Peter Haglund, Conny Danielsson, Erik Spinnel, Ulrika OlofssonUmeå University, Sweden

David Alonso, Joe Binkley, Liz Humston‐Fulmer, Kevin SiekLeco Corp.

2

Presentation Outline

ChallengesChromatography

SolutionsChromatography TOF-MS

GCxGC-HRMS

Comprehensive Identification

ConclusionComprehensive characterization

Isomer/enantiomerseparations

3

Trace Environmental Analysis: ChallengesChallenges Low concentrations Low concentrations

Complex matricesp

Numerous classes of organic compounds

Many isomeric target compounds

4

You cannot get it all…Selectivity

Simplicity,$$$

Speed$$$

Sensitivity 5

Trace Mixture AnalysisSelectivity

Simplicity,$$$

Speed$$$

Sensitivity 6

GC SolutionsSelectivity

Front end: GCxGCBack end: ECDBack end: ECD

ToF-MS

Simplicity,$$$

Speed$$$

Sensitivity 7

GC SolutionsSelectivity

Front end: GCxGCBack end: ECD

Screening/IdentificationBack end: ECD

ToF-MSIdentification

TargetTargetQuantification

Simplicity,$$$

Speed$$$

Sensitivity 8

Screening/ Identification

GCxGC‐ToF‐MS GCxGC ToF MS

Non‐target Screening of House Dust

Non‐target Screening of City Dump Leachate

Di t d S i f H l t t d C d i Bi t Directed Screening for Halogentated Compounds in Biota

”Omics”‐Identification of Compounds Poorly Removed in p yMunicipal Sewage Treatment Plants (STPs)

9

GCxGCDet.Inj.

10 seconds modulation period

Cryo-trapGC 1 GC 2

6 min 10 s

6

43

5

0

2

0 10 s1

3

3 6 min

0

0 s 10 s3 min

10

GC×GC

Primary separation based on volatility

Secondary separation based on polarity, polarizability, H‐bonding ability, shape, etc.

High peak capacity

High sensitivityHigh sensitivity

GC×GC‐ToF‐MS 3D data 3D data Deconvolution of peaks and spectra Exceptional separation power Exceptional separation power

11

Non-Target screening ofpollutants in house dustpollutants in house dust

Examples: House dust from Umeå City Hall 

Full‐scan GC×GC‐ToF‐MS

Total Ion Chromatograms – OverviewLibrary search / Selected ion traces – Identification

12

City Hall dust

Kommun

13

City Hall dustPhthalates

Kommun

14

City Hall dustPhthalates

Kommun

15

Organosphosphatescreening/Identifiction (m/screening/Identifiction (m/z = 

99)99)

Name Similarity Reverse Probability CASEthanol, 2-butoxy-, phosphate (3:1) 857 860 8680 78-51-3Ethanol, 2-butoxy-, phosphate (3:1) 839 862 8520 78-51-37-Methyl-Z-tetradecen-1-ol acetate 524 537 85 0-00-0

16

City Hall dustOrganophosphates

Kommun

17

City Hall dustPBDEs

Kommun

18

City Hall dust

SterolsSterols

Kommun

19

Summary of major components

• Petroleum hydrocarbons

• Phthalate plasticizers

• Organophosphate plasticizers/flame retardants

B i t d fl t d t• Brominated flame retardants

• Bacteria sterolsBacteria sterols

20

Non-Target Screening of City Dump LeachateCity Dump Leachate

Liquid‐liquid extraction with DCM

Base‐neutral fraction

Acidic fraction (methylated with diazomethane)

Total Ion Chromatograms Overview Total Ion Chromatograms – Overview

Library search / Selected ion traces – Identificationy /

21

Leachate, Base/Neutral

2-Piperidin-Sulfamide,

N,N-dimethyl-

2(3H)-Benzo-thiazolone

Bi h l A

Caffeine

Column bleedCyclohexan-

dione

2 Piperidinone 1(3H)-Isobenzo-

furanone

N'-phenyl- Bisphenol A

2 C lTriethyl

phosphate

2-methyl-phenol

2H-Indol-2-one, 1,3-dihydro-

Phenol

N-(2-Cyano-ethyl)-benzenesulfonamide

2-Cylco-hexanol

2-Cylco-hexanone

phosphate

Camphor

Phenol, 2,6-dimethyl-4-nitro-

Diisobutyl-phthalate

Tris(1,3-dichloro-isopropyl)phosphate

”Sterols”

1,3-Oxa-thiolane

2-Piperi-dinol DEHP

DINP2,6-Di-tert-butyl-4-nitrophenolPhenol

”Terpenes”

Sterols

CycloalkanesP 1 th 2 th l

C18

C25

C17C16C19 C20 C21 C22 C23 C24 C26 C27 C28 C29 C30 C31

C15C14C

Linear and branchedalkanesPropane, 1,2-dimethoxy-

Propane, 1-ethoxy-2-methyl-

Aliphatic ethers Sulphur (S8)

1,4-DioxaneToluene

C14C13C12

C11

Sulphur (S8)

22

Leachate, Acidic

3H-1,2-Dithiole-3-thione, 4-methyl-

Dimethylf

Carbonodithioic acid, O S

Dimethyl, pentasulfide Terpenoid background

Caffeine3-Methyl-3H-benzothiazol-

2-one

tetrasulfidO,S-dimethyl ester

Dimethyltrisulfide

Methane sulfonic

acid, methyl ester

Sulfuric acid, dimethylester

Benzoic acid, 2,6-dichloro-, methyl ester

Benzeneaceticacid 4 chloro Mecoprop

Acetic acid, (4-chloro-2-methylphenoxy)-,

methyl ester

Dimethyl, disulfide

acid, 4-chloro-, methyl ester

Benzeneacetic acid, à-methyl-

Mecopropmethyl ester

Saturated FAMEs

Monounsaturated FAMEs

4-(2-methylpropyl)-, methyl ester

**

Fatty acid methyl esters (FAMEs)

Saturated FAMEs

Hexanoic acid, 2-ethyl-

Low molecular weight acidsSulfur

2-ethyl-, methyl ester

Hexathiepane

23

Identified compoundsChemical group Substances Concentration in ng/L

Number Median Maximum

Nitrogen-substances 118 130 65 000

Terpenes 81 220 79 000

Ketones 52 92 51 000

Sulfur-substances incl. aromatic sulphonates 52 180 740 000

Aliphatic acids 39 380 200 000

P t d th t 37 270 32 000P-esters and other esters 37 270 32 000

Aromatic and polyaromatic hydrocarbons 34 54 48 000

Phenols (incl. bisphenol A) 33 860 480 000

Aliphatics 28 150 92 000

Other aromatic acids 27 950 170 000Other aromatic acids 27 950 170 000

Other alcohols 32 180 170 000

Ethers 22 105 14 000

Phthalates and adipates incl. metabolites 16 570 190 000

Other halogenated hydrocarbons 14 340 46 000Other halogenated hydrocarbons 14 340 46 000

Phenoxy acids 13 1200 180 000

Aldehydes 12 42 1 700

Chlorophenols 5 2 450 15 000

Cyclic hydrocarbons 5 94 20 000

Siloxanes 4 44 19 000

Sum: 611

24

Hazard Assessment (PBT)Hazard Assessment (PBT)Substances

Detected 600Assessed (2/6; >100 ng/L) 140PropertiesHighly persistent 3Persistent 47

Highly bioaccum. 1g yBioaccumulating 3Highly toxic 26g y to c 6Toxic 50

25

Directed Screening of Br-Compounds in BiotaCompounds in Biota Identification of persistent compoundsde t cat o o pe s ste t co pou ds

Identification of metabolites

Example: Diloma snail from New Zealand

26

Sea Snail extract (non-polar)

27

Sea Snail extract (non-polar)

28

Isotope Cluster Search

100

Br

100

Br2

100

Br3

100

Br4

60

80

100

60

80

100

60

80

100

60

80

100

20

40

20

40

20

40

20

40

0

M +2 +4 +6 +8 +10

0

M +2 +4 +6 +8 +10

0

M +2 +4 +6 +8 +10

0

M +2 +4 +6 +8 +10

29

Script Proceedure1. Search High toHigh toLow mass

3.Relative

2.

Relative abundance

30

Intensity

Script Proceedure

44.Evaluation of IsotopeClusters

31

2 Br-ScriptRules:

Intensity > 100 auFunction Bromine2()

Bromine2 = FalseMass = EndMass() Finish = StartMass()

Abundance > 20% Ion ratios M+/(M+2)+ 48‐58%

Finish = StartMass()Trappmass = 0Do While Mass>Finish

If intensity(Mass) > 100 thenIf Trappmass = 0 and /( )

(M+4) +/(M+2) + 43‐53% (M+3) +/(M+2) +     < 25%

If Trappmass 0 and _intensity(Mass) > 100 and _abundance(Mass) > 20 thenTrapmass = Mass

End ifR = Ratio(Mass–2 , Mass)R1 = Ratio(Mass+1, Mass)R2 = Ratio(Mass+2, Mass)If R>0.48 and R<0.58 and _

R2<0 53 and R2>0 43 and 80

100

Br2

Donald C. Hilton, LecoCurrent Trends in Mass SpectrometryJuly 2007

R2<0.53 and R2>0.43 and _R1<0.25 then Bromine2 = TrueExit do

End ifEnd if20

40

60

80

End ifMass = Mass – 1 Loop

End Function0

20

M +2 +4 +6 +8 +1032

Sea Snail extract (non-polar)

22 Br

3 Br

4 Br

33

Tribromoanisole (Cork Smell)

900

1000 62

Peak True - sample "3241_14 Diloma :1", peak 3379, at 1274 , 0.300 sec , sec

800

900

600

700 74

400

500 329 141

200

300 344

301

155 250

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

100250

222 118 90 197

171 266

34

MeO-TeBDE

900

1000 75

Peak True - sample "3241_14 Diloma :1", peak 9574, at 2414 , 0.770 sec , sec

800

900

600

700 62

356 420

400

500

516 204 178 154 313 131

200

300

341 235

102

50 100 150 200 250 300 350 400 450 500 550

100102 260 90

277 297 404

35

Sea Snail extract (non-polar)

2

MeO-TeBDEMeO-TrBDE

454-Br4

TeBDE

2 Br

3 Br

4 Br

MeO-TeBDDs ?446-Br4

TrBADiBA

Br4

TrBDDTeBDD

DiBA

36

MeO-TeBDD ?

900

1000 74

Peak True - sample "3241_14 Diloma :1", peak 10649, at 2750 , 0.770 sec , sec

800

900

600

700

400

500 530

281 61

93

200

300

155 124 193

265 487 327 434

50 100 150 200 250 300 350 400 450 500 550

1003

249 515 218 408 299 355 378

37

MeO-TeBDD: GC-HRT confirmation

Exp: 509.7096Theory: 509.7103Diff 1 3 ppmDiff. - 1.3 ppm

38

Natural Products in Marine Sponge

Utkina, N.K. et al. J. Nat. Prod., 2001, 64 (2), pp 151–153. DOI: 10.1021/np000354439

GC-HR-ToF-MSFolded Flight Path of up to 40 m yields ultra-high resolution

Vernchikov et.al.US Patent 7385187

Allows ultra-fast capture of high resolution spectra

40

Chlorine-filter Isolates peaks that may contain chlorines Retains spectral peaks spaced by the mass difference p p p yof 35Cl and 37Cl, i.e. 1.997 +/‐ 0.001

Tested on an marine bird egg sample (50 to 60 min)Tested on an marine bird egg sample (50 to 60 min)

41

Low intensity HxCB

TIC

*

XIC, low resolution

* XIC, high resolution*

42

Low intensity HxCB

43

STP efficiency evaluation

Focus on POPs and Semi‐POPs

GC×GC analysis of, e.g. raw and treated water

All peaks (compounds) are detected and integrated All peaks (compounds) are detected and integrated

Treated water used as template to which the samples are dcompared

Area ratio = 1, treatment failed Area ratio << 1 treatment successful Area ratio << 1, treatment successful Area ratio > 1, transformation product

44

Expected STP Removal vs. RT

2 0

1.5

2.0 s

y

0 5

1.0

Polarity

0

0.5

0 1000 2000 3000 4000 s(67 min)(67 min)

Volatility

45

Expected STP Removal vs. RT

2 0

1.5

2.0 s

y

Polar, Non volatile

Volatile, Polar

0 5

1.0

Polarity Non‐volatile

Non‐Polar  

Polar

Volatile  

0

0.5

0 1000 2000 3000 4000 s(67 min)

Non Polar, Non‐volatile

Volatile, non‐polar

(67 min)

Volatility

46

Bubble Plot of % STP Removal

2 0

1.5

2.0 s

y

0 5

1.0

Polarity

0

0.5

0 1000 2000 3000 4000 s(67 min)(67 min)

Volatility

47

STP Breakthrough % 

1.5 

1.0 

2 0

0.0 

0.5 

1.5 

2.0 

seconds

) 

> 35%

0.5 

1.0 

Polarity

2D (

 35% 

0.0 0  500 1000 1500 2000 2500 3000 3500 4000

Volatility 1D (seconds)48

Library IdentificationTentative name   

 

Effluent conc. 

(ng/L) 

Functional group(s)  Retention time  Break‐through (%) 

% in water  Origin 

Acid Am

ine/amArom

atic HalogenateKetone/estNitro 

OH 

PhosphateS/O

‐ether S,N

,O‐hete

Other 

1D (s)  2D (s) 

Influent 

Effluent 

ide 

ed ter 

  erocyclic

2,4,7,9‐Tetramethyl‐5‐decyn‐4,7‐diol  12000 X X   1042  0.46 73 85 97 Defoamer in paintBenzenesulfonamide, N‐butyl‐  5500 X X   1770  1.53 100 38 89 PlasticizerTris(butoxyethyl) phosphate 3600 X X 2586 0.75 100 94 98 Floor polishTris(butoxyethyl) phosphate  3600 X X   2586  0.75 100 94 98 Floor polishBenzothiazole, 2‐(methylthio)‐  2200 X S NS   1454  1.70 100 88 99 Rubber industryTricyclo[5.2.1.0(2,6)]dec‐3‐en‐10‐one  1500 X   842  1.17 77 100 100 NaturalTris(3‐chloropropyl) phosphate (TCPP 1)  1500 X X   1758  1.04 67 85 98 Flame retardantBenzophenone 930 X X   1494  1.53 100 87 94 UV initiatorBenzothiazole, 2‐(methylthio), methyl  650 X S NS   1820  0.23 56 100 100 Rubber industryEthyl citrate 520 X X 1506 0 97 71 100 100 Plasticizer food additiveEthyl citrate 520 X X   1506  0.97 71 100 100 Plasticizer, food additive Caffeine  460 X N   1916  1.82 100 87 100 Coffee, soft drinksTris(3‐chloropropyl) phosphate (TCPP 2)  390 X X   1780  1.05 63 81 98 Flame retardantTris(2‐chloroethyl) phosphate  360 X X   1726  1.44 100 97 98 Flame retardant2,2,2‐Trichloro‐1‐phenylethanol  320 X X X   1268  1.34 100 100 100 Flavor and fragranceOxybenzone 230 X X O   2144  1.54 100 68 97 Cosmetics, sunscreen4‐tert‐butyl‐cyclohexanone  220 X   750  0.71 100 100 100 Cosmetics, fragranceEthosuximide 150 X N   812  1.08 98 79 98 PharmaceuticalTris(1,3‐dichloroisopropyl) phosphate (TCDPP) 150 X X X   2534  1.39 72 76 98 Flame retardantIsoquinoline 140 X N   770  1.18 100 97 100 Traffic4‐tert‐octyl‐phenol 140 X X   1410  0.96 69 37 86 Surfactants; resinsHexadecenoic acid, Z‐11‐*  120 X   1944  0.53 37 0 40 Natural Diethyltoluamide (DEET)  110 X X   1386  1.20 100 100 100 Insect repellentBenzenesulfonamide, N‐ethyl‐2‐methyl‐  100 X X   1554  1.70 100 99 100 Plasticizer2,3,6,7‐Tetramethylquinoxaline  100 X N   1586  1.16 56 73 100 Traffic

  Tentative structures – HR‐ToF‐MS verification in progress ! 49

GC-HRT: Modes of Operation

High ResolutionNominal

Mirr

Ultra‐High Resolution

AA LensAAD

AD

Lens

Mirr

D

Mirr

L = 2mR = 1 800

L = 20mR = 25 000

L = 40mR = 50 000R = 1,800 R = 25,000 R = 50,000

50

GCxGC-HRMS validation

One false positive detected

Remaning compounds were in excellent agreement!

%

40%

10%

20%

30%

0%

10%

‐2 to ‐3 ‐1 to ‐2 0 to ‐1 0 to 1 1 to 2 2 to 3

ppm deviation51

Identification of unknown unknown

800

1000

ance

)

181.00167

151.

0091

014

8.02

210

0041

3

860

69 595

Caliper – Effluent sample

200

400

600

800

Area

(Ab

unda

108.

0

153.

978

134.

0066

196.

9965

90.0

3545

68.9

8131

63.0

2519

122.

0068

8

81.9

8893

60 80 100 120 140 160 180 2000

M/Z

1000

nce)

181Library: NIST- Benzothiazole, 2-(methylthio)-

Similarity 680

200

400

600

800

Area

(Ab

unda

n

148

108

69 134

63 122

165

60 80 100 120 140 160 180 2000

M/Z

52

Identification of unknown unknown

800

1000

ance

)

181.00167

151.

0091

014

8.02

210

0041

3

860

69 595

Caliper – Effluent sample+ 0.8 ppm

15.99492 amu(oxygen 15 99491 amu)

200

400

600

800

Area

(Ab

unda

108.

0

153.

978

134.

0066

196.

9965

90.0

3545

68.9

8131

63.0

2519

122.

0068

8

81.9

8893

(oxygen 15.99491 amu)

60 80 100 120 140 160 180 2000

M/Z

1000

nce)

181Library: NIST- Benzothiazole, 2-(methylthio)-

Similarity 680OH

200

400

600

800

Area

(Ab

unda

n

148

108

69 134

63 122

165

60 80 100 120 140 160 180 2000

M/Z

53

b l d d hMBT bacterial degradation pathway

54

Alternative ”Dioxin” Analysis HRMS – The Golden Standard 2 GC injections (non‐polar + polar column)j ( p p )

Alternatives/ GCxGC‐ECD/LRMS

GC‐MS‐MS GC‐HR‐ToF‐MS

Analysis of 17 2,3,7,8‐PCDD/F and 12 WHO‐PCBs in Food and FeedFood and Feed

Full Congener‐Specific Analysis of PCBs and PCDD/Fs for u Co ge e Spec c a ys s o C s a d C / s oSource Tracking

55

Selection of GCxGC columns

The non‐polar J&W DB‐XLB gives the best 1D‐resolution !

What 2D‐column to use ?

56

DB-XLBxNN correlationsLiquid crystalCyanopropyl (dipolar)

SolwaxSil88LC50

CP-C18DB210HP-35

O t lA1701DB1HP1301

SPB-20DB5CP-13Octyl

Optima6HT-8PMCDSPB-20

0.94 0.95 0.96 0.97 0.98 0.99 1.00

DBXLBOpt a6

57

209 PCBsec

LC-50 (liquid crystal)TCN

81

77105 126 169

1574

5sec

28 128156

170

194

206

31 47

5666 85

99 110

118 138153

87

74137

180

114123

167

189

2

3

4020 50 60 7030 80

44

49

52101 141

149151

9787 180

1872021

4020 50 60 7030 80

SP-2340 (polar, cyanopropyl) 7781 16912656

4

sec

81

194

189

169

170180

156157128

167

141138

105114118

149110

85

8797

99

56

66

74

31

47

52

44

TCN28

2

3

4020 50 60 70 min30 80

167

202187

137123151101

7449

1

58

Ortho-Effect on Liquid CrystalsR i l b i

Tetra Tri o o’ Di o o Di Mono Non

Rotational energy barriers:

Tetra Tri o,o’-Di o,o-Di Mono Non

60 40 17 - 20 14 - 17 7 - 8 ca. 2

kcal/mol

59

PCB enantiomer separation

P

Permethylated -cyclodextrin (PMCD)

60

PCBs i Seal Blubber

61

All 209 resolved except... DB-XLB / SP2340 (cyanopropyl) 15 PCB pairs 7 Aroclor PCB pairs7 Aroclor PCB pairs

DB-XLB / LC-50 (liquid crystal) 15 PCB pairs15 PCB pairs 5 Aroclor PCB pairs All WHO and indikator PCBs resolved!

Dual second columns 6 PCB pairs

All A l PCB All Aroclor PCBs Chirasil-Dex / LC-50 or 100% cyanopropyl (VF-23MS) 9 chiral PCBs resolved 7 separated from Aroclor PCBs

62

Quantification of PCB/dioxins

Fish Oil

63

Quantification of PCB/dioxins

Compound feed

64

Summary• DBXLB×LC50 provides separation of: WHO‐PCBs and 2,3,7,8‐PCDD/Fs from each other , , , /

and from matrix WHO‐PCBs from other PCBs  16/17 Toxic PCDD/Fs from other PCDD/Fs

• The results agree well with GC‐HRMS data 

GC GC h t ti l ti th d f th• GC×GC has potential as a routine method for the determination of TEQs in food and feed samples

• GCxGC may also provide PCB enantiomer signatures65

Conclusions/ Recommendations

ToF‐MS and HR‐ToF‐MS are powerful techniques!p q

Balance front‐end and back‐end selectivity!y(Don’t forget the chromatography part)

Adopt to the problem at hand – Simplify if possible!

GCxGC‐soft ionization‐HRMS would be very useful for identification work involving complex mixtures

66

The End...

Contact information:P t  H l d

Thank you for your Peter Haglund

Umeå UniversityD   f Ch i

your attention

Department of Chemistry90187 Umeå, Sweden

h l d hpeter.haglund@chem.umu.se+46‐90‐7866667

67