America n Journal of Analy tic al Chemistry, 2011, 2, 352-362
doi:10.4236/ajac.2011. 23043 Published Online July 2011 (
Copyright © 2011 SciRes. AJAC
A Validated Liquid Chromatography-Mass Spectromet ry
Method for the Detection and Quantification of Oxidative
Metabolites of 2,2',4, 4'-Tetrabromodiphenyl Ether in Rat
Hepatic Micro som es
Sarah Catherine Moffatt1, Patrick R obert Edwards2, András Szeitz1, Stelvio Mario Bandiera1*
1Faculty of Pharmaceutical Sciences, The University of British Colum bia, Vancouver, Canada
2Faculty of Pharmacy, University of Toronto, Toronto, Canada
Received January 5, 2011; revised March 4, 2011; accepted March 15, 2011
In the present study, we developed and validated an analytical method using ultra performance liquid chro-
matography-mass spectrometry (UPLC/MS) for the quantitative determination of
2,2',4,4'-tetrabromodiphenyl ether (BDE-47) metabolism by rat hepatic microsomes. BDE-47 is a brominated
flame retardant that was widely used in a variety of consumer products and has subsequently been identified
as a ubiquitous environmental contaminant. Hydroxy-bromodiphenyl ethers (OH-BDEs) were isolated from
rat hepatic microsomes by liquid-liquid extraction. Chromatographic separation was achieved by UPLC on a
C18 column with gradient elution using a mobile phase consisting of methanol and water, each containing
0.1% formic acid, at a flow rate of 0.2 mL/min. Detection and quantification were performed using a mass
spectrometer in single ion recording mode with negative electrospray ionization. The UPLC/MS method was
validated for linearity, limit of quantification (LOQ), accuracy, precision and recovery. The weighted cali-
bration curves (1/X2) were linear over a concentration range of 5 - 250 nM with LOQ values between 5 nM
and 5 0 nM for t he ind ivid ual OH -BDEs. Intra - and inter- day accuracy (%DEV) and precision (%RSD ) val-
ues ranged from 11.7% to 9.5% and 5.9% to 16.5%, respectively. Recovery values of 70% to 90% were
obtained for all OH-BDEs. The validated method allowed us to successfully analyze metabolite formation
following incubation of BDE-47 with hepatic microsomes prepared from phenobarbital-treated rats. Results
demonstrate that the UPLC/MS method has sufficient sensitivity and reproducibility to fully characterize t he
in vitro metabolism of BDE-47 and possibly other PBDEs.
Keywords: BDE-47, Hepatic Metabolism, Polybrominated Diphenyl Ethers, Rat Hepat ic Mic rosom es, Ultra
Performance Liquid Chromatography-Mass Spectrometry.
1. Introduction
Polybrominated diphenyl ethers (PBDEs) are haloge-
nated aromatic hydrocarbons that have been used as ad-
ditive flame retardants on a variety of consumer products
since 1965 [1]. PBDEs were marketed as commercial
mixtures containing a limited number of the 209 possible
brominated diphenyl ether (BDE) congeners [2]. The
penta-BDE mixture, which was used extensively in
North America [3], was composed predominantly of 2,2',
4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pen-
tabromodiphenyl ether (BDE-99),
2,2',4,4',6-pentabromodiphenyl ether (BDE-100),
2,2',4,4',5,5'-hexabromo - diphenyl ether (BDE-153) and
2,2',4,4',5,6'-hexabromo- diphenyl ether (BDE-154) [4].
Penta-BDE was applied to epoxy resins, textiles, paints
and flexible polyurethane foam, which was used in up-
holstered furniture, mattresses and carpet padding [5,6].
PBDEs are not chemically bound to the polymer com-
ponents of the products to which they are applied and
can be released into the environment during manufacture
[5], use [7] and disposal [8] of these products. This fac-
tor, together with the high lipophilicity, chemical stabil-
ity and the widespread use of BDE mixtures has resulted
Copyright © 2011 SciRes. AJAC
in the ubiquitous distribution of PBDEs in the environ-
ment [1]. BDE-47, for example, has been detected in air
[9], sediment [10], fish [10,11], marine mammals [12,13]
and in human blood [14], adipose tissue [15] and breast
milk [16] and is frequently the predominant PBDE con-
gener found in biotic samples [1]. Studies with labora-
tor y animal s have sho wn tha t de velop mental expo sure to
BDE-47 caused alterations in neuromotor activity [17]
and e xposur e in utero produced changes to the reproduc-
tive system and thyroid gland of female rat pups [18].
Laboratory studies have shown that PBDEs can be
metabolized by hepatic cytochrome P450 enzymes to
hydroxy-BDEs (OH-BDEs) [19-22]. OH-BDEs are of
toxicological interest as OH-BDEs show a greater affin-
ity for the thyroid hormone receptor than the natural li-
gand or PBDEs themselves [19]. OH-BDEs have been
detected in blood and feces of rodents treated with
BDE-47 [21] and various OH-BDEs have been found in
human plasma [23]. Several studies, including those
mentioned above, examined the formation of OH-BDEs;
however, the specific enzymes and enzyme kinetics of
OH-BDEs formation are poorly understood. Characteri-
zation of BDE-47 metabolism in vitro is needed to de-
velop a better understanding of the role of metabolism in
the bioaccumula tion and toxicity of BDE -47.
The most common method for the detection and quan-
tification of PBDEs and OH-PBDEs in environmental
samples has been gas chromatography-mass spectrome-
try (GC/MS) or gas chromatography coupled with elec-
tron capture detection (GC/ECD) [19]. In a study that
examined the endocrine disrupting activity of BDEs fol-
lowing hepatic biotransformation, Hamers et al. [19]
identified six hydroxylated metabolites of BDE-47 using
a GC/MS method. GC-based methods are sensitive, but
require derivatization of OH-BDEs, additional sample
preparation time, the use of harmful derivatizing agents
and possible underestimation of OH -BDE concentrations
due to incomplete der ivatization. Liquid chro matograph y
coupled with mass spectrometry (LC/MS) provides an
alternative analytical technique that does not require de-
rivatization. Mas et al. d e monstrated that LC/MS can be
used to detect and quantify OH -BDEs in soi l, fish, sludge
and particulate matter that was spiked with a mixture of
OH-BDEs [24]. However, their LC/MS method was not
validated in a biological matrix [24] and its applicability
to the detection and quantification of oxidative metabo-
lites of BDE-47 generated in vitro or in vivo is unknown.
The aim of the present study was to develop and vali-
date a UPLC/ MS-based analytical method to detect and
quantify OH-BDEs and apply this method to investigate
the in vitro biotransformation of BDE-47 by rat hepatic
2. Materials and Methods
2.1. Chemicals and Reagents
BDE-47 (neat, 99% purity) was obtained from Chiron
(Trondheim, Norway). 4'-Hydroxy-2,2',4-tribromodi-
phenyl ether (4'-OH-BDE-17), 2'-hydroxy-2,4,4'-tribro-
modiphenyl ether (2'-OH-BDE-28), 4-hydroxy-2,2',3,4'-
tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,
2',4,4'-tetrabromodiphenyl ether (3-OH-BDE -47), 5-hy-
droxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-
47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-
OH-BDE-47), 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl
ether (4'-OH-BDE-49) (10 µg/mL or 50 µg/mL in aceto-
nitrile, purity of at least 98%) and 4'-hydroxy-2,2',4,6'-
tetrachlorobiphenyl (4'-OH-CB-50, neat, 99% purity)
were purchased from AccuStandard (New Haven, Con-
necticut, USA). Magnesium chloride, sucrose and nico-
tinamide ade nine dinucleotide p hosphate (NADPH) were
purchased from Sigma-Aldrich (Oakville, Ontario, Can-
ada). Methanol, methyl-tert-butyl ether, hexane, isopro-
panol, sodium hydroxide, hydrochloric acid and mono-
and di-basic potassium phosphate were purchased from
Fisher Scientific (Ottawa, Ontario, Canada). Hydroch-
loric acid and all organic solvents were HPLC- grade or
higher. Ultra pure water was obtained using a Milli-Q
Synthesis syst em (Millipore, Billerica, MA, USA).
2.2. Rat Hepati c Micro so mes
Adult male Long Evans rats (body weight between 160-
190 g) were purchased from Charles River Laboratories
(Montreal, PQ, Canada). Rats were cared for in accor-
dance with the principles and guidelines outlined by the
Canadian Council of Animal Care. Rats (
) were
treated with sodium phenobarbital (PB, 80 mg/kg/day)
for 3 days, as previously described by Edwards et al.
[25]. Pooled hepatic microsomes were prepared by dif-
ferential centrifugation as previously described [26]. He-
patic microsomes were aliquoted and stored at 80˚C
until use. Protein concentration was determined by the
method of Lowry, et al. with bovine serum albumin as
the standard [27].
2.3. Standard Solutions
A stock solution of BDE-47 at a concentration of 2.5
mM was prepared in methanol. A stock solution of
OH-BDE standards containing 4'-OH-BDE-17,
2'-OH-BDE-28, 4-OH-BDE-42, 3-OH-BDE-47,
5-OH-BDE-47, 6- OH-BDE-47 and 4'-OH-BDE-49 (at
1.25 µM each) was prepared in methanol. A second
America n Journal of Analy tic al Chemistry, 2011, 2, 352-362
doi:10.4236/ajac.2011. 23043 Published Online July 2011 (
Copyright © 2011 SciRes. AJAC
Figure 1 . Chemical stru ct ures of BDE-47 a nd seven possibl e hydroxy-metabolites.
stock solution of OH-BDE standards at 0.125 µM each
was prepared by diluting an aliquot of the first stock so-
lution 10-fold in methanol. A stock solution of internal
standard containing 4'-OH-CB-50 at a concentration of
125 µM wa s pr epared in methanol. Three quality control
(QC) solutio ns co ntaini ng OH -BDE standards at concen-
trations of 7.5, 37.5 and 175 nM each and internal stan-
dard at a concentration of 1.25 µM were prepared in me-
thanol. These QC solutions were used for system suita-
bility tests and recovery determination. All stock solu-
tions were stored in amber vials at –20˚C until needed
and eac h via l was vi go ro u sl y vor te x -mixed before use.
2.4. Sample Preparation
Stock solutions of OH-BDE standards were spiked into
rat hepatic microsomes to prepare calibration standards
(CS) samples for the generation of calibration curves. CS
samples were prepared by mixing 1 mg of hepatic mi-
crosomal protein, 50 mM potassium phosphate buffer
containing 3 mM magnesium chloride (pH 7.4) and an
appropriate volume of OH-BDE stock solution in a fina l
volume of 1 mL. Final concentrations of the OH-BDE
standards in the CS samples were 2.5, 5, 10, 25, 50, 100
and 250 nM. QC samples at three concentrations were
prepared in the same manner. Final concentrations of
OH-BDE standards in the QC samples were 7.5, 37.5
and 175 nM (low, medium and high, respectively). The
QC samples were used for the deter mination of accuracy
and precision. Blank samples containing only hepatic
microsomal protein and phosphate buffer were also pre-
CS, QC and blank samples were incubated for 5 min at
37˚C in a shaking water bath. Following incubation, 1
mL of ice-cold 0.5 M sodium hydroxide was added, and
each tube was immediately vortex-mixed. An aliquot
of internal standard (10 µL of 125 µM) was added to
each tube. Tubes were capped and placed in a 70˚C water
bath for 10 min. After cooling to room temperature, 2
mL of 6 M hydrochloic acid and 1 mL of isopropanol
Copyright © 2011 SciRes. AJAC
was added to each tube. Tubes were then vigorously
vortex-mixed for 1 min. Two mL of a methyl-tert-butyl ether: hexane (1:1 v/v) mixture was then added to each
Figure 2. Representative UPLC/MS ion chromatograms showing peaks obtained by spiking rat hepatic microsomes (at a
conce ntr ati on of 1 mg/ mL) w ith a cali brat ion sta ndar d st oc k sol utio n (f inal co nce ntr atio n of OH-B DE standards w as 50 nM)
and the internal standard solution (final concentration was 1.25 µM. (a) Chromatogram of OH-tetra BDEs standards
(3-OH-BDE-47, 4-OH-BDE-42, 5-OH-BDE-47, 4'-OH-BDE-49 and 6-OH-B DE-47) at m/z 500.3; (b) chromatogram of
OH-tri-BDE standards (2'-OH-BDE-28 and 4'-OH-BDE-17) at m/z 421.3; (c) chromatogram of internal standard
(4-OH-PCB-50) at m/z 306.8; (d) total ion current of a blank sample.
tube. Tubes were vi gorousl y vor tex-mixed for 1 min and
spun in a centrifuge at 2,500 rpm for 5 min. The top or-
ganic layer was carefully removed and transferred into
clean test tubes. The extraction procedure was repeated
two more times. The organic phase from each extraction
of the same sample was pooled and evaporated under a
gentl e flo w of nit rogen. The res idue wa s reconsti tuted in
250 µL of met hano l, vort ex-mi xed and filtere d thro ugh a
syringe filter (polytetrafluoroethylene membrane, 0.45
µM) into a 300-µL HPLC v ial.
2.5. UPLC/MS Conditions
The UPLC/MS system consisted of a Waters Acquity
UPLC Sample Manager and a Waters Acquity UPLC
Binary Solvent Manger connected to a Waters Quattro
Premier XE triple quadrupole mass spectrometer
equipped with a combined Electrospray Ionization (ESI)
and Atmospheric Pressure Chemical Ionization (APCI)
probe (Waters, Milford, MA, USA). Chromatographic
separation was achieved using a Waters Acquity UPLC
BEH C18 (2.1 × 100 mm, 1.7 µm) column, which was
maintained at 50ºC. The autosampler tray was main-
tained at 10ºC and the injection volume was 5 µL. The
mobile phase consisted of water containing 0.1% (v/v)
formic acid (solvent A) and methanol containing 0.1%
(v/v) formic acid (solvent B). Mob ile phase solvents were
filtered through a membraine filter (Millipore Durapore
Copyright © 2011 SciRes. AJAC
Membrane Filters, 0.22 µm GV, B illerica, USA) prior to
use. The gradient program was 35% solvent A and 65% solvent B from 0 to 15 min followed by a linear increase
to 80% solvent B from 15 to 30 min. At 30.1 min, sol-
Table 1. Limit of quantification (LOQ) of individu al OH-BDE standards.
Authentic Standard LOQ (nM) Mea n Measured Concentrati on
(nM) Accuracy (%Dev) Precisi on (%RSD) S/N
4'-OH-BDE-17 50 42.9 ± 6.5 –14.2 15.1 5 .4
2'-OH-BDE-28 5 5.5 ± 0.6 10.0 10.9 4.9
4-OH-BDE-42 10 11.3 ± 0.9 13.0 8.0 4.9
3-OH-BDE-47 10 10.7 ± 1.2 7.0 11.2 4.9
5-OH-BDE-47 5 5.6 ± 0.3 12.0 5.4 3.5
6-OH-BDE-47 10 10.8 ± 1.9 8.0 17.5 4.1
4'-OH-BDE-49 5 5.6 ± 0.4 12.0 7.1 3.9
n=6, mean ± SD
vent B was increased to 100% and maintained for 5 min.
The co lu mn wa s t he n r e-equilibrated with 35% solvent A
and 65% solvent B for 5 min. The flow rate was main-
tained at 0.2 mL/min and the total analysis time was 40
min. To protect the mass spectrometer from contamina-
tion, the mobile phase flow was diverted to waste be-
tween 0 and 10 min and 30 and 40 min of each injection.
The mass spectrometer was operated in negative elec-
trospray ionization mode (ESI) using selected ion re-
cording (SIR) at a capillary voltage of 3 kV, cone voltage
of 40 V, source temperature of 120ºC, desolvation tem-
perature of 400ºC and desolvation gas flow of 1005 L/h.
The UPLC/MS system was controlled by MassLynx v.
4.1 software and Windows XP operating system. Chro-
matographic peaks corresponding to the OH-BDEs and
the internal standard were identified by comparing the
mass -to -c harge ratio (m/z) and re tentio n ti me va lue s wi th
those of the authentic standards: m/z 421.3 for 4'-OH-
BDE-17 and 2'-OH-BDE-28; m/z 500.6 for 4-OH-BDE-
42, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47 and
4'-OH-BDE-49; and m/z 306.7 for 4'-OH-CB-50.
2.6. Method Validation
The UPLC/MS method was validated for accuracy, pre-
cision, linearity, limit of quantification (LOQ), selectivi-
ty and recovery. Accuracy and precision were assessed
using the three QC samples. Accuracy was calculated
using the mean measured concentration and expressed as
percent deviation (%Dev) of the nominal concentration.
Precision was expressed as percent relative standard
deviation (%RSD). To determine intra-day accuracy and
precision, six replicates of each QC sample were pre-
pared and analyzed on the same day. To determine in-
ter-day accuracy and precision, QC samples were pre-
pared and analyzed in triplicate on three separate days.
The acceptance criteria for inter- and i ntra - day accurac y
and precision were %Dev ± 20% and %RSD 20%,
respectively, for the low QC samples and %Dev ± 15%
and %RSD 15%, respectively, for the medium and
high Q C sample s .
Linearity of the calibration curve was assessed using
the coefficient of determination (R2). Calibration curves
were generated for each OH-BDE standard by plotting
peak area ratios for each OH-BDE standard and internal
standard (y-axis) against the corresponding nominal
concentration of the OH-BDE standard (nM, X-axis)
using linear regression analysis. To increase accuracy at
the lower end of the calibration curve, a 1/X2 weighting
factor was used. The acceptance criterion for linearity
was R2 0.9.
The LOQ of each OH-BDE standard was determined
by preparing five replicates of CS samples between 2.5
and 50 nM and a calibration curve. The LOQ was set as
the lowest CS concentration that had a signal-to-noise
(S/N) ratio at least three times higher than that of the
Copyright © 2011 SciRes. AJAC
blank sample with a ± 20% Dev and 20% RSD. The
S/N ratio was determined with MassLynx using the peak-to-peak method.
Selectivity was assessed by visually comparing the
Table 2. I ntra-day accurac y (%Dev) and precis ion (%RS D).
Metabolite Nominal Concentration (nM) Mean Measured Concentratio n (nM) Accuracy (%Dev) Precision (%RSD)
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) < LO Q n. d. n. d.
QC-High (175) 167. 0 ± 1 7 .74.6 10.6
QC-Low (7.5) n. d. n. d. n. d.
QC-Med (37.5) 36.1 ± 3.3 –3.7 9 .1
QC-High (175) 162. 3 ± 1 5 .77.3 9 .7
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) 36.7 ± 2.2 –2.1 5 .9
QC-High (175) 153. 9 ± 1 5 .412.1 10.0
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) 36.8 ± 2.8 –2.1 7 .6
QC-High (175) 156. 2 ± 1 4 .910.7 9.5
QC-Low (7.5) 8.2 ± 1 .4 9.3 17.0
QC-Med (37.5) 36.6 ± 3.0 –2.4 8.2
QC-High (175) 155. 7 ± 1 5 .3 11.0 9 .8
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) 38.3 ± 3.0 2.1 7.8
QC-High (175) 178. 2 ± 1 9 .1 1.8 10.7
QC-Low (7.5) 7.8 ± 0 .5 4.0 6.4
QC-Med (37.5) 35.8 ± 3.2 –4.5 8 .9
QC-High (175) 157. 3 ± 1 3 .910.1 8.8
< LOQ = Below limit of quantificati on; n .d., not determined because metabolite concent ration was below the LOQ; n = 6, mean ± SD.
chromatograms obtained from blank samples with chro-
mato grams fro m spiked CS samples at the LOQ value of
each OH-BDE standard. Chromatograms were examined
for the presence of interfering peaks with retention time s
that overlapped those of OH-BDE standard s or the inter-
nal standard.
Recovery rates were determined using QC solutions
and QC samples. Recovery was calculated by comparing
the peak area of each OH-BDE standard in the QC sam-
ple with that in the QC solution at the same concentra-
2.7. In Vitro Biotransformat ion of BDE-47
Following validation, t he UPLC/MS method was applied
to investigate the in vitro biotransformation of BDE-47.
For mation of hydroxy BDE-47 metabolites by rat hepati c
microsomes was determined using a reaction mixture
containing 50 mM phosphate buffer, 0 - 2 mg/mL of rat
hepatic miroso mal prote in and 50 µM BDE-47 (20 µL of
2.5 mM i n met ha no l) i n a fi nal vo lu me o f 0 .9 9 mL. A fter
a 5-mi n pr e-incubation at room temperature, the reaction
was initiated by addition of 10 µL of 100 mM NADPH
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(final concentration 1 mM) and allowed to proceed at
37˚C in a shaking water bath for 0 to 30 min. The reac-
tion was terminated by the addition of 1 mL of ice-cold
0.5 M sodium hydroxide. Extraction and quantification
of the OH -BDE metabolites was performed as described
above. Samples were prepared in duplicate for each as-
Table 3. I nter-day accur acy (%Dev) and precisio n (%RSD).
Metabolite Nominal Concentration (nM) Mean Measured Concentration (nM) Accuracy (%Dev) Precision (%RSD)
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) < LOQ n. d. n. d.
QC-High (175) 164. 3 ± 2 2 .36.1 13.6
QC-Low (7.5) n. d. n. d. n. d.
QC-Med (37.5) 37.3 ± 4.8 –0.4 12.7
QC-High (175) 158. 4 ± 1 2 .09.5 7 .6
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) 37.8 ± 4.0 0.8 10.6
QC-High (175) 155. 4 ± 1 1 .511 .2 7.4
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) 37.6 ±3 .0 0.3 10.1
QC-High (175) 155. 2 ± 1 1 .511 .3 7.4
QC-Low (7.5) 6.9 ±2.58.0 8.0
QC-Med (37.5) 37.4 ±4 .2 0.3 11.3
QC-High (175) 156. 3 ± 1 3 .0 10.7 8.3
QC-Low (7.5) < LOQ n. d. n. d.
QC-Med (37.5) 39.5 ± 4.3 5.3 10.9
QC-High (175) 182. 8 ± 1 4 .8 4.4 8.1
QC-Low (7.5) 7.7 ± 0 .5 2.7 6.5
QC-Med (37.5) 35.7 ± 4.8 –4.8 13.4
QC-High (175) 154. 6 ± 1 1 .011 .7 7.1
< LOQ = Below limit of quantifi cation; n. d., no t deter mined becau se m e t a bo lite concentr ation wa s be l o w the L O Q ; n = 12 (Day 1 n = 6, Day 2 n = 3, Day 3
n. = 3) me an ± SD.
say and experiments were performed 3 times on separate
days. Blank samples contained only rat hepatic micro-
somes and buffer. Negative control samples did not con-
tain BDE-47, NADPH, or rat hepatic microsomes. QC
samples were included in each experiment to assess me-
thod performance.
3. Results and Discussion
3.1. Optimization of UPLC/MS Parameters
APCI and ESI in negative and positive modes were
compared for the detection of OH-BDE standards by
mass spectrometry using SIR. OH-BDE standards were
not detected in positive ion mode. ESI operated in nega-
tive electrospray ion mode produced larger peak area
counts than APCI. BDE-47 was not detected with APCI
Copyright © 2011 SciRes. AJAC
or ESI in either positive or negative ion mode. Flow in-
jection analysis was used to determine the molecular ions
[M-H] and optimal cone voltage (40 V) for all OH-
BDE standards. A source temperature of 120˚C, a desol
vation temperature of 400˚C and a desolvation gas flow
of 1005 L/H were found to be optimal for the detection
of OH-BDE standards. Multiple reaction monitoring
Table 4. Recovery value s of individual OH -BDE stan da rds.
Authentic Standard Retention Time (min) Nominal Concentration (nM) Mea n Recovery (%)
QC-Low (7.5) n. d.
16.4 QC-Med (37.5) n. d.
QC-High (175) 69.9 ± 5.6
QC-Low (7.5) n. d.
15.6 QC-Med (37.5) 86.7 ± 5.3
QC-High (175) 72.3 ± 4.3
QC-Low (7.5)
21.7 QC-Med (37.5) 84.0 ± 6.0
QC-High (175) 74.0 ± 5.3
QC-Low (7.5) n. d.
19.7 QC-Med (37.5) 88.0 ± 4.4
QC-High (175) 76.7 ± 5.0
QC-Low (7.5) 89.2 ± 6.2
23.4 QC-Med (37.5) 88.2 ± 4.6
QC-High (175) 75.2 ± 4.7
QC-Low (7.5) n. d.
25.7 QC-Med (37.5) 88.2 ± 4.3
QC-High (175) 74.0 ± 4.9
QC-Low (7.5) 87.6 ± 5.3
24.9 QC-Med (37.5) 87.6 ± 4.2
QC-High (175) 75.4 ± 5.1
n. d., not determined becaus e metabolite concentration was below th e LOQ; n=6, mean ± SD
(MRM) was also assessed. Product ion scans of the mo-
lecular ions were performed at different collision energy
values. The main product ion was a bromine fragment
(m/z 79 and m/z 81). However, the sensitivity of the
MRM method was much lower than that of the SIR me-
thod. There fore, SIR was used for subse que nt analyses.
Separation of the seven OH-BDE standards was
achieved using a Waters Acquity UPLC BEH C18 (2.1 ×
100 mm, 1.7 µm) column and gradient elution. Several
mobile phase combinations were tested, including mix-
tures of water and acetonitrile or water and methanol. A
mixture of water containing 0.1% formic acid (v/v) and
methanol containing 0.1% formic acid (v/v) yielded the
best peak shape and the highest peak area counts and was
selected for chromatographic separation. Formic acid
was added to enhance ionization of the compounds of
interest. Isocratic and gradient elution were also com-
pared. The best resolution was achieved using the elution
gradient program reported in the experimental section,
which gave baseline separation of all OH-BDE standards
tested, except for 6-OH-BDE-47 and 4'-OH-BDE-49
(Figure 2). Various elution gra dient pr ogra ms, flow rates
Copyright © 2011 SciRes. AJAC
and run times were tried but complete baseline separa-
tion of 6-OH-BDE -47 and 4'-OH-BDE-49 was not
achieved. In comparison, Mas, et al., [25] used a ternary
mixtur e of a m monium aceta te , aceto nitrile a nd methano l,
gradient elution and a shorter run time (20 min compared
to 40 min in the present study) to resolve eight OH-
BDEs but did not attain baseline separation of 5-OH-
BDE-47, 6-OH-BDE-47 and 4'-OH-BDE-49 or of three
hydroxylated tribrominated diphenyl ethers.
3.2. Optimization of Sample Preparation
To achieve efficient extraction of the OH-BDE standards
from the biological matrix (i.e., rat hepatic microsomes)
and to minimize the possibility of unknown peaks that
could interfere with the peaks of interest, variations of
the sample preparation protocol were evaluated. Hexane,
acetone, dichloromethane, me th yl-tert-butyl ether, as
well as, different ratios of acetone and hexane and of
me th yl-tert-butyl ether and hexane were tested as possi-
ble extraction solvents. Three extractions with a mixture
of methyl-tert -butyl ether: hexane (1:1 v/v) yielded the
highest recovery of the OH-BDE standards (Table 4).
The add ition of a ce ntrif ugati on step b efore e xtractio n, to
separate microsomes from supernatant, or replacement of
sodium hydroxide with acetone, methanol, formic acid,
hydrochloric acid, sulfuric acid or trifluroacetic acid, to
terminate the reaction, were tested. Although some of
these modifications reduced the appearance of unknown
non-interfe ri ng pea ks in t he b lank sa mpl es, t he modi fica-
tions reduced the recovery of the OH-BDE standards and
were not incorporated into the assay.
4. Method Validation
Visual inspection of chromatograms obtained from blank
samples and chromatograms obtained from spiked CS
samples s ho wed no interfering peaks at the same m/z and
retention times as the OH-BDE standards or internal
standard. Representative chromatograms obtained from a
CS sample at 50 nM and a blank sample are shown in
Figure 2.
Mean R2 values of the calibration curve constructed for
each individual metabolite were 0.95 ± 0.06 for 4'-
OH-BDE-17, 0.96 ± 0.04 for 2'-OH-BDE-28, 0.94 ± 0.05
for 4-OH-BDE-42, 0.96 ± 0.04 for 3-OH-BDE-47, 0.97
± 0.04 for 5-OH-BDE-47, 0.94 ± 0.06 for 6-OH-BDE-47
and 0.95 ± 0.05 for 4'-OH-BDE-49. The LOQ concentra-
tion of individual OH-PBDEs ranged between 5 and 50
nM (Table 1). The LOQ concentrations determined in
this study, which are in the pg/mL range, are similar to
those reported by Erratico, et al., for OH-penta-BDEs
using a similar UPLC/MS method [28] and comparable
to those reported by Mas, et al. [24], using LC/MS ion
spray operated in negative ion MRM mode.
Intra- and inter-day accuracy and precision values are
reported in Tables 2 and 3, respectively. Both intra-
and inter-day accuracy and precision fell within the es-
tablished acceptance criteria. In contrast to the study by
Mas, et al. [24], our experimental design evaluated ac-
Figure 3. Effect of (a) incubation time and (b) microsomal
protein concentration on OH-BDE metabolite formation.
BDE-47 (50 µM) was incubated with hepatic microsomes
prepared from phenobarbital-treated adult male rats for
(a) 0-30 min at 1 mg microsomal protei n/ mL or (b) 5 min a t
0 2 mg microsomal protein/mL. OH-BDE metabolites
were extracted and analyzed by UPLC/MS as described in
the Exper imental section. Data point s are mea n ± SEM.
curacy and precision using the biological matrix of in-
terest and concentrations at the low, medium and high
range of the calibration curve rather than at a single point
in an organic solvent (i.e., 75 pg/ml, approximately 150
nM). Evaluati on usi ng t he biological matri x and multiple
points on the calibration curve provides a more robust
estimation of accuracy and precision.
Copyright © 2011 SciRes. AJAC
Recovery values for the low and medium QC sample
ranged between 84% and 89% for all OH-BDEs. Recov-
ery values for the high QC sample were slightly lower,
ranging between 70% and 77% (Table 4). The recovery
values are higher than those reported in a previous s tud y
that used GC-based methods [19], but lower than those
reported by Erratico, et a l. [28] using a similar method to
identify the oxida tive metabolites of BDE -99. Increasing
the extraction volume or the number of extractions, did
not increase recovery rates of the OH-PBDE standard s.
The method validation studies confirm that OH-BDEs
are amenable to direct analysis by UPLC/MS in negat ive
electrospray ion mode without the need for derivatization
or extensive fragme ntation and that the UPLC/MS-based
analytical method that we developed can be used for the
separation, detection a nd quantification of OH-BDEs in a
complex biological matrix such as rat liver microsomes.
4.1. Biotr an s for mation of BDE-47 by Rat Liver
Microsom es
The validated UPLC/MS method was applied to analyze
the oxidative metabolism of BDE-47 in vitro. Incubation
of BDE-47 with rat hepatic microsomes prepared from
PB-treated adult male rats produced five OH-tetra-BDE
metabolites (4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-
47, 6-OH-BDE-47 and 4'-OH-BDE-49) which were de-
tected and identified by their retention times and
values. The major metabolite was 4'-OH-BDE-49. There
was no evidence for the fo rmation of 4'-OH-BDE-17 or
2'- OH-BDE-28 by hepatic microsomes prepared from
PB- treated rats. No other metabolite or unidentified
peaks were observed.
The effect of varying incubation time (0 - 30 min) and
protein concentration (0 - 2 mg/mL) on formation of the
five hydroxylated metabolites was investigated using a
substrate (BDE-47) concentration of 50 µM. Formation
of 4 -OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47 and 4'-
OH-BDE-49 was linear for the first 5 min of incubation
at a microsomal protein concentration of 1 mg/mL (Fig-
ure 3a) and approximately linear up to a microsomal
protein concentration of 1 mg/mL at an incubation time
of 5 min (Figure 3b). Under the e xperi mental conditio ns
used, formation of 6-OH-BDE-47 was detected but could
not be quantified because the concentration of this me-
tabolite remained below the LOQ value (10 nM).
5. Conclusions
A UP LC/M S-based analytical method for the separation,
detection and quantification of seven possible oxidative
metabolites of BDE-47 in rat liver microsomes was de-
veloped and validated. The method was applied to ana-
lyze the oxidative metabolism of BDE-47 in vitro and
allowed us to d etect formation of five monohydroxylated
metabolites o f BDE-47 and quantify formation of four of
the metabolites. Our method represents an improvement
of previously published LC/MS methods because valida-
tion was performed using the biological matrix of inter-
est and at the low, medium and high ends of the calibra-
tion curve providing a more complete assessment of the
accuracy, precision, recovery and LOQ of the analytical
method. Quantification of hydroxylated metabolites of
BDE-47 generated by hepatic microsomes following a 5
min incubation at 1 mg microsomal protein/mL demon-
strates the sensitivity and feasibility of the method for
further in vitro metabolic stud ies, includ ing reaction phe -
notyping, analyzing enzyme-catalyzed reaction kinetics
and enzyme inhibition. The selectivity and reproducibil-
ity of the UPLC/MS method combined with the in vitro
metabolism assay will be useful for measuring metabol-
ism of BDE-47 in human liver samples and in liver
preparations from additional species.
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