Chemical synthesis, characterization and analytical method development bromophenols
phase II conjugates as human exposure biomarker for PBDEs
Ka-Lok Ho1, Bonnie Mei-Wah Fong2,3, Yi Wan4, Sidney Tam3,
John P. Giesy1,4,5, Kelvin Sze-Yin Leung2, Michael Hon-Wah Lam1*
1State
Key Laboratory for Marine Pollution, Department of Biology & Chemistry, City University of Hong Kong, Hong Kong SAR,
2Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR,
3Department of Clinical Biochemistry, Queen Mary Hospital, Hong Kong SAR,
4Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada,
5Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
1. Introduction
Table 1. HR-MS characterization
The occurrences of dibromophenols (DBPs) and tribromophenols (TBPs)
glucuronide and sulfate conjugates in urines of mammalian aminals have
been well investigated and established by pharmacokinetic and
toxicokinetic studies. These conjugates are phase II metabolites of the
selected brominated diphenyl ether (BDE) congeners. Until now, there is no
literature to report on the occurrences of these phase II metabolite of BDE
in human urine. It would be ideal if the presence of these phase II
conjugates in human urine be used as the population exposure biomarker
for PBDEs. However, these glucuronide and sulfate conjugates of
bromophenols are not commercially available, so we have to firstly to
synthesize and purify to be an authentic standard before further analytical
experiments. In this study, targeted dibromphenyl- and tribromophenyl
glucuronide and sulfate were synthesized and purified by in house method
and then fully characterized. Also, the stability of these conjugates in
human urine under various preservation were investigated.
Table 2. Elemental Analysis
Compound
Theoretical
Mass (Da)
Experimental
Mass
Error
Mass (Da)
Difference (Da) (ppm)
2,4-Dibromophenyl
Glucuronide
426.88070
426.8843
+0.0036
+8.43
2,4-Dibromophenyl
Sulfate
330.80980
330.8103
+0.0005
+1.51
2,4,6-Tribromophenyl
Glucuronide
408.72031
408.7182
-0.00211
-5.16
410.71825
410.7170
-0.00125
-3.04
2,4,6-Tribromophenyl
Sulfate
504.79119
504.7947
+0.00371
+6.95
506.78920
506.7922
+0.00300
+5.92
Theoretical
%C
%H %N
Compound
2,4-Dibromophenyl
33.61
Glucuronide
2,4-Dibromophenyl
19.47
Sulfate
2,4,6Tribromophenyl
28.43
Glucuronide
2,4,6Tribromophenyl
Sulfate
16.05
Experimental
%C %H %N
2.83
0.00 33.31 2.81 0.02
0.82
0.00 19.26 0.91 0.02
2.19
0.00 28.02 2.21 0.01
0.45
0.00 16.17 0.41 0.08
3. Analytical Method Development
Artificial urine matrix were used throughout whole method development.
Ethyl acetate was used to extract acidified samples and subjected solid phase extraction
clean-up
2. Synthesis, Purification & Characterization
SPE clean-up
 Sample Loading:
SPE cartridge
(Oasis® WAX)
Fig.1a
Conditioning:
1. 5 mL Methanol
2. 5 mL H2O
3. 5 mL 2M CH3COONa
 Sample Washing:
Wash with 5 mL
2M CH3COOH
 Sample Elution:
Elute with 4 mL 0.1%
HCOOH/methanol
(Glucuronide fraction)
Load Urine Sample
(5 mL) mixed with
5 mL 2M
CH3COONa
Wash with 5 mL
H2O
Wash with 5 mL
MeOH
Elute with 4 mL
0.1% NH4OH/methanol
(Sulfate fraction)
Table 3. Method Validation of the developed solid phase extraction
Repeatability Recovery (%) MDL (pg / mL)
2,4-Dibromophenyl Glucuronide
8.54
93.4
12
2,4,6-Tribromophenyl Glucuronide
7.66
100.5
13
2,4-Dibromophenyl Sulfate
7.82
92.2
13
2,4,6-Tribromophenyl Sulfate
7.26
74.0
10
Fig.1b
Relative intensity (A. U.)
Relative Absorbance (mA)
1.0
0.8
0.6
0.4
0.2
0.0
3.5x10
6
3.0x10
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
6
5.0x10
5
4. Stability of bromophenols phase II conjugates

0.0
0
10
20
30
40
0
10
Retention Time (mins)
20
30
40
50
Retention time (mins)
Preservation methods, (1) addition of NaN3, (2) formaldehyde and (3) store under -20oC
were used to investigate stability of synthesized conjugates in human urine
Non-Preserved Human Urine
Formaldehyde Preserved
NaN3 Preserved
(a)
110
90
90
80
70
60
50
40
30
20
0
5
10
15
20
80
60
40
20
0
320
240
245
250
255
260
265 415
420
425
430
435
100
200
300
400
245
250
500
m/z ratio ( a. m. u. )
255
600
260
265 415
700
420
425
800
430
435
330
335
340
345 400
405
410
415
420
30
20
10
5
10
15
20
25
30
Non-Preserved Human Urine
Formaldehyde Preserved
NaN3 Preserved
110
o
100
-20 C Preserved
90
80
70
60
50
40
30
20
10
15
20
25
30
0
5
10
15
20
25
30
Day
Fig. 2 Stability of bromophenols conjugates by chemical and physical measures (a)
2,4-dibromophenyl glucuronide; (b) 2,4,6-tribromophenyl glucuronide (c) 2,4dibrmophenyl sulfate and (d) 2,4,6-tribromophenyl sulfate
425
5. Summary
440
320
240
5
Day
-
325
40
10
0
[M-H]
50
Day
(d)
o
-
[M-H-C6O6H8]
60
0
-20 C Preserved
-20
-
[M-SO3H]
70
30
Relative Response ( % )
Relative Response ( % )
100
[M-H]
25
Non-Preserved Human Urine
Formaldehyde Preserved
NaN3 Preserved
Day
(c)
-
80
0
10
Fig.1d
-20 C Preserved
o
o
Relative Response ( % )
Relative Response ( % )
Fig.1c
100
(b)
-20 C Preserved
100
110
Non-Preserved Human Urine
Formaldehyde Preserved
NaN3 Preserved
325
330
335
340
345 400
405
410
440
100
200
300
400
500
m/z ratio ( a. m. u. )
Fig. 1a. Synthetic pathway of bromophenols glucuronide & sulfate conjugate
Fig. 1b. HPLC-UV chromatogram (Left) & HPLC-MS/MS chromatogram (right) for
purity check
Fig. 1c. NMR characterization
Fig. 1d. ESI-MS characterization
600
700
800
415
420
425
Selected dibromophenol and tribromophenol glucuronide and sulfate conjugate have
been successfully synthesized, purified and characterized. Solvent extraction and SPE
clean-up method of these phase II conjugates was fully developed and validated. The
stability of these conjugates in human urine were also investigated. Low temperature
storage was the best method for to minimize the loss of bromophenols phase II
conjugates in urine sample.
6. Acknowledgement:
This work is supported by a grant from Research Grants Council of the Hong Kong
Special Administrative Region, China [Reference No. CityU 9041623]