Örebro University
School of Science and Technology
Josefin Persson
2009
Development and evaluation of methods for analysis of
TBECH and HBCD using HRGC/HRMS and
UPLC/MS/MS
Abstract
The two additive brominated flame retardants, tetrabromoethylcyclohexane (TBECH) and
hexabromocyclododecane (HBCD) are used to prevent fire to start and spread. They are
simply mixed with material and are most likely to leach out in the environment, because of
non-covalently binding to the material. TBECH can exist as four pairs of enantiomers, α-, β-,
γ- and δ-TBECH. The technical HBCD can exist as three pairs of enantiomers, α-, β- and γHBCD and two meso forms δ- and ε-HBCD. None of these compounds are produced in
Sweden, but they are imported to industries. TBECH has been found in Beluga blubber and
can accumulate in zebrafish. HBCD has been found in water environments and can be toxic to
and bioaccumulate in water-living animals.
In this study, a method was developed for separation and detection of α-, β-, γ- and δ-TBECH
on HRGC/HRMS. All TBECH-isomers could be separated with the developed method. How
much of the TBECH isomers that were recovered after applying existing extraction and cleanup procedures, normally applied for clean-up and extraction of PCBs and PCDD/Fs, was
evaluated. Low recovered amounts (6.8-35.5 %) of TBECH-isomers added in known amounts
to three different whale samples indicate severe evaporation losses and possibly photolytic
degradation. None of the four enantiomers were detected in the three whale samples.
For HBCD analysis, both the chromatography and MS/MS parameters were optimised for δand ε- HBCD yielding good chromatography and sensitivity. However, due to technical
difficulties during the time-period of this project, no whale samples could be analysed for
HBCD on UPLC/MS/MS.
2
Index
1. Introduction ............................................................................................ 4
1.1. Aims ...................................................................................................... 4
1.2. Brominated flame retardants ................................................................... 4
1.3. Tetrabromoethylcyclohexane.................................................................. 5
1.4. Hexabromocyclododecane ...................................................................... 5
2. Materials and methods ............................................................................ 7
2.1. Extraction and cleanup for TBECH analysis ........................................... 7
2.2. Extraction and cleanup for HBCD analysis ............................................. 8
2.3. HRGC/HRMS ........................................................................................ 9
2.4. UPLC/MS/MS ....................................................................................... 10
2.5. Detection of TBECH .............................................................................. 10
3. Result and discussion .............................................................................. 10
3.1. TBECH method development ................................................................. 10
3.2. Evaluation of clean-up procedure............................................................ 12
3.3. Application of the method ...................................................................... 14
3.4. HBCD method development ................................................................... 16
3.5. Application of the method ...................................................................... 18
4. Conclusions and future perspectives....................................................... 19
5. Acknowledgments ................................................................................... 19
6. References ............................................................................................... 20
3
1. Introduction
1.1. Aims
The aims for this work were to evaluate an existing sample clean-up procedure and to
develop methods for detection of two brominated flame retardants. α-, β-, γ- and δtetrabromoethylcyclohexane (TBECH) with high resolution gas chromatography
coupled to high resolution mass spectrometry (HRGC/HRMS) and α-, β-, γ-, δ- and
ε- hexabromocyclododecane (HBCD) with ultra performance liquid chromatography
coupled to triple quadrupole mass spectrometry (UPLC/MS/MS). To evaluate the
develop methods α-, β-, γ- and δ-TBECH and α-, β-, γ-, δ- and ε-HBCD were analysed
in fat samples from Fin whales or Winke whales from Iceland. The whale samples
were extracted and fractionated using open column chromatography and then analysed
on HRGC/HRMS and UPLC/MS/MS, respectively.
1.2. Brominated flame retardants
Brominated flame retardants (BFR) are cyclic carbon compounds which contains
bromine. These compounds have been used for over 30 years to prevent fire to start
and spread in textiles, electronics, building materials and toys [1, 2] and approximately
200 000 tons/year are being produced globally [3]. BFRs are not produced in Sweden,
instead 100 tons of BFRs were imported to the industries in 2007 [4]. BFRs are found
both in the air and biota, because of leakage from industries. Concentrations of BFRs
have been found in birds and fish, mostly in water-living animals. BFRs have also
been found in human tissues and breast milk [1, 2] and in arctic mammalians [9].
Around 70 different BFRs are used today [1, 2]. BFRs are grouped into three different
groups depending on how they are used in polymers; brominated monomers, reactive
and additive. Two additive BFRs are 1, 2-Dibromo-4-(1, 2-dibromoethyl)cyclohexane
(TBECH) and 1, 2, 5, 6, 9, 10-hexabromocyclododecane (HBCD) [5]. Additive BFRs
are mixed with the material (polymer) and are more likely to leach out in nature during
use and disposal, because of their non-covalently binding with the material. Therefore,
research concerning these compounds is of importance [5, 6].
4
1.3. Tetrabromoethylcyclohexane
1, 2-Dibromo-4-(1, 2-Dibromoethyl)cyclohexane or tetrabromoethylcyclohexane
(TBECH) is a BFR with the molecular formula C8H12Br4. TBECH is used as an
additive in polystyrene and polyurethane products [7]. The world production and use
of TBECH are unknown. TBECH can exist as four pairs of enantiomers, α-, β-, γ-, δTBECH (see Figure 1) [8] because of four chiral carbons that can be in R or S
configuration [7]. The nomenclature is based on the elution order from a DB-5
capillary column [9]. The enantiomers are thermal sensitive and can transform to each
other (thermal interconversion) at temperature above 120°C and to detect all four
enantiomers the initial temperature must be 120 °C or below. Despite that, GC/MS is
considered to be the most suitable technique for analysing TBECH enantiomers [7].
Figure 1. The four pairs of enantiomers of TBECH. From left; α- TBECH, β-TBECH, γ-TBECH and δ-TBECH.
Studies have shown that TBECH can bind to the human androgen receptor and
activate it in vitro, which can cause health problem [10]. Today there are few reports
on TBECH in environment but in one study TBECH was found in Beluga blubber
from the Canadian Arctic [9] and has found to accumulate in zebrafish [7].
1.4. Hexabromocyclododecane
1, 2, 5, 6, 9, 10-hexabromocyclododecane or hexabromocyclododecane (HBCD) is a
BFR that is used as an additive in plastic materials and textiles [11, 12]. HBCD is
produced from cyclododeca-1, 5, 9 -triene (CDT) by bromination [5, 6]. The usage of
HBCD in the world is unknown, but the use has decreased in Sweden from 80 ton in
1998 to 6 tons in 2007 [4]. The three pairs of enantiomers, (±) α-, β- and γ-HBCD are
the dominantly forms of HBCD. HBCD has a molecular formula of C12H18Br6 and a
5
structure containing six chiral carbons that can be in R or S configuration [12]. Since
the spatial arrangement is different for the three enantiomers their physical and
chemical properties can vary, such as hydrophobicity and water solubility, which leads
to different ability to accumulate and spread in the environment [13]. In a recent study
16 different possible HBCD has been found in theory, but the technical mixture
contains the three pairs of enantiomers α-, β- and γ-HBCD and two meso forms δ- and
ε-HBCD (see Figure 2) [6].
Figure 2. The three pairs of enantiomers of HBCD and the two meso forms that has been found in technical
mixture. From the top; (±) α-HBCD, (±) β-HBCD and (±) γ-HBCD. At the bottom left is δ-HBCD and at bottom
right is ε-HBCD.
6
HBCD is thermally sensitive and breaks down at temperatures above 160 °C. Even
though GC has been used to separate the enantiomers of HBCD, the most suitable
technique is probably LC [5, 6]. HBCD can cause long-time effects in water
environments and can be toxic to water-living organisms [1]. In food chains studies, it
has been found that HBCD bioaccumulate in water environments [6] and can
bioaccumulate both in terrestrial and aquatic organisms [13]. HBCD has been found in
abiotic samples, such as ambient air and river sediment [6]. HBCD has also been
found in polar bears from Greenland and Svalbard [6]. HBCD can cause human
allergic reactions after physical contact with textiles treated with HBCD [1] and low
levels of HBCD has been found in human breast milk and human blood [6]. The
dominating isomer of HBCD is γ-HBCD in the technical mixture, but if the
temperature has been above 160 °C during production isomerisation of γ-HBCD to αHBCD occurs and α-HBCD is the dominating isomers. This is reflected in samples
and measured concentrations [6, 13].
2. Materials and methods
2.1. Extraction and cleanup for TBECH analysis
Fat from Fin whale or Winke whale from Iceland was first extracted and then going
through a sequential clean-up procedure that normally is applied for PCB and PCDD/F
analysis. To evaluate the extraction and clean-up procedure, three whale samples were
extracted and fractionated with and without the addition of known amounts of all four
TBECH-isomers. Sample 1 (un-spiked) correspond to sample 4 with the only
difference that sample 4 were spiked with all TBECH-sisomers. In the same way,
sample 2 corresponds to sample 5 and sample 3 to sample 6. 5 g of homogenate (fat
from whale grinded with Na2SO4 to remove water) was placed in a glass column.
Before elution 25 µl internal standard ( 13C PCB mix with the following congeners
#28, #52, #70, #101, #105, #118, #138, #153, #156, #170, #180, #194, #202 and #206
with concentrations around 120 pg/µl for each congener) (Wellington Laboratories,
Guelp, Canada) was added to all samples and to the blank sample consisting of
Na2SO4. Samples 4 to 6 were spiked with 50 µl α/β TBECH standard (50 pg/µl)
7
(Wellington Laboratories) and 50 µl γ/δ TBECH standard (50 pg/µl) (Wellington
Laboratories). The homogenate was eluted with n-hexane: dichloromethane (1:1) and
the eluate was collected in glass flasks with known weights. The organic solvents were
evaporated using a rotary evaporator and the flasks were left in the fume hood until
constant weights of the flasks containing whale fat were reached. The fat weights were
registered before the fat was dissolved in a small volume of n-hexane and then
fractioned on a multilayer column which eliminated lipids and other polar molecules
in the sample. The analytes were eluted with n-hexane into glass flasks and the solvent
was evaporated to 1-3 ml on a rotary evaporator. The samples were then fractionated
on an aluminium oxide column into two fractions, a non-planar PCB faction that was
eluted with n-hexane: dichloromethane (49:1) and a planar dioxin fraction that was
eluted with n-hexane: dichloromethane (1:1). The dioxin faction was further
fractionated on a carbon column, yielding a PCB faction (by elution with n-hexane)
and a dioxin faction (by elution with toluene). The PCB fraction from the aluminium
oxide column and the carbon column was collected in the same glass flasks. Before
evaporation to 1 ml on a rotary evaporator, 25 µl tetradecane was added to all flasks.
Then the samples were further treated on a minisilica column to remove remaining
polar compounds in the extract. The PCB fraction and the dioxin fraction were
transferred with n-hexane to 8 ml flasks and evaporated with nitrogen. The samples
were then transferred to GC vials containing 25 µl recovery standard (13C PCB mix
with #81, #114 and #178 congener with concentrations around 120 pg/µl for each) (
Wellington Laboratories). Also two quantifications standards containing internal
standard (13C PCB, 120 pg/µl), recovery standard (13C PCB, 120 pg/µl), α/β TBECH
standard (50 pg/µl), γ/δ TBECH standard (50 pg/µl) and tetradecane were prepared.
For calculations, only the labelled PCB congeners with similar retention times as the
TBECH enantiomers were used. The samples were protected from UV-light by
covering them by aluminium foil throughout the whole analytical procedure.
2.2. Extraction and cleanup for HBCD analysis
The samples for HBCD analysis were fat from Fin whale or Winke whale from
Iceland and were prepared identically to the TBECH extracts (see section 2.1) but by
adding 25 µl internal standard δ-HBCD (5 µg/ml) (Wellington Laboratories) prior to
8
the fat extraction. No tetradecane was added to the final extracts and after transferring
the extract to the 8 ml vial the extract were evaporated to dryness and then dissolved
in 500 µl methanol. The samples were then transferred to LC vials containing 25 µl
recovery standard (ε-HBCD, 5 µg/ml) obtained from Wellington Laboratories. Also,
three quantification standards with different concentrations (25 ng/ml, 75 ng/ml and
150 ng/ml) of δ-HBCD and ε-HBCD (each dissolved in 30 % methanol and 70 %
water) and a standard containing 25 µl internal standard and 25 µl recovery standard
dissolved in methanol were prepared. Standards available for HBCD analysis were
only δ-and ε-HBCD. Since no labelled standards were available δ-HBCD was used as
internal standard and ε-HBCD was used as recovery standard. The probability to find
these enantiomers in biota should be low as α-HBCD is the dominant enantiomer
reported from the literature. If any of the α-, β-, γ-isomers of HBCD were found in the
whale samples their areas would had been summarised and calculated with the relative
response factor 1 against the internal standard (δ-HBCD).
2.3. HRGC/HRMS
A gas chromatograph (6990N Network GC, Agilent Technologies, Waldbron,
Germany) coupled to a Micromass Auto Spec-Ultima (Waters Corporation, Midford,
USA) high resolution mass spectrometer was used for analysing TBECH. 1 µl of
sample was injected by on-column injection on a SilGuard BPX-5 column (30m x
250 µm x 0,1 µm; SGE) equipped with a 3 m long guard column (i.d: 320 µm). As
carrier gas helium was used and a temperature program was developed (see section
3.1). TBECH was ionised using electron impact in positive mode ((+) EI). The
isomers of TBECH were detected by single ion monitoring (SIM) with the m/z
264.9226 [M] and 266.9207 [M+2]. The samples were quantified using isotope
dilution.
2.4. UPLC/MS/MS
For HBCD analysis an Acquity TM Ultra performance LC coupled to a Quattro
Premier XE triple quadrupole mass spectrometer (Waters Corporation) was used. 10
µl of sample was injected to be separated on an Acquity BEH C18 column (2.1 mm x
9
100 mm x 1.7 µm) with a flowrate of 0.125 ml/min. As mobile phase two solutions
were used, methanol (B) and 30:70 % methanol: water (A). These solutions were used
for a gradient. Initial composition was 40 % A and 60 % B. The composition was
changed linearly in 7 minutes to 10 % A and 90 % B. After 7.1 minutes the
composition was reverted to the initial setting and the system was allowed to
equilibrate for 6 minutes. The complete time for analysis was 15 minutes. The samples
were ionised with negative electrospray ((-) ESI) and the cone voltage and collision
energy were set to 15 V and 20 V, respectively. The capillary voltage was set to 2.80
kV. The source temperature and desolvation temperature were set to 120 °C and 300
°C. The cone gas flow was set to 50 L/Hr and the deslovation gas flow was set to 700
L/Hr. HBCD was detected with multiple reaction monitoring (MRM) measuring the
transitions 640.53→78.7 and 640.53→80.8.
2.5. Detection of TBECH
For detect of the TBECH-isomers in the samples the retention times in the
quantification standard and the internal standard were compared. Also, the isotope
ration between the fragment ions, 264 and 266, were compared between the
quantification standard and the spiked and unspiked samples. The limit of detection
(LOD) of the method was estimated by following formula;
3. Result and discussion
3.1. TBECH method development
Method development for detection of α-, β-, γ- and δ-TBECH was performed on high
resolution gas chromatography coupled to high resolution mass spectrometry
(HRGC/HRMS). First the mass spectrometer was set to measure the molecules with
the m/z 264.9226 [M] and 266.9207 [M+2] by single ion monitoring (SIM). These
10
masses are fragments from the precursor molecule with the loss of two bromine
molecules. So, 264.9926 correspond to the fragment [M-HBr2] and 266.9207
correspond to the fragment [M-HBr2+2], which has the highest intensity in the mass
spectra of TBECH.
In a previous study [14] different GC-columns were evaluated for best separation
efficiency of the four TBECH enantiomers. The results indicated that a thin phase (0.1
μm) BPX-5 column would be best suited for the separation of TBECH enantiomers. In
this study a 30 m, thin phase BPX-5 column was evaluated in combination with oncolumn injection. Since these compounds are thermally instable and are known to
suffer from thermal interconversion between the TBECH isomers the on-column
technique seems to be optimal for sample introduction of TBECH.
Different temperature programs were tested both to see which settings resulted in best
resolution of the α-, β-, γ- and δ-enantiomers and also to evaluate how the different
settings affected the degradation of the isomers. Also, the influence of the initial
temperature on the thermal interconversion between the TBECH isomers and
degradation was evaluated for three different temperatures, i.e. 100, 110 and 120°C.
No improvements were seen when varying the initial temperature and the optimal
temperature program giving best separation was the following; 120 °C with a hold for
2 minutes followed by a temperature ramp of 2 °C/minute up to 181 °C. Then the
temperature was increased to 300 °C with the rate of 35 °C/minute and final hold for 6
minutes. In figure 3, the separation of the four TBECH enantiomers obtained with the
described temperature program is shown.
11
DL09-009: std TBECH
09051806
100
IS (PCB # 52)
IS (PCB # 70)
27.37
11955843
RS (PCB # 81)
Voltage SIR 8 Channels EI+
303.9597
1.24e8
Area
34.32
14239150
%
32.40
14071450
27.91 28.23
45060 26915 28.71
5596
0
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
09051806
30.14
29.62 3101862
2428763
100
35.05
477598
33.61 33.87
108821 5845
31.14 31.51
34953 5735
29.33
4617
β-TBECH
γ-TBECH
α-TBECH
34.00
35.00
Voltage SIR 8 Channels EI+
266.9207
2.03e7
Area
δ-TBECH
33.32
1501142
%
33.15
1756660
30.54
21670
29.45
1460
31.45
21414
31.90 32.37
83407 125929
32.87
878809
33.69 34.04 34.36
15351 1580 5668
34.76
3314
0
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
09051806
30.14
1565531
100
Isotope
ratio 2:1
29.63
1316841
34.00
35.00
Voltage SIR 8 Channels EI+
264.9228
1.06e7
Area
33.32
829467
%
33.15
852815
27.18
1369
0
26.00
27.00
30.47
35482
29.48
3094
28.00
29.00
30.00
31.59 31.98 32.40
31.12
28481 56452 87352
7626
31.00
32.00
32.90
440432
33.68 34.05
9770 7139
33.00
34.96
2020
34.69
3687
34.00
Time
35.00
Figure 3. Chromatogram showing the separation of the α-, β-, γ- and δ-isomers of TBECH. Labelled PCB
congeners were used as internal and recovery standard.
3.2. Evaluation of clean-up procedure
To evaluate the existing clean-up procedure the recoveries were calculated for each
sample (see Table 1). Also, to further evaluate the clean-up procedure the recovered
amounts of added TBECH isomers in the spiked whale samples (sample 4 to 6) were
calculated (see Table 2). The recovery in the blank sample was around 30 %. In the
un-spiked whale samples the recoveries varied between 34 to 75% and in the spiked
whale samples the recoveries varied between 97-138%. Acceptable boundaries for
recoveries are normally between 50-120% showing problems with the clean-up
procedure. Because the laboratory ran out of aluminium oxide and of long deliverytimes the blank sample and the un-spiked whale samples were covered with
aluminium foil and kept in the fume-hood for almost two weeks before the clean-up
could be resumed. The low recoveries in these samples could therefore be explained
12
by evaporation losses of the labelled PCB congeners. However, the high recoveries in
sample 6 are still unexplained.
The recovered amounts of added amounts of TBECH isomers varied between 6.8 to
35.5%. The low recovered amounts are probably due to large evaporation losses
during the clean-up procedure and possibly, to some extent, due to photolytic
degradation of the TBECH isomers. The results imply that the adapted method was
insufficient for the TBECH isomers and that the use of labelled TBECH isomers
would be very useful to have better control of the clean-up procedure.
Table 1. Recoveries of PCB #52 and PCB #70 for all whale samples (spiked and un-spiked) and blank (Na2SO4).
Sample
IS (PCB # 52)
IS (PCB # 70)
DL09-009:1
56.4
60.9
DL09-009:2
68.5
75.2
DL09-009:3
34.5
56.5
DL09-009:4 (Spiked)
97.4
106.2
DL09-009:5 (Spiked)
114.5
116.4
DL09-009:6 (Spiked)
137.2
138.9
DL09-009:7 (Blank)
32.4
34.1
13
Table 2. Recovered amounts of the four enantiomers of TBECH in the spiked whale samples after extraction
and clean-up. Results are presented both in concentrations (pg/g lipid) and in percentage (%).
Sample
α-
β-
γ-
δ-
Added
Added
Recovered amount of
TBECH
TBECH
TBECH
TBECH
α/β
γ/δ
added TBECH isomers
(pg/g
(pg/g
(pg/g
(pg/g
TBECH
TBECH
(%)
lipid)
lipid)
lipid)
lipid)
standard
standard
(pg/g
(pg/g
lipid)
lipid)
α
DL09-
β
γ
δ
142.2
177.3
73.9
68.0
500
500
28.4 35.5 14.8 13.6
76.1
145.6
59.2
55.3
500
500
15.2 29.1 11.8 11.1
48.9
135.3
33.8
47.2
500
500
9.8
009:4
DL09009:5
DL09-
27.1 6.8
9.4
009:6
3.3. Application of the method
Three different whale samples were run to apply the evaluated clean-up procedure and
the developed HRGC/HRMS method on real samples. In all of the whale samples one
peak showed the same retention time as the α-TBECH isomer based on retention time
comparisons with the quantification standard and the internal standards, see example
in Figure 4. However, when comparing isotope ratios between the fragment ions, i.e.
mass 264 and 266, the ratios differed between the signals in the quantification
standard and the signals for the suspected peak in the whale samples (see Figures 4
and 5). Since the co-eluting peak did not have the correct isotope ratio the unidentified
peak can not be positively identified as the α-TBECH isomer.
All four TBECH enantiomers could be identified and quantified in the spiked whale
samples, see Figure 5. However, the chromatograms show large contributions of other
14
compounds having the same mass as were monitored in these samples (see Figure 4
and 5).
The LOD (limit of detection) for the method was estimated to 47.3 pg/g. In the Beluga
blubber study the method detction limit (MDL) was determined to 0.8 pg/g with the
signal noise ratio 3:1 [9]. The MDL in the Beluga blubber study is around 500 times
lower compared to the LOD in this study. This indicates that no concentrations of the
four enantiomers of TBECH were detected in the whale samples.
DL09-009: 1
09051809
Voltage SIR 8 Channels EI+
303.9597
1.31e8
Area
34.33
11647301
100
RS (PCB # 81)
IS (PCB # 70)
%
IS (PCB # 52)
27.59
5895963
28.12
12800
27.21
684
25.53
603
0
32.51
7571649
26.00
27.00
28.00
28.70 29.05
1362 1380
29.00
30.06
29.54 29.87
30.49
882
1230 1193
1064
30.00
31.21
6184
31.00
33.84
33.65
5448
51370
31.77 32.17
2600 1400
32.00
33.00
34.00
35.00
Voltage SIR 8 Channels EI+
34.68
266.9207
1348850
3.70e7
Area
09051809
100
%
α-TBECH?
0
25.17
25.62
1519
21922
26.16
12579
26.00
26.94 27.16 27.63 27.82 28.35
91247 3332 15021 3444 7834
27.00
28.00
29.17
173894
29.00
29.93
49419
30.27
182105
30.00
31.39
70009
31.00
31.98
150151
33.69
505783
32.86 33.62;119966
34.18 34.41
144094
31769 70483
32.00
33.00
34.00
35.00
Voltage SIR 8 Channels EI+
34.69
264.9228
530078
1.24e7
Area
09051809
100
Isotope
ratio 1:1
34.98
271482
%
33.69
405986
0
25.64
14673
26.15
11388
26.00
26.94
60688
27.00
27.47 27.83
2111 1523
28.00
28.37
6623
30.19
29.18
29.00
29.53 29.98 60592
59951
45614
9490 54562
31.40
25137
32.22
32.06
5345
22487
33.62
81213
32.84
135910 33.23
32689
34.41
33.89
69483
22890
34.98
77938
35.08
1667
Time
29.00
30.00
31.00
32.00
33.00
34.00
35.00
Figure 4. Chromatogram showing a whale sample without added amounts of TBECH-isomers. Based on
retention time comparisons one peak was tentatively identified as the α enantiomer of TBECH.
15
DL09-009: 4
09051812
Voltage SIR 8 Channels EI+
303.9597
1.23e8
Area
34.32
9865011
100
IS (PCB # 52)
RS (PCB # 81)
IS (PCB # 70)
32.37
10455870
%
27.25
7780832
27.83
9943
31.06
32811
0
26.00
27.00
28.00
29.00
30.00
31.00
33.80
1333968
32.88 33.60
17282 31419
31.60
4790
32.00
33.00
35.07
35.54
36.37
35.25
52225
37604 36.09 5977
75685
42316
34.00
09051812
100
β-TBECH
α-TBECH
γ-TBECH
33.63
645326
34.51
477683
δ-TBECH
%
30.08
865164
34.69
200179
29.56
509190
26.82
209214
27.77
26.99;33487 5885
35.00
36.00
Voltage SIR 8 Channels EI+
35.78
266.9207
686562
9.75e6
Area
30.38
5276
28.67 28.89
15019 61467
31.14
48882
31.96
68307
32.48
46620
33.14
216159
33.44
84055
34.17
46418
35.47
238857
34.95
105278
35.95
31013
0
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
34.00
09051812
33.63
407815
100
Isotope
ratio 2:1
%
33.56
125250
30.09
459741
33.44
67692
29.57
291674
27.03
132904 27.44 27.66 28.26
27445 7671 6480
35.00
36.00
Voltage SIR 8 Channels EI+
264.9228
35.78
6.50e6
391729
Area
30.51
7721
28.77 29.24
13526 12995
31.23
25502
32.47 32.72
31.88
32684 84178
14040
33.77
15298
34.95
34.68 69453
35.16
37509
25111
0
Time
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
34.00
35.00
36.00
Figure 5. Chromatogram of sample (spiked with α/β TBECH standard and γ/δ TBECH standard) where four
peaks was possible identified as α-, β-, γ- and δ-TBECH (see Figures 7 and 8).
3.4. HBCD method development
A method for detection of α-, β-, γ-, δ- and ε-HBCD was developed using ultra
performance liquid chromatography coupled to triple quadrupole mass spectrometry
(UPLC/MS/MS). Detection on the mass spectrometer was optimised by tuning for the
precursor ion and product ions. The bromine trace with the highest intensity [M-H+6](m/z 640.53) (see Figure 6) was chosen as the precursor ion, and the product ions
found were 79Br (m/z 78.7) and 81Br (m/z 80.8). Also, the cone voltage was optimised
for the precursor ion and the collision energy was optimised for the product ions. From
these data a multiple reaction monitoring (MRM) method was developed using
negative electrospray ((-) ESI) ionisation.
16
-
Figure 6. Full scan spectra on UPLC/(-)ESI/MS/MS showing the bromine pattern for the precursor ion [M+4] (
m/z 640.53) with six bromine molecules.
When direct infusion of a standard solution of δ-HBCD (500 ng/ml) was performed,
HBCD formed three adduct clusters, corresponding to m/z +60, +45 and +36.
Tentative structures could be [M+HAc]-, [M+HCOO]- and [M+HCl]-. Since the
mobile phase at first contained NH4Ac it was removed to avoid some of the adduct
formation [15].
Next a method for the liquid chromatograph was created. A 50 mm column was used
at first, but the retention time of δ-HBCD was around 1-2 minutes which was not
sufficient since α, β and γ elutes before δ-HBCD [16]. To obtain a slower system a
longer column (100 mm) was used and the retention time was increased to around 5
minutes (see Figure 7). The instruments limit of detection (LOD) for ε-HBCD was
calculated to 6.8 ng/ml and for δ-HBCD 8.0 ng/ml with the signal to noise ratio 3.
17
Std. 75 dHBCD
09051111 Sm (Mn, 2x3)
MRM of 2 Channels ESTIC (dHBCD)
904
Area
5.23
65
100
%
ε-HBCD
0
1.00
1.50
09051107 Sm (Mn, 2x3)
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
4.76
62
100
8.50
MRM of 2 Channels ESTIC (dHBCD)
740
Area
%
δ-HBCD
0
Time
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
8.50
Figure 7. Chromatogram for standard solution δ-HBCD 75 ng/ml (bottom) and ε-HBCD 75 ng/ml (top).
The mobile phase composition and the flowrate were also changed to optimise the
separation. Further development showed that the chromatography was improved when
adding 30 % water to the vials. The largest problem was that the LC back pressure
increased to max (14 000 psi) during the run. The reason for the increased back
pressure could be instrumental problem and the solution would be to change the
column.
3.5. Application of the method
To evaluate the method whale samples were extracted and fractioned and analysed
(see section 2.3). However, during the run the intensity suddenly decreased. One
reason for the lower intensity can be that the HBCD has been debrominated during
storage, i.e. it breaks down relative fast in methanol. Another reason can be that the
HBCD seemed to have formed adducts with water or methanol which reduce the
signals of the product ions in the mass spectrometry. Due to time limitations further
studies to elucidate this problem were not possible.
18
4. Conclusions and future perspectives
A HRGC/HRMS method was developed for the separation of α-, β-, γ- and δ-TBECH.
Unfortunately, no TBECH-isomers could be detected in the whale samples. The results
from the evaluation of the applied extraction and clean-up procedure showed that only
very low amounts of added TBECH-isomers could be recovered after the whole
procedure. Tentatively, this could be a result from evaporation losses and photolytic
degradation. In a future perspective, a new clean-up procedure ought to be developed to
increase the recovered amounts of TBECH during extraction and clean-up.
A method was developed for separation and detection of δ- and ε-HBCD on
UPLC/MS/MS. However, no samples could be analysed due to instrumental problems.
More effort needs to be directed towards the detection of HBCD, reducing adduct
formation and evaluate storage stability. Moreover, standards for all enantiomers as well
as labelled standards are needed for future studies.
5. Acknowledgments
Professor Bert van Bavel, PhD, Örebro University (MTM), for letting me be a part of your
laboratory group.
Jessika Hagberg, PhD, Örebro University (MTM), for tutoring me during this work and
with all help with the gas chromatography. Also, for the feedback on the report.
Anna Kärrman, PhD, Örebro University (MTM), for tutoring me during this work and
with all help with the liquid chromatography. Also, for the feedback on the report.
19
6.
References
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42, 2008, 543-549
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Chem. 49, 2006, 7366-7372
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20
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21