APCI- and APPI-GC/MS-MS for

Characterization of the Macondo Wellhead

Crude Oil and the Oil Spill

Vlad Lobodin

National High Magnetic Field Laboratory, Tallahassee, FL

Future Fuels Institute, Florida State University, Tallahassee, FL

1
April 20, 2010

Gerald Herbert, AP
April 22, 2010

Gerald Herbert, AP
May 24, 2010

~5 million barrels of crude oil have

leaked from the Macondo well
NASA
Pensacola Beach, Florida June 23, 2010

Michael Spooneybarger, AP
Tarballs collected from beach

the inside of tarballs is saturated

with less weathered petroleum
compounds
 Comprehensive Two-dimensional High Resolution FT-ICR
Gas Chromatography (GC×GC) Mass Spectrometry: 20 < C# < 100
C8-C37, Volatiles
(+) ESI 9.4 FT-ICR MS
Macondo Wellhead Oil

Macondo Wellhead Oil

8
13,700 ± 80 Peaks ≥ 6σ

Biomarker Region
4

1920

6920 2

11920

16920
Pensacola Beach 0
(+) ESI 9.4 FT-ICR MS

6 Pensacola Beach
32,232 ± 488 Peaks ≥ 6σ
4
Biomarker Region
1920

6920 2

1st
Dimension 11920
2nd Dimension
Retention Time
16920 Retention Time
(seconds)
0 (seconds)

400 600 800 1000

B.M. Ruddy et. al., Energy Fuels, 2014, 28 (6), pp 4043–4050 m/z
A) Macondo Well Oil (+) ESI 9.4 T FT-ICR MS
10 Peaks across 250 mDa N1

N1
N1O1 O S 13C
N1O1 N1S1 1 1 1 N1O1

N1O3 O 13C
O313C1 N1O2 2 1 O113C1
O213C1
N1O2 H1C113C1
O113C1

B) Pensacola Beach
32 Peaks across 250 mDa
500.3 m/z 500.4 500.5
GC/MS of “Macondo crude oil” NIST 2779
(Total Ion Chromatogram)
50ºC(3 min)- 3ºC/min- 300ºC

C14H30
C16H34

C18H38

C20H42

C30H62
C25H52
GCxGC/TOF-MS of “Macondo crude oil” NIST 2779
 Simulated Distillation for Macondo Well Petroleum (ASTM D-7169)
100

90 GC Amenable

80 FT-ICR MS

70
% Recovered

60

50
More than 40 % of the Macondo
40 petroleum components cannot be
characterized by conventional
30 GC-based techniques

20

10
Reddy, C.M., et al., PNAS, 2011, 1-6

0
0 100 200 300 400 500 600 700 800
Temperature (°C)
 “Petroleome”
Elemental composition
Structure
aromatic MW distribution

saturated
polar
(resins / asphaltene)

S- 0.3% Macondo well

(Deepwater Horizon)
V- 52 ppm
Ni- 24 ppm 74% saturated
16% aromatic
10% polar
Reddy et al. (2011) PNAS

biomarkers
 FT-ICR FACILITIES
14.5 Tesla
FT-ICR MS

9.4 Tesla
FT-ICR MS
Analyte Ionization (+) ESI and (-) ESI
(+) (-)

CO O H

NH

(4X difference in pKa)

 (+) ESI FT-ICR MS of De-Asphalted Oil”

105,817 peaks > 6σ

500 < m/z < 2000

500 750 1000 1250 1500 1750 2000

m/z
Broadband Positive ESI FT-ICR Mass Spectrum of Crude Oil

C / H12 93.9 mDa

13C
2 / C2H2 8.9 mDa

N13C / C2H3 17.1 mDa

Crude Oil

(+) ESI FT-ICR MS O / CH4 36.4 mDa

C3 / SH4 3.4 mDa

N / 13CH 8.2 mDa

700.40 700.45 700.50 700.55 700.60 700.65 700.70

300 400 500 600 700 800 900 1,000

m/z
Direct Speciation of Metalloporphyrins in Crude Oil
m/z 462.13432 m/z 462.13489
[ C30 H24 N1 S2 ] [ C27 H24 N4 58Ni ]+•
+• Theoretical Abundance 2.6%
(+300ppb) (+80ppb) Experimental Abundance 1.9%

570 µDa
N N
58VNi
O
N N
462.1345
DBE = 18
Mass e- 548 µDa

462 463 464

m/z
 Mass Defect

1H = 1.007825 14N = 14.003074

12C= 12.00000 16O= 15.994915

2
H
1
H 13 14
12 C N 15
C N
Mass defect, mDa

17 18 19
16 O O F
O

31
P 32 33
S S 34 35
Cl 36
S S
37
Cl

79 81
Br Br
127
I
Nuclide
 Workflow for High Resolution “Petroleomics”

m/Δm50%
m/z 704.53510

100 - 400 ppb

*
[C50H72S1]+• S1 Class
400 500 600 700 800
40
m/z

1. Carbon Number 30

DBE
2. Heteroatom Composition 20
3. Aromaticity 10
H N
DBE = C – + + 1 0
20 40 60 80
2 2
McLafferty & Turecek Int. Mass Spectra, 1993 Carbon Number
Relative Abundance (% total)
[Z = -2(DBE) + n + 2]
 Isomeric structure for S-compounds
C1-benzothiophenes (6 isomers)
CH3 CH3
CH3
CH3 S
S S S
S S CH3 CH3
2-methyl- 3-methyl- 4-methyl- 5-methyl- 6-methyl- 7-methyl-
benzothiophene benzothiophene benzothiophene benzothiophene benzothiophene benzothiophene

CH3
C1-dibenzothiophenes (4 isomers)
CH3

CH3
S
S S S CH3
1-methyl-dibenzothiophene 2-methyl-dibenzothiophene 3-methyl-dibenzothiophene 4-methyl-dibenzothiophene

C2-dibenzothiophenes (26 isomers):

22 dimethyl-dibenzotiophene isomers and 4 ethyl-dibenzotiophene isomers

Benzonaphthotiophenes

S
S S
Benzo[b]naphtho[2,1-d]thiophene Benzo[b]naphtho[2,3-d]thiophene Benzo[b]naphtho[1,2-d]thiophene
Petroleum Biomarkers: Hopanes and Steranes
19 20
12 18 E 21 30 32 34
11 22
25 2613 D 17 31 33 35
1 9 C 28
2 16 29
14
10 8 15
A B 27
C35H62O4 3 7
5
4 6
Bacteriohopanetetrol 23 24 Hopanes
(hopanoid in prokaryotes)
============================================================
29
28

21 22 24 26
20 25
18 23
12
11 17 27
19 13
1 9C D 16
2 14
C27H46O 10 8 15
A B
3 7
5
4 6
Cholesterol Steranes
steroid in eukaryotes
EI mass spectrum of 17α (H)-22,29,30-tris-norhopane

C27H46
4% of TIC

M+•
 Petroleum Biomarkers

Hopanes Steranes

m/z 217

m/z 191

M+• → m/z 191 M+• → m/z 217

Multiple reaction monitoring mode

(MRM)
Waters Xevo TQ-S
 Ion Source Diagram of APCI-GC/MS-MS

Ion source
Housing
Ionization
Corona Pin
Chamber

Heated Transfer Line

Mass Spec
Capillary
GC Column
(Atmospheric
Pressure)

N2+• + M M+• + N2 Adapted from Waters Corporation

 Ionization mechanisms:
Charge Transfer vs. Protonation

Charge Transfer

======================================================

Protonation

By courtesy of Waters Corporation

 Ionization mechanisms:
Charge Transfer vs. Protonation
[M+H]+
Phenanthrene 100 pg
179
100
M+•
“wet” source 178
Phenanthrene
%

MW 178
0
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
178
100
[M+H]+
179
“wet” source M+•
%

0
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
100 178

“dry” source M+•

%

179

0
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185

m/z
 APCI-GC/MS-MS of 17α(H)-22,29,30-trisnorhopane

Product (daughter) scan from M+• (m/z 370)

191
100 Collision energy: 15 eV
Collision gas: Ar

95

149
C27H46

MRM transition: m/z 370 → 191

%

109
135

121

163

81
M+•
177
69 355 370

0
50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
m/z
MS/MS spectrum of 17α(H)-22,29,30-trisnorhopane

MS/MS spectrum
The first match
from m/z 370

NIST library EI mass spectrum

17β(H)-22,29,30-trisnorhopane
 APCI-GC/MS-MS of NIST2266
(hopanes & steranes standard)
Sum of 7 MRM transitions
Calibration curve
2000000
17α(H),21β(H)-30-hopane
17α(H)-22,29,30-trisnorhopane C27H46 R² = 0.9998
17α(H),21β(H)-30-norhopane C29H50 1500000
17α(H),21β(H)-30-hopane C30H52
ααα 20R-cholestane C27H48
αββ 20R-cholestane C27H48
1000000
αββ 20R 24S-methylcholestane C28H50
ααα 20R 24R-ethylsholestane C29H52
17α(H),21β(H)-22R-homohopane C31H54
500000
17α(H),21β(H)-22S-homohopane C31H54
αββ 20R 24R- ethylcholestane C29H52
0
0 100 200 300 400 500 600
pg
 APCI-GC/MS-MS of NIST2266. Hopanes.

m/z 370 → 191

17α(H)-22,29,30-trisnorhopane

m/z 398 → 191

17α(H),21β(H)-30-norhopane

m/z 412 → 191

17α(H),21β(H)-hopane

m/z 426 → 191

17α(H),21β(H)-22S-homohopane 17α(H),21β(H)-22R-homohopane
 APCI-GC/MS-MS of NIST2266. Steranes.

m/z 372 → 217

αββ 20R-cholestane ααα 20R-cholestane

m/z 386 → 217

αββ 20R 24S-methylcholestane

m/z 400 → 217

ααα 20R 24R-ethylsholestane

αββ 20R 24R- ethylcholestane
 NIST2779 (Macondo crude oil)

Pricey samples from BP oil spill being sold to scientists

By Mark Schleifstein, NOLA.com | The Times-Picayune. March 08, 2012

It's likely to be one of the oddest ironies to emerge from the BP oil spill: the federal
government is selling tiny containers of oil siphoned from the Macondo well at a price
equal to $76.3 million a barrel. By comparison, a barrel of crude oil was selling for
$106 on Wednesday.

Of course, the BP oil is not being sold by the

barrel.
The National Institute of Standards and
Technology, an agency of the U.S. Department of
Commerce, is selling 1.2 milliliter bottles of the oil
to scientists who need it for comparison with
materials collected as part of the federal Natural
Resources Damage Assessment process. The
price: $480 for a set of five.

http://www.nola.com/news/gulf-oil-spill/index.ssf/2012/03/federal_government_sells_price.html
MS/MS conditions for acquisition of MRM transitions

Compound class MRM transition Dwell time, ms Collision

energy, eV

C27-Hopanes 370.30 > 191.10 50 15

C27-Steranes 372.30 > 217.10 50 20
C27-Steranes 372.30 > 218.10 50 20
C27-Steranes 372.30 > 259.20 50 20
C28-Hopanes 384.30 > 191.10 50 15
C28-Steranes 386.30 > 217.10 50 20
C28-Steranes 386.30 > 218.10 50 20
C28-Steranes 386.30 > 259.20 50 20
C29-Hopanes 398.30 > 191.10 50 15
C29-Steranes 400.30 > 217.10 50 20
C29-Steranes 400.30 > 218.10 50 20
C29-Steranes 400.30 > 259.10 50 20
C30-Hopanes 412.30 > 191.10 50 20
C30-Steranes 414.30 > 217.10 50 20
C31-Hopanes 426.30 > 191.10 50 20
C32-Hopanes 440.40 > 191.10 50 20
C33-Hopanes 454.40 > 191.10 50 20
C34-Hopanes 468.40 > 191.10 50 20
C35-Hopanes 482.40 > 191.10 50 20
 APCI/GC-MS/MS of NIST2779 (Macondo crude oil)
Hopanes: Summed Signals for C27-C35 (M+• → m/z 191)

100
H30 H31S
19 20
12 18 E 21 30 32 34
11 13 H31R
1
25 26 D 17 22 31 33 35
9 C 28 16 29 H32S
2 14
A 10 B 8 27 15
3 5 7
4
23 24
6 H32R H
33S
H33R H
34S
H29 H34RH35S
RA, %

C29Ts H35R

Time
80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00
C31
Ts
DH30 C32
Tm M30
C33 C
34
C35

0
50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00
Time, min
 APCI/GC-MS/MS of NIST2779 (Macondo crude oil)
29
28
100
Sum of C27-C29 Steranes/Diasteranes 21 22 24
20 25 26
18 23
12
11 17 27
19 13 D
1 9C 16
%

2 14 15
A 10B 8
3 7
5
4 6
0
50.00 55.00 60.00 65.00 70.00 75.00 80.00
βα
100
C27 -Diasteranes βα αββR
αββS
C27-Steranes
αααS αααR
m/z 372 → 217
%

αβ
αβ

0
50.00 55.00 60.00 65.00 70.00 75.00 80.00
βα αββR
100
αββS
C28 -Diasteranes βα
αααR
αααS C28-Steranes
αβ αβ
%

m/z 386 → 217

0
50.00 55.00 60.00 65.00 70.00 75.00 80.00
βα
100
αββR
C29 -Diasteranes αββS C29-Steranes
βα
m/z 372 → 217 αααS αααR
%

αβ
αβ

0 Time
50.00 55.00 60.00 65.00 70.00 75.00 80.00
 NIST 2779 (Macondo crude oil)
Ts/Tm
2

1.6
βαC27/βαC29
H29/H30
Diasteranes 1.2

0.8

0.4

αββC27/αββC29
Steranes H32S/H32R

H30/H31+H32+H33+H34+H35 H33S/H33R
 NIST2779 (Macondo crude oil)

Ts/Tm SAM 1-18 collected

2
1-45 months after
1.6 the oil spill
βαC27/βαC29 H29/H30
1.2
Diasteranes
0.8

0.4

αββC27/αββC29 H32S/H32R
Steranes

H30/(H31+H32+H33+H34
H33S/H33R
+H35)
 Natural Oil Seeps (GC600, Megaplume)
"Megaplume" in the GC600 lease block:
Lat: 27° 22.466' N
Long: 90° 30.689'W
water depth: 1382m
 Natural Oil Seeps. The Gulf of Mexico.
Natural oils seeps in the Gulf of Mexico - 140,000 tonnes per year (range of
80,000 to 200,000 tonnes).

from www.sarsea.org
 Megaplume Oil Seep (GC600)
Ts/Tm
2

1.6
βαC27/βαC29 H29/H30
Diasteranes 1.2

0.8

0.4

αββC27/αββC29 H32S/H32R
Steranes

H30/(H31+H32+H33+H34+H3
H33S/H33R
5)
 Blue crude (Anadarko Independence Hub)
Ts/Tm
2

1.6

βαC27/βαC29 H29/H30
Diasteranes 1.2

0.8

0.4

αββC27/αββC29 H32S/H32R
Steranes

H30/(H31+H32+H33+H34+H35) H33S/H33R
 SAM-1 SAM-2 SAM-3
Ts/Tm Ts/Tm
Ts/Tm
2 2
βαC27/βαC29 βαC27/βαC29 2 C27βα/C29βα
Diasteranes 1.5 Diasteranes Diasteranes 1.5
H29/H30 1.5 H29/H30 H29/H30
1 1
1
0.5 0.5 0.5
0 0 0
C27αββ/C29αββ H32S/H32R C27αββ/C29αββ H32S/H32R H32S/H32R
C27αββ/C29αββ
Steranes Steranes
Steranes

H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R

SAM-6
SAM-4 Ts/Tm
SAM-5 Ts/Tm Ts/Tm
C27βα/C29βα 2
C27βα/C29βα 2 C27βα/C29βα 2
Diasteranes 1.5 H29/H30 Diasteranes 1.5 H29/H30 Diasteranes 1.5 H29/H30
1 1 1
0.5 0.5 0.5
0 0 0

C27αββ/C29αββ H32S/H32R C27αββ/C29αββ H32S/H32R C27αββ/C29αββ H32S/H32R

Steranes Steranes Steranes

H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R

SAM-7 SAM-8 SAM-9

Ts/Tm Ts/Tm Ts/Tm
C27βα/C29βα 2 2 C27βα/C29βα 2
C27βα/C29βα
Diasteranes 1.5 H29/H30 1.5 Diasteranes 1.5 H29/H30
Diasteranes H29/H30
1 1 1

0.5 0.5 0.5

0 0 0

C27αββ/C29αββ H32S/H32R C27αββ/C29αββ H32S/H32R C27αββ/C29αββ H32S/H32R

Steranes Steranes Steranes

H30/ΣH31‒H35 H33S/H33R H33S/H33R H30/ΣH31‒H35 H33S/H33R

H30/ΣH31‒H35
 SAM-10 SAM-11 SAM-12
Ts/Tm Ts/Tm
Ts/Tm
C27βα/C29βα 2 C27βα/C29βα 2
Diasteranes C27βα/C29βα 2
Diasteranes 1.5 H29/H30 1.5 H29/H30
Diasteranes 1.5 H29/H30
1 1
1
0.5 0.5
0.5
0 0
0
C27αββ/C29αββ H32S/H32R H32S/H32R
C27αββ/C29αββ H32S/H32R
Steranes C27αββ/C29αββ
Steranes
Steranes

H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R

H30/ΣH31‒H35 H33S/H33R

SAM-13 SAM-14 SAM-15

Ts/Tm
Ts/Tm Ts/Tm
2 C27βα/C29βα 2 C27βα/C29βα 2
C27βα/C29βα
Diasteranes Diasteranes 1.5 H29/H30
Diasteranes 1.5 H29/H30 1.5 H29/H30
1 1 1
0.5
0.5 0.5
0
0 0
H32S/H32R C27αββ/C29αββ H32S/H32R
C27αββ/C29αββ H32S/H32R C27αββ/C29αββ
Steranes Steranes
Steranes

H30/ΣH31‒H35 H30/ΣH31‒H35 H33S/H33R

H30/ΣH31‒H35 H33S/H33R H33S/H33R

SAM-16 SAM-17 SAM-18

Ts/Tm Ts/Tm Ts/Tm
C27βα/C29βα 2 C27βα/C29βα 2 C27βα/C29βα 2
Diasteranes 1.5 H29/H30 Diasteranes 1.5 H29/H30 Diasteranes 1.5 H29/H30
1 1 1
0.5 0.5 0.5
0 0 0
C27αββ/C29αββ H32S/H32R H32S/H32R H32S/H32R
C27αββ/C29αββ C27αββ/C29αββ
Steranes Steranes Steranes

H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R H30/ΣH31‒H35 H33S/H33R

 Overlaid spider diagrams
Ts/Tm
2

1.6
βαC27/βαC29
H29/H30
Diasteranes 1.2

0.8

0.4

αββC27/αββC29
Steranes H32S/H32R

NIST2779 (Macondo crude oil)

Blue crude (Independence Hub)
H30/H31+H32+H33+H34+H35 H33S/H33R Megaplume oil seep (GC600)
SAM-10 (Pensacola Beach)
Correlation coefficients
 Other case studies: Exxon Valdez oil spill

from www.uaf.edu

Prince William Sound, Alaska

March 24, 1989. 258,000 barrels
 (+) APPI FT-ICR MS of Macondo crude oil

25,500 peaks
150 < m/z < 850

150 250 350 450 550 650 750 850

m/z
 (+) APPI FT-ICR MS of Macondo crude oil

HC class (M+•) S class (M+•)

30

25 R
R
20 S
DBE

15
DBE=12
10
DBE=10 DBE=9
5
R S
0
10 20 30 40 50 60 10 20 30 40 50 60
Relative Abundance (% total) Carbon Number
 APPI-GC/MS Ion Source Diagram

Ion source
Housing

UV-lamp Ionization
Chamber
Heated Transfer Line

Mass Spec
Capillary
(Atmospheric GC Column
Pressure)
Atmospheric Pressure PhotoIonization (APPI)
Spectral distribution of a Krypton lamp
hν
M -ē
M+• hν > IE(M)

Kr UV-lamp
E=10.6 eV, λ= 117 nm
E=10.0 eV, λ= 124 nm

Ionization energies, IE (eV)

IE(N2) = 15.6 eV IE(C6H6) = 9.2 eV IE(Alkanes) ~ 10 eV
IE(H2O)= 12.6 eV IE(Toluene)= 8.8 eV
IE(O2) = 12.1 eV IE(Naphthalene) = 8.1 eV
IE (Phenanthrene) = 7.9 eV
IE (Thiophene) = 8.9 eV
IE (DBT) = 8.0 eV
APPI-GC/MS. Mass Spectrum of Phenanthrene

M+•
hν 178
100
M M+•

Phenanthrene (20 pg injected)

%

MW 178
IE = 7.9 eV

0
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190

m/z
APPI-GC/MS vs APCI-GC/MS of Phenanthrene

S/N 4160
100
APCI-GC/MS

m/z 178 →m/z 152

%

0
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00
100

S/N 32830
APPI-GC/MS

m/z 178 → m/z 152

%

0
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00

time
APPI-GC/MS of Aromatic compounds

M+•

MW 168
IE = 8.1 eV

M+•

MW 167
IE = 7.6 eV
M+•

MW 184
IE = 7.9 eV
 APPI-GC/MS of 610 PAH Calibration Mix A

610 PAH Calibration Std (x 1000 dilution)

Acenaphthylene
Fluorene Anthracene
Final concentration: 500-1000 ng/mL
100

Acenaphthene

Benzo[k]fluoranthene
Benz[a]anthracene
Naphthalene
Dibenz[a,h]anthracene

Phenanthrene Benzo[b]fluoranthene
Chrysene
Indeno[1,2,3-cd]pyrene
Pyrene Benzo[g,h,i]perylene
%

Boiling T 500 °C

Benzo[a]pyrene

Fluoranthene

0 Time
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
APPI/GC-MS/MS of NIST2779 (Macondo crude oil)

Me

Me

S
 APPI with Argon lamp
Ar UV-lamp

E=11.6 eV, λ= 106.7 nm

E=11.8 eV, λ= 104.8 nm
Spectral distribution
17α(H),21β(H)-30-hopane of Ar lamp
as internal standard
Environ. Sci. Technol. 1994, 28, 142-145

LiF wavelength transmission range

0.105 ‒ 9 µm
APPI(Ar)-GC/MS-MS. PAHs and PASHs ratios.
 Depletion of PAHs and PASHs in Environmental
samples from AL-MS shore line.
NIST 2779 Jul, 2011 Feb, 2012 Jan, 2014
(DWH)
100

Phen DBT C2-Phen C2-DBT C3-Phen C3-DBT Chrys

Depletion is relative to 17α(H),21β(H)-30-hopane (C30-Hopane)

 SUMMARY

 We first utilized AP-GC/MS for a trace analysis of petroleum

biomarkers from the Macondo crude oil and environmental
samples.

 We describe an Atmospheric Pressure PhotoIonization

(APPI) source that in combination with GC separation and
MS/MS analysis is an efficient method for characterization of
aromatic compounds in wellhead and spilled oil.

 Analysis of petroleum compounds with APGC/MS-MS

provides a sensitive analytical tool for targeted analysis,
source identification of the oil spill, and tracking a fate of oil
spill residues.
FT-ICR Group, NHMFL
Thank you!
 (-) UniSpray TQS mass spectrum of Macondo crude oil
(NIST 2779)

[M-H]-
NEG_UNISPRAY-DWH_1%NH3_B 20 (0.335) Cm (14:143) MS2 ES-
421.26 1.28e5
100

dioctyl sodium sulfosuccinate (DOSS)

%

490.32
566.38

294.15 684.51
790.66
858.68

247.13

0 m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
 (-) UniSpray MS/MS spectrum for [M-H]-
ion of dioctyl sodium sulfosuccinate (DOSS)

[M-H]-
NEG_UNISPRAY-DWH_MS-MS-421 121 (2.028) Cm (116:128) Daughters of 421ES-
421 3.73e4
100

[HSO3]-
81
CID: 20 V; Collision gas: Ar
NEG_UNISPRAY-DWH_MS-MS-421 121 (2.028) Cm (116:128) Daughters of 421ES-
367 904
100
227

291 391
%
%

313 338

187
404
265 298 375
219
279

0 m/z
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

0 m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
 (+) UniSpray TQS mass spectrum of Macondo crude oil
(NIST 2779)

1 mg/mL Tol:MeOH (50:50)- 1% HCOOH

POS_UNISPRAY_DWH_1%HCOOH_A 23 (0.386) Cm (23:84) MS2 ES+
360 1.82e6
100
%

431

227

499
567
635

338 703
450 478 520 586 771 839
305
677

0 m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
 APCI-GC/MS conditions
Mass spectrometer – Waters Xevo TQ-S

Tsource 150ºC
Corona 2.5 μA
Cone voltage 30V
Source offset 50V
Auxilliary gas (N2) 200 L/hr
Cone gas (N2) 250 L/hr
Collision gas Ar

Gas chromatograph –7890

Tinj=300ºC
Column: MXT-5 (or MXT-1), 60m × 0.25mm × 0.25 µm
Carrier gas: He
Flow rate: 1.2 mL/min
Split ratio: 1:10
Oven: 50ºC - 20ºC/min -150ºC- 2ºC/min - 350ºC (25 min)
Transfer Line: 380ºC
Injected volume: 1 μL
 APPI-GC/MS conditions
Mass spectrometer – Waters Xevo TQ-S

Tsource 150ºC
Kr UV lamp 10 eV (or Ar UV lamp 11.7 eV)
Cone voltage 30V
Source offset 50V
Auxiliary gas (N2) 200 L/hr
Cone gas (N2) 150 L/hr
Collision gas Ar
Gas chromatograph –7890
Tinj=300ºC
Column: MXT-5 (or MXT-1), 60m × 0.25mm × 0.25 µm
Carrier gas: He
Flow rate: 1.2 mL/min
Split ratio: 1:10
Oven: 50ºC - 20ºC/min -150ºC- 2ºC/min - 350ºC (25 min)
Transfer Line: 380ºC
Injected volume: 1 μL
APPI(Ar)-GC/MS-MS. PAHs and PASHs ratios.

С2-Phen/C2-DBT and С3-Phen/C3-DBT ratios

NIST2279 (DWH) Jul 17, 2011 Feb 8, 2012 Jan 27, 2014

С2-Phen/C2-DBT 2.9 2.8 2.3 2.8

С3-Phen/C3-DBT 1.9 1.4 1.4 1.5

 Samples’ collection sites and time
SAM-1 (30°17'15.0”N, 87°28'44.6”W))on 04.05.2011,
SAM-2 (30°17'14.6”N, 87°28'50.2”W) on 04.06.2011,
SAM-3 (30°14'25.6”N, 87°44'14.8”W) on 07.17.2011
SAM-4 (29°10'29.3”N, 90°04'33.2”W) on 07.17.2011,
SAM-5 (30°17'18.4”N, 87°28'37.8”W) on 07.19.2011,
SAM-6 (30°14'48.4”N, 87°41'35.2”W) on 11.27.2011
SAM-7 (30°14'25.4”N, 87°44'15.2”W) on 11.28.2011,
SAM-8 (29°56'41.0”N, 88°49'27.0”W) on 11.28.2011,
SAM-9 (29°17'35.5”N, 90°29'17.5”W) on 05.31.2010,
SAM-10 (30°19'32.1”N, 87°10'30.5”W) on 06.23.1010,
SAM-11 (30°14'60.6”N, 88°53'21.1”W) on 02.09.2012,
SAM-12 (30°18'16.4”N, 87°23'20.9”W) on 02.07.2102,
SAM-13 (30°14'34.8”N, 88°42'59.1”W) on 02.08.2012,
SAM-14 (30°14'26.3”N, 87°44'16.9”W) on 08.31.2012,
SAM-15 (30°13'54.4”N, 88°53'47.0”W) on 02.09.21012,
SAM-16 (30°18'30.8”N, 87°22'16.3”W) on 02.07.2012,
SAM-17 (29°10'30.0”N, 90°04'33.6”W) on 07.31.2011,
SAM-18 (30°15'16”N, 88°7'50”W) on 01.27.2014,
Megaplume (27°22'27.96”N, 90°30'41.34”W) - depth – 1200 m, GC600.
Blue crude oil (28°05'89.0”N, 87°59'27.0”W).
 Petroleum Biomarkers. Hopanes.
19 20
12 18 E 21 29 32 34
11 22
25 2613 D 17 31 33 35
1 9 C 28
2 16 30
14
10 8 15
A B 27
C35H62O4 3 7
5
Bacteriohopanetetrol 4 6
23 24 Hopanes
(hopanoid in prokaryotes)
Over 150 distinct, naturally-occurring hopanoids have been identified in soils, sediments, and other
organic matter. Hopanoids have a fixed stereochemistry and differ in the orientation about Carbon-
17 and Carbon-21 (α or β) and Carbon-22 (R or S).

17β, 21β(H) is biological configuration

The order of thermodynamic stability of the 17-21 hopane isomers is
17α(H),21β(H) > 17β(H),21α(H) > 17α(H),21α(H) > 17β(H),21β(H)

17α(H),21β(H) –hopanes are the most stable. 17β(H),21α(H) are called moretanes.

22R is biological configuration. 22S/(22S+22R) ~ 0.58-0.62 for C31-hopane

 Petroleum Biomarkers. Steranes.
29
28

21 22 24 26
20 25
18 23
12
11 17 27
19 13
1 9C D 16
C27H46O 2
10
14
15
A B 8
3 7
5
Cholesterol 4 6
steroid in eukaryotes Steranes

5α,14α,17α(H)–cholestane-20R 5α,14β,17β(H)
biological configuration stable configuration

14β,17β(H)/[14α,17α(H) + 14β,17β(H)] ~ 0.7 – Endpoint configuration

Diasteranes (rearranged steranes) - rearrangement product from sterol precursors through

diasterenes. The rearrangement involves migration of C-10 and C-13 methyl groups to C-5 and
C-14 and is favored by acidic conditions, clay catalysis, and/or high temperatures. Diasteranes
increase relative to steranes with thermal maturation and they are low in clay-poor carbonate
source rocks and related oils.
13β(H),17α – diasteranes 20S or 20R
 Assigned Hopanes

18α(H)-22,29,30-trisnorhopane (Ts),
17α(H)-22,29,30-trisnorhopane (Tm),
17α(H),21β(H)-30-norhopane (H29),
18α(H),21β(H)-30-norneohopane (C29Ts),
17α(H)-diahopane (DH30),
17β(H),21α(H)-hopane (moretane, M30),
17α(H),21β(H)-hopane (H30),
17α(H),21β(H)-22S-30-homohopane (H31S),
17α(H),21β(H)-22R-30-homohopane (H31R),
17α(H),21β(H)-22S-30,31-bishomohopane (H32S),
17α(H),21β(H)-22R-30,31-bishomohopane (H32R),
17α(H),21β(H)-22S-30,31,32-trishomohopane (H33S),
17α(H),21β(H)-22R-30,31,32-trishomohopane (H33R),
17α(H),21β(H)-22S-30,31,32,33-tetrakishomohopane (H34S),
17α(H),21β(H)-22R-30,31,32,33-tetrakishomohopane (H34R),
17α(H),21β(H)-22S-30,31,32,33,34-pentakishomohopane (H35S),
17α(H),21β(H)-22R-30,31,32,33,34-pentakishomohopane (H35R).
 Assigned Steranes and Diasteranes
13β(H),17α(H)-20S-diacholestane (C27βαS),
13β(H),17α(H)-20R-diacholestane (C27βαR),
13α(H),17β(H)-20S-diacholestane (C27αβS),
13α(H),17β(H)-20R-diacholestane (C27αβR),
5α(H),14α(H),17α(H)-20S-cholestane (C27αααS),
5α(H),14β(H),17β(H)-20R-cholestane (C27αββR),
5α(H),14β(H),17β(H)-20S-cholestane (C27αββS),
5α(H),14α(H),17α(H)-20R-cholestane (C27αααR),
13β(H),17α(H)-20S-24-methyldiacholestane (C28βαS),
13β(H),17α(H)-20R-24-methyldiacholestane (C28βαR),
13α(H),17β(H)-20S-24-methyldiacholestane (C28αβS),
13α(H),17β(H)-20R-24-methyldiacholestane (C28αβR),
5α(H),14α(H),17α(H)-20S-24-methylcholestane (C28αααS),
5α(H),14β(H),17β(H)-20R-24-methylcholestane (C28αββR),
5α(H),14β(H),17β(H)-20S-24-methylcholestane (C28αββS),
5α(H),14α(H),17α(H)-20R-24-methylcholestane (C28αααR),
13β(H),17α(H)-20S-24-ethyldiacholestane (C29βαS),
13β(H),17α(H)-20R-24-ethyldiacholestane (C29βαR),
13α(H),17β(H)-20S-24-ethyldiacholestane (C29αβS),
13α(H),17β(H)-20R-24-ethyldiacholestane (C29αβR),
5α(H),14α(H),17α(H)-20S-24-ethylcholestane (C29αααS),
5α(H),14β(H),17β(H)-20R-24-ethylcholestane (C29αββR),
5α(H),14β(H),17β(H)-20S-24-ethylcholestane (C29αββS),
5α(H),14α(H),17α(H)-20R-24-ethylholestane (C29αααR).
 Biomarker ratios (Ts/Tm, H29/H30, H32S/H32R, H30/Σ(H31-H35), H33S/H33R,
C27αββ/C29αββ steranes, and C27βα/C29βα diasteranes)

C27αββ/C29αββ C27βα/C29βα
Sample Ts/Tm H29/H30 H32S/H32R H33S/H33R H30/Σ(H31-H35)
steranes diasteranes
NIST2779 1.42±0.05 0.52±0.04 1.48±0.03 1.46±0.06 0.65±0.03 0.68±0.06 0.94±0.02
SAM-1 1.49±0.02 0.56±0.03 1.36±0.01 1.42±0.07 0.65±0.06 0.68±0.02 0.95±0.03
SAM-2 1.43±0.05 0.58±0.02 1.41±0.13 1.35±0.05 0.62±0.05 0.66±0.04 0.89±0.04
SAM-3 1.49±0.10 0.55±0.01 1.36±0.03 1.34±0.10 0.66±0.02 0.72±0.03 0.91±0.04
SAM-4 1.47±0.03 0.59±0.02 1.39±0.03 1.40±0.12 0.61±0.01 0.73±0.03 0.93±0.02
SAM-5 1.49±0.08 0.57±0.01 1.31±0.07 1.37±0.05 0.62±0.03 0.69±0.02 0.90±0.02
SAM-6 1.56±0.03 0.50±0.05 1.32±0.03 1.33±0.03 0.68±0.01 0.71±0.01 0.97±0.02
SAM-7 1.53±0.09 0.53±0.06 1.35±0.09 1.30±0.01 0.60±0.01 0.67±0.05 0.94±0.05
SAM-8 1.30±0.09 0.62±0.06 1.40±0.09 1.32±0.10 0.46±0.06 0.42±0.02 0.56±0.03
SAM-9 1.43±0.11 0.49±0.03 1.39±0.11 1.49±0.11 0.67±0.05 0.74±0.06 0.96±0.02
SAM-10 1.46±0.07 0.53±0.05 1.42±0.07 1.46±0.08 0.66±0.06 0.64±0.07 0.90±0.08
SAM-11 1.42±0.10 0.52±0.05 1.41±0.08 1.32±0.12 0.60±0.02 0.66±0.07 0.91±0.09
SAM-12 1.48±0.08 0.56±0.06 1.50±0.12 1.39±0.11 0.54±0.05 0.58±0.06 0.89±0.08
SAM-13 1.50±0.07 0.51±0.05 1.46±0.08 1.36±0.09 0.60±0.07 0.62±0.06 0.96±0.06
SAM-14 1.49±0.09 0.49±0.05 1.45±0.13 1.52±0.14 0.63±0.06 0.64±0.05 1.00±0.07
SAM-15 1.33±0.05 0.59±0.05 1.57±0.15 1.52±0.15 0.55±0.05 0.58±0.06 0.92±0.09
SAM-16 1.49±0.10 0.54±0.06 1.46±0.14 1.40±0.12 0.60±0.06 0.54±0.05 0.92±0.07
SAM-17 1.37±0.05 0.54±0.03 1.41±0.09 1.49±0.07 0.46±0.04 0.40±0.03 0.60±0.04
SAM-18 1.47±0.12 0.59±0.06 1.52±0.09 1.45±0.14 0.55±0.05 0.59±0.06 0.81±0.80
Megaplume 1.00±0.05 1.01±0.05 1.48±0.05 1.44±0.08 0.52±0.04 0.84±0.07 0.82±0.08
Blue crude 0.15±0.04 0.74±0.05 0.54±0.05 0.94±0.05 2.00±0.10 0.11±0.03 0.84±0.06
EI mass spectrum of 17α (H)-22,29,30-tris-norhopane

C27H46
4% of TIC

M+•
EI mass spectrum of ααα 20R-cholestane

7% of TIC

C27H48
M+•
GC/MS of NIST 2779 (Macondo crude oil)
(Ion Chromatogram at m/z 191 and 217)
C30

Ion Chromatogram at m/z 191 Hopanes

C29
C31
Ts C32 m/z 191
Tm
C33
C34
C35

Ion Chromatogram at m/z 217

C27 Steranes
C
C27 C29 29
C28 C29 C29
C27 C C29
28
C29 C30 m/z 217
 MS/MS Modes

Product (daughter) ion scan

Precursor (parent) ion scan

Multiple reaction monitoring mode

Constant neutral loss scan

 Fragmentation scheme for steranes
CH3 CH3 CH3
CH3
CH3 CH3 +
CH3

R
•
CH3
R
M+•

CH3

CH3 H3C

CH3 CH3 +
CH3 +
•
R
R R= H m/z 217 H3C
R= CH3 m/z 231
CH3
R= C2H5 m/z 245
H3C
R= C3H7 m/z 259
 APCI-GC/MS-MS of 17α(H)-22,29,30-trisnorhopane

Product (daughter) scan from M+• (m/z 370)

191
100 Collision energy: 20 eV
Collision gas: Ar

95

149
C27H46
%

109
135

121

163

81
M+•
177
69 355 370

0
50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
m/z
 APCI-GC/MS-MS of ααα 20R-cholestane
Product (daughter) scan from M+• (m/z 372)

217
100

Collision energy: 20 eV
Collision gas: Ar 357

C27H48
%

121
95

107 135
81
149
175
M+•
161
73 262 372
189 203

0
50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
m/z
MS/MS spectrum of ααα 20R-cholestane

MS/MS spectrum
from m/z 372
The first match

NIST library EI mass spectrum

of Cholestane
GC/MS of “Macondo crude oil” NIST 2779
(Total Ion Chromatogram)
C14H30
Isoprenoid indices
Pr/Ph=1.33
C16H34
n-C17/Pr=1.58
C18H38 n-C18/Ph=1.67
C20H42
Pr
Reported Pr/Ph ratio for MC252 is 0.9
Ph Environ. Res. Lett. 2012, 7, 035302
C25H52

C30H62

C35H72
 Characteristic ions for steranes
CH3 CH3
CH3
CH3
CH3

R
M+•

CH3 CH3
CH3 + CH3

+
•

R R

R= H m/z 217 R= H m/z 218

R= CH3 m/z 231 R= CH3 m/z 232
R= C2H5 m/z 245 R= C2H5 m/z 246
R= C3H7 m/z 259 R= C3H7 m/z 260
 (+) APPI FT-ICR MS of Macondo crude oil

25,500 peaks
150 < m/z < 850

150 250 350 450 550 650 750 850

m/z
 (+) APPI FT-ICR MS of Macondo crude oil

C1-phenathrene has 5 isomers

HC class (M+•) CH3
30
CH3

25 C#= 25-30 1-methylphenanthrene 2-methylphenanthrene

R
20
DBE

15
CH3 CH3
3-methylphenanthrene 4-methylphenanthrene
10
DBE=10 CH3

0 9-methylphenanthrene
10 20 30 40 50 60
C2-phenanthrene has 30 isomers:
Carbon Number
25 isomers for dimethyl-
Relative Abundance (% total) phenanthrenes
5 isomers for ethyl-phenanthrenes
DBE Distribution
HC class (M+•)

DBE
 (+) APPI FT-ICR MS of Macondo crude oil

S class (M+•)
30
C3
25 R
S
C#=19
20 DBE=12 S
DBE

15
DBE=12 R
10 DBE=9 S
DBE=6
5
R
0 S
10 20 30 40 50 60
Carbon Number C2-dibenzothiophenes (26 isomers):
Relative Abundance (% total) 22 dimethyl-dibenzotiophene isomers and
4 ethyl-dibenzotiophene isomers
 Isomeric structure for S-compounds
C1-benzothiophenes (6 isomers)
CH3 CH3
CH3
CH3 S
S S S
S S CH3 CH3
2-methyl- 3-methyl- 4-methyl- 5-methyl- 6-methyl- 7-methyl-
benzothiophene benzothiophene benzothiophene benzothiophene benzothiophene benzothiophene

CH3
C1-dibenzothiophenes (4 isomers)
CH3

CH3
S
S S S CH3
1-methyl-dibenzothiophene 2-methyl-dibenzothiophene 3-methyl-dibenzothiophene 4-methyl-dibenzothiophene

C2-dibenzothiophenes (26 isomers):

22 dimethyl-dibenzotiophene isomers and 4 ethyl-dibenzotiophene isomers

Benzonaphthotiophenes

S
S S
Benzo[b]naphtho[2,1-d]thiophene Benzo[b]naphtho[2,3-d]thiophene Benzo[b]naphtho[1,2-d]thiophene
 DBE Distribution
S class (M+•)
R
R
S
DBE=9 S

R DBE=12
S
DBE=6

R
S
DBE=3

DBE
 Petroleum Biomarkers

Prokaryotes Eukaryotes

Cholesterol
Hopanes
Steranes
APCI. Mechanism of Ionization (I)
Charge Transfer

• The nitrogen in the source is ionized by corona discharge by the

following series of reactions:
N2 + e ¯ → N2+• + 2e ¯
N2+• + 2N2 → N4+• + N2
• If the nitrogen is dry the N2+• and N4+• act as reagent ions with charge
transfer being the most likely pathway for ionization.
N2+•/ N4+• + A → A+• + xN2 charge transfer
Where A represents an analyte molecule
• Charge transfer results in the formation of radical cations and is
particularly useful for the ionization of non-polars.
APCI. Mechanism of Ionization (II)
Proton Transfer
• In the presence of water vapour the following reactions then occur to
generate ionized water clusters:
N4+• + H2O → H2O+• + 2 N2
H2O+• + H2O → H3O+ + OH•
H3O+ + H2O + N2 → H+(H2O)2 + N2
H2O + H+(H2O)2 → H+(H2O)3 + N2
• The last reaction can then proceed further with successive additions
of water
• Ionization of the analyte then occurs by proton transfer
H3O+ + A → AH+ + H2O proton transfer
• Proton transfer is the main ionization pathway associated with APCI
in LC/MS.
 APPI-GC/MS-MS of Phenanthrene (m/z 178)
Product (daughter) scan from M+• (m/z 178)
[M-C2H2]+•
100
152
M+•
178

C14H10 -C2H2

Collision energy (Ekin) = 27 eV

Collision gas - Ar
%

Mgas
Eint = Ekin
Mion + Mgas
[C10H8]+•
128

0
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
m/z
MS/MS spectrum of Phenanthrene (m/z 178)

MS/MS spectrum
The first match
from m/z 178

NIST library EI mass spectrum

of Phenanthrene
Wavelength Transmission/Absorption Range
Gas Absorption wavelengths (μm)
N2 < 0.1 O3 0.17-0.35 0.45-0.75
O2 < 0.245 H2O < 0.21 0.6-0.72

MgF2 (window) wavelength transmission range

0.11 ‒ 7.5 µm
 APPI(Ar)-GC/MS-MS of NIST2266
(hopanes & steranes standard)
Sum of 7 MRM transitions
IE(Alkanes) ~ 10 eV

100
17α(H),21β(H)-30- 17α(H),21β(H)-30-
hopane norhopane
ααα 20R-cholestane

αββ 20R 24R-

ethylcholestane
%

αββ 20R 24S- 17α(H),21β(H)-

methylcholestane 22S-homophane

ααα 20R 24R-

17α (H)-22,29,30- ethylsholestane
trisnorhopane

17α(H),21β(H)-22R-
αββ 20R-cholestane homopane
0
20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
time
 APPI(Ar)-GC/MS-MS of Phenanthrene,
Dibenzothiophene, and 17α(H),21β(H)hopane

100
Ar-lamp
hν =11.7 eV

17α(H),21β(H)-hopane

m/z 184 → m/z 152 585 ng/mL

Peak area - 4900
S dwell time – 250 ms
50 ng/mL m/z 412 → m/z 191
%

Peak area - 218

dwell time – 250 ms

m/z 178 → m/z 152

dwell time – 250 ms
50 ng/mL
Peak area - 218

0
20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
time
 Double Bond Equivalent (DBE)

n-hexane, C6H14, DBE=0 Cyclohexane, C6H12, DBE=1 Benzene, C6H6, DBE=4

120

100

H N
80 DBE = C – + + 1
2 2
DBE

60

40
C20

C# = 112
20 DBE = 100

C# = 24 C# = 54
DBE = 19 DBE = 46
0
0 10 20 30 40 50 60 70 80 90 100 110 120
Carbon number V.V. Lobodin, et al. Anal. Chem. 2012, 84, 3410-3416