GC-MS and LC-MS methods for Flame Retardant (FR) … spectrum of endosulfan (C9H6Cl6O3S, m. wt. 406 g/mole, CAS number 115-29-7) in EI (upper panel) and ECNI ... problem GC-EI-MS or

Sicco Brandsma

GC-MS and LC-MS methods for

Flame Retardant (FR) analysis

2

Outlines

• Gas Chromatograph

• Different detectors ECD FID NPD and MS

• Focus on Mass spectrometry

• Different GC parameters

• BFRs PBDEs, BDE209, TBBP-A and HBCD

• “New” compounds PFRs, DBDPE, BTBPE, TBPH,

TBB and, PBT

3

Gas chromatograph

Flow controller

Carrier gas

Injector port

Chromatogram

Detector

GC column

Temperature programmed

column oven

Multiplier

4

GC-Detectors

• Flame Ionisation detector (FID)

• Sensitive for detecting hydrocarbons

• Not really selective measures all flammable compounds

• Electron Capture Detector (ECD)

• Sensitive to halogens and nitro compounds

• Low linear range compared to FID

• Nitrogen Phosphorous Detector (NPD)

• Very sensitive but selective detector that responds almost exclusively to

nitrogen and phosphorous compounds

5

ECD chromatogram

• Low selectivity: coeluting compounds can not be separated

6

• Electron Impact (EI)

• Hard ionisation fragmentation of the molecular ion

• Library search for EI spectra

• Chemical ionisation (CI)

• Soft ionisation clear spectrum of the Molecular ion

• Use of reagent gas (Methane)

• Positive PCI or negative ECNI mode

• ECNI very sensitive for PBDEs

Mass Spectrometry (1)

7

Mass Spectrometry (2)

• MS

• Quadrupole (SIM or SCAN)

• Ion trap

• Time Of Flight (TOF)

• Sector instruments (magnet)

• Tandem Mass spectrometry (MS/MS)

• Two analysers coupled by an collision cell

8

327.0145

327.0009

327.0081

Full scan

SIM m/z 327

MS/MS 327 251

HRMS

TCPP TCPP

TCPP

Mass Spectrometry (3)

9

• Selection of the Stationary Phase

• Column Dimension

• Temperature Program

• Linear Gas Velocity

• Type of Carrier Gas

GC parameters

10

Resolution

R = 0.5

R = 0 R = 1

R = 1.5

R = ΔtR × 2 / (W1 + W2)

R = √N / 4

or

Changing mobile phase composition

Changing column temperature

Changing composition of stationary phase

Changing the column length

• Improving the resolution:

11

• The most widely used stationary phases for halogenated pollutants

are: DB-1, DB-5, BPX-5, HT-8, CP-Sil8 CB or CP-Sil 19 CB

polarity scale Stationary phase brand and type names

5 100% Dimethyl polysiloxane ZB-1 MS, CP-Sil 5 CB, DB-1, HP-1 MS, RTX-1

8 5% Phenyl-(arylene)- 95% methyl polysiloxane ZB-5 MS, CP-Sil 8 CB, DB-5 MS, HP-5 MS, RTX-5 MS

17 50% Phenyl-50% methyl polysiloxane ZB-50, DB-17 MS, HP-17, RTX-50

19 14% cyanopropyl-phenyl/86% dimethylpolysiloxane CP-Sil 19 CB

24 75% Phenyl-25% methyl polysiloxane ZB-50, CP-Sil 24 CB

52 polyethylene glycol ZB-WAX, DB-WAX, CP-WAX 52 CB

88 100% 3-cyanopropylpolysiloxane BPX70, CP-Sil 88 CB, RTX-2330

Selection stationary phase

12

•Effect of Diameter (ID): Stronger than that of Length

• Length: > 100m: to much pressure required, too long retention

times, < 25: serious loss of separation • ID: <0.10 mm: hardly possible (columns >20m) >0.25 mm: serious loss of separation • Film Thickness: < 0.1 mm: risk for degradation due to poor coating > 0.3 mm: long retention times, risk for tailing peaks

Column Dimensions

13

Temperature Program

Temperature program: 4 min, 80oC;

5oC/min to 270; 48 min 270oC.

Temperature program: 4 min, 70oC;

30oC/min to 215oC; 40 min 215oC; 5oC

/min to 270oC; 33 min 270oC

14

The Optimum mobile phase velocity

Optimum resolution

The H-u curve is very useful to determine the optimum mobile phase velocity

uopt at which the highest column efficiency will be attained

H = HETP (plate height)

A = Eddy diffusion term

B = Longitudinal diffusion term

u = Linear velocity

C = Resistance to mass transfer coefficient

N = Plate number

L = Column length

Van deemster equation

15

N2

He H2

U

H

Selection of carrier gas

•Hydrogen provide optimum resolution at the highest carrier gas velocity; optimum separation within an the sorter time

16

PBDEs

17

• PBDEs

• GC-EI-MS, GC-ECNI-MS, GC/HRMS or LC-MS/MS

• Advantage and Disadvantage

• EI versus ECNI

• Elution of interfering compounds

• Different GC-columns

• Internal standards

18

GC/LR-ECNI-MS GC/LR-EI-MS GC/HRMS LC-MS/MS

LOD 30 fg - 1.7 pg* 0.5 - 32 pg* 0.1 - 4.8 pg* 12 - 30 pg*

Sensitivity ＋＋ － ＋ －－

Selectivity No yes yes yes

Labeled standards No (only for BDE209) yes yes yes

Thermal degradation yes yes yes no

Expensive ＋－ － ＋＋ ＋

Expert training － － ＋＋ ＋

Libary search No yes yes No*Eljarrat et al, (2002) *Eljarrat et al, (2002) *Alaee et al, (2001) *Abdallah et al, (2009)

J Mass Spectrom 37: 76-84 J Mass Spectrom 37: 76-84 Chemosphere 44: 1489-1495 Anal. Chem., 81, 7460–7467

Advantages and Disadvantages

19

Mass spectrum of endosulfan (C9H6Cl6O3S, m. wt. 406 g/mole, CAS number 115-29-7) in EI

(upper panel) and ECNI (lower panel). EI produces extensive fragmentation with very little molecular

ion formed (M+), but the molecular anion (M–) is the most abundant peak in ECNI. The fragment at 372

m/z is formed by loss of chlorine which also appears at 35 and 37 m/z.

Agilent Technologies

GC-EI MS versus GC-ECNI MS

20

Interfering compounds

•PBBs: BB-153 may interfere with

BDE-154 when ECNI-MS is applied.

•TBBPA may interfere with BDE-153

when ECNI-MS is applied.

•MeO-PBDEs: 5-Cl-6-MeO-BDE47

and 6’-Cl-2’-MeO-BDE68 may

interfere with BDE-99 when ECNI-

MS is applied.

•DiMeO-PBDE: 2’,6-DiMeO-BDE68

may interfere with BDE-99 when

ECNI-MS is applied.

21

This database is useful for determining the most suitable column for quantitative,

congener-specific PBDE analysis.

Resolving power of seven different GC- columns

Korytár et al,(2005) J Chromatogr A 1065:239–249

22

15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Time-->

BDE190

54.1

BB169

49.4

BDE138

46.1

HBCD

45.4

TBBP-A+

BDE153

43.8

BB153+BDE154

42.3

me-TBBP-A

42.2

BDE85

41.2

BDE99

39.1

BDE119

38.3

BDE100

37.8

BB101

35.8

BDE77

35.3

BDE66

33.6

BDE47

32.6

BDE71

31.5

BDE75

30.5

BB49

25.3

BB52

24.2

BDE28

21.2

Internal standard

CB112

17.5

BB1

5

13.8

CP-Sil8 CB MS 50 m x 0.25 mm x 0.25 µm

GC-ECNI MS chromatogram

23

Internal standards

•Internal std for GC-ECNI-MS

• BDE58

• BDE77

• BDE128

• 13C12 BDE209

• F-PBDEs

24

Conclusions

• GC-ECNI-MS is the best all-round method to

analyze PBDEs

• Be aware of coeluting of “new” brominated

compounds (only measuring m/z 79 and 81 in

ECNI mode)

• However when higher detection limits are not a

problem GC-EI-MS or LC-MS/MS are good

alternatives

• Advantage of EI over ECNI mode is the use of

labelled internal standards

25

BDE209

26

BDE209

BDE206

BDE207

BDE208

DB-5, 15 m x 0.20 mm x 0.2 µm

Pulsed splitless injection

degradation

of decaBDE

>15m

BDE209 GC/ECNI-MS

27

Loss of response of higher brominated PBDE

due to matrix

decaBDE

Matrix effects

28

MS spectra (ECNI) of BDE209

29

492

496 498

500

13C12 decaBDE

482

484

486 488

490

494

492

496

MS spectra (ECNI) of 13C12 BDE209

Bjorklund et al, 2003 J. Mass Spectrom. 38, 394-400

30

200°C

250°C

300°C

15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00

Ion source (ECNI) temperature

31

• Use 13C decaBDE as internal standard

• Use <15 m GC column

• Use short injector residence times (pulsed splitless) or

on-column injection

• Reduce sample exposure to glassware and reduce if

possible number of pieces of glassware used

• If possible physically segregate sample by type and

analysis in separate areas

• Reduce UV-light exposure (UV-filters or amber

glassware)

• Reduce and avoid dust

Summary for BDE209

32

HBCD GC or LC ?

33

15.00 20.00 25.00 30.00 35.00 40.00 45.00

2000 4000 6000 8000

10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000 44000 46000 48000 50000

Rt (min)

Abundance

GC-MS (ECNI) HBCD (I)

34

15.00 20.00 25.00 30.00 35.00 40.00 45.00

500

1000

1500

2000

2500

3000

3500

Rt (min)

Abundance

TetraBCDe

PentaBCDe

HBCD

GC-MS (ECNI) HBCD (II)

35

m/z 79

28 75

71

49

HBCD

15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 0

500

1000

1500

2000

2500

3000

Rt (min)

Abundance

47

58

66

77

100

119

99

85 Dime

-TBBP

-A

154

153 138

183 190

GC-MS (ECNI) HBCD and PBDEs

36

32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

Rt (min)

Abundance

BD

E1

53

BD

E1

54

BD

E8

5 B

DE

99

BD

E1

00

BD

E6

6

BD

E5

8

BD

E4

7

BD

E4

9

HBCD

Sediment sample, Scheldt estuary

37

43.00 43.20 43.40 43.60 43.80 44.00 44.20 44.40 44.60 44.80 0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

Rt (min)

Abundance α-HBCD

β-HBCD

γ-HBCD

GC-MS ECNI response HBCD isomers

38

4x10

0

1

2

3

4

Abundance vs. Acquisition Time (min)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

- MRM (652.7 -> 79.0) paling 1-1 met water gradient 1.d

1 1

4x10

0

1

2

3

4

- MRM (652.7 -> 81.0) paling 1-1 met water gradient 1.d

1 1

3x10

0

2

4

- MRM (652.7 -> 652.7) paling 1-1 met water gradient 1.d

1 1

3x10

0

2

4

6

- MRM (640.7 -> 79.0) paling 1-1 met water gradient 1.d

1 1

3x10

0

2

4

6

- MRM (640.7 -> 81.0) paling 1-1 met water gradient 1.d

1 1

3x10

0

0.5

1

- MRM (640.7 -> 640.7) paling 1-1 met water gradient 1.d

1 1

α β

γ 13C-labeled

13C-labeled

13C-labeled

Eel, LC-MS/MS, HBCD

39

1x10

0

1

2

3

Abundance vs. Acquisition Time (min)3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8

- MRM (640.7 -> 79.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d

7.575

6.8384.343 5.674 7.307

6.4895.135 7.0113.862

1 1 2 2 3 3

1x10

0

1

2

- MRM (640.7 -> 81.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d

7.548

4.6223.394 7.6947.1354.432 5.172 5.844 6.163 6.7333.679

1 1 2 2 3 3

1x10

0

0.5

1

- MRM (640.7 -> 79.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d

1 1 2 2 3 3

1x10

0

0.5

1

- MRM (640.7 -> 81.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d

1 1 2 2 3 3

1x10

0

2

4

6

- MRM (640.7 -> 79.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d

9.378

10.8368.663 9.601 9.877 11.4479.044 12.64810.271 11.742

1 1 2 2 3 3

1x10

0

2

4

- MRM (640.7 -> 81.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d

9.389

8.838 9.922 11.28310.512 12.699

1 1 2 2 3 3

Zorbax Eclips, 2.1 x 150 mm x 3.5 um

6 pg

2x10

0

1

2

Abundance vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7

- MRM (640.3 -> 79.0) HBCD0012.d

0.725

0.261 1.1440.835

1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

2x10

0

1

2

- MRM (640.3 -> 81.0) HBCD0012.d

0.7151 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

2x10

0

1

2

3

- MRM (640.3 -> 79.0) HBCD0012.d

1.5131 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

2x10

0

0.5

1

1.5

2

- MRM (640.3 -> 81.0) HBCD0012.d

1.5061 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

2x10

0

1

2

- MRM (640.3 -> 79.0) HBCD0012.d

2.292

2.177

1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

2x10

0

0.5

1

1.5

2

- MRM (640.3 -> 81.0) HBCD0012.d

2.3091 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

Zorbax SB-C18, 2.1 x 30 mm x 3.5 um

6 pg

Normal LC vs. Rapid Resolution LC columns

40

0

50

100

150

200

250

0 50 100 150 200 250

LC-MS

GC

-MS

-GC: internal standard conc. (no 13C)

-GC: response factors isomers differ

-isomers transferred into each other

-LC: Ion-suppression

Quantification of HBCD: LC vs. GC

41

Tomy et al. 2005, Rapid Comm. Mass Spectr. 19, 2819-2826

Uncorrected Corrected, 13C-labelled

LC-MS/MS Ion suppression of HBCD signal by matrix compound

42

HBCD • GC: Thermal degradation on GC column • Thermal degradation causes interfering peaks with PBDEs • LC-MS preferred method • Use of 13C-labeled IS is preferred • LC-MS/MS (1 pg) as sensitive as GC-MS (0.5 pg)

Conclusions

43

TBBP-A GC or LC ?

44

GC-MS TBBP-A

• Without derivatization poor calibration curves

• Interference TBBP-A and BDE153

• With derivatization -> dime-TBBP-A

45

GC-MS TBBP-A

43.60 43.80 44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40 45.60 45.80

TBBP-A standard 10 ng/ml, 1 µl injection

43.60 43.80 44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40

Sediment +10 ng/ml TBBP-A

m/z 543

m/z 543

Sediment, Scheldt estuary

46

44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40 0 50

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800

Abundance 44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40 45 50 55 60 65 70 75 80 85 90 95

100 105 110 115 120 125 130 135 140

Abundance TBBP-A m/z 543

BDE153+TBBP-A m/z 79

TBBP-A and BDE153

47

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Rt (min)

TBBP-A

13C-TBBP-A

m/z 543

m/z 555

500 510 520 530 540 550 560 570 580 590 600 610

m/z

543 555

541 553 545

557

539 547 559

TBBP-A 13C-TBBP-A

• LC-MS easy to perform

TBBP-A analysis

48

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Time 0

100

%

0

100

%

C143350 6: SIR of 1 Channel ES-

640.50

6.15e4

Area

30.92

28.73

C143350 3: SIR of 1 Channel ES-

542.90

1.65e6

Area

23.01

a-HBCD g-HBCD

B-HBCD

TBBP-A

LC-MS/MS: HBCD and TBBP-A

49

TBBP-A

• LC-MS preferred method, use 13C-labelled IS

• GC-MS (ECNI) BDE153 and TBBP-A interfere

• 13C-TBBP-A can cause interference with BDE153

• Sample treatment most critical step

Conclusions

50

PFRs

51

PFRs

TiBP TBP TCEP TCPP

TDCPP TBEP TPP EHDP

TEHP TCP DBPhP DPhBP

52

Compound Acronym Retention time Precursor Ion Product Ion Fragmentor Collision Energy

tri-n-butyl phosphate D27 * TBP D27 15,8 294.4 166.1 100 15

294.4 102.1 100 15

tri-n-butyl phosphate TBP 15,6 267.2 155.1 100 10

267.2 99.1 100 10

tris(2-chloroethyl) phosphate TCEP 6,4 287 99.1 100 19

TCEP

285 63.1 110 18

tris-iso-butyl phosphate TiBP 15,8 267.2 155.1 75 8

267.2 99.1 75 8

tris(chloro 2-propyl) phosphate TCPP 11,6 329 99 100 15

tris(1,3-dichloro-2-propyl)phosphate TDCPP 14,4 433 99.4 125 30

tris(2-butoxyethyl) phosphate TBEP 16,4 399.4 299.2 125 15

399.4 199.1 125 15

tris(2-ethylhexyl) phosphate TEHP 22,8 435.4 113.3 125 10

435.4 99.1 125 10

triphenyl phosphate d15 * TPP d15 16,4 342.2 160.2 175 40

342.2 82.1 175 40

triphenyl phosphate TPP 14,5 327.2 152.1 175 35

327.2 77.1 175 35

tricresyl phosphate TCP 17,1 369.2 165.2 175 35

369.2 91.1 175 35

2-Ethylhexyl diphenyl phosphate EHDP 17,5 251.1 152.2 150 28

251.1 77.2 150 28

Butyl diphenyl phosphate BDphP 15 307.2 251.1 100 6

Dibutyl phenyl phosphate DBphP 15,4 287.2 175.1 100 8

HPLC column 150 x 3 mm Luna C18 (2) 3 um column (Phenomenex)

Acronyms, Rt and MRM for twelve PFRs

53

GC/EI-MS

Van den eede et al, (2011) Environment International 37 454–461

54

PFRs in Belgian home dust (n=33) μg/g

Van den eede et al, (2011) Environment International 37 454–461

55

2100 5400

ng/g

lw

/TO

C

ng/g

lw

/TO

C

Pelagic food web Benthic food web

Pelagic and Benthic food web

56

Conclusion GC or LC • LC-MS/MS is more selective • Use of labelled Std • Less interference of matrices (biota) GC/EI-MS • Good method for analyzing dust (less matrices) • No ion suppression • Separation of isomers (TCPP and TCP)

57

TBB and TBPH

BTBPE and DBDPE

58 Ali et al., (2011) Anal. Bioanal. Chem., 400, 3073-3083

59

GC/ECNI-MS chromatograms revealing the relative retention times of the

primary BDE congeners, TBB and TBPH on a 15 m DB5-MS column

Stapleton et al, (2008)Environ. Sci. Technol. 42, 6910–6916

GC/ECNI-MS chromatogram

60

TBB was quantified using ion fragment (m/z) 357 (Quant)and 471 (Qual)

TBPH was quantified using ionfragments (m/z) 463 (Quant) and 515 (Qual)

Stapleton et al, (2008)Environ. Sci. Technol. 42, 6910–6916

Full scan spectra of TBB and TBPH

61

GC/ECNI-MS chromatogram (m/z 79-+81-) of an standard solution

Kierkegaard et al, (2004) Environ. Sci. Technol., 38, 3247-3253

DBDPE first detected in the environment

62

Ali et al., (2011) Anal. Bioanal. Chem., 400, 3073-3083

DBDPE and BTBPE

63 Ali et al., (2011) Anal. Bioanal. Chem., 400, 3073-3083

How to separate HCDBCO from TBB

64

Concentrations observed in Dust

65

PBT

GC/ECNI-MS

m/z 79, 81

66

Review about Novel BFRs

67

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