Food Chemistry 313 (2020) 126027

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Development of an analytical method for simultaneously determining T

TBBPA and HBCDs in various foods
⁎
Joon-Goo Lee, Jieun Anh, Gil-Jin Kang, Dongsul Kim, Youngwon Kang
Food Contaminants Division, Department of Food Safety Evaluation, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong-eup,
Cheongwon-gun, Chungcheongbuk-do 363-700, South Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Tetrabromobisphenol A (TBBPA) and Hexabromocyclododecanes (HBCDs) are commonly used as brominated
Tetrabromobisphenol A flame retardants in large volumes, and accumulate in plants and animals in the environment, and people are
Hexabromocyclododecane exposed to them when consuming food. As many countries are monitoring them in food, it is necessary to
Brominated flame retardants develop a method to analyze them simultaneously for cost efficiency. A method was developed and optimized
Simultaneously analytical method
under different conditions using accelerated solvent extraction to extract the lipids from the samples, acid silica
Uncertainty of measurement
Method validation
column to clean the samples and liquid chromatography coupled with tandem mass spectrometry to determine
Food TBBPA and HBCDs. The method was validated in different kinds of food. Uncertainty of measurement was
calculated by combining all uncertainties of contributors. Intermediate precision (reproducibility) was the most
influential contributor to uncertainty. 5 food categories with 115 samples were analyzed with the method, and
mackerels containing high level of fat were highly contaminated by TBBPA and HBCDs.

1. Introduction
Brominated flame retardants (BFRs) are used to prevent fires con-
taining brominated organic compounds (de Wit, 2002). BFRs have been
widely used to reduce the flammability of a variety of consumer pro-
ducts for some decades, and they have had a key role to decrease fire-
related accidents (Barghi, Shin, Kim, Choi, & Chang, 2017). Many types
of BFRs have been invented and used. Some BFRs have been found not
only in abiotic but also in biotic samples in the Arctic (de Wit, Herzke, &
Vorkamp, 2010). Tetrabromobisphenol A (TBBPA) and Hex-
abromocyclododecane (HBCD) are commonly used BFRs used in large
volumes (Darnerud, 2008). In 2001, TBBPA of 119,700 tons, HBCDs of
16,700 tons were used worldwide (Law et al., 2006). TBBPA is a lipo-
philic substance used as an additive or reactive constituent in electronic
devices such as computers and televisions. It is also used for plastic
materials and textiles in cars and airplanes (Strack, Detzel, Wahl, Kuch,
& Krug, 2007). HBCDs are also lipophilic brominated organic com-
pounds commonly consisting of α-HBCD (13%), β-HBCD (16%) and γ-
HBCD (70%), and they are used as flame retardants in extruded and
expanded polystyrene foams, and also in electronics, plastics, textiles
and building materials (Goscinny et al., 2011; Koch, Schmidt-Kőtters,
Rupp, & Sures, 2015). TBBPA has cytotoxicity with effects on cellular
signaling pathways (Strack et al., 2007). It effects on levels of thyroid
hormone and is regarded as a carcinogen in rodents (Lai, Kacew &
Dekant, 2015). Some studies found TBBPA to have immunotoxic effects
in mice and nephrotoxicity in newborn rats (Fukuda et al., 2004;
Watabane et al., 2010). HBCDs also show some toxicity. HBCDs have
cytotoxicity by forming reactive oxygen species (Zhang et al., 2008).
HBCDs cause reproductive toxicity in rats (Ema, Fujii, Hirata-Koizumi,
& Matsumoto, 2008), and effects on levels of thyroid hormone in rats
(Saegusa et al., 2009). HBCDs show effects on the nervous and immune
system (Ema et al., 2008; Van der Ven et al., 2009), and they are re-
garded as a carcinogen in mice (Haseman, Huff, & Boorman, 1984).
HBCDs were selected as a persistent organic pollutant (POP) in the 6th
meeting of the Stockholm Convention on POPs (UNEP, 2013). TBBPA
and HBCDs which were used in products have diffused into the en-
vironment, and they have been found in the environment including
water, air and sediments, etc (Driffield et al., 2008). TBBPA and HBCDs
transfer to plants and animals from the environment through the food
chain. Human are exposed to TBBPA and HBCDs by consuming foods
which are contaminated with TBBPA and HBCDs (Shi, Wu, Li, Zhao, &
Feng, 2009). Some studies have been conducted to determine TBBPA
and HBCDs in a variety of foods. HBCDs have been analyzed in fish,
shellfish, edible oils, eggs, meat products, peanut butter, cereal, vege-
table, fruit, dairy products, milk, cheese and human milk, and higher
levels of HBCDs were found in fish and shellfish (Shi et al., 2009;
Schecter et al., 2010; EFSA, 2011a; Goscinny et al., 2011; Hu, Hu, Song,
Li, & Wang, 2011; Schecter et al., 2012; Ten Dam, Pardo, Traag, van der

⁎
Corresponding author.
E-mail address: youngcloud@korea.kr (Y. Kang).

https://doi.org/10.1016/j.foodchem.2019.126027
Received 22 June 2019; Received in revised form 20 November 2019; Accepted 4 December 2019
Available online 31 December 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
J.-G. Lee, et al.
Lee, & Peters, 2012; Barghi et al., 2016). TBBPA has also been de-
termined in different foods such as meat, eggs, milk, dairy products,
aquatic food, fish and human milk (Shi et al., 2009; EFSA, 2011b; Ten
Dam et al., 2012). The risks of TBBPA and HBCDs through food intake
are of no concern in Korea, China and the EU (Shi et al., 2009; EFSA,
2011a; EFSA, 2011b; Barghi et al., 2016). However, TBBPA and HBCDs
still enter into food and need to be monitored. TBBPA and HBCDs are
monitored to regulate them in some countries. There are some analy-
tical methods to determine TBBPA and HBCDs. To extract TBBPA and
HBCDs, accelerated solvent extraction (ASE), Soxhlet and Untra turrax
have been used and gel permeable chromatography (GPC), acidified
silica, alumina acidified silica, carbon and florisil have been used to
clean up the extract. Liquid chromatography (LC) coupled with elec-
trospray ionization tandem mass spectrometry (ESI-MS/MS), atmo-
spheric pressure photo ionization (APPI-MS/MS), atmospheric pressure
chemical ionization mass spectrometry (APCI-MS) and ESI ion trap
have been used to determine TBBPA and HBCDs (Ten Dam et al., 2012).
However, most of the developed methods are analyzing TBBPA and
HBCDs separately. And one method was developed to analyze TBBPA
and HBCDs simultaneously, and the method is only for fish samples
(Ten Dam et al., 2012). Therefore, we developed an analytical method
for simultaneously determining TBBPA and HBCDs in various foods.
The simultaneous analytical method would be widely used due to its
cost-efficiency and time-efficiency. We compared different ways to
extract them and to separate them in chromatography, and we vali-
dated the developed method. We analyzed some different types of food
samples with the new developed method, and we compared the con-
tents of TBBPA and HBCDs when analyzed separately.
2. Materials and methods
2.1. Chemicals and materials
TBBPA, HBCDs (α-HBCD, β-HBCD, γ-HBCD), 13C12 labelled TBBPA
and 13C12 labelled HBCDs were purchased from Wellington
Laboratories (Guelph, ON, Canada) for standards and the internal
standard. TBBPA and HBCDs in toluene were prepared for stock stan-
dard of 50 μg/mL, and they are diluted with methanol to working
standards of 1, 5, 10, 25, 50, 100 and 250 μg/kg. 13C12 labelled TBBPA
and HBCDs were diluted with methanol to internal standards of 100 μg/
kg. Fish tissue (Lake Michigan fish tissue) was prepared from National
Institute Standard and Technology (NIST) (SRM 1947, MD, USA) for
quality control. Solvents of dioxin analysis grade including di-
chloromethane (DCM), acetone, n-hexane and methanol were pur-
chased from Wako (Osaka, Japan). Sulfuric acid of pesticide analysis
grade was prepared from Wako (Osaka, Japan), and water was filtered
using a Milli-Q System (Bedford, MA, USA). Sodium sulfate anhydrous
(0.63 mm~2.0 mm) from Merck (MA, USA) and Silica gel (60
mesh~200 mesh) from Supelco (PA, USA) were activated at 400 °C and
180 °C, respectively.
2.2. Sample preparation
5 food categories represented by 115 samples were collected from
Korean offline markets in 2015. Pork was selected as a representative
food for the meat category including 12 pork samples, 12 beef samples
and 13 chicken samples for method validation. Mackerel and mussel
were chosen for fish and shellfish categories, respectively. The fish
category included 11 mackerel samples, 11 pacific saury samples and
11 salmon samples, and 11 Mussel sample and 12 abalone samples were
collected for the shellfish category. 12 egg samples and 11 milk samples
were purchased. Each meat, fish and shellfish sample was grinded and
stored in a freezer at −20 °C. Egg and milk samples were dried with a
freeze-dryer (Ilshinbiobase, Korea) and stored in a cool place until
further analysis. Fat was extracted with Hexane-acetone (1:1, v/v) from
samples by ASE under cell pressure of 1500 psi for 5 min and nitrogen
2
Food Chemistry 313 (2020) 126027
purge for 90–180 sec at 90 °C. The fat was separated with ASC packed
with sodium sulfate andydrous of 5 g and acid silica of 30 g, and the
ASC was eluted with hexane-DCM (44:55, v/v) for 20 min and con-
centrated to 100 μL with nitrogen.
2.3. Optimization of analysis conditions
2.3.1. Optimization of extraction
Fat were extracted by ASE-200 (Dionex, USA) from mackerel sam-
ples under different conditions to optimize ASE processing methods.
ASE was extracted with mixtures of hexane and DCM (1:1, v/v), hexane
and acetone (1:1 v/v) and acetone and DCM (1:1, v/v) to optimize the
extracting solvent. Oven temperatures were 90 °C, 100 °C, 110 °C and
extracting was conducted 1, 2 and 3 times, respectively. Cell pressure
was 1500 psi and static time was 5 min. Total flush volume was 60%,
and nitrogen purge time was 90–180 sec (Shi et al., 2009; Goscinny
et al., 2011; Ten Dam et al., 2012).
2.3.2. Optimization of clean-up
2.3.2.1. Gel permeable chromatography. An open column
(24 mm × 40 cm) washed with MeOH, acetone, hexane and DCM
was employed for GPC. Bio-beads, Bio-beadsTM S-X3 support (200-
400mesh, BIO-RAD, CA, USA) of 20 g were activated with DCM-hexane
(1:1, v/v) of 80 mL for 12 h, and they were packed into the open
column. The packed open column was washed with DCM-hexane (1:1.
v/v), and the samples added with standards and internal standards
were loaded on it. It was eluted with DCM-hexane (1:1, v/v), and 6
eluted fractions of 10 mL were collected. The collected fractions were
condensed to 100 μL with a nitrogen evaporator (Oa-SYS Heating
Device 5085, Organomation Associates. Inc., USA) at 20 psi stream of
nitrogen (40 °C), and they were analyzed with LC-MS/MS (Shi et al.,
2009; Goscinny et al., 2011; Ten Dam et al., 2012).
2.3.2.2. Multi-layer column. An open column (25 mm × 30 cm) was
packed from the bottom with sodium sulfate anhydrous of 5 g, neutral
silica of 5 g, acid silica (33%) of 5 g, acid silica (16.5%) of 20 g, sodium
sulfate anhydrous of 5 g. The packed column was activated with hexane
of 100 mL, and it was left for 20 min after the sample was loaded. The
column was washed with hexane of 100 mL and eluted with hexane-
DCM (1:1, v/v) of 250 mL. The eluate was evaporated to 100 μL with a
nitrogen evaporator and analyzed with LC-MS/MS (Goscinny et al.,
2011).
2.3.2.3. Acid silica column. A separate funnel was packed with sodium
sulfate anhydrous of 5 g and acid silica (44%) of 30 g and, the sample
was loaded on the separate funnel. The separate funnel was left for
20 min and eluted with different solvents with hexane-DCM (40:60, v/
v), hexane-DCM (45:55, v/v), hexane-DCM (50:50, v/v), hexane-DCM
(55:45, v/v), hexane-DCM (60:40, v/v) and hexane-DCM (65:35, v/v).
The eluate was evaporated to 100 μL with a nitrogen evaporator and
analyzed with LC-MS/MS.
2.3.3. Optimization of chromatography
To optimize the separation of peaks of TBBPA and HBCDs, QC
samples were analyzed by 2 different LC columns of different length.
LC-MS/MS, Thermo TSQ Quantum Ultra (Thermo, Waltham, MA, USA),
was used with C18 column, Imtakt Cadenza CL-C18 (100 mm × 2 mm,
3 μm) (Imtakt co., Kyoto, Japan). Water and acetonitrile-methanol (3:7,
v/v) with 0.01% acetic acid were used for the mobile phase. A
Symmetry® C18 LC column (150 mm × 2.1 mm, 3.5 μm) (Waters,
Milford, MA, USA) was also used with the same instrument system. For
the gradient program, mobile phase A of 50% was initially used for
0.2 min and decreased to 15% for 1.0 min. And it was decreased to 5%
for 8.0 min and increased to 50% for 8.1 min. It was finally held for
10 min.
J.-G. Lee, et al. Food Chemistry 313 (2020) 126027

2.4. Determination of TBBPA and HBCDs with LC-MS/MS uncertainty and coverage factor, k (eq. (6)) (Eurachem Working Group,
1998; Konieczka & Namieśnik, 2010).
Determination of TBBPA and HBCDs were conducted using a LC −
(Thermo finnigan surveyor, Thermo, Waltham, MA, USA) with MS/MS s 1 1 (C − C )2
u c (combined standard uncertainty) = + + 0
(Thermo TSQ Quantum ultra, Thermo, Waltham, MA, USA). Mobile B1 p n Sxx (1)
phase A and B for LC were water and acetonitrile-methanol (3:7, v/v)
s: standard deviation of residual, P: number of analysis of samples, n:
containing acetic acid of 0.01%. Mobile phase B was increased from
number of analysis of standards, Sxx: variation of standard values, C0 :
50% to 85% for 8.1 min after flowing for 0.2 min, and then it was hold −
for 10 min with flow rate of 300 μL min−1. The temperature of the MS obtained value, C : average of standard values, B1: direction coefficient
Source was 250 °C and argon of 99.99999% purity was used as the of the calibration curve
collision gas. Spectrometry of MS was obtained by using multiple re- s (xi)
action monitoring (MRM) modes with the electrospray ionization (ESI). uA (type A uncertainty) = (s(xi): standard deviation, n: number)
√n
Spray voltage was 4000 V. Vaporizer temperature and capillary tem-
(2)
perature were 300 °C and 320 °C. Sheath gas pressure and aux gas
pressure were 60 psi and 55 psi, respectively. Collision energies were U
uB = (U: given expanded uncertainty, k: coverage factor) or uB
41 eV, 45 eV and 37 eV for TBBPA and 48 eV and 52 eV for HBCDs. The k
parent ion was m/z 542.6 and product ions were m/z 290.9 (quanti- a
= (a: scale of measurement)
tative ion), m/z 80.7, and 79.0 for TBBPA. The parent ion was m/z √3 (3)
640.7 and product ions were m/z 80.7 (quantitative ion) and m/z 79.1 u
for HBCDs. The parent ion was m/z 554.7 and product ions were m/z ur (relative uncertainty) = (u: standard uncertainty,
C
296.8 (quantitative ion), m/z 80.7, and 79.0 for 13C12 labelled TBBPA.
c: obtained resutls) (4)
The parent ion was m/z 652.6 and product ions were m/z 81.2 (quan-
titative ion) and m/z 79.0 for 13C12 labelled HBCDs.
u c (combined standard uncertainty)
2
2.5. Method validation = √∑ u (u: standard uncertainty, c: obtained resutls) (5)
i

Performance parameters of the analytical method were obtained
and compared to the acceptable criteria proposed by the Association of
Official Agricultural Chemists (AOAC) (Taverniers, Loose, & Bocksaele,
2004). Specificity was evaluated by analyzing pork, mackerel, mussel,
milk and egg samples fortified with standards of 1 ng g−1 and 5 ng g−1
and 20 ng g−1. Limit of detection (LOD) and limit of quantification
(LOQ) were calculated by multiplying 3.14 and 10 to a relative stan-
dard deviation of data obtained by analyzing 7 blank samples of pork,
mackerels, messels, milk and eggs, respectively. Calibration curves of
TBBPA and HBCDs were calculated by plotting working standards of 1,
5, 10, 25, 50, 100 and 250 μg kg−1 and using regression equations.
The linearity of the calibration curves of TBBPA and HBCDs stan-
dards was confirmed by statistically calculating the correlation coeffi-
cient (R2). The relative recoveries were obtained by analyzing 3 fat
samples of pork, mackerels, mussels, milk and eggs fortified with
standards of 1 ng g−1 and 5 ng g−1 and 20 ng g−1 and comparing their
concentrations to the fortified levels. Repeatability and reproducibility
were obtained by calculating relative standard deviations of analyzed
data of 3samples of pork, mackerels, mussels, milk and eggs in a day
and for 3 days.
2.6. Uncertainty of measurement
To evaluate the uncertainty of the TBBPA and HBCDs measure-
ments, measurement uncertainties of α-HBCD, β-HBCD, γ-HBCD and
TBBPA were estimated and combined to obtain the measurement un-
certainty of TBBPA and HBCDs. A mathematical model was developed
for calculating the results from analyzed data. Fish bone diagrams were
used to find all possible parameters, and associated standard un-
certainties were estimated. A type and B type uncertainties were cal-
culated by statistical measurements and given uncertainties, respec-
tively. Relative standard uncertainty of calibration and linear
regression was calculated by statistical measurement of working stan-
dards 3 times by Eq. (1).
Relative standard uncertainties were transferred with standard un-
certainties by degree of freedom (ν) Eqs. (2) and (3) show how A type
and B type uncertainty are calculated, and relative uncertainties are
calculated by diving uncertainties with obtained results (Eq. (4)). Eq.
(5) shows how to calculate the combined uncertainties. Expanded un-
certainties were finally calculated by multiplying the combined
U (expanded uncertainty) = uc × k (uc : combined uncertainty,
k: coverage factor) (6)
2.7. Statistical analysis
Results were calculated as means with standard deviations. To
evaluate significant differences in different analysis conditions, the
student’s t-test was used with Microsoft Office Excel 2010 (Microsoft
Corporation, Redmond, WA, USA).
3. Results and discussion
3.1. Optimization of analysis conditions
3.1.1. Optimization of extraction
Extraction rates of the lipids were 12.17 ± 1.96% with Hexan-
DCM (1:1, v/v), 22.28 ± 2.36% with hexane-acetone (1:1, v/v) and
20.44 ± 2.25% with acetone-DCM (1:1, v/v). Therefore, hex-
ane–acetone (1:1, v/v) showed the highest efficient extraction of lipid.
The average recoveries of TBBPA and HBCDs were from 98.6 ± 0.6%
to 106.7 ± 6.7% in 90 °C, from 97.5 ± 2.2% to 110.6 ± 11.9% in
100 °C and 86.0 ± 8.8% to 101.28 ± 1.93% in 110 °C, when ex-
tracted with hexane-acetone (1:1, v/v) (Fig. 1-(A)). 90 °C was the best
temperature for extracting TPPBA and HBCDS. When the extracting
cycle was increased, recoveries of HBBPA and HBCDs were not im-
proved. Therefore, TBBPA and HBCDs extracted one time with hexane-
acetone (1:1, v/v) at 90 °C by ASE was shown to be the most efficient
method.
3.1.2. Optimization of clean-up
Fig. 2 shows the separation of TPPBA and HBCDs by GPC. TBBPA
started being eluted from collection volume of 30 mL to 130 mL, and
HBCDs were separated from elution volume of 60 mL to 130 mL. GPC
was not efficient to clean-up the samples to separate TBBPA and HBCDs
simultaneously. Fig. 1-(C) shows the recoveries of TBBPA and HBCs
when samples were cleaned by multi-layer column (MLC) and acid si-
lica column (ASC). Even though the recoveries were not significantly
different, the process of clean-up with ASC was more efficient than
clean-up with MLC. And the recoveries of TBBPA and HBCDs by

3
J.-G. Lee, et al. Food Chemistry 313 (2020) 126027

Fig. 1. Recoveries of TBBPA and HBCDs according to different conditions of pre-treatments; (A) temperatures of accelerated solvent extraction (ASE), (B) numbers of
cycle of accelerated solvent extraction (ASE), (C) types of separation column; Multi-layer column (MLC) and acid silica column (ASC), (D) ratio of DCM in hexane for
acid silica column (ASC).

Fig. 2. Characterization of GPC volume to isolate peaks; A) TBBPA and B) HBCDs. ① 20 to 30 mL, ② 30 to 40 mL, ③ 40 to 50 mL, ④ 50 to 60 mL, ⑤ 60 to 70 mL, ⑥ 70 to
80 mL, ⑦ 90 to 100 mL, ⑧ 110 to 120 mL, ⑨ 120 to 130 mL.

4
J.-G. Lee, et al.
Fig. 3. Separation of peaks of α-HBCD, β-HBCD and γ-HBCD according to LC column length. A) Imtakt Cadenza CL-C18 (100 mm × 2 mm, 3 μm), B) Symmetry
(150 mm × 2.1 mm, 3.5 μm).
cleaning samples with ASC were not significantly different when the
ratio of DCM in elution solvents was increased from 40% to 65%.
However, DCM of 55% had the most precisely recoveries for simulta-
neous analysis of TBBPA and HBCDs (Fig. 1).
3.1.3. Optimization of chromatography
Fig. 3 shows the separation of peaks of 3 HBCDs by two different LC
columns. The peaks of α-HBCD and β-HBCD were not separately
properly with the short column (100 mm × 2 mm, 3 μm) while they
were fully separated by the longer column (150 mm × 2.1 mm,
3.5 μm). Therefore, the column Symmetry (150 mm × 2.1 mm,
3.5 μm), was chosen for the LC. Water with 0.01% acetic acid and
acetonitrile-methanol (3:7, v/v) with 0.01% acetic acid were used for
mobile phase A and B. For the gradient program, mobile phase A of
50% was initially used for 0.2 min and decreased to 15% for 1.0 min.
And it was decreased to 5% for 8.0 min and increased to 50% for
8.1 min. It was finally held for 10 min. Flow rate was 300 μL/min and
injection volume was 10 μL.
3.2. Method validation
Table 1 shows the performance parameters of the analytical
method. All calibration curves for TPPBA and HBCDs showed accep-
table linearity with R2 value higher than 0.999. LODs for TBBPA and
HBCDs were ranged from 0.004 to 0.047 ng g−1 wet weight and 0006
to 0.028 ng g−1 wet weight for different food. Egg and pork had the
lowest LOD values of 0.004 and from 0.006 to 0.007 ng g−1 wet weight
for TBBPA and HBCDs, respectively. Relative recoveries were 87.8 to
105.5% for TBBPA and 90.7 to 121.0% for HBCDs for different con-
centrations. Egg and milk showed better recoveries from 98.6 to
101.9% rather than other foods in high levels of TBBPA. Recoveries of
α-HBCD and γ-HBCD for milk were from 100.3 to 100.6% and 100.6 to
101.6%, respectively, which were better than those for other foods.
Precisions with repeatability (RSDr) and reproducibility (RSDR) of
TBBPA for pork, mackerel, mussel, egg and milk were 1.0 to 5.4%, 0.5
to 10.6%, 1.1 to 9.7%, 5.4 to 4.6% and 1.6 to 14.1%, respectively. RSDr
5
Food Chemistry 313 (2020) 126027
and RSDR of HBCDs were 1.7 to 11.6% and 2.8 to 11.6% for pork, 0.3 to
6.7% and 3.0 to 10.1% for mackerel, 0.6 to 4.9% and 3.5 to 14.1% for
mussel, 0.8 to 7.2% and 1.6 to 12.7% for egg and 0.4 to 5.5% and 1.4 to
6.9% for milk. RSDrs of TBBPA for mackerel were lower than those for
other foods at low levels of TBBPA. RSDrs of HBCDs for pork were
higher than those for other foods. All performance parameters were
acceptable comparing to the criteria of AOAC (Taverniers et al., 2004).
3.3. Uncertainty of measurement
Fig. 4 shows the fish bone diagram of the analytical method to
determine TBBPA and HBCDs in different food by LC-MS/MS. The
making of the STD and ISTD, sample analysis, recovery, sample pre-
paration, and calculation of the calibration curve were contributors to
uncertainty. Each contributor has type A and B factors affecting un-
certainty.
3.3.1. Uncertainty of sample preperations
The combined standard uncertainty of weighing (uc(w)) for sample
preparation was calculated to 0.00237 g (ν of 55) (eq.7, 8) by com-
bining the given expanded uncertainty of a balance of 0.00200 g (for
p = 95%, k = 2) from calibaration certification, statistical measure-
ment standard uncertainty of stability of 0.00213 g (ν of 36) from re-
peatability and standard uncertainty of resolution of 0.00029 g (ν of ∞)
from balance scale. Standard uncertainty of the balance from calibra-
tion certification was calculated to 0.00100 g (ν of ∞) from its ex-
panded uncertainty.
u c (w) = (0.00100)2 + (0.00213)2 + (0.00029)2 (7)
0.002374
νeff (w) =
0.00100 4 0.002134 0.000294
∞
+ 36
+ ∞ (8)
The combined standard uncertainty of volume measurement (uc(v))
for sample preparation was combined to 0.00175 g (ν of 10) with given
expanded uncertainty of a 0.5 mL volumetric flask of 0.00003 mL (for
p = 95%, k = 2) from calibration certification, the statistical
J.-G. Lee, et al. Food Chemistry 313 (2020) 126027

Table 1
Linear equation, limits of detection and quantitation (LOD and LOQ), recovery and precision obtained for TBBPA and HBCDs.
BFRs Linear equation Matrix LOD LOQ Relative recovery (%) RSD r (%) (n = 3) RSD R
(%) (n = 3)
(ng/g (ng/g
wet wet 1 (ng/g) 5 (ng/g) 20 (ng/g) 1 (ng/g) 5 (ng/g) 20 1 (ng/g) 5 (ng/g) 20
weight) weight) (ng/ (ng/
g) g)

TBBPA Y = 0.00902331X + 0.0832055 (R2 = 0.9999) Pork 0.042 0.139 105.5 87.8 93.3 1.6 1.0 3.0 2.2 2.3 5.4
Mackerel 0.043 0.142 98.0 90.5 97.5 0.8 0.5 1.2 10.6 3.4 4.6
Mussel 0.036 0.119 97.6 96.9 92.3 9.7 3.6 1.1 7.0 2.4 4.2
Egg 0.004 0.013 104.0 101.9 99.3 5.4 1.7 0.6 4.6 3.0 3.6
Milk 0.047 0.155 105.1 98.6 100.3 14.1 5.0 1.6 12.5 4.6 6.3
α-HBCD Y = 0.0157639X + 0.157319 (R2 = 0.9997 Pork 0.019 0.063 108.9 100.0 101.0 11.6 3.6 2.5 7.2 3.3 11.6
Mackerel 0.020 0.066 104.7 101.4 91.0 1.0 6.7 2.1 10.0 10.1 7.8
Mussel 0.019 0.063 115.3 100.2 95.0 0.6 1.5 4.9 14.1 6.0 8.0
Egg 0.020 0.066 108.5 96.1 103.6 2.1 4.0 2.9 11.1 4.4 5.7
Milk 0.024 0.079 104.0 100.6 100.3 3.4 4.4 5.5 2.7 2.9 5.5
β-HBCD Y = 0.0152003X + 0.0559638 (R2 = 0.9999) Pork 0.006 0.020 121.0 97.5 93.7 4.5 3.5 1.7 7.3 3.9 5.7
Mackerel 0.013 0.043 103.0 98.9 100.4 3.6 0.7 0.3 3.6 8.8 3.0
Mussel 0.008 0.026 96.7 101.1 103.3 3.7 0.8 1.6 3.5 4.8 3.8
Egg 0.011 0.036 104.8 101.4 103.7 0.8 3.1 1.0 1.6 3.6 3.5
Milk 0.020 0.066 102.5 95.0 100.9 0.8 1.7 1.3 5.6 1.4 4.7
γ-HBCD Y = 0.0159373X + 0.0620876 (R2 = 0.9998 Pork 0.007 0.023 102.7 98.6 105.5 4.1 2.0 7.5 6.6 2.8 5.3
Mackerel 0.028 0.092 104.6 95.5 90.7 2.3 5.2 1.1 5.4 5.1 3.8
Mussel 0.027 0.089 117.0 105.0 105.7 4.1 2.1 1.2 4.4 3.9 10.6
Egg 0.017 0.056 101.2 101.4 101.7 7.2 0.9 0.8 12.7 4.8 7.0
Milk 0.014 0.046 106.6 100.6 101.6 0.4 1.1 0.9 6.9 5.5 1.7

Fig. 4. Fish bone diagram of the analytical method to determine TPPB and HBCDs in different food by LC-MS/MS.

measurement standard uncertainty of stability of the volumetric flask of
0.00172 mL (ν of 9) and the standard uncertainty of thermal expansion
of the volumetric flask of 0.00035 mL (ν of ∞, ± 5 °C). Standard un-
certainty of the volumetric flask from calibration certification was
calculated to 0.00002 mL (ν of ∞) from its expanded uncertainty.
Therefore, the relative standard uncertainty of sample preparation
(ur(sample)) was calculated to 0.00354 (ν of 10) (eq. (9)) by combining
the combined relative standard uncertainty of weighing of 0.000048 (ν
of 55) and the combined relative standard uncertainty of volume
measurement of 0.000351 (ν of 10).
ur (sample) = 0.0000482 + 0.0003512
0.003544
ν (sample) =
0.0000484 0.003514
+ (10)
55 10
3.3.2. Uncertainty of making standards
The standard uncertainty of making standards was combined with
uncertainty of makingSTD stock solutions, uncertainty of making ISTD
stock solutions and uncertainty of making working solutions with STD
and ISTD. The uncertainties of making stock solutions were calculated
by combining the uncertainty of a 1 mL pipette, the uncertainty of a
0.1 mL pipette and uncertainties of STD/ISTD resolution. The un-
certainty of making working solutions was calculated by combining the
uncertainty of the pipettes. The uncertainties of STD (> 98%) resolu-
tion and ISTD (> 98%) resolution were 0.01154 µg/mL (ν of ∞) and
0.00577 µg/mL (ν of ∞), respectively. The combined standard un-
(9) certainty of a 1 mL pipet for making standards was calculated to
0.00193 mL (ν of 123) by combining the given standard uncertainty of
the 1 mL pipet of 0.0015 mL (ν of ∞), the standard uncertainty of
statistical measurement of 0.00100 mL (ν of 9) and the standard

6
J.-G. Lee, et al.
uncertainty of thermal expansion of 0.00069 mL (ν of ∞). The standard
uncertainty of a 0.1 mL pipet for making standards was calculated to
0.00017 mL (ν of 13) by combining the given standard uncertainty of
the 0.1 mL pipet of 0.000 mL (ν of ∞), the standard uncertainty of
statistical measurement of 0.00015 mL (ν of 9) and the standard un-
certainty of thermal expansion of 0.00007 mL (ν of ∞). And the relative
combined uncertainty of the stock solutions and working solutions were
calculated by dividing the standard uncertainties with volume of stock
solutions and working solutions. Therefore, the relative standard un-
certainty of making standards was calculated to 0.0190 (ν of 22) by
combining the combined relative standard uncertainty of making stock
solutions and the combined relative standard uncertainty of making
working solutions.
3.3.3. Uncertainty of calibration
The standard deviation of residual (s), number of analysis of sam-
ples (p), number of analysis of standards (n), variation of standard
−
values (Sxx), obtained value (C0 ), average of standard values (C ), di-
rection coefficient of the calibration curve (B1) for pork were 1.6776, 5,
18, 33300, 21, 36 and 1.02333, respectively. The relative standard
uncertainty of calibration and linear regression for α-HBCD was cal-
culated to 0.0380 with degree of freedom of 16 (eq. (11), 12) for pork.
1.6776 1 1 (21 − 36)2
u c (calibration) = × + + = 0.838
1.02333 5 18 33300 (11)
0.838
ur (calibration) = = 0.0380
(12)
22
The relative standard uncertainties of calibration and linear re-
gression for α-HBCD were 0.0414 for mackerel, 0.0394 for mussel,
0.0415 for eggs and 0.0428 for milk with degree of freedom of 16. The
relative standard uncertainties of calibration and linear regression for
β-HBCD were calculated to 0.0424 for pork, 0.0434 for mackerel,
0.0450 for mussel, 0.0445 for eggs and 0.0440 for milk with degree of
freedom of 16. The relative standard uncertainties of calibration and
linear regression for γ-HBCD were calculated to 0.0669 for pork, 0.0699
for mackerel, 0.0695 for mussel, 0.0706 for eggs and 0.0714 for milk
with degree of freedom of 16. The relative standard uncertainties of
calibration and linear regression for TBBPA were calculated to 0.0306
for pork, 0.0298 for mackerel, 0.0313 for mussel, 0.0302 for eggs and
0.0296 for milk with degree of freedom of 16.
3.3.4. Uncertainty of sample analysis
5 pork, mackerel, mussel, egg and milk samples were fortified with
standards of 20 ng g−1 and analyzed to evaluate the uncertainty of
sample analysis. The relative standards uncertainty of the intermediate
precision (reproducibility) of pork samples for α-HBCD was 0.1886
with degree of freedom of 8. Mackerel, mussel, egg and milk sample
had relative standard uncertainties of 0.1313, 0.1223, 0.0984 and
0.0940 with degree of freedom of 8 for α-HBCD. Relative standards
uncertainty of intermediate precision (reproducibility) of pork samples
for β-HBCD was 0.0892 with degree of freedom of 8. Mackerel, mussel,
egg and milk sample had relative standard uncertainties of 0.0493,
0.0653, 0.0598 and 0.0787 with degree of freedom of 8 for β-HBCD.
Relative standards uncertainty of intermediate precision (reproduci-
bility) of pork samples was 0.0818 with degree of freedom of 8.
Mackerel, mussel, egg and milk sample had relative standard un-
certainties of 0.0630, 0.154, 0.1149 and 0.0286 with degree of freedom
of 8 for γ-HBCD. Relative standard uncertainties of intermediate pre-
cision (reproducibility) were 0.0869 for pork, 0.0750 for mackerel,
0.0693 for mussel, 0.0580 for egg, and 0.1024 for milk with degree of
freedom of 8 for TBBPA.
3.3.5. Uncertainty of recovery
Relative standard uncertainties of recovery were estimated to
0.0361 for pork, 0.0237 for mackerel, 0.0303 for mussel, 0.01810 for
7
Food Chemistry 313 (2020) 126027
egg, and 0.0239 for milk with degree of freedom of 4 for α-HBCD.
Relative standard uncertainties of recovery were estimated to 0.0152
for pork, 0.0098 for mackerel, 0.0053 for mussel, 0.0066 for egg, and
0.0179 for milk with degree of freedom of 4 for β-HBCD. Relative
standard uncertainties of recovery were estimated to 0.0169 for pork,
0.0131 for mackerel, 0.0220 for mussel, 0.0033 for egg, and 0.0065 for
milk with degree of freedom of 4 for γ-HBCD. Relative standard un-
certainties of recovery were estimated to 0.0155 for pork, 0.0114 for
mackerel, 0.0114 for mussel, 0.0111 for egg, and 0.0111 for milk with
degree of freedom of 4 for TBBPA.
3.3.6. Combined expanded uncertainty of method
Relative combined uncertainties of TBBPA, α-HBCD, β-HBCD and γ-
HBCD were calculated by combining the relative standard uncertainties
of sample preparations, making STD, calibrations, sample analysis and
recoveries. And calculated relative combined uncertainties of α-HBCD,
β-HBCD and γ-HBCD were transferred to combined standard un-
certainties and combined to standard uncertainty of HBCDs. Combined
standard uncertainties for α-HBCD were calculated to 20.5(4.0) ng g−1
for pork, 19.8(2.8) ng g−1 for mackerel, 21.8(2.9) ng g−1 for mussel,
19.3(2.1) ng g−1 for eggs and 19.5(2.1) ng g−1 for milk. Combined
standard uncertainties for β-HBCD were calculated to 21.3(2.2) ng g−1
for pork, 20.3(1.4) ng g−1 for mackerel, 19.4(1.6) ng g−1 for mussel,
19.5(1.5) ng g−1 for eggs and 19.9(1.1) ng g−1 for milk. Combined
standard uncertainties for γ-HBCD were calculated to 21.6(2.3) ng g−1
for pork, 20.1(1.9) ng g−1 for mackerel, 21.8(3.7) ng g−1 for mussel,
20.3(2.8) ng g−1 for eggs and 19.8(1.6) ng g−1 for milk. Therefore,
expanded uncertainties were 63.4 ± 10.2 ng g-1 (for p = 95%, k = 2)
for pork, 60.2 ± 7.3 ng g-1 (for p = 95%, k = 2) for mackerel,
63.0 ± 9.9 ng g-1 (for p = 95%, k = 2) for mussel, 59.1 ± 7.6 ng g-1
(for p = 95%, k = 2) for egg, 59.2 ± 5.7 ng g-1 (for p = 95%, k = 2)
for milk for HBCDs. Expanded uncertainties for TBBPA were calculated
to 19.8 ± 3.9 ng g-1 (for p = 95%, k = 2) for pork, 20.5 ± 3.4 ng g-1
(for p = 95%, k = 2) for mackerel, 20.2 ± 3.2 ng g-1 (for p = 95%,
k = 2) for mussel, 20.7 ± 2.9 ng g-1 (for p = 95%, k = 2) for egg,
20.5 ± 4.5 ng g-1 (for p = 95%, k = 2) for milk from relative com-
bined uncertainties. Uncertainties of intermediate precision was the key
influential contributor for analyzing HBCDs (37.9%-56.7%) and
TBBPA(14.9%-56.9%) followed by calibration curve, making STD, and
recovery. Intermediate Reproducibility is normally important con-
tributor for analyzing contaminants in foods with calibration. And un-
certainty of making STD is also high because the used standards did not
have high purified qualities in this study. A study estimating un-
certainty for chemical analytical methods figured out that standards are
the key contributor to uncertainty (Horwitz & Albert, 1997). Improving
proficiency of individual analysts and using highly purified standards
are important to reduce uncertainty.
3.4. Sample analysis
Food samples were analyzed with the developed analytical method.
Meat samples of 3 g, fish samples of 3 g, shellfish of 3 g, egg samples of
4 g, milk samples of 20 mL and freeze-dried milk samples of 2 g were
analyzed. Every sample batch has blank and lake Michigan fish tissue of
National Institute of Standard and Technology (NIST) as QC samples
(SRM 1947) for quality assurance. All analyzed QC samples were within
10% precision. Table 2 shows the levels of TBBPA and HBCDs in food.
Levels of TBBA, α-HBCD, β-HBCD and γ-HBCD ranged from Not De-
tected (N.D) to 0.228 ng g−1 wet weight, N.D to 0.342 ng g−1 wet
weight, N.D to 0.084 ng g−1 wet weight in meat, respectively. Fish
contained TBBPA of N.D to 1778 ng g−1 wet weight and 3 HBCDs of
N.D to 1.485 ng g−1 wet weight. Levels of TBBPA and HBCDs in
shellfish were N.D to 0.053 ng g−1 wet weight and N.D to 1.823 ng g−1
wet weight. TBBPA contents were from N.D to 1.080 ng g−1 wet weight
in eggs and N.D to 0.233 ng g−1 wet weight in milk, and α-HBCD
contents were N.D to 0.042 ng g−1 wet weight in eggs. β-HBCD and γ-
J.-G. Lee, et al. Food Chemistry 313 (2020) 126027

Table 2
Contents of TBBPA and HBCDs in various foods.
Food n TBBPA α-HBCD β-HBCD γ-HBCD Ref.
(fat, %) (ng/g wet weight) (ng/g wet weight) (ng/g wet weight) (ng/g wet weight)

Mean Range Mean Range Mean Range Mean Range

Meat Pork 12 0.009 ± 0.036 N.D.~0.138 0.004 ± 0.016 N.D.~0.060 N.D. N.D. 0.015 ± 0.031 N.D.~0.084 This study
(17.8%)
Beef 12 0.032 ± 0.068 N.D.~0.228 N.D. N.D. N.D. N.D. N.D. N.D.
(22.2%)
Chicken 13 0.012 ± 0.046 N.D.~0.176 0.023 ± 0.088 N.D.~0.342 N.D. N.D. 0.004 ± 0.017 N.D.~0.065
(6.1%)
Fish Mackerel 11 0.140 ± 0.458 N.D.~1.778 3.608 ± 2.796 0.137~9.563 N.D. N.D. 0.189 ± 0.162 N.D.~0.441
(10.4%)
Pacific Saury 11 0.016 ± 0.033 N.D.~0.089 0.365 ± 0.344 N.D.~1.485 0.007 ± 0.020 N.D.~0.076 0.028 ± 0.076 N.D.~0.268
(4.7%)
Salmon 11 0.026 ± 0.067 N.D.~0.251 0.141 ± 0.260 N.D.~1.016 N.D. N.D. N.D. N.D.
(1.9%)
Shellfish Mussel 11 0.004 ± 0.014 N.D.~0.053 0.421 ± 0.498 N.D.~1.823 0.044 ± 0.094 N.D.~0.345 0.041 ± 0.054 N.D.~0.168
(1.2%)
Abalone 12 N.D. N.D. 0.009 ± 0.036 N.D.~0.140 0.032 ± 0.047 N.D.~0.107 N.D. N.D.
(0.7)
Egg 12 0.078 ± 0.278 N.D.~1.080 0.003 ± 0.011 N.D.~0.042 N.D. N.D. N.D. N.D.
(7.4%)
Milk 11 0.016 ± 0.060 N.D.~0.233 N.D. N.D. N.D. N.D. N.D. N.D.
(3.3%)
Pork 36 – – 0.072 ± 0.022 – 0.014 ± 0.004 – 0.013 ± 0.005 – Barghi et al.,
Beef 74 – – 0.064 ± 0.008 – 0.017 ± 0.002 – 0.017 ± 0.006 – 2016
Chicken 14 – – 0.008 ± 0.004 – 0.005 ± 0.002 – 0.010 ± 0.005 –
Chub mackerel 5 – – 1.57 ± 0.40 – N.D. – 0.09 ± 0.02 –
Pacific saury 5 – – 0.58 ± 0.06 – 0.01 ± 0.01 – 0.05 ± 0.01 –
Mussel 9 – – 0.789 ± 0.0177 – 0.061 ± 0.015 – 0.157 ± 0.042 –
Abalone 11 – – 0.018 ± 0.010 – 0.009 ± 0.004 – 0.073 ± 0.061 –
Mackerel 3 4.8 – – – – – – – EFSA, 2011b
Mussel 2 0.6 – – – – – – –

HBCD were not detected in eggs and milk. The highest levels of TBBPA,
HBCDs except β-HBCD were determined in mackerel. Mussels contain
the highest level of β-HBCD. Eggs also contain high level of TBBPA but
not high levels of HBCDs. BFRs including TBBPA and HBCDs are
eventually accumulated in the aquatic environment (Covaci et al.,
2006; Sun et al., 2014). Therefore, fish contains higher levels of HBCDs
than meat. On the other hand, meat contains similar levels of TBBPA as
fish except mackerel. It is because TBBPA is strongly lipophilic and
meat contains high level of fat even though TBBPA finally accumulates
in the ocean. Table 2 shows the fat contents of meat, fish, shellfish, eggs
and milk (NIAS, 2016). Fat contents are 17.8% in pork, 22.2% in beef,
6.1% in chicken, 10.4% in mackerel, 4.7% in pacific saury, 1.9% in
salmon, 1.2% in mussel, 0.7% in abalone, 7.4% in eggs, 3.3% in milk.
Mackerel contains high level of fat and comes from the ocean. There-
fore, mackerel contains the highest levels of TBBPA and HBCDs. Beef
shows the highest level of TBBPA in meat because beef contains the
highest fat content in meat. Barghi (Barghi et al., 2016) determined the
HBCDs of different food in Korea. Chub mackerel and pacific saury
contained α-HBCD of 1.57 ± 0.40 ng g−1 wet weight and
0.58 ± 0.06 ng g−1 wet weight. They were contaminated by β-HBCD
of N.D and 0.01 ± 0.01 ng g−1 wet weight and γ-HBCD of
0.09 ± 0.02 ng g−1 wet weight and 0.05 ± 0.01 ng g−1 wet weight.
Those results are similar to the levels of HBCDs or higher than those in
this study’s results. In Barghi’s study (Barghi et al., 2016), Chub
mackerel showed the highest level of α-HBCD among the samples
tested, and it did not contain β-HBCD too. In the case of shellfish, eggs
and milk, levels of HBCDs in this study were a little less than those in
Barghi’s study. Not many studies determined TBBPA in food samples.
EFSA (2011b) reported data analyzing TBBPA in fish and shellfish. The
levels of TBBPA in mackerel and mussels were less than 4.8 ng g−1 wet
weight and 0.6 ng g−1 wet weight, and those are higher than levels of
this study. And mackerel which contains high level of fat is con-
taminated by TBBPA compared to mussels.
4. Conclusion
TBBPA and HBCDs are persistent BFRs, which are prevalent in the
environment since they were used to reduce the flammability of a
variety of consumer products. Even though they were banned from use
recently, they still exist in the environment and are taken up by human
through the food chain. A number of countries are trying to monitor
them in the environment and food. However, it is very expensive to
analyze TBBPA and HBCDs regularly. Therefore, it is necessary to de-
velop an analytical method for analysing TBBPA and HBCDs simulta-
neously. Previously, a method had been developed to analyze them
simultaneously in only fish. A method to analyze a variety of food with
optimized conditions to extract and to clean samples and to determine
TBBPA and HBCDs in a liquid chromatograph was developed. LC-MS/
MS was used to determine TBBPA and HBCDs. The method was vali-
dated with performance parameters such as linearity of calibration
curves, detection limit, accuracy, and they were all acceptable in
comparison to the criteria of AOAC. Measurement Uncertainty was also
calculated and intermediate precision (reproducibility) was the key
influential contributor. To assure the quality of data, improving profi-
ciency of individual analysts and using a highly purified STD/ISTD are
important. A variety of food samples were analyzed with the developed
method, and mackerels which contained high level of fat contained
high level of TBBPA and HBCDs. In future study, it would be possible to
develop an analytical method to simultaneously analyze more BFRs
including HBCDs and TBBPA in food.
CRediT authorship contribution statement
Joon-Goo Lee: Conceptualization, Data curation, Writing - original
draft, Writing - review & editing. Jieun Anh: Validation, Investigation.
Gil-Jin Kang: Visualization, Formal analysis. Dongsul Kim:
Supervision. Youngwon Kang: Methodology, Project administration.

8
J.-G. Lee, et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
This research was supported by a grant (15161MFDS013) from
Ministry of Food and Drug Safety in 2015.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.126027.
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