Method development: Simultaneous determination of ... · Since HBCD, and TBBPA have a high affinity...

Method development: Simultaneous determination of

tetrabromobisphenol-A and the hexabromocyclododecane

diastereoisomers

Student name : Guillaume ten Dam1,2

Student ID : 5752604

Master program : Analytical Sciences

Master coordinator : dr. W. Th. Kok

Supervisor : Wim Traag

1) RIKILT – Institute of Food Safety, Wageningen UR, Akkermaalsbos 2, 6708WB, Wageningen

2) Faculty of Science – Van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam,

Science Park 904, 1098XH Amsterdam

3

Samenvatting TBBPA en HBCD zijn veel gebruikte vlamvertragers in computerboards, kleding en isolatiemateriaal.

Door diffusie en emissie kunnen deze stoffen in het milieu terecht komen, waar deze zich kunnen

opstapelen in de bodem en door hun hydrofobe karakter kunnen accumuleren in de voedselketen.

Doordat weinig bekend is van de aanwezigheid van deze componenten in het milieu heeft de Europese

voedsel en waren autoriteit laboratoria opgeroepen tot het analyseren van deze stoffen in het milieu en

de voedselketen. Hoewel diverse laboratoria methodes hebben ontwikkeld zijn er nog steeds afwijkende

resultaten niet verklaard. In deze studie zijn de ontwikkelde methoden onderzocht en kritische

variabelen in kaart gebracht. Lage extractie en zuivering recovery’s van TBBPA bleken te worden

veroorzaakt door waterstofbrug binding tussen de hydroxy-groepen en de gebruikte materialen terwijl

de hydroxy-groepen ook verantwoordelijk zijn voor schuivende retentietijden als gevolg van een

veranderende pH. Bij hoge pH verliezen de hydroxy-groepen een proton, waardoor het molecuul polair

wordt. Deze effecten konden worden voorkomen door een juiste selectie van materialen en pH.

Uiteindelijk is een semiautomatische ontwikkeld en toegepast op een gevoelige groep in de

voedselketen. In deze gevoelige groep, paling, worden regelmatig hoge gehaltes aan contaminanten

gevonden en ook HBCD is in relatief hoge gehaltes aanwezig. Hoewel TBBPA wel in hoge gehaltes wordt

gevonden, wordt TBBPA mogelijk doordat het makkelijker kan worden gemetaboliseerd nauwelijks in

paling gevonden.

4

Abstract Since the EFSA enquired a call for data for TBBPA and HBCD in 2009, the analytical determination of

these compounds became of regulatory interest. Therefore, a method for the simultaneous

determination of TBBPA and the three major HBCD stereoisomers was developed. Conventional

techniques like soxhlet, ASE, GPC, sulphuric acid digestion, and acidified silica SPE are generally used in

sample pre-treatment while detection is mostly performed by LC-MSMS. The hydroxyl groups on TBBPA

troubles the extraction and purification. However, using sodium sulphate (0.63 -2.0 mm mesh) in ASE

and a large acid silica (63-200 mesh) column in combination with a Sep-pack Plus silica cartridge on the

Power-PrepTM

result in recoveries of 80 ± 25% for all compounds. Typical limits of detection are

between 1 to 10 pg/g product or 0.02 pg to 0.2 pg on column. Levels of TBBPA and HBCD isomers were

determined in samples eel and a proficiency test. TBBPA was occasionally detected marginally above the

quantification limit of 0.05 ng/g, whereas total amounts of HBCD were between 0.2 and 150 ng/g. The

relative contribution of the α-, β- and γ-HBCD isomer in eel was 89%, 3% and 8%.

Content

1. Introduction ........................................................................................................................................ 7

1.1 Extraction ...................................................................................................................................... 8

1.2 Clean-up ...................................................................................................................................... 10

1.3 Separation ................................................................................................................................... 12

1.4 Detection ..................................................................................................................................... 14

1.5 Occurance.................................................................................................................................... 15

2. Experiments ...................................................................................................................................... 16

2.1 Extraction .................................................................................................................................... 17

2.1.1 Materials and methods ......................................................................................................... 17

2.1.2 Results .................................................................................................................................. 19

2.1.3 Conclusion ............................................................................................................................ 22

2.2 Clean-up ...................................................................................................................................... 23

5

2.2.1 Gel Permeation Chromatography .......................................................................................... 23

2.2.2 Sulphuric acid digestion ........................................................................................................ 25

2.2.3 Solid Phase Extraction ........................................................................................................... 27

2.2.4 Powerprep™ Solid Phase Extraction ...................................................................................... 31

2.2.4 General procedure tests ........................................................................................................ 36

2.2.5 Conclusion ............................................................................................................................ 37

2.3 Separation ................................................................................................................................... 38


2.3.2 Results .................................................................................................................................. 39

2.3.3 Conclusion ............................................................................................................................ 40

2.4 Detection ..................................................................................................................................... 44


2.4.2 Results .................................................................................................................................. 44

2.4.3 Conclusion ............................................................................................................................ 48

3. Sample analysis ................................................................................................................................. 48

4. Conclusion ......................................................................................................................................... 51

References ............................................................................................................................................ 52

Appendix 1 Extraction methods ............................................................................................................. 55

Appendix 2 Purification methods ........................................................................................................... 57

Appendix 3 LC methods ......................................................................................................................... 59

Appendix 4 Simultaneous determination of TBBPA and the three major HBCD diastereoisomers .......... 62

Appendix 4.1 Method ........................................................................................................................ 62

Appendix 4.2 ASE method.................................................................................................................. 63

Appendix 4.3.1 SPE Power-PrepTM

method ..................................................................................... 63

Appendix 4.3.2 SPE sep-pack silica method .................................................................................... 64

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Appendix 4.3.3 SPE acid silica method ........................................................................................... 64

Appendix 4.3.4 GPC method .............................................................................................................. 64

Appendix 4.4 LC method .................................................................................................................... 65

Appendix 4.5 MSMS method ............................................................................................................. 65

7

1. Introduction Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) (Fig. 1) are commonly used

flame retardants in a variety of products textile, fabrics, isolation foam and computer boards. In fact,

TBBPA is since several years the most produced brominated flame retardant (BFR) with worldwide

production volumes over 100,000 tons a year [1, 2]. In Europe over 10,000 tons of TBBPA is annually

used, whereas annual HBCD usage is approximately 6,000 tons [3].

TBBPA is used in a wide range of electrical and electronic devices such as TV sets, PC’s, washing

machines etc., but its main application is in printed circuit boards where it is used in more than 95% of

the most common type of boards. In this application, TBBPA is integrated into the epoxy resin and

therefore no longer exists as a free chemical in the final board. Conversely, TBBPA is also an additive in

acrylonitrile butadiene styrene as a free chemical [4]. The main use of HBCD is in expanded and

extruded polystyrene isolation foams and in textile coatings [5].

Although the World Health Organisation (WHO) [6] and the European Scientific Committee on Health

and Environmental Risks (SCHER) [3] concluded that TBBPA and HBCD did not present risks to human

health, scientific studies have demonstrated a potential hazards of these compounds [7, 8]. Moreover,

TBBPA and HBCD have been classified as very toxic to aquatic species.

In 2008 the European Commission (EC) classified HBCD as a Persistent, Bio accumulative, and Toxic (PBT)

compound [9]. As a result HBCD and all major diastereoisomers became a substance of very high

concern and although TBBPA does not seem to bio accumulate, both compounds are classified as acute

and long-term hazardous (R50/R53). In addition, a call for data was commended by the European Food

Safety Agency (EFSA) in 2009, which included the major HBCD diastereoisomers as well as TBBPA and

other emerging and common BFR [10].

As a result, the development of a quantitative analytical method for the determination of the HBCD

diastereoisomers and TBBPA is not only of scientific interest, but also potentially a regulatory need. Until

now, several groups describe the analysis of the three major diastereoisomers, α-, β- and γ-HBCD, in a

variety of environmental and biological samples. A recent study reports the analyses with liquid

chromatography atmospheric pressure photon ionization tandem mass spectrometry (LC-APPI-MS/MS)

[11-13], but liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MSMS)

seems to be the standard (Ref….).

8

A B

Figure 1 A The structure of the three major HBCD diastereoisomers and B the structure of TBBPA

1.1 Extraction

With a Log Pow of 5.6, HBCD [14] is likely to absorb to sediments in a water column or to accumulate in

fatty tissue [14], whereas TBBPA with a Log Pow of 4.5-5.3 [6] is more polar and dissolves better in

aqueous solutions and muscular tissue [15]. So extraction of HBCD will be similar to that of conventional

contaminants, while for TBBPA a more polar solvent would be required.

ASE, as well as soxhlet have often been used to extract HBCD from solid samples and conditions are

quite consistent throughout the studies, especially for soxhlet. Soxhlet extractions involve the use of a

hexane:aceton mixture varying form 1:1 to 3:1, but also dichloromethane has been used as extraction

solvent. For the ASE comparable mixtures are used, but more alternations were introduced regarding

temperature, pressure and the use of an inert material e.g. hydromatrix. Hydromatrix was sometimes

also used for clean-up while florisil [16], acid silica [17] and polyacrylic acid [18] were added to remove

fat or interfering compounds from the sample. These differences do not seem to affect the recovery of

the HBCD isomers, but in a comparative study ASE could not fully extract spiked TBBPA from the matrix

using a solvent mixture of 3:1 hexane:acetone , resulting in recoveries lower than 15% with optimized

parameters [19]. Since lower temperatures did not seem to enhance the recovery, degradation of the

compound is unlikely to cause these low recoveries. On the other hand, with soxhlet, TBBPA could be

fully extracted from the matrix, and extraction by ASE might not be exhaustive enough. In addition, this

assumption seems to be supported by the results of Granby [18]. Though the extraction of TBBPA from

9

fish by ASE showed better recoveries, the second extraction of the sample still contained a small amount

of TBBPA, <10%, therefore it is concluded that TBBPA seems to adsorb stronger to the matrix [18].

Liquid/liquid extraction is an attractive option for milk samples. Because of the high Log Pow values of the

compounds they will accumulate into the fat. Fangstrom et al. performed liquid/liquid extraction using

formic acid and 2-propanol to modify the matrix and the mixture was extracted with 1:1 n-

hexane/diethyl ether [20]. In addition, SPLE has been performed on freeze dried milk [21] and sediments

[22, 23] using mixtures of dichloromethane:acetone, pentane:acetone and pure acetone with possible

additional sonication or centrifugation. Finally, Ultra turrax has been used for biotic samples [22, 24].

Hexane or pentane was used in combination with acetone or acetone:water to extract the compounds.

Summarizing the studies, ASE or soxhlet is mainly used for the extraction of HBCD and/or TBBPA from all

kinds of matrices, ranging from sediments to oil. ASE might have the disadvantage of incomplete

extraction of TBBPA, but this still needs to be determined. On the other hand, soxhlet is time and

solvent consuming, making it an undesirable for routine analysis. Therefore, an attempt will be made to

develop one or more ASE methods to extract the different matrices. However, for milk samples an

easier liquid/liquid extraction or for freeze dried milk a SPLE might be more convenient. A table of

possible extraction methods for different matrices is presented below (Tab. 1).

When fractions are collected after extraction, care should be taken when using glassware. Hiebl et al.

reported loss of standard compound when dissolved in n-hexane and iso-octane due to adsorption to

the glassware [25] and additionally the crystallization of γ-HBCD has been demonstrated in acetonitrile

(Fig. 2) [22, 23, 26].

10

Figure 2 Temporal trend in individual HBCD isomer concentrations over 30 days in methanol (top) and acetonitrile

(bottom). [26]

Table 1 Sample type and suitable extraction methods

Method Matrix

ASE Sediment, feed, freeze dried milk, biota, oil

Soxhlet Sediment, feed, freeze dried milk, biota, oil

Ultra turrax Biota

SPLE Sediments, freeze dried milk

Liquid/liquid extraction Milk

1.2 Clean-up

Since HBCD, and TBBPA have a high affinity towards a-polar media, they disperse into lipid content or

adsorb onto particles. Extraction methods are designed to extract the compounds out of the matrix, but

these methods also extract the lipid content and interfering components from the sample. In order to

remove the lipid content and interferences, several clean-up treatments have been developed and are

routinely used in contaminant analysis. Gel permeation chromatography (GPC), or also called size

exclusion chromatography (SEC), is an established technique to remove lipids from sample extracts.

Though, Frederiksen et al. found that GPC does not sufficiently remove the lipids from biotic samples

[19]. To remove the residual lipids they used sulphuric acid, and also other groups used additional

treatments like silica and florisil solid phase extraction (SPE) after GPC [26-28], but also only GPC clean-

11

up prior to analysis has been used [22, 29]. The treatment with sulphuric acid is a popular approach and

is the most applied treatment [16-20, 22, 24, 30]. Although the use of sulphuric acid raised concerns

about the recovery of the compounds, lower recoveries (63%) were only observed for methyl-TBBPA

(Me-TBBPA) [19]. In addition, sulphuric acid thoroughly removes the lipid content from extracts and few

or no additional clean-up seems to be required for quantitative analysis. Another way of removing lipids

is via acid silica digestion [13, 14, 31]. This is a less exhaustive lipid removal than sulphuric acid and can

be more easily automated. But acidified silica has been used in fewer studies and initial experiments in

the institute resulted in bad recoveries by this mean.

Figure 3 Fractionation of HBCD diastereomers on a silica column with, A 10% diethylether in hexane as eluent, B

25% DCM in hexane as eluent and C HBCD diastereomers mixed wtih 50μl fish-oil on a silica column with 25% DCM

in hexane as eluent, D florisil column with 10% diethyl ether in hexane as eluent. Each bar represents mean (±SD)

from three separate experiments. [32]

Additional clean-up for extracts without significant lipid content or for non-fatty samples involves SPE

with florisil, silica, silica alumina or carbon. Of these, florisil and silica have been used more often and

A

B

C

D

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the elution patterns have been studied by Mariussen et al. (Fig. 3) [32]. It was found that the HBCD

isomers have quite different retention characteristics, which also seems to be matrix depended.

Therefore a large collection window should be used, resulting in a relatively large solvent consumption.

These findings might also explain the bad recoveries in the initial experiments at the institute.

Based on the literature and aim of the study, a simple clean-up by removal of the lipid content with

sulphuric acid seems to fulfill the analytical needs. For non-fatty samples this step might also be skipped

and simple centrifugation or filtration should result in suitable extracts. When experiments show an

insufficient clean-up, further clean-up steps in the form of a silica or florisil SPE will be studied. The

selected clean-up procedures are displayed in table 2.

In order to assure a reproducible lipid removal, the sample intake needs to be adjusted so that the lipid

content will be in a suitable range for digestion. Another important part of the analysis that also

depends on the amount of matrix, is ion suppression. Therefore, the final extract volume should be

sufficient to minimize these effects. In addition, isotope dilution should be used for proper

quantification [26].

Table 2 Sample types and suitable clean-up procedures .

Treatment Matrix

GPC, Florisil, silica Biota, milk, oil, fatty samples

Centrifugation (silica, florisil) Soil, non-fatty samples

1.3 Separation

Separation of the three main diastereoisomers of HBCD by LC (appendix 3) seems to be a rather straight

forward application. Most analysis have been performed on C18 columns from different brands or with

different modifications. In all cases baseline, or sufficient resolution was obtained (Fig. 4). Also two

groups [23, 28] published results obtained with a C30 stationary phase, but this does not seem to change

the resolution significantly. Other columns tested were C8, phenyl, amide, and ether modified silica, but

C18 turned out to be the most suitable one to separate the different diastereoisomers [14, 31]. The

length of the columns varies between 100 to 5000mm. Shorter columns have been applied but do not

have enough separation power to separate the HBCD isomers [19]. A column length of 150mm has

shown to be sufficient for baseline resolution, while maintaining short analysis times. The separation of

13

the optical enantiomers, can be achieved on a chiral column [14, 33], but this falls outside the scope of

this study.

Mobile phases were mostly kept simple with gradients starting with a simple water/organic mixture to a

higher organic content. Although a fully organic content would not be necessary for the separation of

the HBCD diastereoisomers, it will be necessary for the elution of more apolar components in the matrix

[31]. The organic constituent of the mobile phase is mostly methanol, acetonitrile, or a mixture of them.

When optimizing the mobile phase Janák et al. discovered an improved resolution between the β- and γ-

diastereoisomers with addition of acetonitrile in the mobile phase, but at the same time this resulted in

a lower sensitivity for the α-isomer [14]. Since separation between the α- and β-isomer is the most

critical one, the extra resolution between the β- and γ-isomer would not be such an objective.

While often non-buffered mobile phases have been used for the analysis of HBCD, addition of

ammonium acetate has shown to increase the response of this compound [22, 31]. For optimal

sensitivity and selectivity an optimum seems to be found in a water, methanol and acetonitrile mixture

gradient with an addition of ammonium acetate, which is demonstrated by Yu et al. (Fig. 4) [31].

Since HBCD and TBBPA have a high Log Pow, reconstitution solvents need to be sufficiently a-polar to

properly dissolve the compounds. Therefore, HBCD and TBBPA have been dissolved in relatively strong

organic solutions. Since γ-HBCD has been found to precipitate in acetonitrile (Fig. 2) [22, 23, 26], this

solvent has been found to be unsuitable for reconstitution of sample extracts. Precipitation of this HBCD

isomer has been confirmed by re-dissolving a precipitated extract in methanol, resulting in recoveries

above 95% [26]. The use of methanol as reconstitution solvent has however the disadvantage of

creating a temporary strong band of mobile phase in the column. Therefore, injected volumes will have

an optimum in sensitivity and chromatographic performance and 15μl has been found as a good

compromise [19]. Consequently, a lot of analysis have been performed with injection volumes between

1 and 5 μl, but also volumes up to 20 μl have been injected successfully.

Flow rates between 0.1ml/min and 0.5ml/min have been applied, mostly depending on column

diameter. The influence of this setting has however not been evaluated yet. Therefore, experiments will

set off from standard flow rates for the selected column.

14

Figure 4 Chromatographic separation of three HBCD diastereoisomers on a Zorbax SB-C18 reversed-phase column

(250mm×4.6mm I.D., 5_m, Agilent) by different mobile phases. (a) 9:1 methanol/water, (b) 9:1 acetonitrile/water

and (c) gradient compositions described in Yu et al.. [31]

1.4 Detection

Analysis of HBCD has mostly been performed with ESI-MS/MS, but also APPI-MS/MS, APCI –and ESI-MS,

and electrospray ionization quadrupole linear ion trap (ESI-QqLIT) have been used for this purpose. For

the MS/MS determination in the ESI negative mode, the transition mostly measured for HBCD is M-H

(Fig. 5), m/z 640.6, to bromine, m/z 79, and for TBBPA this is m/z 542.6 (Fig. 5) to 79. However, one

15

article describes the transition of a chlorine adduct when using ammonium chloride buffer, M-Cl, m/z

674.6, 676.6, and 678.6 to m/z 638.6, 640.6, 642.6 [34]. However, this method resulted in higher limits

of detection (LOD’s) compared to the M-H to Br transition, 30-80 pg on-column, against around 0.5-20

pg on column. The lowest limits of detection reported were obtained by ESI-QqLIT, but the difference

with ESI-MS/MS are slim, 0.1-1.2 pg on column [35]. MS detection seems to be insufficient for HBCD and

TBBPA confirmatory analysis, since limits of detection, 50-150 pg [22, 24], and selectivity do not meet

the desired detection limits [36]. The choice between ESI and APCI is less pronounced, Budakowski et al.

[27] reported higher sensitivity with ESI, whereas Suzuki et al. [23] reported the opposite. Additionally,

Suzuki et al. [23] reported a worse sensitivity for TBBPA with APCI, furthermore they also mentioned a

lower selectivity with this ionization technique. Therefore, the choice for ESI and APCI seems to be a

more practical choice than one of performance. Also APPI has been used, but the method applied was

for a multi-target analysis of flame retardants which show better results with APPI or APCI [13]. Since ESI

is a more common ionization method and generally available at the institute, this will be the technique

of choice. For MS/MS optimization the settings of Granby and Nielsen [18] would be a suitable set off

since the Micromass Quattro Ultima is utilized in the institute. The infusion standard will be dissolved in

LC solution in order to mimic the LC mobile phase and to find optimized ESI-MS/MS parameters for the

applications. Since γ-HBCD seems to crystallize and precipitate in acetonitril [26], a water-methanol

solution, additionally a standard with addition of ammonium acetate will be used to optimize the system.

After the LC-method development the values will be optimized again for an optimal application.

Figure 5 Isotope clusters TBBPA and HBCD in APCI negative mode. [23]

1.5 Occurrence

Commercial HBCD is a mixture of mainly α-, β- en γ-HBCD, but also δ- and ε-HBCD have been detected at

lower levels [33]. Although the commercial mixture of HBCD contains mostly γ-HBCD, α-HBCD is the

predominant isomer in biota due to biotransformation and its water solubility [24, 37]. In Europe HBCD

16

production facility can be found in Terneuzen, the Netherlands, while TBBPA is not produced in Europe.

As a result it can be expected that residues of HBCD will be present in the local environment. In previous

studies, the presence of HBCD in the Dutch environment has already been demonstrated [38]. Although,

the HBCD plant is located in the south west of The Netherlands and HBCD in precipitation has only been

found in the vicinity of the HBCD plant [39], high levels of HBCD have been found more east, upstream,

of the HBCD plant [38]. As a consequence, it is likely that HBCD contamination in the upstream regions

of The Netherlands is caused by other, possibly diffuse sources. In general, HBCD concentrations seem

to correspond well with overall environmental pollution. Contrary to HBCD, TBBPA is not abundant

throughout the environment which could be caused by several reasons. First, TBBPA is often chemically

bound to the product [40], secondly, TBBPA is more polar and water soluble than HBCD which might

result in lower bio-accumulation [24], and thirdly TBBPA is more easily metabolized and eliminated from

organisms [2].

Figure 6 Concept analysis for the simultaneous determination of the three major HBCD diastereoisomers and

TBBPA in fish samples.

17

2. Experiments

Based on literature three experimental methods , consisting of an Accelerated Solvent Extraction (ASE),

GPC, acid digestion or SPE based clean up followed by detection using LC-MSMS , were proposed (Fig. 6).

Standard solutions used for the experiments were purchased at Greyhound Chromatography (α-,β- and

γ-HBCD and 13

C α-,β- and γ-HBCD) and Cambridge laboratory (TBBPA and ring labelled 13

C TBBPA). The

solvents were purchased at Biosolve LTD and were of Pesti-S or higher grade.

2.1 Extraction

Regarding literature the three major HBCD diastereoisomers and TBBPA can be extracted using

traditional methods like soxhlet and ultra turrax, but also with the newer ASE approach. Since ASE has

several advantages compared to the other techniques, ASE was tested for the extraction of the three

major HBCD diastereoisomers and TBBPA.

2.1.1 Materials and methods

The initial experiments arise from literature and the methods used in the institute for the extraction of

common contaminants (Tab. 3). An 125 ml extraction cell was filled with approximately 10 grams of

diatomaceous earth and spiked with a mix of native α-,β-, and γ-HBCD, and TBBPA. The sample was

placed in the ASE350 and extracted according the parameters described in table 3. The extract was

transferred over a funnel containing sodium sulphate to a Turbovap tube and evaporated until dryness.

The compounds were reconstituted in 0.5 ml 13

C TBBPA and 13

C HBCD labelled methanol/water (4:1v/v)

and transferred to a vial. Further experiments focussed on the use of other solvents, hydro matrix,

different extraction temperatures and different pH.

18

Table 3 ASE settings

Parameter Setting

ASE ASE350 (Accelerated Solvent Extractor) , Dionex

Temperature 40 °C

60 °C

80 °C

100 °C (initial)

Pressure 1500 psi

Preheat 0 min

Heat 5 min

Static 5 min

Flush 40%

Purge 300 sec

Cycle 3

Solvent n-Hexane : acetone 1:1 (initial)

n-Hexane : acetone 4:1

Dichloromethane

n-Hexane

n-Hexane : dichloromethane 4:1

n-Hexane : acetone 1:1 0.01% acetic acid (pH 4), Merck 1.00063.1000

n-Hexane : acetone 1:1 0.01% diethyl amine (pH 11), Sigma-Aldrich 100973797

Hydro matrix Diatomaceous earth, Sigma-Aldrich D-5509 (initial)

Hyflo Super cel ®, Sigma-Aldrich 56678

Isolute, Biotage 9800-5000

Sodium sulphate, Merck 1.06649.5000

Sodium sulphate mesh 0.63 -2.0 mm, Merck 1.06637.1000

Basic alumina, Sigma-Aldrich A1522

19

2.1.2 Results

The applied method in the institute was similar to methods in literature and

using this in house method HBCD and TBBPA should be extracted from the

sample with a satisfactory recovery. Although recoveries for HBCD were

satisfactory, TBBPA could not be extracted from a procedure blank (Fig. 7), which

is complementary to the results obtained by Frederiksen et al. [19]. Losses of

TBBPA could be reasoned to insufficient strength of the solvent, adhesion to the ASE hydro matrix [18],

and degradation. To determine the extraction strength of the solvents, several solvents were examined.

Although hindered by a large deviation, it could be concluded that no apparent difference occurred in

extraction efficiency between the solvents (Fig. 7). However, when using different hydro matrices the

loss of TBBPA could be accounted to the hydro matrix. When using diatomaceous earth, and the similar

Hyflo super cel, TBBPA was not or to lower extend recovered from the ASE (Fig. 7). These hydro

matrices are based on silica and active sites might induce hydrogen bonding between TBBPA and the

active sites in these materials. Isolute is also based on silica, however the larger particle size and the

modification of this material probably shields the active sites better, preventing adhesion of TBBPA. In

addition, the use of sodium sulphate also resulted in increased recoveries for TBBPA, though lower ones

for HBCD. Once more, basic alumina can form hydrogen bonds with TBBPA, explaining the observed

recoveries. In addition, together with the solvent, another liquid which is not retained by sodium

sulphate was extracted that cannot be easily evaporated.

Since the recovery was not optimal and the variations between replicates were relatively large,

experiments were performed to optimize the extraction temperature. For HBCD no significant

differences were observed, but for TBBPA an increase in recovery was observed at lower temperatures

(Fig. 8).

Since Isolute initially resulted in the relatively best recoveries, Isolute was selected instead of

hydromatrix. However, in general sample analysis Isolute did not result in satisfactory recoveries (Fig. 9).

Since the active sites on the silica based Isolute are thought to cause these recoveries, not silica based

hydromatrix were sought. An obvious alternative would be sodium sulphate, but previous experiments

resulted in unsatisfactory recoveries. On the other hand, larger sodium sulphate particles might improve

the extraction, which was also seen with diatomaceous earth and Isolute. So, sodium sulphate mesh

0.63 – 2.0 mm was tested and satisfactory recoveries were observed (Fig. 7).

ASE350, Dionex

20

Figure 7 Extraction recoveries after ASE, using diatomaceous earth and different solvents at 100 °C and 1500 psi

(left) and using different hydromatrices and hexane : acetone 4:1 at 100 °C and 1500 psi (right)

Figure 8 Extraction recoveries after ASE, using Isolute and hexane : acetone 4:1 at different temperatures (top left)

and 1500psi, Injection recovery after ASE, using Isolute and hexane : acetone 4:1 at different temperatures and

1500psi (top right), and Extraction recoveries after ASE, using Isolute and hexane : acetone 4:1 at 60 °C and 100 °C

and 1500psi (bottom)

21

Figure 9 Procedures recoveries in samples eel (n=37), using ASE (Isolute, hexane : acetone 4:1, 100 °C, 1500psi) and

a mixed bed (acid silica 16.5% H2SO4, neutral silica and Na2SO4).

Figure 10 Theoretical dissociation of TBBPA.

The low recoveries of TBBPA might also been explained by the acidic groups. These acidic groups lead to

multiple dissociation states in neutral environments (Fig. 10). Due to unfavourable polar interactions

losses in clean-up steps might occur in combination with polar solvents [15]. So, in order to obtain more

satisfactory recoveries and to create a more controlled extraction, the solvent was acidified with acetic

acid pH 4. A control procedure was performed in the same sequence at pH 7.

The resulting recoveries at pH 4 were remarkable (Fig. 11). At a pH 4 TBBPA should be fully protonated

and should theoretically have a larger affinity towards the solvent. However, recoveries at pH 4 were

lower than in the neutral control analysis and another effect seems to dominate the extraction

recoveries of TBBPA. TBBPA behaved different compared to HBCD, and based on the molecular

structure this was most likely caused by the hydroxyl groups on TBBPA. These functional groups can

make a significant difference on their behaviour towards silica. Silica based materials contain two forms

depending the pH, silanol (SiOH) and siloxane (SiO) [41]. Since these alcoholic groups can support

22

hydrogen bonding, they are able to form hydrogen bonds with the hydroxyl groups of TBBPA. At low pH,

TBBPA as well as the silica is in its protonated form, so hydrogen bonding is the strongest at low pH.

The higher recoveries at pH 7 support the theory that the extraction recovery is dominated by the

hydrogen bonding of TBBPA with silanol/siloxane groups. At low pH both the hydroxyl groups on TBBPA

and the alcoholic groups on silica are un-dissociated, resulting in maximum hydrogen bonding capacity,

whereas in partial or fully dissociated form hydrogen bonding is decreased or removed (Fig. 12).

In light of these findings an extraction at basic pH (pH 11) was performed using diethyl amine.

Recoveries at pH 11 were comparable to the recoveries at pH 7, however variations were much smaller.

This might be due to the elimination of hydrogen bonding during extraction, resulting in quantitative

extractions. However, injection recoveries were low and the whole preparation procedure needs to be

evaluated in order to determine the suitability of a basified extraction. Since the use of large particle

sodium sulphate seems to result in satisfactory extraction recoveries and does not require base resistant

extraction cells, the basified extraction was discarded and further analysis were performed with large

particle sodium sulphate.

Figure 11 Recovery after extraction, using ASE, Isolute and heaxane:acetone 1:1 at 100 °C and 1500 psi at different

pH.

2.1.3 Conclusion

Due to hydrogen bonding TBBPA can adhere to alcoholic groups in hydro matrix, and depending on the

hydro matrix and pH of the solvent, 100% to 0% recovery can be observed due to this effect. By

modification of the hydro matrix this adhesion can be prevented, and by using the modified

diatomaceous earth “Isolute”, better recoveries for HBCD as well as for TBBPA were observed. However,

23

the best recoveries were observed for not silica based materials and optimum extraction recoveries

were found with large particle sodium sulphate, in combination with a solvent mixture of 1:1 hexane :

acetone, at an extraction temperature of 100 °C conform appendix 4.2.

Figure 12 Hydrogen-bonding between TBBPA and diatomacious earth.

2.2 Clean-up

The clean-up for the determination of HBCD and TBBPA implicates the removal of the lipid content of

the extract. Popular methods to remove the lipid content from fatty sample extracts are GPC, acid

digestion, SPE or a combination of these procedures. All three procedures were tested for their

suitability .

2.2.1 Gel Permeation Chromatography

GPC separations are mainly based on compound size. Large compounds elute earlier since they cannot

enter the pores of the packing material, whereas small molecules can enter these sites and will exhibit a

smaller average velocity. Clean-up by GPC might not result in complete separation of the lipid content

and the compounds of interest and additional procedures might be required [19, 24, 29].

24

2.2.1.1 Materials and methods

To determine the elution pattern a standard mixture of TBBPA and HBCD was dissolved in 15 ml

ethylacetate : cyclohexane 1:1 and 12.5 ml of this solution was injected on the GPC (Tab. 4), Between 30

ml and 55 ml fractions of 5 ml were collected. After this window a fraction of 20 ml was collected to

ensure that no TBBPA and HBCD was lost due to late elution form the GPC column. The collected

fractions were evaporated until dryness with the Turbovap and reconstituted in 13

C TBBPA and 13

C HBCD

labelled methanol : water 4:1.

To determine the recovery of TBBPA and HBCD in fish, 2.5 g of fish oil was spiked with a standard

mixture of TBBPA and HBCD and dissolved in 15 ml ethylacetate : cyclohexane 1:1. 12.5 ml of the

solution was injected on the GPC and a the fraction between 35 ml and 75 ml was collected. The

collected fraction was evaporated till dryness with the Turbovap after which the amount of residual oil

was determined. Next the residue was reconstituted in 0.5 ml 13C TBBPA and 13C HBCD labelled

methanol : water 4:1 and transferred to a vial.

Table 4 GPC settings

Parameter Setting

Column 2.5 x 60 cm, Spectrum USA Omnilabo

Stationary phase Biobeads SX3, 200-400 mesh, Biorad (nr. 1522750)

Mobile phase Ethyl acetate : cyclohexane 1:1

HPLC pump Gilson 305, Gilson, Rijswijk, The Netherlands

Autosampler Gilson 231-401, Gilson, Rijswijk, The Netherlands

Fraction collector Gilson 202, Gilson, Rijswijk, The Netherlands

Software Unipoint v3.3

Flow 0-5 min, from 0 ml/min to 1 ml/min

5-10 min, 1 ml/min

10-118 min, 5 ml/min

118-122min, 1ml/min

2.2.1.2 Results

The elution patterns of milk fat and TBBPA and the three major HBCD diastereoisomers in a blank and

fish oil were determined (Fig. 13). In the blank the majority of the compounds were eluting between 35

and 55 m and therefore, smaller fractions were collected in the sample fish oil. The elution pattern of

the fish oil (Fig. 13) showed that the elution window is even smaller.

25

The majority of the fat is eluting before 35 ml and approximately 5 % of the fat content elutes between

35 ml and 40 ml. However, in this window a significant amount of TBBPA and αHBCD is also eluting.

Therefore, the compounds of interest cannot be fully separated from the fat. However, matrix and

stationary phase depended changes in elution patterns of the HBCD diastereoisomers have been

described in literature [32], so the pattern most likely differs per sample and system.

Although the sum of the recoveries in the elution pattern experiment were promising, recoveries of

spiked samples after GPC were low and not satisfactory for trace analysis (Fig. 14). However, this

approach might still be suitable for screening purposes.

Figure 13 (left) Elution pattern of TBBPA and the three major HBCD diastereoisomers in a sample fish oil on a 2.5 x

60 cm, Biobeads SX3, 200-400 mesh GPC column with a flow of 5 ml/min.

Figure 14 (right) Recovery of TBBPA and the three major HBCD diastereoisomers spiked to 2.5 g fish oil on a 2.5 x

60 cm, Biobeads SX3, 200-400 mesh GPC column with a flow of 5 ml/min.

2.2.2 Sulphuric acid digestion

Sulphuric acid is used to digest and remove organic matter from sample extracts. These kind of

treatments can affect the compounds of interest and therefore it can only be applied for stable

compounds. The reaction that occurs removes the hydrogen and oxygen atoms from the carbon

skeleton by dissolving them into solution leaving carbon atoms:

Example : C12H22O11(s) + 11H2SO4 � 12C(s) + 11H2SO4.H2O


Samples containing 0.5 g of fish oil spiked with native TBBPA and HBCD were prepared in a reagent tube.

The spiked fat was dissolved in 2 ml hexane after which 1 ml of concentrated sulphuric acid (97 %)

26

(purchased at VWR (Merck)) was added. The mixture was shaken gently and rested till the phases were

separated. The organic layer was transferred to an Turbovap tube, and the sulphuric acid was two times

extracted with 2 ml hexane. The organic phase was than evaporated till dryness and reconstituted in 0.5

ml 13

C TBBPA and 13

C HBCD labelled methanol : water 4:1, after which the extract was transferred to a

vial.

2.2.2.2 Results

Recoveries of the native compounds seem to be reasonable, but when taking the internal standards into

account it becomes clear that the sulphuric acid treatment heavily disturbs the LC-MS/MS analysis,

resulting in very low recoveries (Fig. 15). Regarding the accuracy, sulphuric acid clean-up is not especially

suitable for quantitative analysis, even with the use of labelled internal standards.

On basis of these results it cannot be concluded that compounds are lost during the clean-up. However,

due to matrix suppression reduction of sensitivity occur in the MS. By applying additional clean-up steps

this suppression might be decreased and lead to better recoveries. Other opportunities for

improvement lie in a better phase separation by centrifugation, filtering the final extract, and in

increasing the final extract volume. However, this last opportunity leads to lower theoretical detection

limits.

Figure 15 Recovery of TBBPA and the three major HBCD diastereoisomers spiked to 0.5 g fish oil after sulphuric

acid clean-up.

27

Na2SO

4

Acid silica 16.5 %

H2SO

4

Acid silica 33 %

H2SO

4

Silica gel

Na2SO

4

SPE column design 2 (Tab.6).

2.2.3 Solid Phase Extraction

SPE can be used to remove interference and even fat from a sample. For removal of

interferences and small amounts of fat silica is an often used material, whereas acidified silica is

used for the removal of large amounts of fat. Silica cartridges are commercially available, but also

handmade columns can be prepared. Since several silica based materials showed to have a

negative influence on compound recovery in the ASE experiments, experiments were performed

to establish the influence of the material on the compounds of interest. Afterwards acidified

silica columns were prepared to remove approximately 2.5 g of fat from a sample.


Sep-pack Plus silica cartridges were purchased from Waters (WAT020520), silica gel (70-230

mesh) and concentrated sulphuric acid (97 %) were purchased at VWR (Merck). For the silica gel

experiments glass columns were filled with glass wool, 0.5 g anhydrous sodium sulphate and

different amounts of silica gel (Tab. 5). The columns were conditioned with 10 ml hexane after

which a spiked solution containing native TBBPA and HBCD in 0.5 ml hexane was applied to the

column. After washing with hexane, the compounds were eluted with hexane : ethylacetate 1:1

and collected in a Turbovap tube (Tab. 5). The collected extract was evaporated till dryness in the

Turbovap and re-dissolved in 0.5 ml 13

C TBBPA and 13

C HBCD labelled methanol : water 4:1, after

which the extract was transferred to a vial. The procedure for the silica cartridges was the same

as for the 2 g silica column.

Table 5 SPE columns and SPE procedures

Silica (g) Wash (ml) Elute (ml)

2 12 16

4 24 32

8 48 64

12 96 128

28

Table 6 SPE mixed bed column design and SPE procedure

Packing/step Design 1 Design 2

Sodium sulfate 0.5 g 10 g

16.5% H2SO4 8 g 40 g

33% H2SO4 6 g 10 g

Silica 2 g 10 g

Sodium sulfate 0.5 g 10 g

Condition with hexane 25 ml 100 ml

Wash with hexane 100 ml 400 ml

Elution hexane dichloromethane 130 ml 500 ml

For the acid silica experiments, silica gel was acidified (33 %(w/w)) by adding concentrated sulphuric acid

(97 %) to silica gel in a glass bottle. The mixture was shaken firmly till no observable clotting was present

and mixed head over head during one night. The columns were prepared according to table 5, but

instead of using silica, acid silica was used. Samples containing 0.2 g of fish oil spiked with native TBBPA

and HBCD were prepared in 10 ml hexane. The procedure was according the silica gel SPE, but next to

hexane : ethyl acetate 1:1 as eluent, also experiments with hexane : dichloromethane 1:1 as eluent were

performed.

For the mixed bed columns, silica was acidified to 33 %(w/w) and 16.5 %(w/w). The column designs and

procedures were according table 6. Samples in design 1 contained 0.25 g fat, spiked with native TBBPA

and HBCD in 10 ml hexane, whereas samples in design 2 contained 2.5 g of fish oil spiked with native

TBBPA and HBCD in 25 ml hexane. The final extract was evaporated till dryness and reconstituted in 0.5

ml 13

C TBBPA and 13

C HBCD labelled methanol : water 4:1, after which the extract was transferred to a

press fit vial.

2.2.3.2 Results

SPE clean-up with silica has been described in literature [19, 28, 29] and a standard method was applied.

The experiments confirmed the results (Fig. 16) in literature and scaling of the method is possible

without affecting recoveries. When applying the method to acidified silica problems occurred when the

extract was evaporated. The last part of the solvent was not prone to volatilization and did not

evaporate. The residue seemed to be sulphuric acid which co-eluted from the column together with the

29

Na2SO

4

Acid silica 16.5 %

H2SO

4

Acid silica 33 %

H2SO

4

Silica gel

Na2SO

4

SPE column design 2 (Tab.6)

after application

solvent. Therefore, the residue was extracted three times with 2 ml hexane and the general

procedure was continued again. Recoveries in this experiment were low and varied a lot, which

is in agreement with the observations in the experiment with sulphuric acid digestion followed

by a liquid/liquid extraction.

Since a more polar solvent than hexane is needed to elute TBBPA effectively from the column

and a less polar solvent than ethyl acetate (polarity index 4.4) is necessary to prevent elution of

the sulphuric acid, dichloromethane (polarity index 3.1) was added to the eluent solvent instead

of ethyl acetate. This time, no sulphuric acid seemed to be eluted from the column and the

recoveries and variations were significant better than when ethyl acetate was used in the eluent

(Fig. 17).

Although the recoveries seem promising, problems occurred when a sample was applied. The

small column diameter and high acid concentrations resulted in clogging, and in most columns

the flow rate was tediously slow. Some columns even clogged in such extend that the solvent

could not flow through. By applying pressure to the entrance of the column the procedure could

be speeded or continued, but for general sample procedures this is not desired. Therefore, the

column design was improved and instead of a high acid concentration at the beginning of the

column, a mixed bed design was applied with a lower acid concentration in the first part of the

column and a higher acid concentration at the end. The final part of the column still consisted of

silica gel for additional clean-up.

The improved column showed to be capable of handling the desired amount of fat without

losing excessive column permeability while procedure recoveries remained satisfactory together

with a small variation (Fig. 18). Since the design was capable of dealing with the set amount of

fat, the design was enlarged for the removal of 2.5 g of fat (Tab. 6 design 2).

Together with the expansion of the column, also the amount of necessary solvent increases

which also troubles the following evaporation step. To establish the necessary amount of elute,

the elution pattern of the compounds was determined (Fig. 19). Since the polarity lengthens the

elution of TBBPA to the end of the collection window it was not possible to shorten this step.

However, the observed capacity of the column showed to be a larger than expected. Based on

30

the observations (photo page 29) and the acid concentration, the column seems to be able to handle

amounts of fat up to 6 g.

Since the capacity of the second column design was more than enough for the selected amount of fat, a

column design with a first layer 18.4 % H2SO4 (easier to produce) and without the 10 g of 33 % H2SO4

was applied for a proficiency test. The observed column capacity showed to be capable of handling 20 g

of salmon (8.5 % fat), while maintaining good recoveries (Fig. 20).

Figure 16 (left) Recovery of TBBPA and the three major HBCD diastereoisomers after silica SPE clean-up.

Figure 17 (right) Recovery of TBBPA and the three major HBCD diastereoisomers after acid silica SPE clean-up using

hexane : dichloromethane 1:1 as eluent.

Figure 18 (left) Recovery of TBBPA and the three major HBCD diastereoisomers after mixed bed acid silica SPE

clean-up conform design 1.

Figure 19 (right) Elution pattern of TBBPA and the three major HBCD diastereoisomers from mixed bed acid silica

SPE clean-up conform design 2.

31

Figure 20 Recoveries in samples proficiency test Folkehelse 2011

2.2.4 Powerprep™ Solid Phase Extraction

The Power-Prep™ is an alternative for gravitational SPE. Instead of

using gravity to accomplish a flow through the column, a pump

generates the flow. Besides maintaining a constant flow and an

automated pump system, the Power-Prep™ is programmable, so

complex SPE can be performed automatically. In these experiments an

attempt is made to replace the manual and slower SPE clean-up by the

automated Power-Prep™ SPE system.


The Power-Prep™ can be equipped with a large acid silica (63-200

mesh) column, an acid : base : neutral silica (63-200 mesh) mixed bed

column, an alumina column, a carbon column, and various other

columns. For the analysis of TBBPA and HBCD the large acid silica, mixed bed, and a neutral silica column

were tested. The large acid silica consist of a Teflon column of which a part is filled with Teflon chips and

the rest with 45 g of acid silica. The acid silica contains 44 %(w/w) of sulphuric acid and has a prescribed

Power-PrepTM

, FMS

32

capacity of 4 g of fat. The mixed bed column consist of 4 g acid silica (44 %(w/w) H2SO4), 2 g basic silica (27

% (w/w) 1N NaOH), and 1.5 g neutral silica (5 %(w/w) distilled water), the neutral silica column consist of 6 g

silica.

In the first experiment, the large acid silica column and the acid : base mixed bed column were tested .

The method consisted of conditioning the columns with 50 ml hexane at a flow of 10 ml.min-1

after

which a sample consisting of 2.5 g fish oil spiked with native TBBPA and HBCD in 20 ml hexane was

applied at a flow rate of 5 ml.min-1

. The Power-Prep™ was programmed to draw 25 ml from the sample,

so when the sample was almost completely applied, an additional 5 ml was added to ensure a

quantitative sample transfer. Subsequently, the column was eluted with 500 ml hexane divided into

fractions of 50 ml. The fractions were evaporated till dryness and reconstituted in 0.5 ml methanol :

water 4:1, after which the extract was transferred to a vial.

In the second experiment, only the acid : base silica column was used with a sample intake of 0.5 g. The

method consisted of conditioning the columns with 40 ml hexane : dichloromethane at a flow of 10

ml.min-1

after which a sample spiked with native TBBPA and HBCD in 20 ml hexane was applied at a flow

rate of 5 ml.min-1

. The Power-Prep™ was programmed to draw 25 ml from the sample, so when the

sample was almost completely applied, an additional 5 ml was added to ensure a quantitative sample

application. Next, the column was eluted with 250 ml hexane : dichloromethane divided into fractions of

50 ml. The fractions were evaporated till dryness and reconstituted in 0.5 ml 13

C TBBPA and 13

C HBCD

labelled methanol : water 4:1, after which the extract was transferred to a vial.

In the third experiment, the large acid silica column was tested with a blank. The method consisted of

conditioning the columns with 100 ml hexane at a flow of 10 ml.min-1

after which a 25 ml hexane spiked

with native TBBPA and HBCD was applied at a flow rate of 5 ml.min-1

. The Power-Prep™ was

programmed to draw 35 ml from the sample, so when the sample was almost completely applied, three

times 5 ml was added to ensure a quantitative sample application. Subsequently, the column was

washed with 300 ml hexane and eluted with 500 ml hexane : dichloromethane 1:1 divided into fractions

of 100 ml. The fractions were evaporated till dryness and reconstituted in 0.5 ml 13

C TBBPA and 13

C

HBCD labelled methanol : water 4:1, after which the extract was transferred to a vial.

33

Table 7A Power-PrepTM

method without wash for the simultaneous determination of TBBA and the three major

HBCD diastereoisomers.

Step flow volume valves event Solvent

1 10 200 01122006 condition Hexane : dichloromethane 50:50

2 5 35 06122006 add sample Hexane : dichloromethane 50:50

3 10 100 03122001 elute Hexane : dichloromethane 50:50





8 10 12 01122006 end Hexane : dichloromethane 50:50

Table 7B Power-PrepTM

method with wash for the simultaneous determination of TBBA and the three major HBCD

diastereoisomers.

Step flow volume valves event Solvent

1 5 50 01112006 condition hexane


3 5 35 06112006 add sample hexane

4 10 150 01112006 wash hexane

5 10 500 03112001 elute hexane:dichloromethane 50:50

6 10 12 01112006 end hexane:dichloromethane 50:50

In the fourth experiment the wash step was discarded. The method (Tab. 7A) consisted of conditioning

the columns with 200 ml hexane : dichloromethane 1:1 at a flow of 10 ml.min-1

after which a 25 ml

hexane spiked with native TBBPA and HBCD was applied at a flow rate of 5 ml.min-1

. The Power-Prep™

was programmed to draw 35 ml from the sample, so when the sample was almost completely applied,

three times 5 ml was added to ensure a quantitative sample application. Afterwards, the column was

eluted with 500 ml hexane : dichloromethane 1:1 divided into fractions of 100 ml. The fractions were

evaporated till dryness and reconstituted in 0.5 ml 13

C TBBPA and 13

C HBCD labelled methanol : water

4:1, after which the extract was transferred to a vial.

In the fifth experiment an additional neutral silica column was installed after the large acid silica column.

The method was applied according table 7B. In the sixed experiment the wash step was reduced till 150

ml and the neutral silica column was discarded. Additionally, an elution pattern was determined with

and without addition of 0.01% acetic acid (pH 4) to the elution solvent.

34

2.2.4.2 Results

When using the large acid silica and the mixed bed column no recoveries were observed for any of the

compounds. This was probably caused by the low elution power of the solvent and the retention power

of the columns. Because of this complication, TBBPA and HBCD cannot be analysed together with

current contaminant analysis on the Power-Prep™ at the institute.

Since it was thought that the large acid silica column was leading to degradation and hexane was

supposed to be too a-polar, only the mixed bed column was tested, while dichloromethane was used for

elution. The recoveries in this experiment were good for HBCD, but TBBPA was not or nearly not

recovered from the column (Fig. 21). This observation is again in line with the previous results obtained

with the ASE, together with basic materials. Therefore, the mixed bed column would not be suitable for

the analysis of TBBPA.

In the third experiment the large acid silica column was tested conform the design of the manual SPE

experiments. So, the column was conditioned and washed with hexane, and eluted with hexane :

dichloromethane 1:1. The elution pattern obtained shows that TBBPA is slowly eluting from the column,

whereas the HBCD diastereoisomers eluted already partly during the wash step.

In the experiment without wash step it became clear that HBCD was send to the waste. Overall

recoveries were approximatly 100 % and an automatisation of SPE step seems to be possible (Fig. 22).

The elution pattern shows again that TBBPA is slowly eluting from the column (Fig. 23). When the

method was applied to real samples recoveries were again good (Fig. 22). However, injection recoveries

were low and were most probably caused by matrix effects. These effects might raise from interferences

that were removed by the wash step, but since this was not possible for this column these could not be

removed. Therefore, the washstep was introduced again, but at reduced volume. In addition, to reduce

the elution time of TBBPA the elution solvent was acidified with 0.01 % acetic acid. The reduction of the

volume of the washstep wash sufficient to recover all of the compounds and addition of the acid

modifier seemd to shorten the elution band of HBCD. However, TBBPA was not reovered from the

column, which supports the theory of hydrogen binding to free silanol groups.

The method with washstep was applied to samples, but yet again low to no recoveries were observed

due to matrix interferences. So, the extracts were purified once more on a 2 g silica SPE cartridge which

resulted in satisfactory recoveries (Fig. 24).

35

Figure 21 Recovery of TBBPA and the three major HBCD diastereoisomers from mixed bed Power-Prep™ column.

Figure 22 Recovery of TBBPA and the three major HBCD diastereoisomers from the acid silica Power-Prep™

column without wash step eluting with hexane dichloromethane in blanks (left) and spiked samples (right)

Figure 23 (left) Elution pattern of TBBPA and the three major HBCD diastereoisomers from the acid silica Power-

Prep™ column without wash step, elution with hexane dichloromethane 1:1..

Figure 24 (right) Procedure recovery after ASE extraction with sodium sulphate 0.63 – 2.0 mm mesh, and clean-up

with a large acid silica and and an sep-pack silica column.

36

2.2.4 General procedure tests

Between the main sample pre-treatment steps, two minor procedures are applied. After the extraction

the extract is dried using sodium sulphate, and later evaporated till dryness. The evaporation step also

occurs after the clean-up stage. Due to these procedures losses might occur which are not related to the

tested parameters, so recoveries of these steps were determined to establish their influence on the

overall recovery.


For the evaporation experiment native TBBPA and HBCD was added to 50 ml hexane : acetone 1:1 and

evaporated till dryness in the Turbovap. The compounds were then reconstituted in methanol : water

4:1 and transferred to a vial.

In the experiment with sodium sulphate native TBBPA and HBCD was added to 50 ml hexane : acetone

1:1. The mixture was homogenized and passed through a funnel filled with glass wool and sodium

sulphate after which 100 ml hexane was passed through. The collected extract was evaporated till

dryness and reconstituted in 0.5 ml 13

C TBBPA and 13

C HBCD labelled methanol : water 4:1, after which

the extract was transferred to a vial.

2.2.4.2 Results

The recoveries obtained after the evaporation step are much higher than the added amount for TBBPA

and slightly above the added amount for the HBCD diastereoisomers (Fig. 25). This deficiency might

arise from the LC-MSMS separation and measurement and it is believed that during this step no

significant losses occur.

The recoveries after the sodium sulphate treatment are slightly below the added amount (Fig. 26).

Sodium sulphate was not believed to cause losses of the compounds of interest, and since only a single

measurement has been performed the lower recoveries are not assumed to be caused by the sodium

sulphate treatment.

37

Figure 25 (left) Recovery after evaporating hexane : acetone 1:1 and reconstitution in methanol : water 4:1.

Figure 26 (right) Recovery after extract drying using sodium sulphate and hexane.

2.2.5 Conclusion

The clean-up for the simultaneous determination of TBBPA and the three major HBCD diastereoisomers

is troubled by the difference in polarity of the two compounds. Whereas HBCD is strong a-polar, TBBPA

is moderately polar and needs a polar solvent to be extracted from column material.

SPE with acid silica has proven to be a suitable method to remove fat and oil from samples. This

analytical technique can either be performed manually or the automated Power-PrepTM

SPE system.

However, due to differences in column material, the applied method differs and cannot be interchanged

between the manual approach and the Power-PrepTM

.

Next to SPE, also GPC was capable of separating the fat or oil from the compounds. However, where in

SPE good recoveries were observed, recoveries by GPC were only reasonable. Therefore GPC does not

seem to be suitable for quantitative trace analysis, but might still be suitable for screening purposes

since it is a non-invasive separation technique. Lastly, since sulphuric acid has a high capacity for

removing fat, a sulphuric acid treatment was tested. However, this method resulted in dirty extracts

leading to low injection recoveries and high limits of detection.

Concluding, for sample pre-treatment two approaches are suggested for the simultaneous analysis of

TBBPA and HBCD; firstly, ASE extraction followed by acid silica and silica SPE, and secondly ASE

extraction followed by GPC (Fig. 27).

38

Figure 27 Supposed sample pretreatment for the simultenenous analysis of TBBPA and the three major HBCD

diastereoisomers.

2.3 Separation

The separation of the three major HBCD diastereoisomers is well described in

literature and often involves simple mobile phases with a small and simple

gradient. The influence of acetonitrile and methanol was evaluated, as well as

the gradient, pH and injection volume.


Standard mixtures of TBBPA and α-, β-, γ-, δ- and ε- HBCD were prepared to

optimize the LC separation on a Waters Symmetry C18, 150 mm x 3 mm, 5 µm

column using an Agilent 1200 series HPLC. In a later phase the column was

changed to a Waters Symmetry C18, 150 mm x 2.1 mm, 3.5 µm column, and

several other HPLC systems were used. Detection was performed by a Waters

Quattro Micro MS/MS.

To determine the influence of methanol and acetonitrile, two gradients were developed; one from

methanol : water 8:2 to methanol, and another from acetonitrile : water 8:2 to acetonitrile. In further

experiments isocratic methods with an organic (methanol : acetonitrile 7:3) : water ratio of 8:2 and a

39

ratio of 9:1 were evaluated to simplify the analysis. Additionally, the injection capacity and the retention

times of δ- and ε-HBCD were determined.

Since peak widths and retention times of TBBPA might be influenced by pH, the pH dependence of

TBBPA was evaluated. Considering the pka (7.6), the protonation of TBBPA is prone to pH changes in a

neutral environment (Fig. 10.). Since LC-columns are preferentially operated at low pH, the pH was

lowered to pH 4 woth acetic acid (0.01%) to obtain fully protonated TBBPA.

2.3.2 Results

As derived from literature, α- and β-HBCD can be separated by adding methanol to the mobile phase,

whereas β- and γ-HBCD can be separated by adding acetonitrile to the mobile phase (Fig. 28 A, B) [14,

31]. Additionally, these solvents have influence on the response height of the different compounds.

When using methanol, TBBPA exhibits a lower response than the HBCD isomers, but when using

acetonitrile TBBPA becomes more sensitive.

Based on literature a mixture of 7:3 methanol : acetonitrile was used in order to obtain the best

separation and sensitivity for the HBCD isomers [31]. The results obtained using this composition

confirms the reporting in literature and additionally, a small gradient is necessary to separate the HBCD

isomers without peak broadening (Fig. 28 C-F). When an isocratic mode was applied, peaks became

broad for the HBCD isomers using a mixture of organic : water 8:2 and at a higher organic content (9:1)

the peak of TBBPA broadened, whereas the peaks for the HBCD isomers were narrow, though baseline

resolution was not obtained. Finally, using a gradient optimal separation of the three major isomers

were obtained.

Since TBBPA is not fully protonated at neutral pH, TBBPA peaks were somewhat broader than the

peaks of HBCD. Even worse, when applying different matrices retention times started to change (Fig.

29). For this reason, the mobile phase was acidified till pH 4, resulting in stable retention times and

narrow peak width.

Next to the major HBCD isomers, also δ-HBCD and ε-HBCD can be found, though in lower amounts. The

retention times of these isomers interfere with the retention times of α-HBCD and γ-HBCD. Where α-

HBCD and δ-HBCD are still approximately for 50% separated, γ-HBCD and ε-HBCD are not separated at

all (Fig. 30). The optimal separation was achieved with a gradient from 70 % organic till 95 % organic in

40

18 min. Nevertheless, since γ-HBCD and ε-HBCD are not separated, γ-HBCD cannot be reported as being

only γ-HBCD. So combined reporting is necessary for γ-HBCD and ε-HBCD.

Although the injection volume, the absolute respnse and the signal to noise ration show a linear

correlation (Fig. 30), the injection volumes cannot be increased excessively. While, graphical illustrations

do not clearly show the consequence of large inejction volumes, chromatograms display excessive

peakbroadening at injections volumes overl 20 µl.

2.3.3 Conclusion

The LC separation of TBBPA and the three major HBCD diastereoisomers is straightforward and easily

developed based on literature. Using a Waters Symmetry C18 150 mm x 2.1 or 3 mm x 3.5 or 5 µm

column, a flow rate of 0.4 ml.min-1 and the gradient presented in Table 8, in which A is water 0.01%

acetic acid and B methanol : acetonitrile 0.01 % acetic acid (7:3) , TBBPA and the three major HBCD

diastereoisomers could be separated with baseline resolution. Injections up to 20 µl do not lead to

excessive peak broadening and will provide in suitable limits of detection. Though, the analysis

developed and also as described in literature can be interfered by δ-HBCD and ε-HBCD

41

A

Mix 50 ng\ml

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

%

0

100

HBCD-20101018-9-MeOH MRM of 4 Channels ES- TIC

7.08e411.25

10.87

9.976.23

B

Mix 50 ng\ml

Time2.00 4.00 6.00 8.00 10.00

%

0

100

HBCD-20101018-10-ACN MRM of 4 Channels ES- TIC

5.96e43.84

7.16

6.61 8.72

C

Mix 50 ng\ml

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

%

0

100

HBCD-20101018-3 MRM of 4 Channels ES- TIC

4.91e48.55

20.32

16.27

25.09

D

Mix 50 ng\ml

Time2.00 3.00 4.00 5.00 6.00 7.00

%

0

100


1.40e54.32

2.94

4.05

4.63

E

Mix 50 ng\ml

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

%

0

100


9.16e45.85

11.8410.66

9.48

F

Mix 50 ng\ml

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

%

0

100


8.31e45.98

12.67

10.83

14.57

Figure 28 A Gradient from 8:2 (Methanol: H2O) to 1:0 (Methanol:H2O) in 15 minutes, B Gradient from 8:2 (ACN:

H2O) to 1:0 (ACN:H2O) in 15 minutes C Isocratic 8:2 (Methanol/Acetonitrile 7/3: H2O), D Isocratic 9:1

(Methanol/Acetonitrile 7/3: H2O) E Gradient from 8:2 (Methanol/Acetonitrile 7/3: H2O) to 1:0 (A:B) in 15 minutes,

F Gradient from 8:2 (Methanol/Acetonitrile 7/3/: H2O) to 9:1 (A:B) in 15 minutes

42

Powerprep 1 wk 15 fraction 1

Time2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75

%

0

100

HBCD-20110427-30 Sm (Mn, 2x1) 1: MRM of 4 Channels ES- 552.9 > 79.05

8.55e34.58

Wastewater dilutionStandardFish sampleafter clean-up

Figure 29 Chromatograms of 13

C TBBPA for a standard, fish sample and waste water sample using a mobile phase

of A water 0.01 % acetic acid, B methanol 0.01 % acetic acid, and a gradient from 80 % B till 100 % B in 9 min.

Time4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

%

0

HBCD-20110623-70 2: MRM of 4 Channels ES- 638.8 > 79

1.41e56.38

delta

gamma /epsilon

betaalpha

Figure 30 Chromatograms of 13

C TBBPA for a standard, fish sample and waste water sample using a mobile phase A

water 0.01 % acetic acid, B methanol 0.01 % acetic acid, and a gradient from 70 % B till 95 % B in 18 min.

43

A B

C D

E F

Figure 31 The injection volume versus the signal to noise ratio A αHBCD and B TBBPA, the injection volume versus

the absolute response C αHBCD and D TBBPA, and the absolute response versus the signal to noise ratio E αHBCD

and F TBBPA.

Table 8 LC gradient

Time (min) Mobile phase composition

0 20 % A, 80 % B

9 100 % B

12 100 % B

13 20 % A, 80 % B

17 20 %, 80 % B

44

2.4 Detection

Confirmatory target analysis is often employed by tandem MS. The advantage of this

technique is it specificity which results in low backgrounds and therefore in low limits of

detection. In addition, tandem MS can be used to confirm the presence of a substance

conform EU legislation [36, 42].


Solutions of 5 µg.ml-1

native α-HBCD and TBBPA were infused at a flow rate of 10 µl.min-1

into a flow of

0.4 ml.min-1

methanol generated by an Agilent 1200 series UPLC pump on a Waters Quattro Micro

MSMS. The MSMS was optimised on the parent ion and MS and MSMS spectra were obtained over the

range of m/z 70 to m/z 700 for HBCD and from m/z 70 to m/z 600 for TBBPA. In a later stage the MSMS

parameters were also optimized for an Waters Quattro Ultima MSMS.

2.4.2 Results

The optimized MSMS parameters are displayed in Table 9 for the Waters Quattro Micro for the

transition parent to bromine and for the Waters Quattro Ultima for the parent to other daughters. The

parameters on the Waters Quattro Micro deviate from general users specifications, but with these

settings the highest signals were obtained. When the settings were transferred to the Waters Quattro

Ultima a low sensitivity was observed. After a new optimization the sensitivity was significantly

improved and it was demonstrated that MSMS parameters cannot be easily interchanged between

MSMS models.

To determine the correct masses a MS scan of α-HBCD and TBBPA was performed and the expected

isotope cluster for HBCD at m/z 641 was observed (Fig. 32). Additionally, at m/z 79 and m/z 81 loss of

bromine is evident as it has approximately the same intensity as the parent molecule. The peak

observed at m/z 89 originates from toluene in which the purchased standard is dissolved. In general two

distinct transitions would be preferred to measure, but for HBCD only the parent to bromine transition

is strong in the MSMS scan (Fig. 33).

In the MS spectrum of TBBPA (Fig. 34) the parent ion cluster is the most abundant and few degradation

is observed, compared to HBCD. The parent MSMS scan shows three transitions at m/z 448, m/z 418

45

and m/z 79 (Fig. 35). For quantitative analysis the transition to bromine is again the most suitable one,

but for confirmatory reasons the transition to m/z 448 or m/z 418 would be preferential since these

transitions are more compound specific (Fig. 36).

Table 9A Optimized MSMS parameters for the detection of TBBPA and HBCD with a Waters Quattro Micro.

Parameter Waters Quattro Micro TBBPA

Parent to bromine

HBCD

Parent to bromine

Capillary 3

Cone voltage 40 13

Source temperature °C 150

Desolvation temperature °C 400

Cone gas flow l/h 10

Desolvation gas flow l/h 550

Collision energy eV 31 12

Pirani pressure mbar 2.83E-03

Table 9B Optimized MSMS parameters for the detection of TBBPA and HBCD with a Waters Quattro Ultima.

Parameter Waters Quattro Ultima TBBPA

Parent to 417.8 / 447.8

HBCD

Parent to bromine

Capillary 3 2.5

Cone voltage 130 50





Collision energy eV 41 / 33 13


46

Figure 32 ESI-MS spectrum of HBCD

Figure 33 ESI-MSMS spectrum of HBCD

47

Figure 34 ESI-MS spectrum of TBBPA

Figure 35 ESI-MSMS spectrum of TBBPA

48

2.4.3 Conclusion

Sensitivity of the ESI MSMS detection of HBCD is troubled by the labile bromine carbon bond in the

molecule. As a results bromine dissociation already occurs in the ion source leading to a lower intensity

for the parent ion cluster. The parent MSMS spectrum also shows few specific fragments and MSMS has

to be performed on the parent to bromine transition.

TBBPA is less prone to dissociation in the ion source, leading to a more abundant parent ion cluster. The

MSMS spectrum shows molecule specific fragments of which m/z 418 and m/z 448 are most abundant.

3. Sample analysis During method developed the proficiency test Folkehelse 2011 was executed. The samples in the

proficiency test consisted of egg (white and yoke), mozzarella and salmon and a standard solution.

Additionally, samples eel from a relatively clean and contaminated area were analysed for HBCD isomers

and TBBPA. The samples were extracted by ASE at 60 °C (Appendix 4.1) , purified by acid silica SPE

(Appendix 4.3) and analysed by LC-MSMS conform Table 8, 9 and 10. Recoveries of the internal

standards were good for mozzarella and salmon, but for the egg the recoveries were low (Fig. 20).

However, the accuracies in an spiked and two days incubated egg sample were around 100 %, so

quantitative results could be obtained. Recoveries in samples eel were 80 ± 25 % for all compounds.

As well as in the egg, as in the mozzarella no TBBPA or HBCD was detected, but in the salmon different

amounts of α-HBCD, β-HBCD and γ-HBCD were detected (Fig 36) many times above the detection limits

(Tab. 11).

HBCD could be determined in samples salmon and eel (Fig. 37). Since α-HBCD is most prone to

bioaccumulation [43] relative high levels of α-HBCD were found compared to β- and γ-HBCD. In known

HBCD polluted regions total levels of HBCD up to 134 ng/g were found, whereas in clean areas levels

between 0.1 and 1 ng/g were observed. These results are complementary to earlier studies on HBCD,

and over the years the potential of HBCD to bio accumulate has been demonstrated, which supports the

EFSA commandment [10]. While δ-HBCD and ε-HBCD are present in commercial HBCD mixtures [44],

they are not observed in the samples since ε-HBCD co-elutes with γ-HBCD while the relative high

concentration of α-HBCD and the limited separation between α- and δ-HBCD make identification of δ-

HBCD impossible.

49

Although HBCD was widely found in water samples collected throughout the Netherlands, TBBPA was

found only in a few samples of eel at levels up to 1 ng/g. Previous studies also reported low levels and

mostly non-detect results for TBBPA. There are a number of reasons that only low TBBPA concentrations

are found in aquatic biota. Firstly, TBBPA emissions are probably low since TBBPA is chemically bound in

most applications, secondly, TBBPA has a lower bioaccumulation potential compared to other BFR’s as

PBDE’s, thirdly, the polar nature of TBBPA can subject it to metabolism and elimination from the

organisms [37].

Figure 36 Chromatogram of sample salmon in proficiency test Folkehelse 2011

LOD Egg Mozzarella Salmon

TBBPA 112 13 7

αHBCD 300 30 2

βHBCD 29 4 3

γHBCD 17 10 1

Table 11 Limits of detection (pg/g) for the in the analysis of the proficiency test samples Folkehelse 2011

50

Figure 37 Levels of HBCD isomers

and TBBPA in eel in The Netherlands

51

4. Conclusion

A method for the simultaneous determination of TBBPA and the three major HBCD

stereoisomers was developed and a platform for the screening of polar contaminants was

created (Fig. 38, appendix 4). The method has proven to be effective in the proficiency test

organized by the Folkehelse institute in 2011, in which α-HBCD, β-HBCD and γ-HBCD were

detected in a salmon samples. Additionally, HBCD contamination in eel

has been demonstrated using the developed method

Samples can be extracted using the ASE with hexane : acetone (1:1) and sodium sulphate 0.63 –

2.0 mm mesh at a temperature of 100 °C and a pressure of 1500 psi. Adhesion of TBBPA to the

hydromatrix through hydrogen bonding might occur when other materials are used. Hydrogen

bonding can also occur during clean-up steps with silica or basic materials.

After the ASE extraction, a solvent switch, consisting of evaporation of the ASE solvent by the

Turbovap evaporation system and reconstitution in hexane is needed for the further clean-up of

the sample. The clean-up consist of a acid silica combined with neutral silica SPE for

quantitative analysis and a GPC separation could be applied for screening purposes.

After SPE or GPC separation, a solvent switch to methanol is needed to provide the LC

with a compatible solvent, and with the use of a Waters

Symmetry C18, 150 mm x 3.1 mm, 3.5 µm column, a

gradient (Tab. 8) of acidified water, acidified methanol and

acetonitrile, and a flow rate of 0.4 ml.min-1

, separation of

the compounds was achieved, while obtaining stable

retention times and narrow peaks. However, interferences

might occur from δ-HBCD and ε-HBCD at the

retention time of α-HBCD and γ-HBCD.

Quantitative detection of the compounds after

LC was achieved by monitoring compound

specific transitions on

a Waters Quatro

Ultima.

Figure 38 Schematics of the

simultaneous determination of

TBBPA and the three major HBCD

diastereoisomers

52

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30. Bethune, C., K. Julshamn, and A.-K. Lundebye, A preliminary comparison of polybrominated diphenyl ether concentrations relative to lipid content and to levels of dioxins and dioxin-like polychlorinated biphenyls in Norwegian farmed Atlantic salmon (<i>Salmo salar</i>). International Journal of Food Science & Technology, 2005. 40(2): p. 143-148.

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32. Mariussen, E., et al., Relevance of 1,2,5,6,9,10-hexabromocyclododecane diastereomer structure on partitioning properties, column-retention and clean-up procedures. Journal of Chromatography A, 2010. 1217(9): p. 1441-1446.

33. Heeb, N.V., et al., Structure elucidation of hexabromocyclododecanes--a class of compounds with a complex stereochemistry. Chemosphere, 2005. 61(1): p. 65-73.

34. Gomara, B., et al., Development of a new method for the enantiomer specific determination of HBCD using an ion trap mass spectrometer. Anal Chim Acta, 2007. 605(1): p. 53-60.

35. Guerra, P., et al., Identification and trace level determination of brominated flame retardants by liquid chromatography/quadrupole linear ion trap mass spectrometry. Rapid Commun Mass Spectrom, 2008. 22(7): p. 916-24.

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37. Brandsma, S.H., et al., Identification of Hydroxylated Metabolites of Hexabromocyclododecane in Wildlife and 28-days Exposed Wistar Rats. Environmental Science & Technology, 2009. 43(15): p. 6058-6063.

38. van Leeuwen, S.P. and J. de Boer, Brominated flame retardants in fish and shellfish - levels and contribution of fish consumption to dietary exposure of Dutch citizens to HBCD. Mol Nutr Food Res, 2008. 52(2): p. 194-203.

39. Peters, R.J.B., H. Beeltje, and R.J. van Delft, Xeno-estrogenic compounds in precipitation. Journal of Environmental Monitoring, 2008. 10(6): p. 760-769.

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55

Appendix 1 Extraction methods Method Matrix Solvents Condition 1 Condition 2 Condition 3 Condition 4 LOD Comments Ref

ASE dust 1:1 DCM:Hexane 90C 1500psi Florisil, hydromatrix

Flush V 50%

5-50 ng/g Abdallah 2008

ASE oil, fish, feed 8:2 DCM:Hexane 40C 1500psi acid silica 2-3 pg/g Berntssen 2010

ASE fish 4-6 pg Budakowski 2003

ASE biota, egg, liver, addipose tissue

3:1 Hexane:Acetone 60/100/125/150C

1-90 ng/g Low recovery TBBPA

Frederiksen 2007

4:1 Hexane:Acetone

1:1 Hexane:DCM

ASE fish 1:1 Hexane:Acetone polyacrylic acid, hydromatrix

0.25 pg/ul Granby

ASE biota DCM 100C 2000psi 0.3 ng/g Stapleton 2006

ASE biota, sediments DCM 100C 2000psi Flush V 75%

Tomy 2005

Soxhlet biota, egg, liver, addipose tissue

3:1 Hexane:Acetone 75C 7 hours 0.5-1.5 pg/ul Frederiksen 2007

Soxhlet fish 1:1 Hexane:Acetone 0.25 pg/ul Granby

Soxhlet Marine biota 3:1 Hexane:Acetone Janak 2005

56

Soxhlet Marine biota 3:1 Hexane:Acetone van Leeuwen 2008

Soxhlet Sediment, biota 3:1 Hexane:Acetone 50-150 pg Morris 2004/2006

1:1 Hexane:Acetone

Soxhlet biota DCM 1-4 pg/g Ueno 2006

Soxhlet Soil 1:1 Hexane:Acetone 0.3-0.5 pg Yu 2008

Ultra turrax biota 1:1 Hexane:Acetone Morris 2004

Ultra turrax biota 1:1:1 Pentane:Acetone:Water Morris 2006

liquid/liquid extraction

milk Formic acid, 2-propanol, n-hexane/diethylether

90-650 pg/g (fat) Fängstrom 2008

liquid/liquid extraction

Suzuki 2006

Solid/liquid extraction

milk (freeze dried)

1:1 DCM:Acetone 500 pg/g Cariou 2005


sediments 1:1 Pentane:acetone Morris 2006

Acetone


sediments Acetone sonication 10 min

3000 rpm 10 min

Suzuki 2006

57

Appendix 2 Purification methods Treatment Ref Matrix

GPC Budakowski 2003 fish

Frederiksen 2007 biota egg liver adipose tissue

Morris 2004, 2006 sediment biota

Stapleton 2006 biota

Tomy 2005 biota

Ueno 2006 biota

sulphuric acid Abdallah 2008 dust

Berntssen 2010 fish salmon feed fish oil

Bethune 2005 fish

Fängstrom 2008 milk

Frederiksen 2007 biota egg liver adipose tissue

Fängstrom 2008

Granby fish

Morris 2004, 2006 sediment biota

Acidified silica Janák 2005 marine

Yu 2008 soil air

Zhou 2010 fish

silica Frederiksen 2007 biota egg liver adipose tissue

Stapleton 2006 biota

58

Ueno 2006 biota

silica alumina Yu 2008 soil air

Silica gel Morris 2004, 2006 sediment biota

Florisil column Abdallah 2008 dust

Budakowski 2003 fish

Tomy 2005 biota

Carbon Zhou 2010 fish

59

Appendix 3 LC methods

Ref. Compound Matrix Guard Analytical Eluent program Flow Inj. Vol. Inj. Solvent

Abdallah 2008 αβγ-HBCD dust

Varian Pursuit XRS3 C18 (2x150mm, 3um)

A MeOH/H2O 2mM ammonium acetaat, B MeOH

50% A to 0% A to 35% A, 16 min

0.12 mL/min 4ul MeOH

Budakowski 2003 αβγ-HBCD fish

C18 (2.1x5000mm, 4um) A H2O, B MeOH

30% A to 0% A, 6.5 min

0.3 ml/min MeOH

Cariou 2005

αβγ-HBCD, TBBPA, BDE

blood, addipose tissue, milk

Symmetry C18 octadecyl grafted silica (2.1 x 10mm)

Symmetry C18 octadecyl grafted silica (2.1x150mm, 3.5um)

A MeOH, B ACN, C H2O 0.5% acetic acid

30:10:60 1 min, 50:50:0 4.5 min

0.25 ml/min 20ul

MeOH:ACN 1:1

Frederiksen 2007

αβγ-HBCD, TBBPA, Me-TBBPA

biota, egg, liver, adipose tissue

Zorbax C18 (150mm) ,

0.20 mL/min

Granby αβγ-HBCD, TBBPA fish

Gemini C-18 (2x200mmx3um) A MeOH, B H2O

50% A to 95% A 10 min

0.20 mL/min

MeOH:H2O 4:1

Guerra 2008

BFR, αβγ-HBCD, TBBPA, tri-,

sediment, biota

C18 (2.1 x 10mm) Symmetry C18

(2.1x150mm,

A H2O/MeOH (1:3), B MeOH 100% A to

10% A 17

0.25 mL/min 4ul MeOH

60

di-, mono-BBPA, BPA

5um) min

Heeb 2005 HBCD isomers

HBCD mixture

a-chiral C18-RP, Nucleosil 100-5 (4x125mm) MeOH H2O (78:22),

1 mL/min 20ul hexane:DCM

Janak 2005 αβγ-HBCD Marine

Symmetry C18 (2.1x150mm, 5um)

A H2O/MeOH/ACN(6/3/1), B MeOH/ACN (5/5)

A to B in 5 min, hold 6 min

0.25 ml/min ACN

van Leeuwen 2008 αβγ-HBCD Marine

Zorbax XDB-C18 (2.1x150mm, 3.5um)

A ACN, B 0.01mM Ammoniumchloride

70% A 4 min, 90%A in 0.1 min hold 3.9 min MeOH

Morris 2004 Sediment, biota Phenomex

Luna C18 (2x150mm, 5um)

A H2O 10mM ammonium acetate, B ACN 10mM ammonium acetate

80% A to 13% A in 25 min, to 80% A at 36 min

0.25 ml/min 15ul MeOH

Morris 2006 Sediment, biota Phenomex

Luna C18 (2x150mm, 5um)

A H2O 20mM ammonium acetate, B ACN 20mM ammonium acetate

0.2 ml/min 15ul MeOH

Stapleton 2006 αβγ-HBCD Sea lion

C30 YMC Caotenoid S-5 (4.6x250mm)

A H2O:MeOH 20:80, B MeOH

A to B in 35 min 5ul MeOH

Suzuki 2006 αβγ-HBCD, TBBPA

sediment H2O

Develosil C30-UG-5 (2x150mm) A 5% DCM, B 100% DCM

5% DCM to 100% DCM in 20min hold 10 min

0.2 ml/min ACN

Tomy 2005 αβγ-HBCD biota, sediments Vydac 218MS

polymeric rp

A ACN:H2O:MeOH 65:23:12 10mM ammonium acetate, B

A 5 min 150ul/min, B 7 min

1 ul MeOH

61

(2.1x150mm) ACN 200ul/min

Ueno 2006 αβγ-HBCD biota

Vydac 218MS polymeric rp (2.1x150mm)

A ACN:H2O:MeOH 65:23:12 10mM ammonium acetate, B ACN

A 5 min 150ul/min, B 7 min 200ul/min 1 ul MeOH

Yu 2008 αβγ-HBCD air, soil

Zorbax SB-C18 rp (4.6x250mm, 5um)

A MeOH, B ACNe, C H2O 10mM ammonium acetate

80:10:10 to 50:40:10 in 18min, to 30:70:0 at 23 min hold 7 min

0.5 ml/min MeOH

Zhou 2010 αβγ-HBCD, BDE fish

Restek C18 (2.1x100mm, 2.2um)

A MeOH:H2O 85:15, B MeOH

100% A to 0% A in 6 min hold 8 min

0.4 ml/min 10min, 0.5 ml/min 4min

Isopropanol : toluene

62

Appendix 4 Simultaneous determination of TBBPA and the three

major HBCD diastereoisomers

Appendix 4.1 Method

1. Weigh 10 g of sample in a beaker glass and add approximately 40 g sodium sulphate

0.63 – 2.0 mm mesh

2. Add 500 µl of standard mixture containing 50 ng.ml-1

13

C labelled TBBPA, α-HBCD, β-

HBCD and γ-HBCD

3. Homogenize the sample, together with the sodium sulphate and standard mixture

4. Wait half an hour for incubation of the standard mixture into the sample material

5. Transfer the mixture to an 125 ml ASE extraction cell

6. Load the ASE350 with the extraction cell and an collection flask

7. Run the ASE program

8. Add sodium sulphate to the collected extract so that all water is bound

9. Transfer the extract over a funnel filled with glass wool and sodium sulphate into a

Turbovap tube

10. Add 20 ml hexane to the ASE collection flask and shake firm for about 10 seconds

11. Tranfer the extract over the funnel filled with glass wool and sodium sulphate into the

Turbovap tube

12. Repeat step 10 and 11

13. Evaporate the extract in the Turbovap till 0.5 ml

14. Transfer the extract to a 25 ml cylinder flask

15. Rinse the Turbovap tube with approximately 1 ml hexane

16. Transfer the extract again to the 25 ml cylinder flask

17. Repeat step 15 and 16

18. Add hexane to the 25 ml cylinder flask till the 25 ml line

63

19. Load the Power-PrepTM

with the acid silica column, the cylinder flask and two collection

Turbovap tubes in position one and two

20. Start the program

21. Evaporate the collected extracts in the Turbovap till 0.5 ml

22. Combine the extracts from both tubes in one of the two tubes

23. Evaporate the collected extracts in the Turbovap till 0.5 ml

24. Apply the sep-pack silica SPE procedure

25. Evaporate the extracts till dryness

26. Reconstitute the residue in 375 µl methanol and 125 µl milipore water

27. Homogenize

28. Transfer the extract to an LC press filter vial

29. Press the filter through the vial

30. Analyse with LC-MSMS

Appendix 4.2 ASE method

Parameter Setting

ASE ASE350 (Accelerated Solvent Extractor) , Dionex

Temperature 100 °C

Pressure 1500 psi

Preheat 0 min

Heat 5 min

Static 5 min

Flush 40%

Purge 300 sec

Cycle 3

Solvent n-Hexane : acetone 1:1

Hydro matrix Sodium sulphate mesh 0.63 -2.0 mm, Merck 1.06637.1000

Appendix 4.3.1 SPE Power-PrepTM method

step flow volume valves event Solvent


64


3 5 35 06112006 add sample hexane

4 10 150 01112006 wash hexane

5 10 500 03112001 elute hexane:dichloromethane 50:50

6 10 12 01112006 end hexane:dichloromethane 50:50

Appendix 4.3.2 SPE sep-pack silica method

Silica (g) Wash (ml) Elute (ml)

2 12 16

Hexane Hexane : dichloromethane 1:1

Appendix 4.3.3 SPE acid silica method

Packing/step Amount

Sodium sulfate 10g

18.4% H2SO4 40g

silica 10g

Sodium sulfate 10g

Condition with hexane 100ml

Wash with hexane 400ml

Elution hexane dichloromethane 500ml

Appendix 4.3.4 GPC method

Parameter Setting

Column 2.5 x 60 cm, Spectrum USA Omnilabo

Stationary phase Biobeads SX3, 200-400 mesh, Biorad (nr. 1522750)

Mobile phase Ethyl acetate : cyclohexane 1:1

HPLC pump Gilson 305, Gilson, Rijswijk, The Netherlands

Autosampler Gilson 231-401, Gilson, Rijswijk, The Netherlands

Fraction collector Gilson 202, Gilson, Rijswijk, The Netherlands

Collecting 35-110

Software Unipoint v3.3

Flow 0-5 min, from 0 ml/min to 1 ml/min

5-10 min, 1 ml/min

10-118 min, 5 ml/min

118-122min, 1ml/min

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Appendix 4.4 LC method

Parameter Setting

LC system Agilent 1200 series (or others)

Column Waters symmetry C18, 150 mm x 2.1 mm, 3.5 µm

Mobile phase A water 0.01% acetic acid

B methanol : acetonitril (7:3) 0.01% acetic acid

Gradient 0 min, 80 % B

9 min, 100 % B

12 min, 100 % B

13 min, 80 % B

17 min, 80 % B

Flow 0.4 ml.min-1

Injection volume 20 µl

Appendix 4.5 MSMS method Function 1 TBBPA

Type MRM

Ion mode EI-

Inter channel delay 0.050

Start time 0.0

End time 5 min

Ch Prnt(Da) Dau(Da) Dwell(s) Cone(V) Coll(eV) Delay(s)

1 542.80 417.80 0.200 100.00 41.00 0.020

2 542.80 447.80 0.200 130.00 33.00 0.020

3 554.80 428.80 0.200 100.00 41.00 0.020

4 554.80 459.80 0.200 130.00 33.00 0.020

Function 2 HBCD

Type MRM

Ion mode EI-

Inter channel delay 0.050

Start time 5

End time 17 min

Ch Prnt(Da) Dau(Da) Dwell(s) Cone(V) Coll(eV) Delay(s)

1 638.60 79.00 0.200 50.00 13.00 0.020

2 640.60 79.00 0.200 50.00 13.00 0.020

3 650.60 79.00 0.200 50.00 13.00 0.020

4 652.60 79.00 0.200 50.00 13.00 0.020

Parameter Waters Quattro Ultima

TBBPA

Parent to 417.8

Parent to 447.8

HBCD

Parent to bromine

Capillary 2.5

Cone voltage 100/130 50


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Collision energy eV 41/33 13


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