2/13/2015

Comprehensive Two‐Dimensional
Gas Chromatography‐Mass
Spectrometry in Environmental
Sciences and POPs analysis
Shunji HASHIMOTO
National Institute for Environmental
Studies,Tsukuba, JAPAN

Global (non‐target) analysis byComprehensive Two‐Dimensional
Gas Chromatography‐Mass
Spectrometry12/13/2015

SELECTIVE AND COMPREHENSIVEANALYSIS OF ORGANOHALOGENCOMPOUNDS BY GC×GC‐HRTOFMS
AND MS/MS

Developed instrument (1)
GC×GC

Comprehensive
multidimensional
gas chromatographZoex KT‐2004 or KT‐2006+HRTofMS

High resolution
time of flightmass spectrometer

JEOL JMS‐T100GC or 4G Precise mass measurement
 High separation capability
 Enhancement of S/N ratio of peaks  Constant full‐range mass measurement Skip cleanup process (acceleration, comprehensive analysis) Separation targets (precision enhancement) Accurate mass measurement (compositional estimation)2

2/13/2015

Developed instrument (2)
GC×GC

Comprehensive
multidimensional
gas chromatographZoex KT‐2004 or KT‐2006+

qMS/qMS

Tandem quadrapole
mass spectrometer

Agilent QQQ 7000A

 High selectivity
 High separation capability
 Enhancement of S/N ratio of peaks  Various function (MRM, scan, NLS…)

 Skip cleanup process (acceleration, comprehensive analysis) Separation targets (precision enhancement) Selective detection (functional group analysis, structural estimation)

Selective detection of organohalogens
by NLS on GC×GC‐MS/MS

3

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Measurement methods with MS/MS
Ion
sourceMethodPrecursor scan

MS1

Behavior

Collision
cellMS2

Usage

Detector

Application in this study

MS1

MS2

scan

fix

Identification, searching

Partially use
Partially use

Product scanfix

scan

Identification, searching

Neutral loss scan(NLS)

scan1

scan1

Screening for specific
For global detection ofsubstances, which hascompounds
specific functional group(s) (e.g. organochlorines…)

Multiplereactionmonitoring(MRM)

fix2

fix2

Quantification

For multi‐component
concurrent
measurement

1) scanning MS1 and MS2 simultaneously while maintaining constant the difference
between their masses
2) Rapidly switching a pair of MS1 and MS2 mass numbers (@ <500ch/sec)
7

Instruments & the parameters
GCxGC
Agilent 7890 GC
Zoex KT2006
GL Science InertCap 5MS/Sil (60 m long, 0.25 mm i.d., 0.1 μm film thickness)
SGE BPX‐50 (1.5 m long, 0.1 mm i.d., 0.1 μm film thickness)from 70 °C holding for 1 min
to 180 °C at rate 50 °C min‐1 holding for 0 min
to 230 °C at rate 3 °C min‐1 holding for 0 min
to 300 °C at rate 5 °C min‐1 holding for 16.133 min
Injection
volume: 1 μl, temp: 280 °C, method: splitless
Carrier gas
type: He, mode: constant flow, initial head pressure: 488.8 kPa at 70 °C
Modulation
period: 4 sec, releasing: 0.35 sec
MS/MS
Instrument
Agilent 7000A (7000B equivalent)
Ion source
mode: EI+, temp: 250 °C, ionizing voltage: 38 eV, ionizing current: 35 µA
Analyzer
mode: neutral loss scan, monitoring loss*: 19, 35, 37, 79, 81 m/z
scan range: 150 ‐ 530 m/z, cycle: 20 Hz
Collision energy 25 V

Instrument
GCxGC
1st column
2nd column
Oven program4

2/13/2015

Organohalogens (1)Compound nameConc.
ID *(ng/ml)

RT 1st(min)2378TCDD
12378PeCDD
123478HxCDD
123678HxCDD
123789HxCDD
1234678HpCDD
OCDD
2378TCDF
12378PeCDF
23478PeCDF
123478HxCDF
123678HxCDF
123789HxCDF
234678HxCDF
1234678HpCDF
1234789HpCDF
OCDF

2001000
1000
1000
1000
1000
2000200
1000
10001000
1000
1000
1000
1000
1000
2000

D2D3D4D5D6D7D8F1F2F3F4F5F6F7F8F9F1024.406 2.309
27.939 2.237
30.739 2.525
30.873 2.453
30.939 2.309
33.673 2.453
36.339 3.752
23.939 2.525
26.939 2.381
27.739 2.453
30.139 2.381
30.206 2.453
31.206 2.381
31.473 2.742
32.739 2.381
34.139 2.886
36.539 0.144

HCBa‐HCH
b‐HCH
g‐HCH
d‐HCH
HpCHL
c‐HpEPOt‐HpEPO
c‐NoCHL
t‐NoCHL
c‐CHL
t‐CHL
OxyCHL
opDDD
ppDDD
opDDE
ppDDE
opDDT
ppDDT
ALD
DLD
END
MRX

800
800800
800
800
800
800
800
800
800
800
800
800
800
800
800
800
800
800
800
800
800
800

P2P1P4P3P5P6P9P10P19P14P13P11P8P17P21P12P15P20P22P7P16P18P23

13.205 722
13.005 0.866
13.805 1.227
13.605 1.371
14.472 1.804
15.938 1.227
18.605 2.02
18.805 2.093
22.805 2.165
20.205 1.804
20.072 2.165
19.538 2.02
18.538 1.732
21.272 2.598
22.938 2.38119.605 2.381
20.938 2.237
22.805 2.453
24.405 2.309
17.205 1.443
21.272 2.237
22.205 2.526
28.605 2.309

Abbrev.

RT2nd(sec)Dioxins (17 compounds)
2,3,7,8‐tetrachlorodibenzo‐p‐dioxin
1,2,3,7,8‐pentachlorodibenzo‐p‐dioxin
1,2,3,4,7,8‐hexachlorodibenzo‐p‐dioxin
1,2,3,6,7,8‐hexachlorodibenzo‐p‐dioxin
1,2,3,7,8,9‐hexachlorodibenzo‐p‐dioxin
1,2,3,4,7,8,9‐heptachlorodibenzo‐p‐dioxin
octachlorodibenzo‐p‐dioxin
2,3,7,8‐tetrachlorodibenzofuran
1,2,3,7,8‐pentachlorodibenzofuran
2,3,4,7,8‐oentachlorodibenzofuran
1,2,3,4,7,8‐hexachlorodibenzofuran
1,2,3,6,7,8‐hexachlorodibenzofuran
1,2,3,7,8,9‐hexachlorodibenzofuran
2,3,4,6,7,8‐hexachlorodibenzofuran
1,2,3,4,6,7,8‐heptachlorodibenzofuran
1,2,3,4,7,8,9‐heptachlorodibenzofuran
octachlorodibenzofuran

Abbrev.

Conc.
(ng/ml)ID *RT 1st(min)RT2nd(sec)

400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400

B1B2B3B6B5B11B7B16B15B10B21B19B18B17B24B12B2711.272
11.93812.338
14.33813.60517.60515.07221.47221.072
17.23823.53822.93822.47222.33824.93819.40526.538

0.289
0.433
0.649
1.155
1.082
2.02
1.66
2.453
2.381
1.804
2.526
2.381
2.237
2.237
2.309
1.948
2.309

PCBs (35 compounds)

POPs (23 compounds)
hexachlorobenzene
alpha‐hexacyclohexane
beta‐hexacyclohexane
gamma‐hexacyclohexane
delta‐hexacyclohexane
Heptachlorcis‐Heptachlor epoxidetrans‐Heptachlor epoxide
cis‐Nonachlortrans‐Nonachlorcis‐Chlordane
trans‐Chlordane
Oxychlordane
2,4'‐DDD
4,4'‐DDD2,4'‐DDE
4,4'‐DDE
2,4'‐DDT
4,4'‐DDT
AldrinDieldrin
EndrinMirex

Compound name2‐chlorobiphenyl
4‐chlorobiphenyl
2,2'‐dichlorobiphenyl
4,4'‐dichlorobiphenyl
2,2',6‐trichlorobiphenyl
3,4,4'‐trichlorobiphenyl
2,2',6,6'‐tetrachlorobiphenyl
3,3',4,4'‐tetrachlorobiphenyl
3,4,4',5‐tetrachlorobiphenyl
2,2',4,6,6'‐pentachlorobiphenyl
2,3,3',4,4'‐pentachlorobiphenyl
2,3,4,4',5‐pentachlorobiphenyl
2,3',4,4',5‐pentachlorobiphenyl
2',3,4,4',5‐pentachlorobiphenyl
3,3',4,4',5‐pentachlorobiphenyl
2,2',4,4',6,6'‐hexachlorobiphenyl
2,3,3',4,4',5‐hexachlorobiphenyl

PCB1
PCB3
PCB4
PCB15
PCB19
PCB37
PCB54
PCB77
PCB81
PCB104
PCB105
PCB114
PCB118PCB123
PCB126
PCB155
PCB156

2,3,3',4,4',5'‐hexachlorobiphenyl

PCB157400

B2826.7382.381

2,3',4,4',5,5'‐hexachlorobiphenyl
3,3',4,4',5,5'‐hexachlorobiphenyl
2,2',3,4',5,6,6'‐heptachlorobiphenyl
2,3,3',4,4',5,5'‐heptachlorobiphenyl
2,2',3,3',5,5',6,6'‐octachlorobiphenyl
2,3,3',4,4',5,5',6‐octachlorobiphenyl
2,2',3,3',4,4',5,5',6‐nonachlorobiphenyl
2,2',3,3',4,5,5',6,6'‐nonachlorobiphenyl
Decachlorobiphenyl
2,4,4'‐trichlorobiphenyl
2,3,3',5,5'‐pentachlorobiphenyl
2,2',3,3',5,5',6‐heptachlorobiphenyl
2,5‐dichlorobiphenyl
2,2',5,5'‐tetrachlorobiphenyl
2,2',4,5,5'‐pentachlorobiphenyl
2,2',3,4,4',5'‐hexachlorobiphenyl
2,2',3,3',4,4',5,5'‐octachlorobiphenyl

PCB167
PCB169PCB188PCB189PCB202
PCB205
PCB206
PCB208PCB209
PCB28
PCB111
PCB178PCB9PCB52
PCB101
PCB138PCB194

400
400
400
400
400
400
400
400
400
100
100
100
100
100
100
100
100

B25
B29B20
B30
B26
B33
B34
B31
B35
B8B14B23
B4B9B13B22
B32

25.67228.00523.00529.33826.20530.67231.73829.67237.73815.53820.87224.67212.73816.60519.80524.47230.538

2.093
2.165
2.093
2.237
2.02
2.237
2.309
2.165
2.309
1.372
1.948
2.02
0.722
1.66
2.165
2.309
2.237

RT 1st(min)RT2nd(sec)Organohalogens (2)Compound name

Abbrev.

Conc.
ID *(ng/ml)

RT 1st(min)RT2nd(sec)

Cl‐PAHs (21 compounds)
9‐chlorofluorene
9‐chlorophenanthrene
2‐chloroanthracene
9‐chloroanthracene
3‐chlorofluoranthene
8‐chlorofluoranthene
1‐chloropyrene
6‐chlorochrysene
7‐chlorobenz[a]anthracene
3‐chlorobenzanthrone
6‐chlorobenzo[a]pyrene
3,9‐dichlorophenanthrene
1,9‐dichlorophenanthrene
9,10‐dichlorophenanthrene
9,10‐dichloroanthracene
1,3‐dichlorofluoranthene
3,8‐dichlorofluoranthene
3,4‐dichlorofluoranthene
6,12‐dichlorochrysene
7,12‐dichlorobenz[a]anthracene
3,9,10‐trichlorophenanthrene

9‐Cl‐FLE
9‐Cl‐PHE
2‐Cl‐ANT
9‐Cl‐ANT
3‐Cl‐FLA
8‐Cl‐FLA
1‐Cl‐PYR
6‐Cl‐CHR
7‐Cl‐BaA
3‐Cl‐BA‐O
6‐Cl‐BaP
39‐Cl2‐PHE
19‐Cl2‐PHE
910‐Cl2‐PHE
910‐Cl2‐ANT
13‐Cl2‐FLA
38‐Cl2‐FLA
34‐Cl2‐FLA
612‐Cl2‐CHR
712‐Cl2‐BaA
3910‐Cl2‐PHE

4.6
3.6
146.3
4.8
5.4
5.8
5.6
1023107.7
3.0
2.2
5.1
6.3
6.9
6.7
4.8
5.0
5.4

C1C2C3C4C9C10C11C16C17C18C21C5C6C8C7C13C14C15C19C20C1213.606
17.07317.33917.33923.00623.13924.27330.07330.27330.33936.33921.07321.33921.67321.40626.13927.13928.27332.93933.13925.139

1.587
2.67
2.742
2.814
3.319
3.247
3.463
3.391
3.391
3.968
1.876
3.103
3.103
3.247
3.175
2.958
3.103
3.319
3.319
3.535
2.886

2‐Br‐FLE
9‐Br‐PHE
9‐Br‐ANT
910‐Br2‐ANT
1‐Br‐PYR
7‐Br‐BaA
711‐Br2‐BaA
712‐Br2‐BaA
47‐Br2‐BaA
57‐Br2‐BaA
6‐Br‐BaP

11139.7
10111010109.5
9.511R1R2R3R4R5R615.539 1.948
19.406 30175
19.806 3.247
26.139 30175
26.606 3.319
32.206
3.39

Br‐PAHs (11 compounds)
2‐bromofluorene
9‐bromophenanthrene
9‐bromoanthracene
9,10‐dibromoanthracene
1‐bromopyrene
7‐bromobenz[a]anthracene
7,11‐dibromobenz[a]anthracene
7,12‐dibromobenz[a]anthracene
4,7‐dibromobenz[a]anthracene
5,7‐dibromobenz[a]anthracene
6‐bromobenzo[a]pyrene

Compound name

Abbrev.

Conc.
ID *(ng/ml)

PBDEs (27 compounds)
4‐bromodiphenyl ether
2,4‐dibromodiphenyl ether
4,4'‐dibromodiphenyl ether
2,2',4‐tribromodiphenyl ether
2,4,4'‐tribromodiphenyl ether
2,2',4,4'‐tetrabromodiphenyl ether
2,2',4,5'‐tetrabromodiphenyl ether
2,3',4,4'‐tetrabromodiphenyl ether
2,3',4',6‐tetrabromodiphenyl ether
3,3',4,4'‐tetrabromodiphenyl ether
2,2',3,4,4'‐pentabromodiphenyl ether
2,2',4,4',5‐pentabromodiphenyl ether
2,2',4,4',6‐pentabromodiphenyl ether
2,3',4,4',6‐pentabromodiphenyl ether
3,3',4,4',5‐pentabromodiphenyl ether
2,2',3,4,4',5‐hexabromodiphenyl ether
2,2',4,4',5,5'‐hexabromodiphenyl ether
2,2',4,4',5,6'‐hexabromodiphenyl ether
2,3,3',4,4',5‐hexabromodiphenyl ether
2,2',3,4,4',5',6‐heptabromodiphenyl ether
2,2',3,4,4',6,6'‐heptabromodiphenyl ether

BDE3
BDE7
BDE15
BDE17
BDE28
BDE47
BDE49
BDE66
BDE71
BDE77
BDE85
BDE99
BDE100
BDE119
BDE126
BDE138
BDE153
BDE154
BDE156
BDE183
BDE184400400
400
400
400
400
400
400
400
400
400
400
400
400
400
800
800
800
800
800
800

E1E2E3E4E5E8E6E9E7E10E14E13E11E12E15E18E17E16E19E21E20

12.20615.87317.33921.673
22.53927.33926.60628.13926.80629.20633.20631.60630.60631.00633.60637.13935.27333.93938.00639.40638.539

0.722
1.876
2.165
2.886
2.597
2.67
2.67
2.67
2.886
2.67
3.103
2.742
2.742
2.814
2.886
1.659
3.896
3.103
2.381
3.896
3.175

E2240.939

1.587

2,3,3',4,4',5',6‐heptabromodiphenyl ether

BDE191800

2,2',3,3',4,4',5,6'‐octabromodiphenyl ether
2,2',3,3',4,4',6,6'‐octabromodiphenyl ether
2,2',3,3',4,4',5,5',6‐nonabromodiphenyl ether
2,2',3,3',4,4',5,6,6'‐nonabromodiphenyl ether
decabromodiphenyl ether

BDE196
BDE197
BDE206
BDE207
BDE209

800
800
2000
2000
2000

Total: 129 compounds

52/13/2015

Organohalogen STD Mixture<single scan>Two-dimensional TIC of standard organohalogens measured in a single scan(m/z=150-530) with the GCxGC-MS/MS (MS1 scan).

D9C20C21F10F6F8F9D4B32

D8C16C17C18C19F4D3B28F7C15F3C11C12C13C14B27B24C5C6C7C8C9C10B17P18B19 B18B21 P20
D2C4P13P15 B15P17B12B13P14B14B16P16P219
B20F1P21 D1P22B23B22B25
B26F2B29P23B30
B31 F5B33
D6D7 B34
B35

P11 P12P7P8P9P10B7B9B10B11P2P4B6 B8P6B2B4B1 B3P1B5P3C1P5C2C3Organohalogen STD Mixture<NLS of m/z=35 (‐35Cl)>

D: PCDDs, F: PCDFs, B: PCBs, P: POPs & C: Cl-PAHs

Two-dimensional TIC of standard organohalogens measured in a neutral loss ofm/z=35 scan (NLS) with the GCxGC-MS/MS.

6

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Organohalogen STD Mixture<NLS of m/z=79 (‐79Br)>

E: PBDEs, R: Br-PAHs

Two-dimensional TIC of standard organohalogens measured in a neutral loss ofm/z=79 scan (NLS) with the GCxGC-MS/MS.

Samples
Environmental samples; fly ash extract (NIES CRM
No.17)
sediment (NIES CRM No.20), soil (NIES CRM
No.21)
Biological Extraction
samples; human urine,
fish oil, pine
SamplePretreatment
needlesFly ashToluene solution

n.a.

Sediment

Soxhlet extraction withtoluene

44%(w/w)‐H2SO4/silica gelcolumn chromatography

Humanurine

Shaking with hexane

deconjugation with β‐glucronidase7

2/13/2015

Sediment<single scan>molecular sulfurhydrocarbons

Two‐dimensional TIC of sediment sample (NIES CRM20) measured in a single scan(m/z=150‐530) with the GCxGC‐MS/MS (single MS mode).

Sediment<NLS of m/z=35 (‐35Cl)>

About 200 peaks
were detectedTwo‐dimensional TIC of sediment sample (NIES CRM20) measured in a neutral loss of
m/z=35 scan (NLS) with the GCxGC‐MS/MS.

Journal of Chromatography A, 1218: 3799–3810

8

2/13/2015

Fly ash extract<Neutral Loss Scan>
About 800 peaks
were detected.

single scan

NLS‐79About 600 peaks
were detected.

About 900 peaks
were detected.

NLS‐35

NLS‐19Two‐dimensional TIC of fly ash extract (NIES CRM17) measured in a neutral loss of
m/z=35, 79&19 scan (NLS) with the GCxGC‐MS/MS.

Journal of Chromatography A, 1218: 3799–3810

Selective extraction oforganohalogens by softwarefrom GCxGC‐HRTofMS data9

2/13/2015

How to extract required informationfrom GC×GC‐HRTofMS?

9.E+08

TIC of TD‐GC‐HRTOFMS

8.E+087.E+086.E+085.E+084.E+083.E+082.E+081.E+080.E+007142027344047546067

RT (min)Huge number ofpeaks detected
Large data size
(10‐20 gigabytes)
TIC of TD‐GCxGC‐HRTOFMS
About 3m3 of air collected with a Gerstel Tenax‐TA tube at NIES in May,2007

19Developing novel software for extracting
organochlorines or organobromines from
HRTofMS data
This software can…
 directly read a netCDF file, which is a common format forMS data, and extract mass spectra including a chlorine or bromine
isotopic profile from whole data without setting a target
mass number, under various mass accuracy (mass resolution) conditions, optionally simulate neutral loss scan (NLS) of extracted
data as a post‐filter,

Interface of original software ‘CBEｘ’ thatwas developed by Visual C# 2010
(Microsoft) and that can be run onWindows

 pre‐filter data by checking for mass defects, which areusually observed if the compound includes atoms such as
halogens, and by narrowing the range of mass,
 and finally, output netCDF.

Journal of Chromatography A, 1282 (2013) 183‐189102/13/2015

Composition of ‘CBEx’
Pre‐filter

checking threshold, mass range, mass deficiency
Mass spectra un‐matched conditions are removed from data.

35Cl…

79

34.96885268

78.9183371‐0.03114732‐0.0816629

Main programiii)s(mi+Δm)ii)

smi
mm
1 279Br35

finding Cl or Br ‘s isotopic pattern
Mass spectra including part of the chlorine or
bromine isotopic pattern is left in the data.

mi+1

mi mi+2Δm

Δm*2 mass accuracy (MA)i)

Post‐filterMnsimulating NLS of Cl or Br
Mass spectra including mass fragments possibly
caused by loss of Cl or Br are left in the data.

Mxmmm/zm/z

Journal of Chromatography A, 1282 (2013) 183‐189

Results of selective extraction of organochlorines by software
2D‐TIC & mass spectra example <indoor air>original data

NLS filter effectseems limited to
low resolution data

×0.1u, NLS=onhigh mass accuracy(resolution) is effective to×
reduce the number ofunneeded peaks But isotope clusters of Cl (Br)are buried in mass spectra

0.01u

0.01u, NLS=on×
MD=on (only MD)

××
0.01u, MD=on×
Mass defect filter is very effective to reduce the numberof peaks and remove spectrum of hydrocarbons

Mass spectra@23.333min,1.310sec

11

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Effectiveness of mass defect filter (MDF)
MDF: OFFMDF: ON

Indoor airvery effective

MDF: OFFMDF: ON
molecular sulfur

fairly effective

Sediment

MDF: OFF

Human urine

Especiallyeffective tohydro carbons

MDF: ONpoorly effective
Original

Data of samples that were measured by TD‐GCxGC‐HRTofMS were processed under several mass resolution conditions.

Selective & sensitive detectionof organohalogens by negative
chemical ionization12

2/13/2015

Features of major ionization methods
Methods

Features (advantages & disadvantages)

EI Easy to use Popular
 Enable to estimate the molecular structure from fragments
 Upgrading database Hardly distinguish mixtureCI A few fragmentation
 Negative CI allows selective detection of special group (e.g.,organohalogen) Divers effectivity of ionization depending on compounds

FI Produce only molecular ion
 Some compounds can not be ionized Ionization efficiency is low (1/100 – 1/1000 of EI+）

Measurement of soil by several ionization methods
EI+

CI‐EI+> CBEx

FI+

Intensity enhanced x1000

TICs of soil sample measured by GCｘGC‐HRTOFMS on EI+, CI‐ and FI+ mode, and EI+
data processed with CBEx

13

2/13/2015

Comparison of mass spectra with various ionization mode
about typical peaks in soil sample
EI+

CI‐
some molecular ionsare absent

a lot of fragmentsa few fragments

EI+> CBEx

FI+

refined fragments

only molecular ions

How to use each ionization methods
for global (non‐target) analysis
 Enhancing compound search accuracy bydeveloping exact mass library Searching compound in library (present: nominal mass)  Assessment of isolationof components
known

EI+

CI‐FI+

unknown Obtaining accurate mass
 Obtaining accurate massof molecular ionof fragment ions
 Selective detection of compounds having
electrophilic functional group (halogen, nitro group)
Identification of unknown compounds142/13/2015

Future issues
 Provision of accurate exact mass library (compensatedmeasurement mass error) for reliable identification ofcompounds
 Alignments of peaks on 2D‐chromatograms to compare
data directly

Thank you for your attention.

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