Analysis and Significance of HBCD and TBBP-A · 2011-07-26 · stereoisomers & lack of isomer...

Analysis and Significance of HBCD and TBBP-A

Colin Allchin & Philippe BersuderCefas Burnham Laboratory

HBCD (Hexabromocyclododecane)

• HBCD is a high volume (20,000t worldwide), additive brominated flame retardant i.e. directly applied to the product requiring flame retardancy

• HBCD has been used for 20 years

• Major applications:• Extruded or expanded polysterene foam e.g rigid insulation

panels, blocks for construction• Textile back coatings e.g upholstery• Electrical housing

• Exhibiting all the classic characteristics of a POP HBCD is suspected of disrupting endocrine system by impairing thyroid function and of causing liver cancer

Complex stereochemistry

• HBCD is a technical mixture containing 16 possible stereoisomers, 6 pairs of enantiomers and 4 meso forms. (See Heeb et al Chemosphere 61 (2005) 65-73).

• For practical reasons we need only be concerned with the three dominant HBCD diasteromeric pairs (racemic mixtures) designated as α-, β- and χ-HBCD.

Composition of technical HBCD*

0.3%ε (meso)3/4/9/10b

3/4/9/10b

8a/b

7a/b

6a/b

Structure

δ (meso)

χ (racemic)

β(racemic)

α(racemic)

Designation

0.5%

70.35 - 81.6 %

5.8 – 12.83 %

11.8 – 16.20 %

Proportion in tech mixture

* After Heeb et al

The chemical structure of α-, β- and γ-HBCD diastereoisomers

α- HBCD β-HBCD γ-HBCD

Molecular Weight = 641.6

Common BFR structures

Analytical method development

• Inherent problems associated with traditional techniques (GC) used to quantify persistent organic compounds i.e. thermal rearrangements and decomposition of stereoisomers & lack of isomer resolution

• Use of LC-MS circumvents these problems

Isotopic distribution for Bromine (Br)Br 20/09/2004 17:59:59

Br1SimulationBrProfileResolution:

Daltons 0.25at 5% height

Charges 1Chrg dist 0Ions 2Min Ion Ab. 1e-020Min Ions 5000Max Ions. 20000

77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5m/z

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Abun

danc

e

78.9280.91

Isotopic distribution for HBCD anion [M-H]

C12H17Br6 16/04/2003 11:08:26 C12 H17 Br6

SimulationC12H17Br6ProfileResolution:

Daltons 0.25at half height

Charges 1Chrg dist 0Ions 517Min Ion Ab. 1e-020Min Ions 5000Max Ions. 20000

633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649m/z

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

Abun

danc

e

640.63

638.63642.63

636.63644.62

641.63639.63 643.63

634.64 646.62637.64 645.63635.64 647.63 648.63 649.63

Ion Trap MS Detector parameters optimisation

Figure 5. Effect of Ion Transfer Capillary Temperature on HBCD ion abundance

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

4.00E+09

120 140 160 180 200 220 240 260

Ion Transfer Capillary Temperature (in °C)

Rela

tive

ion

abun

danc

e(T

IC)

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

3.00E+07

Rela

tive

ion

abun

danc

e(m

/z 6

40.7

Da)

TIC

m/z 640.7

Figure 2. Effects of mobile phase modifiers on HBCD parent ion [M-H]-

(m/z 640.7) intensity

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

3.00E+07

3.50E+07

ACN:H2O

ACN:FA

ACN:AA

ACN:AH

MeOH:H

2OMeO

H:FAMeO

H:AA

MeOH:A

H

Modifiers combinations

Rel

ativ

e io

n ab

unda

nc

m/z 640.7

Ion trap MS Detector parameters optimisation

Key: ACN: acetonitrile; FA: formic acid; AA: Ammonium acetate; AH: Ammonium hydroxide; MeOH: Methanol

γ-HBCD DIMERIC ADDUCT ION SPECTRA

1342.0

1357.9[2M+59]-

[2M+75]-[2M+37]-

1320.3

13801280 m/z

γ-HBCD DIMERIC ADDUCT ION SPECTRA

1342.0

1357.9

[2M+C2H3O2]-

[2M+C2O3H3]-[2M+Cl]-

O

OH

1320.3

13801280 m/z

Control of dimer formation by promoting HBCD-Clclusters (m/z 677 and m/z 1318) using NH4Cl as a

mobile phase additive

Mass spectra for γ-HBCD separated by HPLC in the presence of 10 µM NH4Cl

[M+Cl]- [2M+Cl]-

ITMS CHARACTERISATION OF ANALYTES

LCQ Advantage in negative ESI and SIM mode

TBBP-A (MW = 543.9) m/z 541 & m/z 543

α-, β- & γ- HBCD (MW = 641.7) m/z 677 & m/z 1318[M+Cl]- and [2M+Cl]-

13C12 TBBP-A (MW = 555.8) m/z 555 & m/z 553

d18 α-, β- & γ- HBCD (MW = 659.7) m/z 695 & m/z 1354[M+Cl]- and [2M+Cl]-

LIQUID CHROMATOGRAPHIC SEPARATION OFTBBP-A & HBCDs

LC ThermoFinnigan Surveyor Quarternary PumpColumn Phenomenex ‘Luna’ C18 (2 x 150 mm; 3µm)

+ (2 x 4 mm) Security Guard cartridgeColumn Temp 40 °CFlow Rate 200 µL min-1

Injection vol. 20 µLMobile Phase

Time (minutes)

20 mM ammonium

acetate (%)

methanol (%)

0.05 mM ammonium

chloride (%)

acetonitrile (%)

0.0 70 20 10 0 3.5 19 66 10 5

18.0 19 66 10 5 18.1 0 0 0 100 30.0 0 0 0 100 30.1 70 20 10 0 40.0 70 20 10 0

Calibration curves obtained for α-, β-, γ- HBCD using d18-HBCDs internal standards and 5 µM NH4Cl in the mobile

phase

HBCDs produce 2nd order calibration curves with a linear range from 10 to ca 500 ng/ml

α-HBCD([M+35]-+[2M+35]-)

0.0

0.5

1.0

1.5

2.0

2.5

0 100 200 300 400 500

Concentration (in ng/ml)

Are

a R

atio

β-HBCD([M+35]-+[2M+35]-)

0.00.2

0.40.6

0.81.0

1.21.4

1.61.8

2.0

0 100 200 300 400 500


Area

Rat

io

γ-HBCD ([M+35]-+[2M+35]-)

0.00.20.40.60.81.01.21.41.61.82.0

0 100 200 300 400 500


Are

a R

atio

R^2 = 0.999R^2 = 0.997R^2 = 0.995

Calibration curves obtained for TBBP-A using 13C12 TBBP-A as an internal standards and 5 µM NH4Cl in the mobile

phase

TBBP-A produces linear calibration curves over the 10 - 1000 ng/ml range

R^2 = 0.999

TBBP-A

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 100 200 300 400 500 600 700 800 900 1000


Are

a R

atio

Analytical scheme for HBCD & TBBP-AS o x h le t/M A S E /A S E

1 0 g a ir d rie d s e d im e n t/5 g b io ta1 3C 1 2 T B B P -A

d 1 8 H B C D s (a lp h a , b e ta & g a m m a )

2 g e q u iva le n t a liq u o t s e d im e n t/ 0 .2 5 ge q u ivq a le n t fa t

R e d u c e d to 1 .5 m l u n d e r O F N

C le a n u p b y H P G P C u s in g e th y l/a c e ta te :c yc lo h e x a n e 1 :1

A d d itio n a l c le a n u p w ith c o n cs u lp h u ric if n e c e s s a ry

R e c o n s titu te in m e th a n o l to 1 2 0 µ l

L C -M S

Trends in Analytical Chemistry, Vol. 25, No.4, 2006

HBCD QC using a Cod LRM (n=10)

α-HBCD β-HBCD γ-HBCD Σ-HBCDLRM concentration (µg/kg) 4.0 3.2 17.6 24.8Concentration (µg/kg) 3.5 3.0 13.4 19.9SD 0.7 0.3 2.7 2.2% RSD 21 9 20 11% Recovery 87 93 76 80

LOQ = 1 ng/g

HBCD Recovery from Silica(3g 3% Deactivated)

0

20

40

60

80

100

120

0-25ml Hexane 25-30ml 20%Ether/Hexane

30-35ml 20%Ether/Hexane




AHBCDBHBCDGHBCD

HBCD Recovery from Alumina(5% deactivated)

0

20

40

60

80

100

120

0-25ml Hexane 25-30ml 20%Ether/Hexane





AHBCDBHBCDGHBCD

Eel sample from the river Thames (2005)RT: 0.00 - 29.97 SM: 7G

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

14.74

12.60

10.58 25.55 28.6720.67 27.5024.0522.0315.72 19.24

12.86

20.9323.01 26.20 28.8727.3110.51 14.94 19.4317.22

NL:2.69E4m/z= 694.10-695.10+1353.60-1354.60 MS 05-4493

NL:5.30E4m/z= 676.00-677.00+1317.30-1318.30 MS 05-4493

d18 α-HBCD

d18 β-HBCD d18 γ-HBCD

α-HBCD

ΣHBCD= 60 ng/g

RT: 0.00 - 29.99 SM: 5G

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

13.71

12.93

11.96

10.99 15.59 25.1117.94 28.0423.4820.09 22.3712.15

13.90 22.1110.79 16.83 23.5418.0715.27 25.3720.94 28.8227.19

NL:3.53E5m/z= 694.10-695.10+1353.60-1354.60 MS 2655

NL:6.40E5m/z= 676.00-677.00+1317.30-1318.30 MS 2655

Stranded porpoise - Gibraltar point, Lincolnshire (2003)

d18 α-HBCD

d18 β-HBCD

d18 γ-HBCD

α-HBCD

ΣHBCD= 8223 ng/g

RT: 0.00 - 29.94 SM: 7G

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

14.61

13.70

21.15 24.6012.47

25.8419.2015.49 28.9017.6411.17

14.86

12.73

20.76 24.0822.00 26.94 28.6418.4215.9411.75

NL:4.01E4m/z= 694.10-695.10+1353.60-1354.60 MS 2523-01

NL:1.61E6m/z= 676.00-677.00+1317.30-1318.30 MS 2523-01

d18 α-HBCD

d18 β-HBCDd18 γ-HBCD

α-HBCDβ-HBCD

γ-HBCD

ΣHBCD= 239 ng/g

Cork Sewage Sludge sample

SELECTED CHROMATOGRAMS OF TEES SEDIMENT EXTRACTS - TBBP-A

20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00Time0

100

%

0

100

%

0

100

%

0

100

%

C1433216 3: SIR of 1 Channel ES540.901.21e4

Area

25.66

20.94


Area

25.66


Area

25.70


Area

25.93

TBBP-A=12 ng/g

TBBP-A=57 ng/g

TBBP-A=55 ng/g

13C12 TBBP-A

Standard

-

-

-

-

SEWAGE SLUDGE EXTRACTS

20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00Time0

100

%

0

100

%

0

100

%

0

100

%

C1433271 3: SIR of 1 Channel ES- 540.902.47e5

Area

26.04


Area

26.17


Area

34.98

32.59 33.05


Area

35.0132.6833.14

13C12 TBBP-A

Standard

Sewage sludge

HBCD tech Standard

TBBP-A=192 ng/g

ΣHBCD=7,519 ng/g

Sewage sludge

Summary• LC-MS in single quad , triple quad or ion trap

configuration allows robust determination of TBBP-A & isomeric based HBCD measurement and is to be preferred over GC-MS techniques without isomeric resolution.

• The techniques presented has been fully validated and applied to a range of environmental matrices from biota (fish, shellfish, marine mammals) to abiotic materials (sediments and sewage sludge)

Analysis and Significance of HBCD and TBBP-A · 2011-07-26 · stereoisomers & lack of isomer...

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Transcript of Analysis and Significance of HBCD and TBBP-A · 2011-07-26 · stereoisomers & lack of isomer...