Chemosphere 133 (2015) 6–12

Contents lists available at ScienceDirect

Chemosphere

journal homepage: www.elsevier .com/locate /chemosphere

Urinary bromophenol glucuronide and sulfate conjugates: Potentialhuman exposure molecular markers for polybrominated diphenyl ethers

http://dx.doi.org/10.1016/j.chemosphere.2015.03.0030045-6535/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors at: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong Special Administrative Reg+852 3442 6888; fax: +852 3442 7406 (M.B. Murphy). Department of Biology & Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong KonAdministrative Region. Tel.: +852 3442 7329; fax: +852 3442 0522 (M.H.-W. Lam).

E-mail addresses: mbmurphy@cityu.edu.hk (M.B. Murphy), bhmhwlam@cityu.edu.hk (M.H.-W. Lam).1 Present address: Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing, People’s Republic of China.

Ka-Lok Ho a, Man-Shan Yau a, Margaret B. Murphy a,⇑, Yi Wan b,1, Bonnie M.-W. Fong c,d, Sidney Tam c,John P. Giesy a,b,e,f,g,h, Kelvin S.-Y. Leung d, Michael H.-W. Lam a,⇑a State Key Laboratory for Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong Special Administrative Regionb Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Canadac Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong Special Administrative Regiond Department of Chemistry, Hong Kong Baptist University, Hong Kong Special Administrative Regione Department of Zoology and Center for Integrative Toxicology, Michigan State University, USAf School of Biological Sciences, The University of Hong Kong, Hong Kong Special Administrative Regiong Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USAh State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China

h i g h l i g h t s

� Parallel blood and urine samples werecollected from 100 donors in HongKong.� Levels of selected BP glucuronide and

sulfate conjugates in urine weredetermined.� Their levels were found to correlate

well with that of PBDEs in bloodplasma.� Our results suggest that BP

conjugates can be useful markers forPBDE exposure.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 October 2014Received in revised form 18 February 2015Accepted 1 March 2015Available online 24 March 2015

Handling Editor: Andreas Sjodin

Keywords:BromophenolsPolybrominated diphenyl ethersMetabolitesExposure molecular markers

a b s t r a c t

One possible source of urinary bromophenol (BP) glucuronide and sulfate conjugates in mammalian ani-mal models and humans is polybromodiphenyl ethers (PBDEs), a group of additive flame-retardantsfound ubiquitously in the environment. In order to study the correlation between levels of PBDEs inhuman blood plasma and those of the corresponding BP-conjugates in human urine, concentrations of17 BDE congeners, 22 OH-BDE and 13 MeO-BDE metabolites, and 3 BPs in plasma collected from 100voluntary donors in Hong Kong were measured by gas chromatograph tandem mass spectrometry(GC–MS). Geometric mean concentration of RPBDEs, ROH-BDEs, RMeO-BDEs and RBPs in human plasmawere 4.45 ng g�1 lw, 1.88 ng g�1 lw, 0.42 ng g�1 lw and 1.59 ng g�1 lw respectively. Concentrations of glu-curonide and sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP) inpaired samples of urine were determined by liquid chromatography tandem triple quadrupole massspectrometry (LC–MS/MS). BP-conjugates were found in all of the parallel urine samples, in the rangeof 0.08–106.49 lg g�1-creatinine. Correlations among plasma concentrations of RPBDEs/ROH-BDEs/

ion. Tel.:g Special

K.-L. Ho et al. / Chemosphere 133 (2015) 6–12 7

Human blood plasmaHuman urine

RMeO-BDEs/RBPs and BP-conjugates in urine were evaluated by multivariate regression and Pearsonproduct correlation analyses. These urinary BP-conjugates were positively correlated with RPBDEs inblood plasma, but were either not or negatively correlated with other organobromine compounds inblood plasma. Stronger correlations (Pearson’s r as great as 0.881) were observed between concentrationsof BDE congeners having the same number and pattern of bromine substitution on their phenyl rings inblood plasma and their corresponding BP-conjugates in urine.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Polybrominated diphenyl ethers (PBDEs), a class of brominatedflame retardants (BFRs), have aroused considerable public concernbecause of their resistance to environmental degradation, espe-cially for lower brominated congeners, their tendency tobioaccumulate and potential adverse effects on health of humans(de Boer et al., 2000; Hooper and McDonald, 2000; Alaee et al.,2003; Guvenius et al., 2003; Henrik and Birger, 2010). In 2009,the Penta- and Octa-BDE commercial mixtures were listed as per-sistent organic pollutants (POPs) under the Stockholm Convention(Eljarrat and Barceló, 2011). Despite international efforts on therestriction of their production and usage, PBDEs are likely toremain in the global ecosystem for a considerable period of timebecause of their slow rate of degradation for lower brominatedcongeners, and the fact that large amounts of manufactured goodscontaining PBDEs are still in use (Harrad et al., 2006; Betts, 2008).Thus, the continuous monitoring of accumulation of PBDEs inhumans is still important for the accurate assessment of their riskto public health at national and international levels.

The most frequently adopted approach to monitor human expo-sure to PBDEs is the direct quantification of selected BDE congenersand their hydroxylated (OH-BDEs) and methoxylated (MeO-BDEs)species in blood/serum (Athanasiadou et al., 2008; Turyk et al.,2008; Roosens et al., 2009; Wang et al., 2012), and human breastmilk (Sudaryanto et al., 2008; Schuhmacher et al., 2009, 2013;Toms et al., 2009; Shi et al., 2013). Other human tissues such ashair, kidney, lung, liver and adipose tissues have also been used(Covaci et al., 2008; Zhao et al., 2008, 2009; Zheng et al., 2014).However, collecting human tissue samples from people for chemi-cal/biochemical analysis and risk assessment is an intrusive opera-tion and difficult to achieve in large-scale population-wide ornational surveys. While sampling of human hair and breast milkcan be considered non-intrusive processes, they face other lim-itations, such as the ease of exogenous contamination of hair sam-ples (Morris et al., 2012; Barbosa et al., 2013) and the gender andage distribution restrictions of sampling breast milk (Landriganet al., 2002). Alternatively, sampling of human urine is simple,quick and non-intrusive, making it much easier to obtain urinesamples from a large number of voluntary donors within a commu-nity for large-scale surveys.

Occurrence of metabolites of selected BDE congeners in urine ofmammalian animal models has already been well established inseveral toxicokinetic studies (Hakk and Letcher, 2003; Chen et al.,2006; Sanders et al., 2006). These metabolites are mainly glu-curonide and sulfate conjugates of dibromophenols (DBPs) and tri-bromophenols (TBPs), probably because of their lower molecularweight (relative to their parent BDE congeners) that facilitatestheir renal removal. Our research team has previously reportedthe synthesis, purification, and characterization of glucuronideand sulfate conjugates of bromophenols (BP) and has developedan analytical protocol for their determination in human urine(Ho et al., 2012). A preliminary survey on 20 voluntary donorsrevealed the presence of at least one of these BP-conjugates intheir urine. In this study, we examined the correlation between

concentrations of PBDEs/OH-BDEs/MeO-BDEs/BPs and in bloodplasma and BP-glucuronide and -sulfate conjugates in urine ofhumans. A total of 100 matched samples of plasma and urine werecollected from volunteer donors in Hong Kong, China. The objectiveof this work was to evaluate whether glucuronide and sulfate con-jugates of BPs in human urine are suitable molecular markers forthe assessment of population exposure to PBDEs.

2. Experimental

2.1. Safety precautions

All necessary precautions were taken during the handling ofsamples of blood and urine. Double latex gloves, facemasks andeye-protection goggles were worn at all times during handling,spiking and transfer of samples from humans. All spent samplesof urine were collected after analysis in separate capped containerswith proper clinical waste labels. Both spent samples and usedpersonal protection items were treated as clinical waste andwere collected and disposed of in accordance with the ‘‘Code ofPractice for the Management of Clinical Waste’’ issued by theEnvironmental Protection Department of the Hong Kong SARGovernment.

2.2. Sample collection

All studies that involved human tissues and body fluids wereconducted in accordance with guidelines of the Research EthicsCommittee of City University of Hong Kong after proper approvalswere given by the Committee. Parallel samples of human plasmaand urine (n = 100; 50 from male and 50 from female donors) werecollected from voluntary donors during March to July 2010 byregistered doctors and nurses at Queen Mary Hospital, HongKong. Besides their gender and age, no other personal informationof those voluntary donors was collected. The age range of thevolunteers was from 16 to 93 years of age (mean ± SD:54.9 ± 21.9 years). These donors were subdivided into differentage groups for comparison: age 16–25 (n = 11); age 26–35(n = 15); age 36–45 (n = 12); age 46–55 (n = 14); age 56–65(n = 15); age 66–80 (n = 13) and age > 80 (n = 20).

Samples of whole blood were collected using the standard phle-botomy technique in vacutainer tubes containing sodium heparinanticoagulant (Vacuette, Greiner bio-one, GmbH, Austria). Wholeblood was then centrifuged at 1500g for 25 min. Plasma wasremoved from the top of the tube. Morning-first urine sampleswere collected in 100 mL sterilized glass bottles and stored at�80 �C within 15 min of sampling until analysis. Urine from eachdonor was subdivided into three replicate samples before low-temperature storage. All samples were carefully labeled anddocumented. Upon analysis, samples were thawed, and 10 mL ofeach sample was retained for creatinine content determination(D’Haese et al., 1985). Creatinine determination was conductedby a kinetic colorimetric assay based on the modified Jaffe methodusing the Roche Modular System (Roche Diagnostics, IN, USA), withan analytical range between 360 and 57500 mmol L�1.

8 K.-L. Ho et al. / Chemosphere 133 (2015) 6–12

2.3. Sample extraction and preparation

Synthesis, purification and characterization of the glucuronideand sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP), and the analytical protocols for thequantification of PBDEs, OH-BDEs, MeO-BDEs and BPs in humanplasma and the BP-conjugates in human urine have been describedin our previous publications (Hovander et al., 2000; Qiu et al.,2009; Ho et al., 2012). Details on materials, instrumentation,extraction methods, and QA/QC protocols of this study are givenin the Supporting Information.

2.4. Analysis of data

All statistical analyses were performed using SPSS 16 (SPSS Inc.,Chicago, IL), Prism 2.01 (GraphPad Software, Inc.) and Sigmastat3.5 (Sigmastat, Jandel Scientific, CA). Normality of data waschecked by the Klomogorov-Smirnov test. Logarithm, natural-loga-rithm, arcsine, square root, reciprocal square root or cubic roottransformations were used whenever fit to obtain normallydistributed data sets for parametric statistical testing. Student’st-test was used to compare concentrations of PBDEs, MeO-BDEs,OH-BDEs and BPs, in human plasma samples, and BP-conjugates,in human urine samples, between male and female donors. If datawere not normally distributed, a non-parametric Mann–WhitneyRank Sum test was used for the comparison. One-way ANOVA(parametric) or ANOVA on Ranks (non-parametric) tests were usedto compare concentrations of the target brominated species inplasma and urine among different age categories. Multivariatelinear regression and Pearson product moment correlation analysiswere used to examine the influence of concentrations of PBDEs,MeO-BDEs, OH-BDEs, BPs in blood plasma on concentrations ofBP-conjugates in urine. A P < 0.05 was considered statisticallysignificant for all statistical measurements.

3. Results and discussion

3.1. Concentrations of PBDEs, OMe-PBDEs, OH-PBDEs and BPs inhuman plasma

Concentrations of RPBDEs, RMeO-PBDEs, ROH-PBDEs andRBPs in plasma, normalized by the plasma lipid weight (lw), aresummarized (Table 1). PBDEs were detected in all of the 100human plasma samples, with RPBDEs ranging from 0.01 to18.2 ng g�1 lw. These concentrations of RPBDEs are quitecomparable to those reported in previous studies in other Asiancountries (Tan et al., 2008; Zhu et al., 2009; Uemura et al., 2010;Kim et al., 2012), New Zealand (Harrad and Porter, 2007) and someEuropean countries (Thomas et al., 2006; Gómara et al., 2007;Antignac et al., 2009; Kalantzi et al., 2011), but are lesser thanthose in the population of Northern America (Schecter et al.,2005; Sandanger et al., 2007; Lunder et al., 2010) and those wholive near sites where BFR are produced or used in large quantities(Jin et al., 2009), or e-waste disposal/dismantling areas (Biet al., 2007) (Fig. S1, Supporting Information). Similar to manyprevious studies on the occurrence of PBDEs in human plasma,BDE-47, �99 and �209 were the most abundant BDE congenersdetected in this study, with geometric mean concentrations of0.55 ng g�1 lw; (95% confidence interval: 0.39–0.79 ng g�1 lw),0.33 ng g�1 lw (95% confidence interval: 0.23–0.48 ng g�1 lw), and0.47 ng g�1 lw (95% confidence interval: 0.35–0.62 ng g�1 lw),respectively. There was no significant difference between concen-trations of PBDEs in plasma among all age categories (p = 0.736)or gender (p = 0.143). BDE-47 and �99 were the two most fre-quently detected congeners with a detection frequency of >90%,

while <20% of the plasma samples contained BDE-209. This fre-quency of occurrence of BDE-209 is much less compared to surveyscarried out in the US, European countries and Japan. A recent studyby Wang et al. (2011) reported greater concentrations and occur-rence frequencies of BDE-47 and �99 in seafood from fish marketsin Hong Kong. This suggests that food consumption rather thaninhalation of indoor dust, an environmental matrix with greaterconcentrations of BDE-209, might be a more significant exposureroute to the residents of Hong Kong.

OH-BDEs and BPs were detected in human blood at concentra-tions similar to those of PBDEs. This is consistent with findings ofprevious studies (Athanasiadou et al., 2008; Roosens et al., 2009;Qiu et al., 2009; Wan et al., 2010). 6-OH-BDE-47 and 50-OH-BDE-99 were the two most abundant OH-BDE congeners in the humanblood plasma samples, with geometric mean concentrations of0.32 ng g�1 lw; (95% confidence interval: 0.23–0.45 ng g�1 lw)and 0.06 ng g�1 lw (95% confidence interval: 0.03–0.11 ng g�1 lw),respectively. Three different BPs, namely 2,4-DBP, 2,4,5-tribro-mophenol (2,4,5-TBP) and 2,4,6-TBP, were detected in 80% of theplasma samples. The geometric mean concentration of RBPs was1.59 ng g�1 lw (95% confidence interval: 1.34–1.89 ng g�1 lw). Theoccurrence of these three BPs in mouse plasma after exposure tothe commercial Penta-BDE mixture DE-71 has been reported byQiu et al. (2007). In an in vitro study of the metabolism of BDE-99 by human hepatocytes, Stapleton et al. (2009) also determined2,4,5-TBP in the cell extracts, which was deemed to be generatedby the catabolic cleavage of the diphenyl ether linkage of theBDE congener. These studies suggest that the diphenyl ether cleav-age of PBDEs constitutes one of the sources of BPs (including 2,4-DBP and 2,4,6-TBP) in blood plasma. Other sources may includefood consumption and exposure to other BFRs. 2,4-DBP and2,4,6-TBP have been detected in fresh fish samples commonly con-sumed in Hong Kong (Chung et al., 2003). 2,4,6-TBP has been usedas a flame retardant, and population may be exposed to it in asimilar way as to PBDEs. There was no significant difference inthe concentrations of OH-BDEs and BPs in human plasma betweenmale and female donors (p = 0.749 for OH-BDEs, p = 0.165 for BPs).There were also no significant differences in OH-BDE and BPcontent in human plasma samples among all the age groups(p = 0.449 for OH-BDEs, p = 0.571 for BPs).

MeO-BDEs were also found in human plasma samples. The geo-metric mean concentration of RMeO-BDEs was 0.42 ng g�1 lw (95%confidence interval: 0.31–0.56 ng g�1 lw). This concentration iscomparable to that revealed in a previous study carried out inHong Kong (Wang et al., 2012). 6-MeO-BDE-47 and 4-MeO-BDE-17 were the two most abundant MeO-BDE congeners, with meanconcentrations in human plasma of 0.88 and 0.73 ng g�1 lw,respectively. This pattern of occurrence of MeO-BDEs in humanplasma samples is different from that observed in the US (Qiuet al., 2009), perhaps because some abundant MeO-BDE congeners(6-MeO-BDE-47, 4-MeO-BDE-17, 2-MeO-BDE-28) were notincluded in the previous study. There were no significant differ-ences among concentrations of MeO-BDEs in human plasmaamong all the age groups (p = 0.210), and no statistically significantdifferences in the concentrations of MeO-BDEs were foundbetween male and female donors (p = 0.702).

3.2. Concentrations of BP-glucuronide and -sulfate conjugates inhuman urine samples

To the best of our knowledge, BP-glucuronide and –sulfate con-jugates are not commercially available. Authentic standards of theglucuronide and sulfate conjugates of 2,4-DBP and 2,4,6-TBP forLC–MS/MS determination in this work were obtained via chemicalsynthesis and liquid chromatographic purification. On the otherhand, the presence of conjugates of 2,4,5-TBP in urine samples

Table 1Concentrations (ng g�1 lw) of RPBDEs, RMeO-BDEs, ROH-BDEs, and RBPs in human plasma samples (n = 100) from Hong Kong, China.

Congeners Human plasma samples

Total (n = 100) Male (n = 50) Female (n = 50)

GMa (95% CI)b Min–max % of detection GMa (95% CI)b Min–max % of detection GMa (95% CI)b Min–max % of detection

RPBDEs 4.45 (3.66–5.41) 0.01–18.20 100 5.13 (4.10–6.41) 0.12–18.20 100 3.82 (2.77–5.28) 0.01–14.18 100RMeO-BDEs 0.42 (0.31–0.56) N.D.–6.87 89 0.44 (0.30–0.65) N.D.–5.15 90 0.40 0.25–0.63 N.D.–6.87 90ROH-BDEs 1.88 (1.53–2.29) N.D.–8.88 83 1.82 (1.40–2.36) N.D.–5.19 86 1.94 (1.43–2.64) N.D.–8.88 80RBPs 1.59 (1.34–1.89) N.D.–7.18 84 1.79 (1.43–2.25) N.D.–5.16 92 1.40 (1.08–3.00) N.D.–7.18 78

N.D.: not detected.a GM: geometric mean.b CI: confidence interval.

K.-L. Ho et al. / Chemosphere 133 (2015) 6–12 9

was not determined in this study because of the unavailability of2,4,5-TBP starting material for the synthesis of its conjugates.

One or more of the 2,4-DBP- and 2,4,6-TBP-glucuronide and –sulfate conjugates were detected in all of the urine samples, withRBP-conjugates ranging from 0.08 to 106.49 lg g�1-creatinine(Table 2). Conjugates of 2,4,6-TBP were the most frequentlydetected, while the frequency of detection of 2,4-DBP conjugateswas around 70%. BP-glucuronides were 5- to 10-fold moreabundant than their corresponding BP-sulfates. There were no sta-tistically significant differences in concentrations of BP-conjugatesin urine among age groups (p = 0.378 for 2,4-DBP-sulfate; p = 0.558for 2,4,6-TBP-sulfate; p = 0.25 for 2,4-DBP-glucuronide; and p= 0.976 for 2,4,6-TBP-glucuronide). Alternatively, a statisticallysignificant difference was observed in the urine concentrationsof 2,4-DBP-glucuronide between male and female donors(p = 0.038), with concentrations of 2,4-DBP-glucuronide found tobe greater in men. A previous study on human urinary bisphenolA (BPA) in Korea also found a similar phenomenon where concen-trations of BPA-glucuronides in men were greater than those inwomen.

3.3. Correlations between BP-glucuronide and -sulfate conjugates andRPBDEs, ROMe-PBDEs, ROH-PBDEs and RBPs

Multivariate linear regression analysis was employed to explorethe relationship among different forms of BDEs/BPs in bloodplasma and BP-conjugates in urine. The standardized regressioncoefficient b was used to quantify the relationship between BP-conjugates and the various determinants. The first regressionmodel was built using the natural log-transformed summation ofconcentrations of PBDEs, OH-BDEs, MeO-BDEs and BPs, i.e.lnRPBDEs, lnROH-PBDEs, lnRMeO-PBDEs and lnRBPs, in plasmasamples as independent variables, and the sum of all four BP-glucuronide and -sulfate conjugates, i.e. lnRBP-conjugates, in urinesamples as the dependent variable (Table 3). The coefficient ofdetermination (R2) of the regression model established wasonly 0.223, and the model was significantly affected by bothlnRPBDEs and lnRBPs, with their standardized regression coeffi-cients (b) being 0.716 and �0.585 respectively (p < 0.001 forlnRPBDEs and p = 0.013 for lnRBPs).

While there might not be simple relation between total concen-trations of PBDEs in human blood and that of total BP-conjugates inhuman urine, we explored correlations among structurally-relatedorganobromine species in blood and in urine. Based on numerousprevious in vivo studies on laboratory mice and in vitro studieson human cells, catabolic cleavage of PBDEs at the ether linkagecan produce water-soluble glucuronide and sulfate BP-conjugateswith the number and relative position of the bromine-substituentsresembling that of the corresponding parent BDE congeners.Thus, correlations between concentrations of PBDEs/MeO-BDEs/OH-BDEs/BP with 2,4-dibromo and 2,4,6-tribromo substitution in

the blood plasma samples and the glucuronide and sulfate conju-gates of 2,4-DBP/2,4,6-TBP in urine were investigated (Tables 4aand 4b). The R2 values increased to 0.744 (lnR2,4-DBP-conjugates)and 0.707 (lnR2,4,6-TBP-conjugates), which suggests that therefined regression models better explained the variability of thedependents. They also gave better standardized coefficients (b):4.34 for lnR2,4-dibromo-BDEs and 3.23 for lnR2,4,6-tribromo-BDEs, revealing much stronger relationships between R2,4-di-bromo-BDEs/R2,4,6-tribromo-BDEs in human plasma and theircorresponding BP-conjugates in urine. All the variance inflationfactors (VIFs) of the independent variables were <2, indicatingthe absence of multicollinearity in the regression models. Thus,our results revealed strong relationships between BP-glucuronideand -sulfate conjugates in human urine and R2,4-dibromo-BDEsand R2,4,6-tribromo-BDEs in human blood plasma. On the otherhand, their relationships with RMeO-BDEs, ROH-BDEs, or RBPsin blood plasma were of less significance. These results suggestthat urinary BP-conjugates in human originated from exposure toPBDEs rather than MeO-BDEs, OH-BDEs or BPs.

Pearson product moment correlation was used to evaluate thecorrelations between natural-log-transformed RBP-conjugatesand RPBDEs, RMeO-BDEs, ROH-BDEs and RBPs. Strong relation-ships were observed between lnR2,4-dibromo-BDEs in humanblood vs lnR2,4-DBP-conjugates in human urine and lnR2,4,6-tri-bromo-BDEs in human blood vs lnR2,4,6-TBP-conjugates in humanurine (Pearson’s r = 0.881 and 0.823, respectively; Tables 5a and5b). These results demonstrate the correlation between urinaryconcentration of BP-glucuronide and -sulfate conjugates and thelevel of PBDEs in blood plasma (Figs. 1 and 2). On the other hand,concentrations of urinary BP-glucuronide and -sulfate conjugatesdo not correlate well with those of lnRMeO-BDEs, lnROH-BDEsand lnRBPs in blood plasma. This suggests that the glucuronideand sulfate conjugates of BPs in human urine may be useful asmolecular markers for human exposure to PBDEs. It is arguablethat these BP-conjugates may only be able to reflect human expo-sure to PentaBDEs, but not OctaBDEs and DecaBDE. The apparenthalf-life of OctaBDEs and DecaBDE in human serum are less than91 days (Thuresson et al., 2006), which is much shorter than thatof 2 years for PentaBDEs (Geyer et al., 2004). Thus, even thoughthe bromophenol conjugates may be more related to PentaBDEs,they can still be useful in revealing long term exposure to PBDEs.

Owing to the unavailability of 2,4,5-TBP, correlation betweenR2,4,5-tribromo-BDEs in human blood and R2,4,5-TBP-conjugatesin human urine was not explored in this study. Also, the presenceof BDE-glucuronide and -sulfate conjugates in human urine wasnot determined because of the unsufficient quantity of thecorresponding OH-BDEs available for the chemical synthesis ofthe metabolites. Their aptness as molecular markers for populationexposure to PBDEs will have to be addressed in the future. Infantsand children were not included in this study because of ethicalconsiderations associated with the collection of their blood and

Table 2Concentrations (lg g�1 creatinine) of BP-glucuronide and -sulfate conjugates in human urine samples (n = 100) from Hong Kong, China.

Compound Samples of human urine

Total (n = 100) Male (n = 50) Female (n = 50)

GMa (95% CI)b Min–max % of detection GMa (95% CI)b Min–max % of detection GMa (95% CI)b Min–max % of detection

2,4-DBP glucuronide 0.32 (0.23–0.44) N.D.–23.81 71 0.42 0.27–0.64 0.01–23.81 76 0.21 (0.13–0.34) N.D.–7.52 662,4-DBP sulfate 0.11 (0.08–0.14) N.D.–2.08 86 0.10 (0.07–0.15) N.D.–2.08 88 0.11 (0.08–0.17) N.D.–2.08 842,4,6-TBP glucuronide 0.87 (0.58–1.30) N.D.–102.21 68 0.98 (0.51–1.73) N.D.–102.21 54 0.80 (0.47–1.38) N.D.–44.08 822,4,6-TBP sulfate 0.10 (0.08–0.13) N.D.–2.93 94 0.10 (0.07–0.15) N.D.–2.93 94 0.10 (0.07–0.14) N.D.–2.93 94

N.D.: not detected.a GM: geometric mean.b CI: confidence interval.

Table 3Significant independent variables of urinary concentrations of BP-conjugates revealedby multivariate linear regression analysis.

Model summaryR2 0.223

Independent variable ba Pb VIFc

lnRPBDEs 0.716 <0.001 1.056lnRMeO-BDEs 0.063 0.632 1.059lnROH-BDEs �0.23 0.239 1.041lnRBPs �0.585 0.013 1.056

Dependent value was urinary lnR[BP-conjugates].a b: Standardized regression coefficients, slope from the analysis of the model

regression of lnRBP-conjugates versus independent variables.b P-value for the term in the multiple linear regression, P < 0.05, statistically

significant.c VIF: variance inflation factor.

Table 4bSignificant independent variables of urinary concentrations of 2,4,6-TBP-conjugatesrevealed by multivariate linear regression analysis.

Model summaryR2 0.707

Independent variable ba Pb VIFc

lnR2,4,6-Tribromo-BDEs 3.226 <0.001 1.005lnR2,4,6-TBP 0.135 0.428 1005

Dependent value was urinary lnR[2,4-DBP-conjugates].a b: Standardized regression coefficients, slope from the analysis of the model

regression of lnR2,4-DBP-conjugates versus independent variables.b P-value for the term in the multiple linear regression, P < 0.05, statistically

significant;c VIF: variance inflation factor.

Table 5aCorrelation coefficients between urinary 2,4-DBP-conjugates and the various PBDEs/MeO-BDEs/OH-BDEs/BPs in human plasma.

Total (n = 100) Male (n = 50) Female(n = 50)

r P r P r P

lnR2,4-Dibromo-BDEs 0.881 <0.05 0.871 <0.05 0.884 <0.05lnR2,4-Dibromo-MeO-BDEs �0.080 0.449 �0.047 0.77 �0.100 0.526lnR2,4-Dibromo-OH-BDEs �0.095 0.452 0.053 0.743 �0.200 0.221lnR2,4-DBP �0.157 0.187 �0.223 0.184 �0.102 0.561

Analysis of urinary BP conjugates were conducted after natural-log transformation.

Table 5bCorrelation coefficients between urinary 2,4,6-TBP-conjugates and the variouscongeners PBDEs and BPs in human plasma.

10 K.-L. Ho et al. / Chemosphere 133 (2015) 6–12

urine samples. However, previous studies have revealed aninverted age-dependent accumulation of PBDEs, perhaps becauseof the dietary preferences, greater frequency of hand-to-monthactivities and greater metabolic rate of children (Fischer et al.,2006; Lunder et al., 2010; Eskenazi et al., 2011; Gari and Grimalt,2013). Thus, it is worthy to further explore the correlation betweenplasma PBDEs and urinary BP-conjugates in children. Another areathat needs further study is potential ethnic differences in the effi-cacy of PBDE metabolism. Previous studies have shown thatgreater glucuronidation of morphine occurred in Chinese peoplecompared to Caucasians (Zhou et al., 1993). Alternatively, ethnicChinese were less able to metabolize codeine by glucuronidation(Yue et al., 1989, 1991). Results from Goldzieher and coworkerhave shown that the pattern of glucuronide conjugation as wellas oxidative metabolism of estrogens (ethinyl estradiol) differedamong Nigerian, Sri Lankan and American populations (Williamsand Goldzieher, 1980; Goldzieher and Brody, 1990). Another studyof the excretion of N-glucuronide conjugates of nicotine and

Table 4aSignificant independent variables of urinary concentrations of 2,4-DBP-conjugatesrevealed by multivariate linear regression analysis.

Model summaryR2 0.744

Independent variable ba Pb VIFc

lnR2,4-Dibromo-BDEs 4.34 <0.001 1.089lnR2,4-Dibromo-MeO-BDEs 0.0138 0.834 1.071lnR2,4-Dibromo-OH-BDEs 0.0682 0.547 1.001lnR2,4-DBP 0.0345 0.617 1.059

Dependent value was urinary lnR[2,4-DBP-conjugates].a b: Standardized regression coefficients, slope from the analysis of the model

regression of lnR2,4-DBP-conjugates versus independent variables.b P-value for the term in the multiple linear regression, P < 0.05, statistically

significant;c VIF: variance inflation factor.

Total (n = 100) Male (n = 50) Female (n = 50)

r P r P r P

lnR2,4,6-Tribromo-BDEs 0.823 <0.05 0.820 <0.05 0.834 <0.05lnR2,4,6-TBP �0.007 0.907 �0.06 0.699 �0.074 0.655

Analysis of urinary BP conjugates were conducted after natural-log transformation.

cotinine has shown that people with African origins excreted sig-nificantly less glucuronide conjugates than Caucasians (Caraballoet al., 1998; Benowitz et al., 1999). Information on the differencesin the metabolism of POPs among ethnic groups is scarce. Mattersare further complicated by the differences in the compositions ofPBDE mixtures used in different parts of the world, and dietaryhabits of people. Zota and coworkers (Zota et al., 2008) have shownthat PBDE exposure at different regions of the US was different.

Fig. 1. Correlation analyses between the concentration of natural logarithm-transformed R2,4-dibromo-BDEs in human plasma and natural logarithm-transformed urinary R2,4-DBP-conjugates.

Fig. 2. Correlation analyses between the concentration of natural logarithm-transformed R2,4,6-tribromo-BDEs in human plasma and natural logarithm-transformed urinary R2,4,6-TBP-conjugates.

K.-L. Ho et al. / Chemosphere 133 (2015) 6–12 11

Acknowledgements

This work is support by a grant from Research Grants Council ofthe Hong Kong Special Administrative Region, China [Reference No.CityU 9041623]. Prof. Giesy was supported by the program of 2012‘‘Great Concentration Foreign Experts’’ (#GDW20123200120)funded by the State Administration of Foreign Experts Affairs, theP.R. China to Nanjing University and the Einstein ProfessorProgram of the Chinese Academy of Sciences. He was also sup-ported by the Canada Research Chair program, a VisitingDistinguished Professorship in the Department of Biology andChemistry and State Key Laboratory in Marine Pollution, CityUniversity of Hong Kong.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2015.03.003.

References

Alaee, M., Arias, P., Sjödin, A., Bergman, Å., 2003. An overview of commercially usedbrominated flame retardants, their applications, their use patterns in differentcountries/regions and possible modes of release. Environ. Int. 29, 683–689.

Antignac, J.P., Cariou, R., Zalko, D., Berrebi, A., Cravedi, J.P., Maume, D., Marchand, P.,Monteau, F., Riu, A., Andre, F., Le Bizec, B., 2009. Exposure assessment of Frenchwomen and their newborn to brominated flame retardants: Determination oftri- to deca- polybromodiphenylethers (PBDE) in maternal adipose tissue,serum, breast milk and cord serum. Environ. Pollut. 157, 164–173.

Athanasiadou, M., Cuadra, S.N., Marsh, G., Bergman, Å., Jakobsson, K., 2008.Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated

PBDE metabolites in young humans from Managua, Nicaragua. Environ. HealthPerspect. 116, 400–408.

Barbosa, J., Faria, J., Carvalho, F., Pedro, M., Queiros, O., Moreira, R., Dinis-Qliveria,R.J., 2013. Hair as an alternative matrix in bioanalysis. Bioanalysis 5,895–914.

Benowitz, N.L., Perez-Stable, E.J., Fong, I., Modin, G., Herrera, B., Jacob, P., 1999.Ethnic differences in N-glucuronidation of nicotine and cotinine. J. Pharmacol.Exp. Ther. 291, 1196–1203.

Betts, K., 2008. Unwelcome guest: PBDEs in indoor dust. Environ. Health Perspect.116, A203–A208.

Bi, X.H., Thomas, G.O., Jones, K.C., Qu, W.Y., Sheng, G.Y., Martin, F.L., Fu, J., 2007.Exposure of electronics dismantling workers to polybrominated diphenylethers, polychlorinated biphenyls, and organochlorine pesticides in SouthChina. Environ. Sci. Technol. 41, 5647–5683.

Caraballo, R.S., Giovino, G.A., Pechacek, T.F., Mowery, P.D., Richter, P.A., Strauss, W.J.,Sharp, D.J., Eriksen, M.P., Pirkle, J.L., Maurer, K.R., 1998. Racial and ethnicdifferences in serum cotinine levels of cigarette smokers – Third NationalHealth and Nutrition Examination Survey, 1988–1991. J. Am. Med. Assoc. 280,135–159.

Chen, L.J., Lebetkin, E.H., Sanders, J.M., Burka, L.T., 2006. Metabolism and dispositionof 2,20 ,4,40 ,5-pentabromodiphenyl ether (BDE99) following a single or repeatedadministration to rats or mice. Xenobiotica 36, 515–534.

Chung, H.Y., Ma, W.C.J., Kim, J.S., 2003. Seasonal distribution of bromophenols inselected Hong Kong seafood. J. Agric Food Chem. 51, 6752–6760.

Covaci, A., Voorspoels, S., Roosens, L., Jacobs, W., Blust, R., Neels, H., 2008.Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls(PCBs) in human liver and adipose tissue samples from Belgium.Chemosphere 73, 170–175.

de Boer, J., de Boer, K., Boon, J.P., 2000. Polybrominated biphenyls anddiphenylethers. In: Paasivirta, J. (Ed.), The Handbook of EnvironmentalChemistry. Springer-Verlag, Berlin, Germany, pp. 61–95. Vol. 3.

D’Haese, P.C., van der Vyver, F.L., de Wolff, F.A., de Broe, M.E., 1985. Measurement ofaluminum in serum, blood, urine and tissues of chronic hemodialyzed patientsby use of electrothermal atomic absorption spectrometry. Chin. Chem. 31, 24–29.

Eljarrat, E., Barceló, D. (Eds.), 2011. Brominated flame retardants Handbook ofenvironmental chemistry, vol. 16, Springer-Verlag, Berlin Heidelberg.

Eskenazi, B., Fenster, L., Castorina, R., Marks, A.R., Sjödin, A., Rosas, L.G., Holland, N.,Guerra, A.G., Lopez-Carillo, L., Bradman, A., 2011. A comparison of PBDE serumconcentrations in Mexican and Mexican-American children living in California.Environ. Health Perspect. 119, 1442–1448.

Fischer, D., Hooper, K., Athanasiadou, M., Athanassiadis, I., Bergman, Å., 2006.Children show highest levels of polybrominated diphenyl ethers in a Californiafamily of four: a case study. Environ. Health Perspect. 114, 1581–1584.

Gari, M., Grimalt, J.O., 2013. Inverse age-dependent accumulation ofdecabromodiphenyl ether and other PBDEs in serum from a general adultpopulation. Environ. Int. 54, 119–127.

Geyer, H.J., Schramm, K.W., Darnerud, P.O., Aune, M., Feicht, E.A., Fried, K.W.,Henkelmann, B., Lenoir, D., Schmid, P., McDonald, T.A., 2004. Terminalelimination half-lives of the brominated flame retardants TBBPA, HBCD, andlower brominated PBDEs in humans. Organohal. Compd. 66, 3820–3825.

Goldzieher, J.W., Brody, S.A., 1990. Pharmacokinetics of ethinyl estradiol andmestranol. Am. J. Obstet. Gynecol. 163, 2114–2119.

Gómara, B., Herrero, L., Ramos, J.J., Mateo, J.R., Fernández, M.A., García, J.F., González,M.J., 2007. Distribution of polybrominated diphenyl ethers in human umbilical.cord serum, paternal serum, maternal serum, placentas, and breast milk fromMadrid population, Spain. Environ. Sci. Technol. 41, 6961–6968.

Guvenius, D.M., Aronsson, A., Ekman-Ordeberg, G., Bergman, Å., Norén, K., 2003.Human prenatal and postnatal exposure to polybrominated diphenyl ethers,polychlorinated biphenyls, polychlorobiphenylols, and pentachlorophenol.Environ. Health Perspect. 111, 1235–1241.

Hakk, H., Letcher, R.J., 2003. Metabolism in the toxicokinetics and fate ofbrominated flame retardants – a review. Environ. Int. 29, 801–828.

Harrad, S., Porter, L., 2007. Concentrations of polybrominated diphenyl ethers inblood serum from New Zealand. Chemosphere 66, 2019–2023.

Harrad, S., Hazrati, S., Ibarra, C., 2006. Concentrations of polychlorinated biphenylsin indoor air and polybrominated diphenyl ethers in indoor air and dust inBirmingham, United Kingdom: Implications for human exposure. Environ. Sci.Technol. 40, 4633–4638.

Henrik, A., Birger, S., 2010. Developmental neurotoxicity of PBDEs, mechanisms andimplications. In: Neuroscience Research Progress Series. Nova SciencePublishers, N.Y..

Ho, K.L., Murphy, M.B., Wan, Y., Fong, B.M.W., Tam, S., Giesy, J.P., Leung, K.S.Y., Lam,M.H.W., 2012. Synthesis and characterization of bromophenol glucuronide andsulfate conjugates for their direct LC–MS/MS quantification in human urine aspotential exposure markers for polybrominated diphenyl ethers. Anal. Chem.84, 9881–9888.

Hooper, K., McDonald, T.A., 2000. The PBDEs: an emerging environmental challengeand another reason for breast-milk monitoring programs. Environ. HealthPerspect. 108, 387–392.

Hovander, L., Athanasiadou, M., Asplund, L., Jensen, S., Klasson-Wehler, E., 2000.Extraction and cleanup methods for analysis of phenolic and neutalorganohalogens in plasma. J. Anal. Toxicol. 24, 696–703.

Jin, J., Wang, Y., Yang, C., Hu, J., Liu, W., Cui, J., Tang, X., 2009. Human exposure topolybrominated diphenyl ethers at production area, China. Environ. Int. 35,1048–1052.

12 K.-L. Ho et al. / Chemosphere 133 (2015) 6–12

Kalantzi, O.I., Geens, T., Covaci, A., Siskos, P.A., 2011. Distribution of polybrominateddiphenyl ethers (PBDEs) and other persistent organic pollutants in humanserum from Greece. Environ. Int. 37, 349–353.

Kim, J., Kang, J.H., Park, H., Beak, S.Y., Kim, Y.H., Chang, Y.H., 2012. Assessment ofpolybrominated diphenyl ethers (PBDEs) in serum from the Korean generalpopulation. Environ. Pollut. 164, 46–52.

Landrigan, P.J., Sonawane, B., Mattison, D., McCally, M., Garg, A., 2002. Chemicalcontaminants in breast milk and their impacts on children’s health: anoverview. Environ. Health Perspect. 110, A313–A315.

Lunder, S., Hovander, L., Athanassiadis, I., Bergman, Å., 2010. Significantly higherpolybrominated diphenyl ether levels in young US children than in theirmothers. Environ. Sci. Technol. 44, 5256–5262.

Morris, J.S., Spate, V.L., Crane, S.B., Gudino, A.J., 2012. Determination of seleniumstatus using the nail biologic monitor in a canine model. Radioanal. Nucl. Chem.291, 409–414.

Qiu, X., Mercado-Feliciano, M., Bigsby, R.M., Hites, R.A., 2007. Measurement ofpolybrominated diphenyl ethers and metabolites in mouse plasma afterexposure to a commercial pentabromo diphenyl ether mixture. Environ.Health Perspect. 115, 1052–1058.

Qiu, X., Bigsby, R.M., Hites, R.A., 2009. Hydroxylated metabolites of polybrominateddiphenyl ethers in human blood samples from the United States. Environ.Health Perspect. 117, 93–98.

Roosens, L., Abdallah, M.A.E., Harrad, S., Neels, H., Covaci, A., 2009. Factorsinfluencing concentrations of polybrominated diphenyl ethers (PBDEs) instudents from Antwerp, Belgium. Environ. Sci. Technol. 43, 3535–3541.

Sandanger, T.M., Sinotte, M., Dumas, P., Marchand, M., Sandau, C.D., Pereg, D.,Bérubé, S., Brisson, J., Ayotte, P., 2007. Plasma concentrations of selectedorganobromine compounds and polychlorinated biphenyls in postmenopausalwomen of Quebec, Canada. Environ. Health Perspect. 115, 1429–1434.

Sanders, J.M., Chen, L.J., Lebetkin, E.H., Burka, L.T., 2006. Metabolism and dispositionof 2,20 ,4,40-tetrabromodiphenyl ether following administration of single ormultiple doses to rats and mice. Xenobiotica 36, 103–117.

Schecter, A., Päpke, O., Tung, K.C., Jean, J., Robert, H.T., James, D., 2005.Polybrominated diphenyl ether flame retardants in the US population:Current levels, temporal trends, and comparison with dioxins, dibenzofurans,and polychlorinated biphenyls. J. Occup Environ. Med. 2005 (47), 199–211.

Schuhmacher, M., Kiviranta, H., Ruokojärvi, P., Nadal, M., Domingo, J.L., 2009.Concentrations of PCDD/Fs, PCBs and PBDEs in breast milk of women fromCatalonia, Spain: a follow-up study. Environ. Int. 35, 607–633.

Schuhmacher, M., Kivirant, H., Ruokojavi, P., Nadal, M., Domingo, J.L., 2013. Levels ofPCDD/Fs, PCBs and PBDEs in breast milk of women living in the vicinity of ahazardous waste incinerator: assessment of the temporal trend. Chemosphere93, 1533–1540.

Shi, Z.X., Jiao, Y., Hu, Y., Sun, Z.W., Zhou, X.Q., Feng, J.F., Li, J.G., Wu, Y.N., 2013. Levelsof tetrabromobisphenol A, hexabromocyclododecanes and polybrominateddiphenyl ethers in human milk from the general population in Beijing, China.Sci. Total Environ. 452, 10–18.

Stapleton, H.M., Kelly, S.M., Pei, R., Letcher, R.J., Gunsch, C., 2009. Metabolism ofPolybrominated Diphenyl Ethers (PBDEs) by Human Hepatocytes in vitro.Environ. Health Perspect. 117, 197–202.

Sudaryanto, A., Kajiwara, N., Takahashi, S., Muawanah, Tanabe, S., 2008.Geographical distribution and accumulation features of PBDEs in humanbreast milk from Indonesia. Environ. Pollut. 151, 130–138.

Tan, J., Loganath, A., Chong, Y.S., Obbard, J.P., 2008. Multivariate data analyses ofpersistent organic pollutants in maternal adipose tissue in Singapore. Toxicol.Environ. Chem. 90, 837–859.

Thomas, G.O., Wilkinson, M., Hodson, S., Jones, K.C., 2006. Organohalogen chemicalsin human blood from the United Kingdom. Environ. Pollut. 141, 30–41.

Thuresson, K., Höglund, P., Hagmer, L., Sjödin, A., Bergman, Å., Jakobsson, K., 2006.Apparent half-lives of hepta- to decabrominated diphenyl ethers in humanserum as determined in occupationally exposed workers. Environ. HealthPerspect. 114, 176–181.

Toms, L.M.L., Hearn, L., Kennedy, K., Harden, F., Bartkow, M., Temme, C., Mueller,M.F., 2009. Concentrations of polybrominated diphenyl ethers (PBDEs) inmatched samples of human milk, dust and indoor air. Environ. Int. 35, 864–869.

Turyk, M.E., Persky, V.W., Imm, P., Knobeloch, L., Chatterton, R., Anderson, H.A.,2008. Hormone disruption by PBDEs in adult male sport fish consumers.Environ. Health Perspect. 116, 1635–1641.

Uemura, H., Arisawa, K., Hiyoshi, M., Dakeshita, S., Kitayama, A., Takami, H.,Sawachika, F., Yamaquchi, M., Sasai, S., 2010. Congener-specific body burdenlevels and possible determinants of polybrominated diphenyl ethers in thegeneral Japanese population. Chemosphere 79, 706–712.

Wan, Y., Choi, K., Kim, S., Ji, K., Chang, H., Wiseman, S., Jones, P.D., Khim, J.S., Park, S.,Park, J., Lam, M.H.W., Giesy, J.P., 2010. Hydroxylated polybrominated diphenylethers and bisphenol a in pregnant women and their matchingfetuses: placental transfer and potential risks. Environ. Sci. Technol. 44, 5233–5239.

Wang, H.S., Du, J., Ho, K.L., Leung, H.M., Lam, M.H.W., Giesy, J.P., Wong, C.K.C., Wong,M.H., 2011. Exposure of Hong Kong residents to PBDEs and their structuralanalogues through market fish consumption. J. Hazard. Mater. 192,374–380.

Wang, H.S., Chen, Z.J., Ho, K.L., Ge, L.C., Du, J., Lam, M.H.W., Giesy, J.P., Wong, M.H.,Wong, C.K.C., 2012. Hydroxylated and methoxylated polybrominated diphenylethers in blood plasma of humans in Hong Kong. Environ. Int. 47, 66–72.

Williams, M.C., Goldzieher, J.W., 1980. Chromatographic patterns of urinary ethynylestrogen metabolites in various populations. Steroids 36, 255–282.

Yue, Q.Y., Svensson, J.O., Sawe, J., Bertilsson, L., 1989. Interindividual and interethnicdifferences in the demethylation and glucuronidation of codeine. Br. J. Chin.Pharmacol. 28, 629–637.

Yue, Q.Y., Svensson, J.O., Sjoqvist, F., Sawe, J., 1991. A comparison of thepharmacokinetics of codeine and its metabolites in healthy Chinese andCaucasian extensive hydroxylators of debrisoquine. Br. J. Chin. Pharmacol. 31,643–647.

Zhao, G., Wang, Z., Dong, M.H., Rao, K., Luo, J., Wang, D., Zha, J., Huang, S., Xu, Y., Ma,M., 2008. PBBs, PBDEs, and PCBs levels in hair of residents around e-wastedisassembly sites in Zhejiang Province, China, and their potential sources. Sci.Total Environ. 397, 46–57.

Zhao, G., Wang, Z., Zhou, H., Zhao, Q., 2009. Burdens of PBBs, PBDEs, and PCBs intissues of the cancer patients in the e-waste disassembly sites in Zhejiang,China. Sci. Total Environ. 407, 4831–4837.

Zheng, J., Chen, K.H., Luo, X.J., Yan, X., He, C.T., Yu, Y.J., Hu, G.C., Peng, X.W., Ren, M.Z.,Yang, Z.Y., Mai, B.X., 2014. Polybrominated diphenyl ethers (PBDEs) in pairedhuman hair and serum from e-waste recycling workers: source apportionmentof hair PBDEs and relationship between hair and serum. Environ. Sci. Technol.48, 791–796.

Zhou, H.H., Sheller, J.R., Nu, H., Wood, M., Wood, A.J.J., 1993. Ethnic-differences inresponse to morphine. Clin. Pharmacol. Ther. 54, 507–513.

Zhu, L., Ma, B., Hites, R.A., 2009. Brominated flame retardants in serum from thegeneral population in Northern China. Environ. Sci. Technol. 43, 6963–6968.

Zota, A.R., Rudel, R.A., Morello-Frosch, R.A., Brody, J.G., 2008. Elevated house dustand serum concentrations of PBDEs in California: unintended consequences offurniture flammability standards. Environ. Sci. Technol. 42, 8158–8164.

Page

1

1

Urinary Bromophenol Glucuronide and Sulfate Conjugates: Potential Human 2

Exposure Molecular Markers for Polybrominated Diphenyl Ethers 3

4

Ka-Lok Ho,a Man-Shan Yau. a Margaret B. Murphy, *a Yi Wan,†b Bonnie M. –W. Fong,c,d Sidney Tam,c 5

John P. Giesy,a,b,,e,f,g,h Kelvin S. –Y. Leung,d Michael H. –W. Lam*a 6

7

8

9

10

Supporting Information 11

12

13

14

15

16

17

18

19

20

Page

2

Table of Content 21

Methods 22

Materials and General Procedures 23

Instrumentation 24

Identification and Quantification 25

Quality Assurance and Quality Control 26

27

Figures 28

Figure S1. Median concentrations of PBDEs in blood plasma of human blood in various 29

countries. 30

31

References 32

Page

3

Methods 33

Materials and General Procedures. 34

All starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as 35

received unless stated otherwise. Oasis WAX® (6 mL / 150 mg) and HLB® (6 mL / 200 mg) 36

cartridges were obtained from Waters Corp. (Milford, MA, USA). Dihexylammonium acetate 37

(DHAA) was obtained from Sigma-Aldrich. Standards for polybrominated diphenylethers 38

(PBDE), including BDE-3, BDE-15, BDE-28, BDE-47, BDE-66, BDE-85, BDE-99, BDE-100, 39

BDE-153, BDE-154, BDE-183, BDE-184, BDE-197, BDE-202, BDE-207, BDE-208, BDE-209; 40

recovery spike standard (13C12-BDE-77 and 13C12-BDE-138); 13C12-BDE-139, 13C12-BDE-209 41

and 13C6-2,4-dibromphenol were purchased from Wellington Labs (Ontario, Canada). Three 42

standards for bromophenols (BP), 2,4-dibromophenol (2,4-DBP), 2,4,5-tribromophenol 43

(2,4,5-TBP) and 2,4,6-tribromophenol (2,4,6-TBP), and five hydroxylated-PBDE (OH-BDE) 44

(4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 5’-OH-BDE-99 and 6’-OH-BDE-99) standards 45

were purchased from Accustandard (New Haven, Connecticut, USA). All other OH-BDEs, 46

MeO-BDEs (2’-OH-BDE-7, 3’-OH-BDE-7, 4’-OH-BDE-17, 6’-OH-BDE-17, 2’-OH-BDE-28, 47

6-OH-BDE-47, 4’-OH-BDE-49, 2’-OH-BDE-66, 2’-OH-BDE-68, 6-OH-BDE-85, 48

4-OH-BDE-90, 6-OH-BDE-90, 5’-OH-BDE-99, 6’-OH-BDE-99, 3-OH-BDE-100, 49

2-OH-BDE-123, 6-OH-BDE-137, 4’-MeO-BDE-17, 6’-MeO-BDE-17, 2’-MeO-BDE=28, 50

5-MeO-BDE-47, 6-MeO-BDE-47, 4’-MeO-BDE-49, 2’-MeO-BDE-68, 6-MeO-BDE-85, 51

3-MeO-BDE-90, 6-MeO-BDE-90, 3-MeO-BDE-100, 2-MeO-BDE-123, 6-MeO-BDE-137) and 52

glucuronide and sulfate conjugates of 2,4-DBP and 2,4,6-TBP were synthesized by the authors at 53

City University of Hong Kong. Purities of all of the OH-BDEs were greater than 98%.1 All 54

phenolic compounds were derivatized by reacting with N,O-bis(trimethylsilyl) 55

Page

4

trifluoroacetamide (BSTFA) with 1% trimethylchlorosaline (TMCS) obtained from Acros 56

Organics (Geel, Belgium). 57

58

Page

5

Instrumentation. 59

HPLC-MS/MS 60

Quantification of glucuronide and sulfate conjugates of bromophenols was performed by 61

HPLC–ESI-MS/MS (Agilent 1200 Series HPLC, Agilent Technologies, Waldbronn, Germany) 62

coupled to a MDS Sciex API 3200 QTrap triple quadrupole / linear ion trap mass spectrometer 63

with a Turbo V ion spray source (Applied Biosystems, Foster City, CA, USA). In order to 64

improve sensitivity and selectivity, analytes were detected in Multiple Reaction Monitoring 65

(MRM) mode with a dwell time of 150 ms. The ionization source parameters were as follow: ion 66

spray voltage: -4500kV; curtain gas (N2): 15 psig; collision gas (N2), high; temperature of 67

ionization source, 600oC; ion source gas 1 (nebulizer gas), 60 psig; ion source gas 2 (heater gas), 68

50 pisg. Declustering potential (DP), entrance potential (EP), collision energy (CE) and collision 69

cell exit potential (CXP) of all analytes were optimized to obtain maximum sensitivity. The 70

analytical column was a Waters XBridgeTM C18 2.5 μm 3.0 mm × 50 mm column. A guard 71

column XBridgeTM C18 2.5 μm 3.0 × 20 mm was placed in front of the analytical column. 72

LC separation was accomplished by use of gradient elution at a flow rate of 300 μL/min, 73

with solvent A (5 mM DHAA in Milli-Q) and solvent B (5 mM DHAA in methanol) at the 74

composition of A:B (90:10, v/v) at t = 0 to t = 2 min, changed linearly to A:B (30:70, v/v) over a 75

period of 18 min, then held at such composition for a further 10 min. After the separation, the 76

eluent composition was switched back to A:B (90:10 v/v) and held for 20 min before the next 77

injection. The injection volume was 10 μL. 78

79

GC-NCI-MS 80

Page

6

Identification and quantification of all targeted PBDEs, MeO-BDEs, OH-BDEs and BPs 81

were performed by use of gas chromatography (GC, Agilent 7890A) with mass-selective 82

detection (MS, Agilent 5975C with triple axis detector) in electron-capture negative ionization 83

(ECNI) mode, by monitoring at m / z = 79 and 81 for most of the congeners, and at m / z = 486.6 84

for BDE-207, BDE-208 and BDE-209. The GC injector was set to 285 oC with an injecting 85

volume of 2 μL. Lesser brominated BDE congeners (mono- to hexa-substituted BDEs), 86

MeO-BDEs, OH-BDEs and BPs were analyzed by use of a 30 m × 0.25 mm × 0.25 μm DB-5MS 87

column, whereas higher BDE congeners (hepta- to deca-substituted BDEs) were analyzed by a 88

15 m × 0.25 mm × 0.1 μm DB-5HT column. The temperature program for the analysis of lesser 89

brominated BDE congeners and MeO-BDEs was as follows: 110 oC for 5 min; 30 oC / min to 90

240 oC; held for 20 min; 30 oC / min to 280 oC held for 2 min and 30 oC / min to 300 oC; held for 91

30 min. The temperature program for the analysis of more brominated BDE congeners was as 92

follows: 60 oC for 5 min; 10 oC / min to 290 oC; held for 2 min; 20 oC / min to 300 oC; held for 9 93

min. The temperature program for the analysis of OH-BDEs and BPs was as follows: 110 oC for 94

5 min; 20 oC / min to 240 oC for 20 min; 30 oC / min to 280 oC; held for 10 min and finally 30 oC 95

/ min to 300 oC; held for 20 min. Total concentrations of different groups of analytes (ΣPBDEs / 96

ΣOH-BDEs / ΣMeO-BDEs / ΣBPs) were reported as the sum of the individual PBDEs / 97

OH-BDEs / MeO-BDEs / BPs congeners quantified. 98

99

Quantification 100

Targeted PBDEs, OH-BDEs, MeO-BDEs and BRs in human plasma 101

To avoid photo-degradation, samples were kept in amber vials or sampling tubes wrapped 102

with aluminum foil. Neutral and phenolic fractions of extracts of human blood plasma were 103

Page

7

slightly modified from previously described methods.2 Each sample of plasma was transferred to 104

a clean glass tube and known amounts of PBDE recovery standards (13C12-BDE-77 and 105

13C12-BDE-138), and 13C12-BDE-209 and 6’-OH-BDE-17 as surrogate standards were added. 106

Hydrochloric acid (2 mL, 6 M) and iso-propanol (6 mL) were added, followed by 107

homogenization. Each sample was extracted by 3 × 6 mL hexane / methyl tert-butyl ether 108

(MTBE). The organic extracts were combined and washed with 1% potassium chloride solution 109

(3 mL). The combined organic extract was evaporated over a gentle steam of nitrogen and lipid 110

content was determined gravimetrically. Lipid was re-dissolved in hexane (4 mL) and partitioned 111

with potassium hydroxide (2 mL, 0.5 M in 50% ethanol) to ionize the phenolic analytes. PBDEs 112

and MeO-BDEs were separated by 3 × 4 mL of hexane. The aqueous layer was acidified by 113

hydrochloric acid (2 mL, 0.5 M), then phenolic compounds were extracted by 3 × 4 mL of 114

hexane / MTBE (9:1, v/v). The neutral and phenolic fractions were blown down to dryness and 115

reconstituted in 5 mL hexane. The neutral fraction was treated with 5 mL of concentrated 116

sulfuric acid twice to remove any lipids. 117

The neutral fraction was concentrated over a gentle stream of nitrogen and cleaned-up by 118

passing the concentrate through multilayer column chromatography with 1 g of anhydrous 119

sodium sulfate on top, followed by 8 g of silica and 8 g of alumina. PBDEs and MeO-BDEs were 120

eluted with 50 mL of a hexane / dichloromethane mixture (3:2, v/v). The organic solvent was 121

evaporated to dryness and the sample was reconstituted to 100 μL with 13C12-BDE-139 added as 122

an internal standard for GC / MS analysis. 123

The phenolic fraction was also concentrated under a gentle stream of nitrogen and clean-up 124

by Florisil column chromatography with 1 g of anhydrous sodium sulfate on top of 5 g of Florisil. 125

OH-BDEs and BPs were eluted with 30 mL of a mixture of dichloromethane and hexane (1:1, 126

Page

8

v/v). The organic solvent was evaporated to dryness and the phenolic fraction was derivatized 127

with 100 μL of BSTFA with 1% TMCS at 70 oC for an hour. 13C12-BDE-139 was added as the 128

internal standard for GC / MS analysis. 129

130

Bromophenol conjugates in human urine 131

The extraction method was similar to that reported in our previous work.1a A human urine 132

sample (5 mL) was partitioned with 3 × 5 mL ethyl acetate. The combined organic solution was 133

evaporated to dryness under a gentle stream of nitrogen. Residues were dissolved in 15 mL of 134

0.67 M sodium acetate buffer at pH 5.2. The resultant solution was applied, at a rate of 1 drop 135

s–1, to an Oasis WAX solid-phase extraction (SPE) cartridge that had been preconditioned 136

sequentially by 5 mL of methanol, 5 mL of Milli-Q water, and 5 mL of 2 M sodium acetate 137

buffer at pH 5.2. The loaded WAX SPE cartridge was then washed in turn by 5 mL of 2 M 138

sodium acetate buffer at pH 5.2, followed by 5 mL of methanol. The glucuronide fraction was 139

then eluted with 4 mL of a formic acid/methanol (1:9, v/v) mixture, and the sulfate fraction was 140

eluted with 4 mL of an aqueous ammonia/methanol (1:9, v/v) mixture. The eluates were 141

evaporated to around 100 μL under a gentle stream of nitrogen. 13C6-2,4-dibromphenol (200 μL, 142

500 ng mL–1) was added as an internal standard for LC-MS/MS quantitation. 143

144

Quality Assurance and Quality Control. 145

Surrogate standards were used to quantify the concentration of all the BDE congeners using 146

mean relative response factors determined from standard calibration during analysis of human 147

plasma samples. PBDE recovery standards (13C12-BDE-77 and 13C12-BDE-138) were used as 148

surrogates for mono- to hexa-substituted BDEs and MeO-BDEs, 13C12-BDE-209 was used as the 149

Page

9

surrogate for hepta- to deca-substituted BDEs, and 6’-OH-BDE-17 was used as the surrogate for 150

OH-BDEs and BPs. All equipment was rinsed with acetone and hexane to avoid contamination. 151

One laboratory blank and one matrix spike were analyzed for each batch of 18 samples to 152

check for interferences or contamination from solvent and glassware. The method detection limit 153

(MDL) was established by use of lesser concentrations and a consecutive analysis of the series of 154

n spiked samples (Equation 1). 155

156

MDL = t × σ (1) 157

158

where: σ is the standard deviation of the data and t is the compensation factor from the Student’s 159

t-Table with n – 1 degrees of freedom at a confidence interval of 95%. Method detection limits 160

(MDLs) for BDEs, MeO-BDEs, OH-BDEs and BPs ranged from 0.007 to 0.24 ng/g l.w. For 161

BDE-207, -208, -209, MDLs ranged from 0.15 to 0.75 ng/g l.w. Recoveries of all targeted 162

analytes were within 88 – 103% while the matrix-spiked recoveries were within 78 – 114%. 163

In the analysis of BP conjugates, procedural blanks and matrix spikes were included in each 164

batch of 10 samples. None of the synthesized BPs conjugates were detected in procedural blanks. 165

MDLs of targeted analytes were assessed by use of the same method as that used for plasma: 166

MDLs for 2,4-dibromophenyl glucuronide, 2,4,6-tribromophenyl glucuronide, 167

2,4-dibromophenyl sulfate and 2,4,6-tribromophenyl sulfate were 2.7, 2.9, 2.9 and 2.2 ng/g 168

creatinine, respectively. Recoveries of the analytes were within the range of 72 to 102% and the 169

%RSD ranged from 4 to 9%. 170

171

172

Page

10

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

Figure S1. Median concentrations of PBDEs in blood plasma of human blood in various 188

countries. 189

190

191

192

193

194

403 310

*arithmetic mean

Page

11

References: 195

1. (a) Ho, K.L.; Murphy, M.B.; Wan, Y.; Fong, B.M.W.; Tam, S.; Giesy, J.P.; Lam, M.H.W. 196

Anal. Chem. 2012, 84, 9881-9888. (b) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X.; Jones, 197

P.D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J.; Lam, M.H.W.; Giesy, J.P. Environ. Sci. 198

Technol. 2008, 43, 7536-7542. 199

2. (a) Hovander, L.; Athanasiadou, M.; Asplund, L.; Jensen, S.; Klasson-Wehler, E. J. Anal. 200

Toxicol. 2000, 24, 696-703. (b) Qiu, X.; Bigsby, R.M.; Hites, R.A. Environ. Health Prespect. 201

2009, 117, 93-98. 202