APCI and APPI GC/MSMS for Characterization of the Macondo Wellhead Crude Oil and the Oil Spill

APCI- and APPI-GC/MS-MS for
Characterization of the Macondo Wellhead
Crude Oil and the Oil Spill
Vlad Lobodin
1
National High Magnetic Field Laboratory, Tallahassee, FL
Future Fuels Institute, Florida State University, Tallahassee, FL

Gerald Herbert, AP
April 20, 2010

April 22, 2010
Gerald Herbert, AP

NASA
May 24, 2010
~5 million barrels of crude oil have
leaked from the Macondo well

Michael Spooneybarger, AP
Pensacola Beach, Florida June 23, 2010

the inside of tarballs is saturated
with less weathered petroleum
compounds
Tarballs collected from beach

400 600 800 1000
m/z
Macondo Wellhead Oil
13,700 ± 80 Peaks ≥ 6σ
(+) ESI 9.4 FT-ICR MS
Pensacola Beach
32,232 ± 488 Peaks ≥ 6σ
(+) ESI 9.4 FT-ICR MS
High Resolution FT-ICR
Mass Spectrometry: 20 < C# < 100
Biomarker Region
Biomarker Region
1920
6920
16920
11920
8
6
4
2
0
1920
6920
16920
11920
8
6
4
2
0
1st Dimension
Retention Time
(seconds)
2nd Dimension
Retention Time
(seconds)
Pensacola Beach
Macondo Wellhead Oil
Comprehensive Two-dimensional
Gas Chromatography (GC×GC)
C8-C37, Volatiles
B.M. Ruddy et. al., Energy Fuels, 2014, 28 (6), pp 4043–4050

m/z 500.5500.4500.3
N1O1
N1
O1S1
13C1
N1O1
N1
N1O1 N1S1
A) Macondo Well Oil
10 Peaks across 250 mDa
O1
13C1
O2
13C1N1O2
H1C1
13C1O1
13C1
O2
13C1
N1O2
N1O3
O3
13C1
B) Pensacola Beach
32 Peaks across 250 mDa
(+) ESI 9.4 T FT-ICR MS

C14H30
C16H34
C18H38
C25H52
C30H62
C20H42
50ºC(3 min)- 3ºC/min- 300ºC
GC/MS of “Macondo crude oil” NIST 2779
(Total Ion Chromatogram)

GCxGC/TOF-MS of “Macondo crude oil” NIST 2779

Simulated Distillation for Macondo Well Petroleum (ASTM D-7169)
Temperature (°C)
%Recovered
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500 600 700 800
More than 40 % of the Macondo
petroleum components cannot be
characterized by conventional
GC-based techniques
FT-ICR MS
Reddy, C.M., et al., PNAS, 2011, 1-6
GC Amenable

biomarkers
74% saturated
16% aromatic
10% polar
Macondo well
(Deepwater Horizon)
saturated
aromatic
polar
(resins / asphaltene)
Reddy et al. (2011) PNAS
“Petroleome”
Elemental composition
Structure
MW distribution
S- 0.3%
V- 52 ppm
Ni- 24 ppm

9.4 Tesla
FT-ICR MS
14.5 Tesla
FT-ICR MS
FT-ICR FACILITIES

N
HN
N
S
COOH
(+) (-)
(4X difference in pKa)
Analyte Ionization (+) ESI and (-) ESI

500
m/z
105,817 peaks > 6σ
500 < m/z < 2000
750 1000 1250 1500 1750 2000
(+) ESI FT-ICR MS of De-Asphalted Oil”

700.70700.65700.60700.55700.50700.45700.40
93.9 mDa
m/z
1,000900800700600500400300
(+) ESI FT-ICR MS
Crude Oil
36.4 mDa
8.2 mDa
3.4 mDa
17.1 mDa
C3 / SH4
N / 13CH
C / H12
O / CH4
13C2 / C2H2
N13C / C2H3
8.9 mDa
Broadband Positive ESI FT-ICR Mass Spectrum of Crude Oil

462.1345
m/z
464463462
m/z 462.13489
[ C27 H24 N4
58Ni ]+•
(+80ppb)
m/z 462.13432
[ C30 H24 N1 S2 ]+•
(+300ppb)
570 µDa
Theoretical Abundance 2.6%
Experimental Abundance 1.9%
N
N
N
N
OV
DBE = 18
58Ni
Mass e- 548 µDa
Direct Speciation of Metalloporphyrins in Crude Oil

1H = 1.007825
12C= 12.00000
Mass Defect
1
H
2
H
13
C
14
N 15
N12
C
16
O
19
F
17
O
18
O
31
P 32
S
33
S 34
S 36
S
35
Cl 37
Cl
79
Br
81
Br
127
I
Nuclide
Massdefect,mDa
14N = 14.003074
16O= 15.994915

1. Carbon Number
2. Heteroatom Composition
3. Aromaticity
m/z 704.53510
[C50H72S1]+•
800700600500400
*
m/z
m/Δm50%
100 - 400 ppb
DBE = C –
H
2
N
2
+ + 1
McLafferty & Turecek Int. Mass Spectra, 1993
[Z = -2(DBE) + n + 2]
Carbon Number
DBE
S1 Class
Relative Abundance (% total)
40
30
20
10
0
20 40 60 80
Workflow for High Resolution “Petroleomics”

S
CH3
Isomeric structure for S-compounds
S
CH3
C1-dibenzothiophenes (4 isomers)
S
CH3
S
CH3
1-methyl-dibenzothiophene 4-methyl-dibenzothiophene3-methyl-dibenzothiophene2-methyl-dibenzothiophene
C1-benzothiophenes (6 isomers)
C2-dibenzothiophenes (26 isomers):
22 dimethyl-dibenzotiophene isomers and 4 ethyl-dibenzotiophene isomers
S
CH3
S
CH3
2-methyl-
benzothiophene
S
CH3
S
CH3
3-methyl-
benzothiophene
4-methyl-
benzothiophene
5-methyl-
benzothiophene
SCH3
6-methyl-
benzothiophene
S
CH3
7-methyl-
benzothiophene
Benzonaphthotiophenes
S
S S
Benzo[b]naphtho[1,2-d]thiopheneBenzo[b]naphtho[2,3-d]thiopheneBenzo[b]naphtho[2,1-d]thiophene

Petroleum Biomarkers: Hopanes and Steranes
Bacteriohopanetetrol
(hopanoid in prokaryotes)
Hopanes
A B
C
D
E
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19 20
21
22
23 24
25 26
27
28
30
29
31
32
33
34
35
C35H62O4
Cholesterol
steroid in eukaryotes
Steranes
A B
C D1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21 22
23
24
25 26
27
28
C27H46O
============================================================
29

M+•
4% of TIC
C27H46
EI mass spectrum of 17α (H)-22,29,30-tris-norhopane

Petroleum Biomarkers
SteranesHopanes
Multiple reaction monitoring mode
(MRM)
m/z 191
m/z 217
M+• → m/z 191 M+• → m/z 217

Ion Source Diagram of APCI-GC/MS-MS
Corona Pin
Capillary
GC Column
Ionization
Chamber
Adapted from Waters Corporation
Ion source
Housing
Mass Spec
Heated Transfer Line
N2
+• + M M+• + N2
(Atmospheric
Pressure)

Charge Transfer
Protonation
======================================================
Ionization mechanisms:
Charge Transfer vs. Protonation
By courtesy of Waters Corporation

Phenanthrene 100 pg
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
%
0
100
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
%
0
100
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
%
0
100
179
178
178
179
178
179
m/z
Ionization mechanisms:
Charge Transfer vs. Protonation
“wet” source
“wet” source
“dry” source
M+•
[M+H]+
MW 178
Phenanthrene
M+•
[M+H]+
M+•

APCI-GC/MS-MS of 17α(H)-22,29,30-trisnorhopane
Product (daughter) scan from M+• (m/z 370)
M+•
Collision energy: 15 eV
Collision gas: Ar
C27H46
m/z
50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
%
0
100
191
95
81
69
149
109
135
121
163
177
355 370
MRM transition: m/z 370 → 191

MS/MS spectrum of 17α(H)-22,29,30-trisnorhopane
MS/MS spectrum
from m/z 370
NIST library EI mass spectrum
17β(H)-22,29,30-trisnorhopane
The first match

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

17α(H)-22,29,30-trisnorhopane
17α(H),21β(H)-hopane
17α(H),21β(H)-22S-homohopane 17α(H),21β(H)-22R-homohopane
17α(H),21β(H)-30-norhopane
APCI-GC/MS-MS of NIST2266. Hopanes.
m/z 370 → 191
m/z 398 → 191
m/z 412 → 191
m/z 426 → 191

αββ 20R-cholestane ααα 20R-cholestane
αββ 20R 24S-methylcholestane
αββ 20R 24R- ethylcholestane
ααα 20R 24R-ethylsholestane
APCI-GC/MS-MS of NIST2266. Steranes.
m/z 372 → 217
m/z 386 → 217
m/z 400 → 217

NIST2779 (Macondo crude oil)
Pricey samples from BP oil spill being sold to scientists
http://www.nola.com/news/gulf-oil-spill/index.ssf/2012/03/federal_government_sells_price.html
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.

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)
Time, min
50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00
RA,%
0
100
Time
80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00
Hopanes: Summed Signals for C27-C35 (M+• → m/z 191)
C35
C34
C33
C32
C31
H30
H29
Ts
Tm
H31S
H31R
H32S
H32R H33S
H33R H34S
H34R
H35R
H35S
A B
C
D
E
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19 20
21
22
23 24
25 26
27
28 29
30
31
32
33
34
35
C29Ts
DH30
M30

m/z 372 → 217
m/z 386 → 217
m/z 372 → 217
βα
βα
αβ
αβ
βα
βα
αβ
αβ
αααS
βα
βα
αβ
αααS
αααR
αβ
Time
50.00 55.00 60.00 65.00 70.00 75.00 80.00
50.00 55.00 60.00 65.00 70.00 75.00 80.00
%
100
50.00 55.00 60.00 65.00 70.00 75.00 80.00
50.00 55.00 60.00 65.00 70.00 75.00 80.00
0
%
100
0
%
100
%
100
0
0
APCI/GC-MS/MS of NIST2779 (Macondo crude oil)
C27 -Diasteranes
C27-Steranes
C28 -Diasteranes
C28-Steranes
C29 -Diasteranes C29-Steranes
Sum of C27-C29 Steranes/Diasteranes
A B
C D1
2
3
4
5
6
7
8
9
10
11
12
13
14 15
16
17
18
19
20
21 22
23
24 25 26
27
28
29
αααS
αββR
αααR
αββS
αββR
αββS
αααR
αββR
αββS

0
0.4
0.8
1.2
1.6
2
βαC27/βαC29
Diasteranes
Ts/Tm
H29/H30
H32S/H32R
H33S/H33R
αββC27/αββC29
Steranes
H30/H31+H32+H33+H34+H35
NIST 2779 (Macondo crude oil)

0
0.4
0.8
1.2
1.6
2
Ts/Tm
H29/H30
H32S/H32R
H33S/H33R
H30/(H31+H32+H33+H34
+H35)
αββC27/αββC29
βαC27/βαC29
Steranes
Diasteranes
SAM 1-18 collected
1-45 months after
the oil spill

"Megaplume" in the GC600 lease block:
Lat: 27° 22.466' N
Long: 90° 30.689'W
water depth: 1382m
Natural Oil Seeps (GC600, Megaplume)

Natural oils seeps in the Gulf of Mexico - 140,000 tonnes per year (range of
80,000 to 200,000 tonnes).
Natural Oil Seeps. The Gulf of Mexico.
from www.sarsea.org

0
0.4
0.8
1.2
1.6
2
Ts/Tm
H29/H30
H32S/H32R
H33S/H33R
H30/(H31+H32+H33+H34+H3
5)
αββC27/αββC29
βαC27/βαC29
Steranes
Diasteranes
Megaplume Oil Seep (GC600)

0
0.4
0.8
1.2
1.6
2
Ts/Tm
H29/H30
H32S/H32R
H33S/H33RH30/(H31+H32+H33+H34+H35)
αββC27/αββC29
βαC27/βαC29
Steranes
Diasteranes
Blue crude (Anadarko Independence Hub)

0
0.5
1
1.5
2
0
0.5
1
1.5
2
0
0.5
1
1.5
2
0
0.5
1
1.5
2
SAM-1 SAM-2 SAM-3
SAM-4
SAM-6
0
0.5
1
1.5
2
SAM-7
0
0.5
1
1.5
2
SAM-8
0
0.5
1
1.5
2
0
0.5
1
1.5
2
SAM-5
βαC27/βαC29
Diasteranes
Ts/Tm
H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27βα/C29βα
Diasteranes
Ts/Tm
H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27βα/C29βα
Diasteranes
Ts/Tm
H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
Ts/Tm
βαC27/βαC29
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
0
0.5
1
1.5
2
SAM-9
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
C27αββ/C29αββ
Steranes

0
0.5
1
1.5
2
SAM-10
0
0.5
1
1.5
2
SAM-11
0
0.5
1
1.5
2
SAM-12
0
0.5
1
1.5
2
SAM-13
0
0.5
1
1.5
2
SAM-14
0
0.5
1
1.5
2
SAM-15
SAM-16
0
0.5
1
1.5
2
0
0.5
1
1.5
2
SAM-17
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm Ts/Tm
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
C27αββ/C29αββ
Steranes
0
0.5
1
1.5
2
SAM-18
C27βα/C29βα
Diasteranes H29/H30
H32S/H32R
H33S/H33RH30/ΣH31‒H35
Ts/Tm
C27αββ/C29αββ
Steranes

0
0.4
0.8
1.2
1.6
2
βαC27/βαC29
Diasteranes
Ts/Tm
H29/H30
H32S/H32R
H33S/H33R
αββC27/αββC29
Steranes
H30/H31+H32+H33+H34+H35
Blue crude (Independence Hub)
Megaplume oil seep (GC600)
SAM-10 (Pensacola Beach)
Overlaid spider diagrams

Other case studies: Exxon Valdez oil spill
from www.uaf.edu
Prince William Sound, Alaska
March 24, 1989. 258,000 barrels

25,500 peaks
150 < m/z < 850
850750650550450350250150
m/z
(+) APPI FT-ICR MS of Macondo crude oil

Relative Abundance (% total) Carbon Number
10
15
5
0
20
DBE
10 20 30 40
S class (M+•)
50 60
DBE=12
S
DBE=9
R
10 20 30 40 50 60
25
30
HC class (M+•)
DBE=10
S
R
R

Mass Spec
UV-lamp
(Atmospheric
Pressure)
Heated Transfer Line
Capillary
GC Column
Ionization
Chamber
Ion source
Housing
APPI-GC/MS Ion Source Diagram

Kr UV-lamp
Atmospheric Pressure PhotoIonization (APPI)
Spectral distribution of a Krypton lamp
E=10.6 eV, λ= 117 nm
E=10.0 eV, λ= 124 nm
M+•M
hν
hν > IE(M)
Ionization energies, IE (eV)
IE(N2) = 15.6 eV
IE(H2O)= 12.6 eV
IE(O2) = 12.1 eV
IE(C6H6) = 9.2 eV
IE(Toluene)= 8.8 eV
IE(Naphthalene) = 8.1 eV
IE (Phenanthrene) = 7.9 eV
IE (Thiophene) = 8.9 eV
IE (DBT) = 8.0 eV
IE(Alkanes) ~ 10 eV
- ē

100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190
%
0
100
178
m/z
APPI-GC/MS. Mass Spectrum of Phenanthrene
M+•
MW 178
IE = 7.9 eV
Phenanthrene (20 pg injected)
M+•M
hν

time
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
%
0
100
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
%
0
100
APPI-GC/MS vs APCI-GC/MS of Phenanthrene
APCI-GC/MS
m/z 178 →m/z 152
APPI-GC/MS
m/z 178 → m/z 152
S/N 4160
S/N 32830

APPI-GC/MS of Aromatic compounds
M+•
M+•
M+•
MW 168
IE = 8.1 eV
MW 167
IE = 7.6 eV
MW 184
IE = 7.9 eV

Time
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
%
0
100
Acenaphthylene
Naphthalene
Acenaphthene
Fluorene Anthracene
Benz[a]anthracene
Fluoranthene
Pyrene
Phenanthrene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Dibenz[a,h]anthracene
Benzo[g,h,i]perylene
Benzo[a]pyrene
Indeno[1,2,3-cd]pyrene
Boiling T 500 °C
APPI-GC/MS of 610 PAH Calibration Mix A
610 PAH Calibration Std (x 1000 dilution)
Final concentration: 500-1000 ng/mL

APPI/GC-MS/MS of NIST2779 (Macondo crude oil)
S
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
of Ar lamp
0.105 ‒ 9 µm
LiF wavelength transmission range
17α(H),21β(H)-30-hopane
as internal standard
Environ. Sci. Technol. 1994, 28, 142-145

APPI(Ar)-GC/MS-MS. PAHs and PASHs ratios.

Depletion of PAHs and PASHs in Environmental
samples from AL-MS shore line.
Phen DBT C2-Phen C2-DBT C3-Phen C3-DBT Chrys
≈
100
NIST 2779
(DWH)
Jul, 2011 Feb, 2012 Jan, 2014
Depletion is relative to 17α(H),21β(H)-30-hopane (C30-Hopane)

 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.
SUMMARY

(-) UniSpray TQS mass spectrum of Macondo crude oil
(NIST 2779)
m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
NEG_UNISPRAY-DWH_1%NH3_B 20 (0.335) Cm (14:143) MS2 ES-
1.28e5421.26
294.15
247.13
490.32
566.38
684.51
790.66
858.68
dioctyl sodium sulfosuccinate (DOSS)
[M-H]-

m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
%
0
100
NEG_UNISPRAY-DWH_MS-MS-421 121 (2.028) Cm (116:128) Daughters of 421ES-
3.73e4421
81
m/z
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
NEG_UNISPRAY-DWH_MS-MS-421 121 (2.028) Cm (116:128) Daughters of 421ES-
904367
227
187
219
291
265
279
338313
298
391
375
404
(-) UniSpray MS/MS spectrum for [M-H]-
ion of dioctyl sodium sulfosuccinate (DOSS)
CID: 20 V; Collision gas: Ar
[M-H]-
[HSO3]-

m/z
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
POS_UNISPRAY_DWH_1%HCOOH_A 23 (0.386) Cm (23:84) MS2 ES+
1.82e6360
227
338
305
431
499
478450
567
520
635
586
703
677
839771
(+) UniSpray TQS mass spectrum of Macondo crude oil
(NIST 2779)
1 mg/mL Tol:MeOH (50:50)- 1% HCOOH

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

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).
Samples’ collection sites and time

Petroleum Biomarkers. Hopanes.
Bacteriohopanetetrol
(hopanoid in prokaryotes)
Hopanes
A B
C
D
E
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19 20
21
22
23 24
25 26
27
28
29
30
31
32
33
34
35
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)
22R is biological configuration.
17α(H),21β(H) –hopanes are the most stable. 17β(H),21α(H) are called moretanes.
22S/(22S+22R) ~ 0.58-0.62 for C31-hopane
C35H62O4

Cholesterol
Petroleum Biomarkers. Steranes.
steroid in eukaryotes Steranes
A B
C D1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21 22
23
24
25 26
27
28
29
C27H46O
5α,14α,17α(H)–cholestane-20R
biological configuration
5α,14β,17β(H)
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)
Sample Ts/Tm H29/H30 H32S/H32R H33S/H33R H30/Σ(H31-H35)
C27αββ/C29αββ
steranes
C27βα/C29βα
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

M+•
7% of TIC
C27H48
EI mass spectrum of ααα 20R-cholestane

GC/MS of NIST 2779 (Macondo crude oil)
(Ion Chromatogram at m/z 191 and 217)
Ion Chromatogram at m/z 191
Ion Chromatogram at m/z 217
Hopanes
C30
C31
C32
C33
C29
C34
C35
Steranes
C28
C29
C29
C27
C29
C27
C29
C30
C27 C28 C29
C29
m/z 217
m/z 191Ts
Tm

Constant neutral loss scan
Multiple reaction monitoring mode
Precursor (parent) ion scan
Product (daughter) ion scan
MS/MS Modes

CH3
CH3
CH3
CH3CH3
R
M+•
+
•
•
+
+
R= H m/z 217
R= CH3 m/z 231
R= C2H5 m/z 245
R= C3H7 m/z 259
CH3
CH3
R
CH3
CH3
CH3
CH3
CH3
R
CH3
CH3
CH3
CH3
CH3
R
H
Fragmentation scheme for steranes

APCI-GC/MS-MS of 17α(H)-22,29,30-trisnorhopane
M+•
Collision gas: Ar
C27H46
m/z
50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
%
0
100
191
95
81
69
149
109
135
121
163
177
355 370

50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
%
0
100
217
121
95
81
73
107 135
149
175
161
189 203
357
262 372
APCI-GC/MS-MS of ααα 20R-cholestane
M+•
Collision gas: Ar
C27H48
m/z

MS/MS spectrum of ααα 20R-cholestane
MS/MS spectrum
from m/z 372
of Cholestane
The first match

C35H72
C14H30
C16H34
C18H38
C25H52
C30H62
C20H42
Pr
Ph
Isoprenoid indices
Pr/Ph=1.33
n-C17/Pr=1.58
n-C18/Ph=1.67
Reported Pr/Ph ratio for MC252 is 0.9
Environ. Res. Lett. 2012, 7, 035302
GC/MS of “Macondo crude oil” NIST 2779
(Total Ion Chromatogram)

+
+
•
R= H m/z 217
R= CH3 m/z 231
R= C2H5 m/z 245
R= C3H7 m/z 259
R= H m/z 218
R= CH3 m/z 232
R= C2H5 m/z 246
R= C3H7 m/z 260
M+•
CH3
CH3
R
CH3
CH3
R
CH3
CH3
CH3
CH3CH3
R
Characteristic ions for steranes

25,500 peaks
150 < m/z < 850
850750650550450350250150
m/z

Carbon Number
10
15
5
0
20
DBE
10 20 30 40 50 60
25
30
HC class (M+•)
DBE=10
C1-phenathrene has 5 isomers
R
CH3CH3
1-methylphenanthrene 2-methylphenanthrene
CH3
3-methylphenanthrene
CH3
CH3
C2-phenanthrene has 30 isomers:
25 isomers for dimethyl-
phenanthrenes
5 isomers for ethyl-phenanthrenes
C#= 25-30

DBE
HC class (M+•)
DBE Distribution

Carbon Number
10
15
5
0
20
DBE
10 20 30 40
S class (M+•)
50 60
C#=19
DBE=12
DBE=6
S
R
25
30
S
C3
S
R
DBE=12
DBE=9
S
R
C2-dibenzothiophenes (26 isomers):
22 dimethyl-dibenzotiophene isomers and
4 ethyl-dibenzotiophene isomers

DBE
S class (M+•)
DBE Distribution
S
R
DBE=12
S
R
DBE=9
DBE=6
S
R
DBE=3
S
R

Steranes
Hopanes
Cholesterol
Petroleum Biomarkers
EukaryotesProkaryotes

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.

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
%
0
100
152
128
178
APPI-GC/MS-MS of Phenanthrene (m/z 178)
m/z
M+•
[M-C2H2]+•
[C10H8]+•
C14H10
-C2H2
Collision energy (Ekin) = 27 eV
Collision gas - Ar
Eint =Ekin
Mgas
Mion + Mgas

MS/MS spectrum of Phenanthrene (m/z 178)
MS/MS spectrum
from m/z 178
of Phenanthrene
The first match

Wavelength Transmission/Absorption Range
0.11 ‒ 7.5 µm
N2 < 0.1 O3 0.17-0.35 0.45-0.75
O2 < 0.245 H2O < 0.21 0.6-0.72
Gas Absorption wavelengths (μm)
MgF2 (window) wavelength transmission range

Sum of 7 MRM transitions
APPI(Ar)-GC/MS-MS of NIST2266
(hopanes & steranes standard)
20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
%
0
100
time
IE(Alkanes) ~ 10 eV
αββ 20R-cholestane
ααα 20R-cholestane
17α (H)-22,29,30-
trisnorhopane
αββ 20R 24S-
methylcholestane
αββ 20R 24R-
ethylcholestane
17α(H),21β(H)-30-
hopane
17α(H),21β(H)-30-
norhopane
ααα 20R 24R-
ethylsholestane
17α(H),21β(H)-
22S-homophane
17α(H),21β(H)-22R-
homopane

20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
%
0
100
time
Ar-lamp
hν =11.7 eV
S
17α(H),21β(H)-hopane
50 ng/mL
Peak area - 218
50 ng/mL
Peak area - 218
m/z 412 → m/z 191
dwell time – 250 ms
m/z 178 → m/z 152
m/z 184 → m/z 152
585 ng/mL
Peak area - 4900
APPI(Ar)-GC/MS-MS of Phenanthrene,
Dibenzothiophene, and 17α(H),21β(H)hopane

DBE
Carbon number
0 70 80 90605040302010 100 110 120
80
60
40
20
100
120
0
C# = 112
DBE = 100
C# = 54
DBE = 46
C# = 24
DBE = 19
C20
DBE = C –
H
2
N
2
+ + 1
Double Bond Equivalent (DBE)
n-hexane, C6H14, DBE=0 Cyclohexane, C6H12, DBE=1 Benzene, C6H6, DBE=4
V.V. Lobodin, et al. Anal. Chem. 2012, 84, 3410-3416

APCI and APPI GC/MSMS for Characterization of the Macondo Wellhead Crude Oil and the Oil Spill

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APCI and APPI GC/MSMS for Characterization of the Macondo Wellhead Crude Oil and the Oil Spill