Synthesis and characterization of bromophenol glucuronide and sulfate conjugates for their direct LC-MS/MS quantification in human urine as potential exposure markers for polybrominated diphenyl ethers

Article abstract of DOI:10.1021/ac302161t

Bromophenol glucuronide and sulfate conjugates have been reported to be products of mammalian metabolism of polybrominated diphenyl ethers (PBDEs), a group of additive flame-retardants found ubiquitously in the environment. In order to explore their occurrence in human urine, four water-soluble bromophenol conjugates, namely, 2,4-dibromophenyl glucuronide, 2,4,6-tribromophenyl glucuronide, 2,4-dibromophenyl sulfate, and 2,4,6-tribromophenyl sulfate, were synthesized, purified, and characterized. An analytical protocol using solid-phase extraction and ion-paired liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) quantification has been developed for the direct and simultaneous determination of these glucuronide and sulfate conjugates in human urine samples. The limit of detections for all analytes were below 13 pg mL^-1, with 73-101% analyte recovery and 7.2-8.6% repeatability. The method was applied to analyze 20 human urine samples collected randomly from voluntary donors in Hong Kong SAR, China. All the samples were found to contain one or more of the bromophenol conjugates, with concentration ranging from 0.13-2.45 μg g^-1 creatinine. To the best of our knowledge, this is the first analytical protocol for the direct and simultaneous monitoring of these potential phase II metabolites of PBDEs in human urine. Our results have also suggested the potential of these bromophenol conjugates in human urine to be convenient molecular markers for the quantification of population exposure to PBDEs.

Full text of DOI:10.1021/ac302161t

Article
pubs.acs.org/ac
Synthesis and Characterization of Bromophenol Glucuronide and
Sulfate Conjugates for Their Direct LC-MS/MS Quantification in
Human Urine as Potential Exposure Markers for Polybrominated
Diphenyl Ethers
Ka-Lok Ho,† Margaret B. Murphy,† Yi Wan,‡,# Bonnie M.-W. Fong,§,∥ Sidney Tam,§ John P. Giesy,†,‡,⊥
Kelvin S.-Y. Leung,∥ and Michael H.-W. Lam*,†
†State Key Laboratory for Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR,
China
‡Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Canada
§Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong SAR, China
∥Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China
⊥Department of Zoology and Center for Integrative Toxicology, Michigan State University, United States
S
* Supporting Information
ABSTRACT: Bromophenol glucuronide and sulfate conju-
gates have been reported to be products of mammalian
metabolism of polybrominated diphenyl ethers (PBDEs), a
group of additive flame-retardants found ubiquitously in the
environment. In order to explore their occurrence in human
urine, four water-soluble bromophenol conjugates, namely, 2,4-
dibromophenyl glucuronide, 2,4,6-tribromophenyl glucuronide,
2,4-dibromophenyl sulfate, and 2,4,6-tribromophenyl sulfate,
were synthesized, purified, and characterized. An analytical
protocol using solid-phase extraction and ion-paired liquid
chromatography−electrospray tandem mass spectrometry (LC-
ESI-MS/MS) quantification has been developed for the direct
and simultaneous determination of these glucuronide and
sulfate conjugates in human urine samples. The limit of detections for all analytes were below 13 pg mL−1, with 73−101% analyte
recovery and 7.2−8.6% repeatability. The method was applied to analyze 20 human urine samples collected randomly from
voluntary donors in Hong Kong SAR, China. All the samples were found to contain one or more of the bromophenol conjugates,
with concentration ranging from 0.13−2.45 μg g−1 creatinine. To the best of our knowledge, this is the first analytical protocol
for the direct and simultaneous monitoring of these potential phase II metabolites of PBDEs in human urine. Our results have
also suggested the potential of these bromophenol conjugates in human urine to be convenient molecular markers for the
quantification of population exposure to PBDEs.
PBDEs. Therefore, monitoring the accumulation of PBDEs in
olybrominated diphenyl ethers (PBDEs) are a class of
Padditive brominated flame retardant used commonly in humans is important to assess the risk of PBDE exposure. This
calls for the reliable estimation of the population exposure to
PBDEs. However, the reliability of many of the current
practices for the measurement of population exposure to
PBDEs is seriously limited by difficulties in the acquisition of
adequate quantities of human tissue samples.
Direct quantification of selected BDE congeners and their
hydroxylated and methoxylated metabolites in human blood or
serum4 and human breast milk5 is the most commonly adopted
approaches for the assessment of human exposure to PBDEs.
polymers, electric and electronic goods, and textiles. As they are
not chemically bonded to materials, PBDEs can be released
into the environment during product usage.1 Their hydro-
phobicity and resistance to degradation and metabolism have
led to their ubiquity in the natural environment and in humans.
PBDEs with four to seven bromine substituents have been
formally recognized as persistent organic pollutants (POPs) by
the Stockholm Convention in 2009 as a result of the strong
evidence showing that these compounds have been entering the
global ecosystem at an alarmingly rapid rate.2,3 Even after the
ban of their production and use, their levels in human may
further increase because of their persistence in the ecosystem
and our continued utilization of manufactured goods containing
Received: August 9, 2012
Accepted: October 16, 2012
Published: October 17, 2012
© 2012 American Chemical Society
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a
Scheme 1. General Synthetic Procedure of Glucuronide and Sulfate Conjugates of Bromophenols
a
(a) Acetic anhydride, NaOAc; (b) HBr in CH3COOH; (c) 2,4-dibromophenol/2,4,6-tribormophenol, tetrabutylammonium bromide, NaOH(aq);
(d) NEt3, MeOH, THF, H2O; (e) TEMPO, NaOCl(aq), NaOH(aq); (f) ClSO3H, CHCl3.
Other less commonly used tissues for exposure estimation
include hair, lung, liver, and adipose tissues.6 However,
sampling human tissues for contaminant analysis is intrusive
and difficult to achieve on a large scale, especially in the case of
healthy subjects who are not hospitalized or required to go
through clinical diagnostic tests. Sampling of breast milk can be
considered a nonintrusive method, but that restricts the
sampling to lactating women within a relatively narrow age
distribution, and therefore, it is not clear whether these samples
can truly reflect population-level exposure.7 Sampling of human
urine is a truly nonintrusive approach, making it much easier to
obtain urine samples from much more voluntary donors for
large-scale population surveys.8 Detection of PBDE metabolites
in human urine as exposure markers for PBDEs enables the
collection of more representative data on population exposure
to the contaminants in order to make comparisons and public
health risk assessment on both nationwide and international
scales.
The occurrence of phase II metabolites of selected BDE
congeners in the urine of exposed mammalian animal models
has already been well established by numerous pharmacokinetic
and toxicokinetic studies (Figure S1 of the Supporting
Information).9 These metabolites are mainly glucuronide and
sulfate conjugates of dibromophenols (DBPs) and tribromo-
phenols (TBPs). To the best of our knowledge, there is no
literature report on the occurrence of these potential phase II
PBDE metabolites in human urine. Authentic standards of
these phase II metabolites are generally not available. Thus, the
aims of this study are to synthesize, purify, and characterize
selected dibromophenyl- and tribromophenyl-glucuronide and
sulfate conjugates as authentic standards for the development of
a liquid chromatography−tandem mass spectrometry (LC-MS/
MS) analytical protocol for their quantification in human urine
samples.
EXPERIMENTAL SECTION
■
Safety Precautions. Extra precaution was practiced in the
handling of human urine samples. Double latex gloves,
facemasks, and eye-protection goggles were worn all the time
during the handling, spiking, and transferring of human urine
samples. All the spent urine samples after analysis were
collected in a separated close-lipped container with proper
clinical waste labels. The spent urine samples and used personal
protection items were treated as clinical wastes and were
collected and disposed of in accordance with the “Code of
Practice for the Management of Clinical Waste” issued by the
Environmental Protection Department of the Hong Kong SAR
Government.
1
Instrumentation. H NMR spectra were recorded by a
Bruker AV400 (400 MHz) FT-NMR spectrometer. Electro-
spray ionization (ESI) mass spectra were measured by a PE
SCIEX API 365 LC-MS/MS system and Applied Biosystems
SCIEX QSTAR ELITE hybrid quadrupole/time-of-flight (Q-
TOF) tandem high-resolution mass spectrometer. Elemental
analyses were carried out on a Vario EL III CHN elemental
analyzer. Purification of the authentic standards was performed
using a Waters 515 high-performance liquid chromatograph
(HPLC) isocratic pump and a Waters 2487 dual λ absorbance
detector (Milford, MA) using a Zorbax Eclipse XDB-C18 5 μm
150 mm × 4.6 mm i.d. analytical column at a flow rate of 1 mL
min−1 with water/methanol (1:1 v/v) as the mobile phase.
Quantification of the bromophenol glucuronide and sulfate
conjugates was performed using an Agilent 1200 Series HPLC
(Agilent Technologies, Waldbronn, Germany) coupled to a
MDS Sciex API 3200 QTrap triple quadrupole/linear ion trap
MS with a Turbo V ion spray source (Applied Biosystems,
Foster City, CA, USA). For improved detection sensitivity and
selectivity, analytes were detected in the multiple reaction
monitoring (MRM) mode with a dwell time of 150 ms. The
ionization source parameters were as follows: ion spray voltage,
−4500 kV; curtain gas (N2), 15 psig; collision gas (N2), high;
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temperature of heater gas, 600 °C; ion source gas 1 (nebulizer
gas), 60 psig; ion source gas 2 (heater gas), 50 pisg.
Declustering potential (DP), entrance potential (EP), and
collision energy (CE) for all analytes were optimized to obtain
maximum sensitivity. The collision cell exit potential (CXP)
was held constant at −1 V.
Chromatographic separations were performed using a Waters
XBridge C18 2.5 μm 3.0 mm i.d. × 50 mm column. A guard
column (XBridge C18 2.5 μm 3.0 mm i.d. × 20 mm) was
placed in front of the analytical column. Separation was
obtained using gradient elution at a flow rate of 300 μL min−1,
with solvent A (5 mM DHAA in Milli-Q water) and solvent B
(5 mM DHAA in methanol) at the composition of 90:10 (v/v)
at t = 0 to t = 2 min, changed linearly to 30:70 (v/v) over a
period of 18 min and then held at such composition for a
further 10 min. After the separation, the eluent composition
was switched back to 90:10 (v/v) and held for 20 min before
the next injection. The injection volume was 10 μL.
RESULTS AND DISCUSSION
■
Synthesis and Purification of the Glucuronide and
Sulfate Conjugates. Synthesis of the sulfate esters of the
bromophenols was relatively straightforward. The literature
method reported by Burkhardt and Lapworth10 was generally
adopted, with slight modifications. Chloroform was used
instead of carbon disulfide because the bromophenols were
not too soluble in the latter solvent, leading to low synthetic
yields. The synthesis of the glucuronide conjugates involved the
nucleophilic substitution reaction between 2,3,4,6-tetra-O-
acetylglucopyranosyl bromide and the bromophenols using
tetrabutylammonium bromide as the phase-transfer catalyst.
Deprotection of the hydroxyl groups on the glucuronide moiety
was carried out using Et3N. The final products were obtained
by the selective oxidation of the primary alcohol by TEMPO
and NaOCl. The glucuronide six-membered rings of the
bromophenol conjugates can have two stereisomeric config-
urations, the α-anomer and β-anomer, depending on the
position of the hydrogen atom on its C1 carbon. In the α-
anomer, the C−H on C1 is cis to the C−H on C2. In the β-
anomer, those two C−H groups are trans to each other. In
human, phenolic compounds are conjugated to form their β-
anomers with glucuronic acid as the conjugation reactions
follow an Sn2 mechanism.11 1H−1H ROESY two-dimensional
(2D) NMR was used to measure the coupling between the C1
and C2 protons of the glucuronide moieties through space so as
to determine their configuration. A crossed signal can be
observed in α-anomers but not in β-anomers. Figure 1 shows
the typical 1H−1H ROESY 2D NMR spectra of the synthesized
2,4-dibromophenyl glucuronide and 2,4,6-tribromophenyl
glucuronide. The absence of any crossed signal between H1
and H2 confirmed the β-anomeric configuration of the
glucuronide moiety on the two synthesized bromophenol
conjugates.
The bromophenol glucuronide and sulfate conjugates were
purified by recrystallization and high-performance liquid
chromatographic separation. Purity of all the resultant products
was checked by HPLC-UV monitored at dual wavelengths (254
and 226 nm). No ghost peak was observed on their
corresponding chromatograms. Q-ToF high-resolution mass
spectrometry (HRMS) and LC-ESI-MS were used to confirm
the identity of their parent ions. Results of elemental analyses
and the exact molecular masses of the synthesized conjugates,
revealed by HRMS, are shown in Table S1 of the Supporting
Information. Differences between the actual m/z values of the
parent ions of the synthesized conjugates to their correspond-
ing theoretical m/z were less than 10 ppm. LC-ESI-MS spectra
of the four bromophenol conjugates showing good matches
between isotope distribution patterns of their parent ions and
major fragments with the theoretical patterns are shown in
Figure S2 of the Supporting Information. These conjugates
were used as authentic standards for the development of the
corresponding analytical protocol for their quantification in the
human urine matrix by LC-MS/MS.
Synthesis, Purification, and Characterization of
Bromophenol Glucuronide and Sulfate Conjugates.
The general synthetic routes for the bromophenol glucuronide
and sulfate conjugates are outlined in Scheme 1. Detail
synthetic and purification procedures and characterization
data are given in the Supporting Information.
Sample Extraction and Cleanup. A human urine sample
(5 mL) was partitioned with 3 × 5 mL ethyl acetate. The
combined organic solution was evaporated to dryness under a
gentle stream of nitrogen. Residues were dissolved in 15 mL of
0.67 M sodium acetate buffer at pH 5.2. The resultant solution
was applied, at a rate of 1 drop s−1, to an Oasis WAX solid-
phase extraction (SPE) cartridge that had been preconditioned
sequentially by 5 mL of methanol, 5 mL of Milli-Q water, and 5
mL of 2 M sodium acetate buffer at pH 5.2. The loaded WAX
SPE cartridge was then washed in turn by 5 mL of 2 M sodium
acetate buffer at pH 5.2, followed by 5 mL of methanol. The
glucuronide fraction was then eluted with 4 mL of a formic
acid/methanol (1:9, v/v) mixture, and the sulfate fraction was
eluted with 4 mL of an aqueous ammonia/methanol (1:9, v/v)
mixture. The eluates were evaporated to around 100 μL under a
gentle stream of nitrogen. 13C6-2,4-dibromphenol (200 μL, 500
ng mL−1) was added as an internal standard for LC-MS/MS
quantitation.
Stability of Glucuronide and Sulfate Conjugates of
Bromophenols in Human Urine. The stability of the
bromophenol glucuronide and sulfate conjugates was examined
by spiking 300 ng mL−1 of the authentic standards into 2 L of
human urine samples to assess the persistence of these
conjugates 14 times over a period of 30 days. Three parameters
related to the storage conditions of the urine samples were
assessed: (i) addition of formaldehyde; (ii) addition of sodium
azide (NaN3) as preservatives; and (iii) storage at −20 °C.
Collection of Human Urine Samples. Human urine
samples were obtained from 20 healthy volunteers randomly
recruited in Hong Kong SAR, China. Urine samples were
collected in 100 mL sterilized glass bottles and stored at −80
°C, within 15 min after sampling, until being analyzed. The
urine sample from each donor was subdivided into three
replicate samples before low temperature storage. All samples
were carefully labeled and documented. Upon analysis, samples
were thawed, and 10 mL of each sample was taken for
creatinine content determination.
Optimization of Chromatographic Conditions. Ion-
pairing liquid chromatography is a commonly adopted
technique to improve peak shape, retention capacity, and
detection sensitivity of highly polar, charged organic analytes.12
The biggest drawback of ion-pairing chromatography is ion
suppression during MS analysis.13 The sensitivity of ESI-MS is
strongly affected by the type, concentration, and pH of the ion-
pairing agents in the mobile phase.14 In this work, we have tried
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optimal mobile phase pH for analyte ionization at the
electrospray ion source was found to be pH 5.4.
Selection of SPE Sorbents. Four different SPE sorbents,
including Oasis HLB, Oasis WAX, Discovery DSX-SAX, and
Supelclean LC-NH2, were tested for the optimal extraction
efficiency of the highly water-soluble glucuronide and sulfate
conjugates from an artificial urine matrix. The spike level used
was 10 ng mL−1. A summary of the relative LC-MS/MS
responses of the conjugates after extraction by the various SPE
sorbents is shown in Figure S4 of the Supporting Information.
Extraction efficiencies of polymeric sorbents HLB and WAX
were higher than those of silica-based NH2 and SAX sorbents.
Of the two polymeric sorbents, WAX out-performed HLB,
probably because of its weak anion exchange properties.12a,16
Therefore, Oasis WAX SPE cartridges were used in all the
subsequent sample extractions.
Validation of Analytical Protocol. Table 1 tabulates all
the multiple reaction monitoring (MRM) transitions adopted
Table 1. MS/MS Transitions and MS Parameters Adopted in
the Analysis
MRM
transitions
(m/z)
entrance
potential
(V)
collision
energy
(V)
bromophenol
conjugate
declustering
potential (V)
2,4-dibromphenyl
glucuronide
427.1 →
−27.5
−24.0
−15.9
−12.0
−28.9
−28.0
−17.0
−20.0
−37.5
−3.8
−4.9
−3.8
−3.0
−4.5
−4.9
−3.8
−3.9
−8.9
−32.7
−22.1
−27.0
−23.9
−28.6
−76.1
−28.5
−99.0
−39.6
251.1
427.1 →
113.2
2,4,6-tribromphenyl 507.5 →
glucuronide
331.2
505.4 →
113.2
2,4-dibromphenyl
sulfate
330.9 →
250.9
330.9 →
81.3
1
Figure 1. H−1H ROESY 2D NMR spectra of 2,4-dibromophenyl
2,4,6-tribrophenyl
sulfate
411.2 →
331.0
glucuronide (upper) and 2,4,6-tribromophenyl glucuronide (lower).
411.2 →
81.3
13C6-2,4-
dibromphenol I. S.
257.1 →
80.8
various alkylamines of different alkyl chain lengths as ion-
pairing agents for the liquid chromatographic separation of the
glucuronide and sulfate conjugates. Retention capacity, k′, and
suppression of the electrospray ionization efficiency of these
conjugates by ammonium acetate (AA), triethylammonium
acetate (TEAA), tributylammonium acetate (TBAA), and
dihexylammonium acetate (DHAA) at a level of 10 mM in
the mobile phase are shown in Figure S3 of the Supporting
Information. All the ion-pairing agents were able to bring about
sufficient retention of the analytes. Alkylamines with longer
carbon chains, that is, TBAA and DHAA, gave higher retention
capacity. Extents of ionization suppression on the analytes by
the four alkylamine salts were comparable. DHAA was
eventually chosen as the ion-pairing agent for subsequent
method development of the LC-MS/MS determination. A
series of concentrations (0.1 mM to 10 mM) of DHAA has
been tried in order to evaluate the separation of the glucuronide
and sulfate conjugates. Our results showed that the conjugates
were effectively separated at a DHAA concentration of greater
than 5 mM.
for the identification and quantification of the bromophenyl
glucuronide and sulfate conjugates. Good linearity of the LC-
MS/MS determination for all the conjugates (with r2 ranging
from 0.9953 to 0.9999) was observed over the spiked
concentration range of 1−500 ng mL−1. Repeatability and
analyte recovery were evaluated by the consecutive analysis of
seven independent artificial urine samples spiked with 0.5 ng
mL−1 of each of the conjugates. Method detection limits
(MDLs) of the SPE LC-MS/MS analytical protocol for the
conjugates were determined based on the lowest spiked levels
of the glucuronide and sulfate bromophenol conjugates in the
artificial urine matrix (5 mL) that were still able to produce a
signal-to-noise ratio of >10 at their corresponding MRM
chromatographic peaks over a series of seven consecutive
analyses. Table 2 summaries the optimize performance of the
analytical protocol for the determination of the bromophenol
conjugates. The MDLs for all the conjugates were ≤13 pg
mL−1. This level of detection sensitivity is considered adequate
for environmental analysis purposes. In fact, even lower MDLs
are achievable by using a larger sample volume of urine. Finally,
robustness of the analytical protocol was assessed by analyzing
the same spiked sample once a week for seven weeks. No
Mobile phase acidity is another important factor in LC-MS
analysis as it has significant influence on analyte ionization in
mass spectrometry.15 In general, capacity factors for the
conjugates decreased with increasing mobile phase pH, as
higher pH reduced the ion-pairing ability of DHAA. The
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sampling, concentrations of the bromophenyl glucuronide and
sulfate conjugates in human urine samples could have dropped
to <30% of their original levels. The 2,4,6-tribromophenyl
glucuronide content fell below the detection limit after storage
at room temperature for five days. Previous studies have
suggested that chemical additives such as NaN3 and form-
aldehyde could prevent the microbial biodegradation of
glucuronide conjugates of testosterone and epitestosterone.17
Controlling storage temperature has also been used extensively
to ensure long-term stability of samples and analytes. In this
study, we compared the effectiveness of the above three
approaches to preserve the bromophenyl glucuronide and
sulfate conjugates in human urine samples. As shown in Figure
2, low-temperature storage (at <−20 °C) is the most effective
way to preserve these conjugates, while the use of chemical
preservatives does not slow down their degradation at all. In all
subsequent experiments, urine samples were stored at −80 °C,
within 15 min after sampling, until being analyzed.
Determination of Urinary Bromophenol Conjugates
in Humans. To assess the applicability of the analytical
protocol and to verify the occurrence of bromophenyl
glucuronide and sulfate conjugates in human, we have collected
and analyzed urines from 20 healthy voluntary donors (10
males and 10 females), selected randomly in Hong Kong SAR,
China. Hong Kong is a Special Administrative Region, with an
area of 1104 km2 and a population of seven million, located in
the southern coast of the Pearl River Delta, one of the most
Table 2. Performance of the SPE LC-MS/MS Analytical
Protocol for the Determination of the Bromophenyl
Glucuronide and Sulfate Conjugates in Artificial Urine
Matrix
relative
recovery
(%)
MDL (pg
bromophenol conjugate
repeatability (%)
mL−1
12
)
2,4-dibromophenyl
glucuronide
8.5
93.4
2,4,6-tribromophenyl
glucuronide
7.7
100.5
13
2,4-dibromophenyl sulfate
7.8
7.3
92.2
74.0
13
10
2,4,6-tribromophenyl
sulfate
variation in the relative retention time (RRT) of the conjugates
whatsoever was observed. Fluctuations in the corresponding
chromatographic peak areas of the conjugates were within 5%.
Stability of the Bromophenyl Glucuronide and
Sulfate Conjugates in Human Urine. One of the criteria
for molecular markers to be useful for population exposure
assessment is their stability in the sample matrices. If a marker
can be further chemically or biochemically transformed or
degraded after sampling, it has to be effectively preserved.
Figure 2 shows the stability of spiked bromophenyl glucuronide
and sulfate conjugates in human urine. Without any sample
preservation, levels of the conjugates drop significantly (for
>50%) within the initial week, and then, the rate of degradation
gradually plateaus off. Nevertheless, within the first month after
Figure 2. Effects of various chemical preservatives and storage temperature on the stability of the bromophenol conjugates in human urine. Spike
level of the conjugates adopted was 300 ng mL−1.
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Figure 3. Typical HPLC-MS/MS chromatograms of human urine sample monitored at the corresponding MRM transitions of selected
bromophenol conjugates: (a) 2,4-dibromophenyl glucuronide; (b) 2,4-dibromophenyl sulfate; (c) 2,4,6-tribromophenyl glucuronide; and (d) 2,4,6-
tribromophenyl sulfate.
a
Table 3. Mean Concentrations ( 1 SD) of Bromphenol Conjugates Detected in Human Urine Samples, μg g−1 Creatinine
sample
2,4-dibromophenyl glucuronide
2,4,6-tribromophenyl glucuronide
2,4-dibromophenyl sulfate
2,4,6-tribromophenyl sulfate
sample 1 (male 1)
sample 2 (female 1)
sample 3 (female 2)
sample 4 (male 2)
sample 5 (female 3)
sample 6 (female 4)
sample 7 (female 5)
sample 8 (male 3)
sample 9 (male 4)
sample 10 (female 6)
sample 11 (male 5)
sample 12 (female 7)
sample 13 (male 6)
sample 14 (male 7)
sample 15 (male 8)
sample 16 (female 8)
sample 17 (male 9)
sample 18 (male 10)
sample 19 (female 9)
sample 20 (female 10)
mean SD
N.D.
0.22
0.27
0.07
0.06
N.D.
4.80
N.D.
0.02
N.D.
0.05
0.29
0.12
0.08
0.01
0.03
0.91
2.96
0.13
0.18
0.63
0.41
0.13
0.15
0.08
0.09
N.D.
N.D.
0.10
0.01
0.16
0.18
0.01
N.D.
0.09
6.85
0.27
0.26
N.D.
0.29
N.D.
0.61
N.D.
2.93
3.52
9.14
0.52
0.50
N.D.
N.D.
0.08
0.24
0.05
0.05
3.17
0.15
0.15
0.86
0.08
N.D.
0.25
0.87
1.29
0.22
0.10
0.41
0.04
0.03
0.49
4.32
0.31
0.87
2.73
10.14
0.06
0.19
0.17
0.23
N.D.
0.13 0.14
0.06
0.50 0.85
2.45 3.15
0.17 0.23
a
N.D. = not detected.
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industrialized regions, of China. Previous surveys have already
demonstrated a relatively high level of PBDE contamination of
the coastal marine environment of Hong Kong.18 The local
population is also suspected to have a high exposed to PBDEs.
In this study, samples obtained were immediately frozen at −80
°C and were analyzed within one week after sampling. Figure 3
shows typical LC-MS chromatograms of urine samples
monitored at the MRM transitions specific for the four
bromophenol conjugates. All the peaks observed on the same
chromatogram possessed similar MRM transitions to the
selected bromophenol conjugates. They were originated most
probably from isomers of the selected conjugate with bromine
and glucuronide/sulfate substitutions at different positions on
the aromatic ring. The method of standard addition was
adopted to identify and directly quantify levels of the
bromophenyl glucuronide and sulfate conjugates in the
samples. These levels of conjugates were then normalized by
the corresponding levels of creatinine in the urine samples. Our
results are tabulated in Table 3. In all the tested samples, one or
more of the bromophenol conjugates were detected, with the
concentration ranging 0.13−2.45 μg g−1 creatinine. Among
these four conjugates, the level of 2,4,6-tribromophenyl
glucuronide was the highest in the human urine samples. In
general, levels of the glucuronide conjugates were higher than
those of the sulfate conjugates. These results confirmed the
occurrence of the potential phase II metabolites of PBDEs in
human urine, which in turn, suggested considerable population
exposure to the fire retardants in Hong Kong and probably the
southern China region.
ACKNOWLEDGMENTS
■
This work is support by the GRF grant CityU 160510 from the
Research Grants Council of the Hong Kong Special
Administrative Region, China.
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ASSOCIATED CONTENT
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* Supporting Information
Additional information as noted in text. This material is
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AUTHOR INFORMATION
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Corresponding Author
*Phone: (852)-3442-7329; fax: (852)-3442-0522; e-mail:
bhmhwlam@cityu.edu.hk.
Present Address
#Dr. Yi Wan Laboratory for Earth Surface Processes, College of
Urban and Environmental Sciences, Peking University, Beijing,
China.
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Notes
The authors declare no competing financial interest.
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