(j)fluoranthene benzo(k)fluoranthene benzo(b)fluoranthene benzo(e)pyrene benzo(a)pyrene perylene

H C H3C 3

H C CH3 3 CH3 Acenaphthylene 2,6-dimethylnaphthalene Fluorene 2,3,5-trimethylnaphthalene C12H8 C12H12 C13H10 C13H14 (MW: 152.062) (MW: 156.094) (MW: 166.078) (MW: 170.110)

H3C CH3 S CH3 Rubrene C42H28 (MW: 532.219) 1-methylphenathrene Dibenzothiophene 7,12-dimethylbenzo(a)anthracene Coronene C15H12 C12H8S C20H16 C24H12 (MW: 192.084) (MW: 184.035) (MW: 256.125) (MW: 300.094)

O OH O O Oxidative PAH Metabolites C10H8O C10H6O2 HO (MW: (MW: O described in Literature. 144.058) 1-Naphthol 2-Naphthol 158.037) 1,2-Naphthoquinone 1,4-Naphthoquinone

OH OH

C13H10O OH C14H10O Difficult to impossible to find (MW: (MW: 182.073) 194.073) OH OH some of these as analytical 2-Hydroxyfluorene 9-Hydroxyfluorene 1-Anthracenol 9-Anthracenol 9-Phenanthrol OH

standards. C18H12O HO C20H12O OH (MW: (MW: 244.089) HO 244.089) Toxicity unknown for many of 2-Hydroxychrysene 3-Hydroxychrysene 6-Hydroxybenzo(a)pyrene 2-Hydroxybenzo(a)pyrene OH O O O O O these. OH

O 1-Acenaphthenol 9-Fluorenone Acenaphthenequinone 2-Hydroxy-9-fluorenone Anthraquinone C12H10O C13H8O C12H6O2 C13H8O2 C14H10O2 (MW: 170.073) (MW: 180.058) (MW: 182.037) (MW: 196.052) (MW: 210.068) O

O OH

OH HO 1-Hydroxypyrene 9,10-Dihydrobenzo(a)pyrene-7(8H)-one Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide C16H10O C20H14O C20H14O3 (MW: 218.073) (MW: 270.104) (MW: 302.094)

 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 ionizable functional group.

8 © 2011 AB SCIEX PAH Analysis - Ionization  Ionization efficiencies investigated using benzo(a)pyrene  [M]+ radical was observed as charged species for all ionization modes.  APPI was tested using: no dopant, toluene, anisole chlorobenz ene  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 monitored 4000QTRAP TM: 5-μL injections in triplicate per concentration (1 pg/mL to 1,000 ng/mL); unit - unit resolution Linear 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 Note detector cps/(ng/mL) cps detected(ng/mL) at LD APCI 98.4 -21.5 1.00 8.9 1 - 1,000 robust ESI 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 background UV (()254 nm) 13.8 152.0 10.00 2.9 10 - 1,000 higggh background FL(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 run

QTRAP scans much faster than QQQ MS2 (QQQ Based Product Ion Scan) resulting in higher quality spectra for ID purposes.

Retro Diels-Alders fragments (loss of H2 and CH2) are observed EPI (QTRAP based Product Ion Scan) for most PAH’s.

Time(min) Module Events Parameter  MPA: H2O 0.01 Pumps Pump B Conc. 40.00 7007.00 Pumps Pump B Conc . 60.00 14.00 Det. A Emission wavelength 352.00  MPB: CH3CN 14.01 Det. A Emission wavelength 440.00 16.00 Pumps Pump B Conc. 100.00 18.90 Pumps Pump B Conc. 100.00  GL Sciences Inertsil ODS-P 18.90 Pumps Total Flow 0.50 19.00 Pumps Total Flow 1001.00 HP 3m, 2. 1x250mm 23.00 Det. A Emission wavelength 440.00 23.10 Det. A Emission wavelength 420.00 25.00 Pumps Pump B Conc. 100.00  Shimadzu Nexera UHPLC 25.00 Pumps Total Flow 1.00 system with RF-20Axs 25.10 Pumps Total Flow 0.50 25.11 Pumps Pump B Conc . 40.00 fluorescence detector. 29.00 Det. A Emission wavelength 420.00 29.10 Det. A Emission wavelength 352.00 30.00 System Controller Stop  Quantitative work done on Excitation Wavelength=260 nm; Lamp=D2; Gains x4; Sensitivity Low API 5000 mass spectrometer Response=1 .5 sec

C8H10 C11H10 C12H10 C14H10 C18H12

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.wiffXIC of +MRM... (66 pairs): 142.060/115.050 Da ID: 1-methylnaphthalene_115 Max. 4.1e5 cps. from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-PXIC of +MRM A... (66 pairs): 154.070/153.070 Da ID: acenaphthene_153 Max. 2.3e5 cps. from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-PXIC ACN-10-.. of +MRM. (66 pairs): 178.090/176.080 Da ID: phenanthrene_176 Max. 4.0e5 cps. from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-PXIC ACN-10-.. of +MRM. (66 pairs): 228.070/226.070 Da ID: tripheneylene_226 Max. 7.2e4 cps. from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-P ACN-10-u... Max. 3.0e5 cps.

8.45 1 11.20 x 40.0 10.400.40 13.69 7.2e4 2e4 18.45 45 10.77

Triphenylene Benzo(a)anthracene

1-methylnaphthalne 16.99 12.55

4.0e5 7.0e4 4.0e5 2.3e5 Acenaphthene 3.0e5 3.8e5 Biphenyl 3.8e5 2.2e5 6.5e4 2.8e5 3.6e5 3.6e5 2.1e5 2-methylnaphthalene 2.0e5 3.4e5 6.0e4 2.6e5 3.4e5 10.22 1.9e5 3.2e5 Anthracene 3.2e5 5.5e4 2.4e5 Chrysene 1.8e5 3.0e5 Phenanthrene 3.0e5 19.25 1.7e5 5.0e4 2.2e5 Azulene 2.8e5 2.8e5 1.6e5 Benzo(b)anthracene 2.6e5 2.0e5 2.6e5 1.5e5 4.5e4 2.4e5 2.4e5 1.4e5 1.8e5 4.0e4 1.3e5 2.2e5 2.2e5 1.6e5 1.2e5 3.5e4 2.0e5 2.0e5 Intensity, cps cps Intensity, Intensity, Intensity, cps cps Intensity, Intensity, Intensity, cps cps Intensity, Intensity, cps cps Intensity, Intensity, InIn te te n n s s1.1e5 ity ity , c , c p p s s 1.8e5 1.4e5 1.8e5 3.0e4 1.0e5 1.6e5 1.6e5 1.2e5 9.0e4

2.5e4 5e4 1.4e5 4e5 8.0e4 0e4 1.4e5 4e5

Naphthalene

24.31 1.0e5 1.2e5 7.0e4 1.2e5 2.0e4 6.0e4 8.0e4 1.0e5 1.0e5 5.0e4 1.5e4 8.0e4 8.0e4 6.0e4 4.0e4 6.0e4 6.0e4 1.0e4 3.0e4 4.0e4 4.0e4 4.0e4 2.0e4 5000.0 2.0e4 2.0e4 2.0e4 1.0e4 19.91 20.87 21.93 23.00 0.0 0.0 0.0 0.0 0.0 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 9.29.7 9.4 9.8 9.6 9.9 10.0 9.8 10.1 10.0 10.2 10.2 10.3 10.4 10.4 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 11.410.0 11.5 10.1 11.6 10.2 11.7 10.3 11.8 11.9 10.4 12.0 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.310.5 11.4 11.5 11.0 11.6 11.511.7 11.8 12.0 11.9 12.5 13.0 13.5 14.0 14.515.0 15.0 16.0 15.5 17.0 16.0 18.0 16.5 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 Time, min Time, min Time, min Time, min Time, min

C20H10 C22H10

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...XIC of +MRM (66 Max.pairs): 1.5e6 276.080/274.080 cps. Da ID: indeno(1,2,3-cd)perylene_274 from Sample 44 (SCIEX MIX 10 ng_uL) of Inertsil_ODS-... Max. 2.5e5 cps. mrm TIC 20.85 30.42 Benzo(j)fluoranthene 1.5e6 2.5e5 1.5e6 21.91 2.4e5 Indeno(1,2,3-cd)pyrene 1.4e6 2.3e5 2.2e5 Dibenzo(a,h)anthracene 1.3e6 2.1e5 cascading 1.2e6 2.0e5 Perylene 1.9e5 1.1e6 Benzo(ghi)perylene 1.8e5 Benzo(e)pyrene 1.7e5 1.0e6 0e6 26.45

28.66

Benzo(a)pyrene 1.6e5 9.0e5 1.5e5 Intensity, cps cps Intensity, Intensity, Intensity, cps Intensity, 1.4e5 8.0e5 1.3e5 1.2e5 7.0e5 24.31 1.1e5 6.0e5 BbF 1.0e5 9.0e4 5.0e5 Benzo(k)fluoranthene 8.0e4

4.0e5 7.0e4 22.97 6.0e4 3.0e5 5.0e4 4.0e4 Fluorescence 2.0e5 3.0e4

1.0e5 2.0e4 1.0e4

000.0 000.0 20.6 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 24.423.5 24.6 24.0 24.8 24.5 25.0 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 34.5 Time, min Time, min

 Optimized compounds, developed chromatography, how else can we increase sensitivity without resorting to sample prep?

1.05e5  For APCI  good correlation with injection1.00e5 volume & s/n 9.50e4

9.00e4

8.50e4

8.00e4

7.50e4

7.00e4BaP 5-ul injection.rdb (252 - 224): "Linear" Regression ("1 / x" weighting): y = 100 x + -2.33 (r = 0.9983) Measurements were in triplicates. S/N, retention time, peak width, theoretical plate are averages of 3 measurements 6.50e4 5‐µL Injections Slope linear correlation Detection S/N at Retention time peak width Theoretical 6.00e4 m/z or UV sensitivity coefficient limit (ng/mL) detection limit (min) base(min) plate 5.50e4 y = 100 X – 2.33 m/z = 252.09 → 224.06 100.0 0.9981 1 8.5 5.64 0.139 479 5.00e4 4.50e4 5-µL injection m/z = 252.09 → 250.09 397.0 0.9986 1 13.8 5.48 0.234 481 4.00e4 ts n u , co

a 3.50e4

UV @ 254 nm 14.5 0.9983 10 7.2 7.19 0.290 446 re A 3.00e4 10‐µL Injections Slope linear correlation Detection S/N at Retention time peak width Theoretical 2.50e41.50e4 2.00e41.00e4 m/z or UV sensitivity coefficient limit(ng/mL) detection limit (min) base(min) plate 5000.001.3e6 1.3e6 m/z = 252.09 → 224.06 221.0 0.9829 1 17.9 5.32 0.169 453 0.00 1.2e6 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z = 252.09 → 250.09 863.0 0.9830 1 26.6 5.33 0.269 454 1.2e6 Concentration, ng/mL BaP 40-ul injection.rdb (252 - 224): "Linear" Regression ("1 / x" weighting): y = 1.22e+003 x + -28.3 (r = 0.9939) UV @ 254 nm 28.6 0.9856 10 21.5 5.28 0.217 447 1.1e6 1.1e6 1.0e6 20‐µL Injections Slope linear correlation Detection S/N at Retention time peak width Theoretical 9.5e5 y = 1.22e3 X -10.1 9.0e5 m/z or UV sensitivity coefficient limit(ng/mL) detection limit (min) base(min) plate 8.5e5 6.0e5 8.0e5 m/z = 252.09 → 224.06 519.0 0.9952 1 33.1 5.43 0.230 472 5.5e5 7.5e5 40-µL injection 5.0e5 m/z = 252.09 → 250.09 1980.0 0.9961 0.1 5.8 5.41 0.157 469 7.0e5 4.5e5 6.5e5 UV @ 254 nm 53.8 0.6848 10 15.5 5255.25 0.229 442 counts 4.0e5 Area, 3.5e5 3.0e5 40‐µL Injections Slope linear correlation Detection S/N at Retention time Peak width Theoretical 2.5e5 2.0e5 m/z or UV sensitivity coefficient limit(ng/mL) detection limit (min) base(min) plate 1.5e5 m/z = 252.09 → 224.06 1220.0 0.9939 0.1 6.5 5.36 0.103 461 1.0e5 5.0e4 m/z = 252.09 → 250.09 4480.0 0.9955 0.1 5.1 5.37 0.171 461 0.0 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 UV @ 254 nm 107.0 0.9996 1 6.2 5.32 0.230 453 Concentration, n g /mL

Oyster extract prepared using slightly modified NOAA method

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. 1.4e4

1.2e4

1.0e4 Scheduled MRM

8000.0

6000.0 tensity,tensity, cps cps nnnn II 4000.0

9.72 2000.0 9.85

0.0 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 24.0 e, min 4.0e5 Tim

3.5e5

Solvent3.0e55.5e5 extracted air sampling disks

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. 2.5e55.0e5

2.0e54.5e5

1.5e5 s p , c ity s n te In

1.0e5

5.0e4 9.72 9.21 9.36 9.91 0.0 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 17.0 Time, min

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 Q3 incubation study_MSMS.wi... Max. 6.4e5 cps. x 25.0 239.1 6.4e5 +14 Da RLM incubation 6.0e5 5.5e5

yielded expoxide 5.0e5

4.5e5

4.0e5 99.0

3.5e5

3.0e5

2.5e5

2.0e5

1.5e5

1.0e5 H2C=CH2 238.3 -28 Da 5.0e4

267.1 0.0 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z, Da

 Developed a method for: – 26 PAH’ s – 6 Alkylated (methyl) species – 11 Oxidized degradants – 5l5 la be le did in terna l s tan dar ds  Chromatographically separates isobaric compounds  SbSub pp bdtb detec tion liitilimit in wa ter – other mat ri ces will be base d on sample prep techniques.

 Developed methods for: – DOSS(ditllfDOSS (dioctylsulfosucc iiinic ac id) – 2-Butoxyethanol – Di(propylene glycol) tert-butyl ether  2-butoxyethanol is present in Corexit EC9527A

dioctylsulfosuccinic acid sodium (DOSS) 2-butoxyethanol Di(propylene glycol) tert-butyl MW: 444.21, 423.24 MW: 118.10 MW: 190.16

O OH

2-Butoxyethanol & D(ipropylene glycol) tert-butyl ether, no strong ionizable + groups. 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) +Q3: 0.100 to 5.710 min from Sample Max. 2 (dipropylene9.6e5 cps. glycol monobutyl ether 100 pg_uL) of dipropylene glycol monobutyl ether posQ3.wif... Max. 1.8e6 cps. MNa+ 141.0 MH+191.1 9.6e5 1.8e6 9.0e5 + 8.0e5 MH 1.6e6 + 1.4e6 7.0e5 0e5 MNH4 119.1 136.1 179.1 + + MNH4 MNa208.1 213.1 1.2e6 6.0e5104.9 1.0e6 Intensity, cps 5.0e5 cps Intensity, 192.1 8.0e5 193.1 4.0e5 129.0 6.0e5 3.0e5 4.0e5 2.0e5 2.0e5 1.0e5

0.0 0.0 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134 136 138150 140 155 142 160 144 146 165 148 170 150 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 m/z, Da m/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)... +MS2 (191.16) CE (10): 0.000 to 0.551 Max. min 5.1e5 from cps. Sample 1 (dipropylene glycol monobutyl ether 10 pg_uL) of dipropylene glycol monobut... Max. 6.9e5 cps.

62.9 115.0

58.9 5.0e5 + 6.9e5 MS/MS of MH+ 4.5e5 MS/MS of MH 6.0e5 4.0e5 5.0e5 3.5e5

3.0e5 cps

cps 4.0e5 191.1 1

119.0 Intensity, 2.5e5 Intensity, 45.0 3.0e5 2.0e5 57.0 1.5e5 2.0e5 41.01.0e5 101.0 1.0e5 117.0 5.0e4 56.9 173.1 97.1 101.0 0.0 0.0 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 10520 110 30 115 120 40 125 50 130 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 m/z, Da , Da m/z 2-butoxyethanol Dipropylene glycol t-butyl ether

DOSS ionizes in both negative mode and positive mode. Positive mode also + works best as an NH4 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. x 20.0 421.0 7.0e5 80.9 6.0e5 . Negativ e m ode .. 4.0e5

- Inten. MS/MS [M-H] 2.0e5 227.0 97.0 160.9 183.3 290.9 0.0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, Da +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.

71.1 8947

8000 57.0 199.1 113.1 Positive mode 6000 414.9 4000 Inten... 423.4 44.9 + 89.1 406.4 MS/MS [M+H] 2000 133.0

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 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.

113.1 9.2e4 8.0e4 199.0

6.0e4 71.0 Positive mode 311.2 4.0e4

Inten... 423.3 + 57.0 440.4 MS/MS [M+NH4] 202.0e4 4 217.1 98.9 181.2 0.0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, Da

Standard compound optimization techniques. Work done on an API 5000 trippqle quadru pole mass s pectrometer.

Negative ion mode MRM Table M1 M3 Dwell Time ID DP EP CE CXP 421.24 80.90 50 dioctylsulfosuccinate_81 -210 -10 -45 -34.0 421.24 183.10 50 dioctylsulfosuccinate_183 -210 -10 -45 -24.0 421.24 227.10 50 dioctylsulfosuccinate_227 -210 -10 -34 -28.0 421.24 290.90 50 dioctylsulfosuccinate_291 -210 -10 -33 -37.0

Positive ion mode MRM Table M1 M3 Dwell Time ID DP EP CE CXP 119.11 45.05 50 2-butoxyethanol 119 _45 52 10 15 676.7 119.11 63.08 50 2-butoxyethanol 119_63 52 10 9 9.8 136.11 45.05 50 2-butoxyethanol 136_45 7 10 21 5.5 136.11 63.08 50 2-butoxyethanol 136_63 7 10 13 6.5 191.16 59.07 50 di(propylene glycol) t -butyl ether_191_59 72 10 17 8.8 191.16 115.09 50 di(propylene glycol) t -butyl ether_191_115 ether 191 115 72 10 10 12.3 208.19 59.07 50 di(propylene glycol) t -butyl ether_208_59 6.5 10 23 8.9 208.19 115.09 50 di(propylene glycol) t -butyl ether_208_115 6.5 10 16 13.0 423.24 113.10 50 dioctylsulfosuccinic acid_423_113 197 10 13.4 13.0 423.24 199.10 50 dioctylsulfosuccinic acid_423_199 197 10 15 15.7 440.27 113.10 50 dioctylsulfosuccinic acid_440_113 152 10 18 18.3 440.27 199.10 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  Zorb ax RRHD SB-C18 co lumn, 50 mm X 2. 1 mm id, 1.8 μm HPLC column

Column Column

A A MS MS 2 1 2 1 3 6 waste 3 6 waste 45 45

Pump C Pump C

Valve Position “0” Valve Position “1”

These compounds – particularly DOSS - are very “sticky”, and thus highly prone to carryover. Used the Shimadzu NEXERA’s autosampler rinse program to dramatically reduce injection to injection carryover.

Fig. 13. Internal/External Rinse steps used R0 = reagent alcohol; R1 = acetonitrile; R2 = 50% water + 50% acetonitrile

Because 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.

3.7e6 4.57 3.0e6 +e 2.0e6 -e In... In... 1.0e6 000.0 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 8.5 Time, min 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.

1.00e4 3.83

5000.00 2-butoxyethanol (RT 3.83min) In... In...

0.00 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Time, min 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.

1.9e6 4.57 1.5e6 Di(propylene glycol) t-butyl ether (4.57 min) 1.0e6 In... In... 5.0e5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Time, min 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.

1.5e5 5.47

1.0e5 .. Dioctylsulfosuccinate (5.47 min)

Int.. Int.. 5.0e4

0.0 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 9.0 Time, min

2-butoxyethanol 136.163.1 LOD 18ng/ml

Dipropy lene g lyco l t-btlbutyl ether 19159 LOD 0.5 ng/ml

DOSS Pos mode 440199.1, 0.9 ng/mL

DOSS Neg mode 421.280.9, 0.8 ng/mL

Neg mode is actually ~13x more sensitive than Pos mode – carryover still a problem!

 Developed reliable, sensitive methods that should be fairly easy ftlbtiltfor 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.  NlNo sample prep requ idftlired for water samples.

 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 present i n the mariti me spill env ironmen t.  Challenges for dispersant analysis – Even though reduced, carryover is still a problem.

Qti?Questions?

Takeo Sakuma ABSCIEX Rebecca Wittrig 71 Four Valley Drive, Concord, ON, L4K Stacy Tremintin 4V8 Timothy L. Hoffman YunYun Zou Carmai Seto Rebecca Wittrig Scott Kragerud Robert I. Ellis Deolinda Fernandes Fouad Khalaf Christopher D. Borton CtiCCurtis Camp bllbell Shimad zu S ci entifi c Ins trumen ts Masatoshi Takahashi 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

 OSU Superfund PAH & OPAH Monitoring http://oregonstate.edu/superfund/oilspill  BaP Metabolism http://herkules.oulu.fi/isbn9514270398/html/x203.html