Glucuronide and Sulfate Conjugates of Bisphenol A: Chemical Synthesis and

Correlation between Their Urinary Levels and Plasma Bisphenol A Content in

Voluntary Human Donors

Ka-Lok Ho, Ka-Ki Yuen, Man-Shan Yau, Margaret B. Murphy, Yi Wan, Bonnie M. –W.

Fong, Sidney Tam, John P. Giesy, Kelvin S. –Y. Leung, Michael H. –W. Lam*

Supplementary Information
Table of Content

Methods

Materials and General Procedures

Instrumentation

Human Blood Sample Collection

Identification and Quantification

Quality Assurance and Quality Control

Figures

Figure S1. ESI-MS of the Bisphenol A phase II conjugates

Figure S2. 1H NMR of Bisphenol A mono-glucuronide

Figure S3. 1H NMR of Bisphenol A di-glucuronide

Figure S4. 1H NMR of Bisphenol A mono-sulfate

Figure S5. 1H NMR of Bisphenol A di-sulfate

Tables

Table S1. LC-MS/MS transitions and MS parameters of BPA-conjugates (upper) and

GC-MS/MS transitions and MS parameters of BPA analysis (lower) adopted

in this study.

Table S1. Proposed fragmentation assignments of mass spectra of BPA analyzed by GC-

MS/MS

Table S2. Proposed fragmentation assignments of mass spectra of BPA phase II

conjugates analyzed by LC-MS/MS.

References
Methods

Materials and General Procedures

All starting materials, bisphenol A (BPA), D-(+)-glucuronic acid γ-lactone, hydrogen bromide

in glacial acetic acid (33%), acetic anhydride, potassium carbonate, tetrabutylammonium

bromide, ammonium hydroxide, potassium hydroxide, sodium acetate, sodium methoxide

(NaOMe), perchloric acid were purchased from commercial sources (Sigma – Aldrich, Acros

Organics, or Wako) and were used as received unless stated otherwise. The solvents used for

synthesis were of analytical grade while solvents used for chromatography analysis were of

HPLC or LC-MS grade. Dichloromethane was distilled from anhydrous calcium hydride

prior to use. Deuterated mass labeled bisphenol A (BPA-d16) and pyrene-d10 were purchased

from Cambridge Isotope Laboratories. Oasis® WAX cartridges (6 mL / 150 mg) and Sep-Pak

Florisil (3 mL / 500 mg) were obtained for Waters Corp. N,O-

Bis(trimethylsilyl)trifluoroacetamide were purchased from Supelco. LabAssay TM Creatinine

was obtained from WAKO. Artificial urine matrix was prepared according to the literature.1

Synthesis of BPA glucuronide and sulfate conjugates

BPA mono-2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid, methyl ester (C1)

The general synthetic routes for the BPA glucuronide and sulfate conjugates are outlined in

Scheme 1. Intermediate A & B were synthesized according to literature methods.2-3 A solution

of intermediate B (2.8 g, 7.05 mmol) in chloroform (30 mL) was vigorously stirred and

heated under reflux at 70 C overnight with an aqueous solution (30 mL) of BPA (4.83 g,

21.1 mmol), tetrabutylammonium bromide (1.14 g, 3.53 mmol) and potassium carbonate

(6.14 g, 44.4 mmol). The reaction mixture was cooled and diluted with water. The organic

layer was separated and washed by saturated K2CO3 solution twice, followed by washing

with distilled water, dried over anhydrous magnesium sulfate, and evaporated in vacuo. The

yellow syrup was purified by flash silica column chromatography eluted with a solvent
system of ethyl acetate : petroleum ether (1:2) affording BPA mono-2,3,4-tri-O-acetyl-β-D-

glucopyranosiduronic acid, methyl ester as a white solid (2.62 g, yield 68.5 %).

BPA di-2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid, methyl ester (C2)

The synthetic method was similar to that of BPA mono-2,3,4-tri-O-acetyl-β-D-

glucopyranosiduronic acid, methyl ester (C1), except that a mole ratio of BPA to 1-bromo-

2,3,4-tri-O-acetyl-α-D-glucopyranuronic acid, methyl ester of 1:3 was used instead. The

yellow syrup was purified by flash silica column chromatography eluted by a solvent system

of ethyl acetate : petroleum ether (1:1) affording BPA di-2,3,4-tri-O-acetyl-β-D-

glucopyranosiduronic acid, methyl ester as a white solid (4.48 g, yield 67%).

BPA mono-glucuronide (D1)

BPA mono-2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid, methyl ester (1.12 g, 2.06

mmol) was dissolved in MeOH (20 mL). Potassium hydroxide (1.156 g, 20.6 mmol) was

added and the mixture was stirred overnight. The precipitate was collected and re-dissolved

in a mixture of formic acid and methanol (1:9). The solution was then collected and

evaporated in vacuo and the crude product of BPA mono-glucuronide (0.6 g) was collected as

a white solid. This was then subjected to HPLC purification. ESI-MS (-ve): 403.4 (M-H-),

227.3 (M-H-C6O6H8-). 1H NMR (400 MHz, acetone-d6): δ Q 7.158 – 7.136 (d, 2H, J = 8.8Hz),

7.055 – 7.033 (d, 2H, J = 8.8Hz), 6.972 – 6.950 (d, 2H, J = 8.8Hz), 6.730 – 6.708 (d, 2H, J =

8.8Hz), 5.077 – 5.058 (d, 1H, J = 7.6Hz), 4.089 – 4.065 (d, 1H, J = 9.6Hz), 3.720 – 3.680 (t,

1H, J = 9.6Hz), 3.600 – 3.480 (m, 2H), 1.600 (s, 6H). HRMS (-ve) calcd for C21H24O8:

403.13926; found 403.1430. Elemental analysis calcd(%) for C21H24O8: C 62.37, H 5.98, N

0.00; found C 62.27, H 5.91 N 0.01.

BPA di-glucuronide (D2)

 The synthetic method was similar to that of BPA mono-glucuronide (D 1) except that

BPA di-2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid, methyl ester (C2) instead of BPA

mono- 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid, methyl ester (C1) was used, and the

number of mole of potassium hydroxide was 2 times larger than that in the synthesis of D 1.

The crude product of BPA mono-glucuronide (0.9 g) was collected as a white solid. ESI-MS

(-ve): 579.4 (M-H-), 403.4 (M-H-C6O6H8-), 227.2 (M-H-C12O12H16 -). 1H NMR (400 MHz,

D2O): δ Q 7.306 – 7.284 (d, 4H, J = 8.8Hz), 7.074 – 7.052 (d, 4H, J = 8.8Hz), 5.095 – 5.080

(d, 2H, J = 6Hz), 3.887 – 3.864 (d, 2H, J = 9.2Hz), 3.611 – 3.591 (m, 6H), 1.656 (s, 6H).

HRMS (-ve) calcd for C27H32O14: 579.17132; found 579.1740. Elemental analysis calcd(%)

for C27H32O14: C 55.86, H 5.56, N 0.00; found C 55.91, H 5.71 N 0.03.

BPA mono-sulfate (E1)

Synthesis of sulfate ester was adopted from our previous reported method with

modifications.4 Diethylaniline (3.65 mL, 22.9 mmol) in chloroform (4 mL) was stirred at -40

C. Chlorosulphonic acid (0.66 mL, 9.9 mmol) was steadily added over 1 hour with the

temperature maintained below 10 C. BPA (2.1 g, 9.2 mmol) in chloroform (10 mL) was then

added all at once and the reaction was stirred overnight. Excess triethylamine was then added

to the reaction mixture and the white precipitate was filtered off as the crude product, which

was re-dissolved in methanol. Potassium hydroxide in methanol solution was added until

white precipitate was formed again. The white precipitated of BPA mono-sulfate mono-

potassium salt (1.6 g) was filtered and subjected to further purification by HPLC. ESI-MS (-

ve): 307.1 (M-K-), 227.0 (M-K-SO3-), 211.2 (M-K-SO4-). 1H NMR (400 MHz, DMSO-d6): δ

Q 7.082 – 7.060 (d, 2H, J = 8.8Hz), 7.023 – 7.001 (d, 2H, J = 8.8Hz), 6.952 – 6.932 (d, 2H, J

= 8.0Hz), 6.586 – 6.566 (d, 2H, J = 8.0Hz), 1.544 (s, 6H). HRMS (-ve) calcd for C15H15O5S:

307.06399; found 307.0666. Elemental analysis calcd(%) for C15H15O5SK: C 49.71, H 4.17,
N 0.00; found C 49.59, H 4.21 N 0.01.

BPA di-sulfate (E2)

The synthesis was similar to that of BPA mono-sulfate (E1) except that the mole ratio of

starting materials was different. The number of mole of chlorosuphonic acid was doubled.

BPA di-sulfate di-potassium salt (1.1 g) was obtained as a white precipitate. ESI-MS (-ve):

425.0 (M-K-), 345.0 (M-K-SO3-). 1H NMR (400 MHz, DMSO-d6): δ Q 7.103 – 7.081 (d, 4H,

J = 8.8Hz), 7.044 – 7.022 (d, 2H, J = 8.8Hz), 1.584 (s, 6H). HRMS (-ve) calcd for

C15H14O8S2K: 424.97668; found 424.9790. Elemental analysis calcd(%) for C15H14O8S2K2: C

37.49, H 2.94, N 0.00; found C 37.59, H 3.01 N 0.02.

HPLC Purification of BPA glucuronide and sulfate conjugates

Crude mono- and di-glucuronide and mono- and di-sulfate conjugates of BPA were purified

by HPLC. The HPLC separation procedure was repeated until sufficient amount of each of

the conjugate were obtained. Purity of all the four BPA conjugates was checked by LC

tandem hi-res mass spectrometry using an Agilent 1200 Series High performance liquid

chromatography system (Agilent Technologies, Santa Clara, CA, USA) with an Applied

Biosystems 3200 Q-Trap triple quadrupole linear ion trap mass spectrometer equipped (ESI)

source and Analyst Software version 1.5.2 (Applied Biosystems, Concord, Ontario, Canada).

Human Sample Collection

Whole blood samples were collected using the standard phlebotomy technique in

vacutainer tubes containing sodium heparin anticoagulant (Vacuette, Greiner bio-one, GmbH,

Austria). Samples were centrifuged at 1500  g for 25 min. Plasma was removed from the top

of the tube. 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. Creatinine determination was carried

out by a kinetic colorimetric assay based on the modified Jaffe method 5 using the Roche

Modular System (Roche Diagnostics, IN, USA), with an analytical range between 360 and

57,500 mmol L-1.

Sample Extraction and Cleanup

BPA in human plasma

BPA-d16 (1 ng) was spiked to 1 mL of human plasma sample. The plasma was allowed

to stand for 10 minutes. After equilibrium, 50 μL of concentrated hydrochloric acid (37%),

Milli-Q water (2 mL) and iso-propanol (3 mL) were added. The mixture was then extracted

by hexane / MTBE (1:1 v/v, 3  5 mL). The organic fractions were combined and partitioned

with a 1% KCl solution (3 mL) followed by evaporation to dryness under a gentle steam of

nitrogen. The residue was determined for lipid content gravimetrically. The residue was re-

dissolved in hexane (4 mL) and partition with potassium hydroxide (2 mL, 0.5 M in 50%

ethanol) to ionize the phenolic analytes. Neutral compounds were separated by hexane (2  4

mL). The aqueous layer was acidified by hydrochloric acid (2 mL, 0.5 M), then the phenolic

compounds were extracted by hexane / MTBE (9:1 v/v, 2  4 mL). Phenolic fraction was

evaporated to dryness under a gentle stream of nitrogen and reconstituted in 1 mL of 5%

acetone in hexane. The mixture was subjected to a SPE clean-up using a Sep-Pak Florisil

cartridge previously conditioned by DCM / MeOH (4:1 v/v, 6 mL) and 5% acetone in hexane

(6 mL) at the flow rate of 1 drop per second. The cartridge then washed by 5% acetone in

hexane (6 mL). The cartridge was the dried under reduced pressure and the BPA and BPA-d16

were eluted by DCM / MeOH (4:1 v/v, 10 mL). The eluate was evaporated to dryness under a

gentle stream of nitrogen. Analytes were reconstituted in 50 μL iso-octane containing 5 ng

pyrene-d10 as an internal standard and the phenolic analytes were derivatized by 50 μL

BSTFA with 1% TMCS at 70 C for an hour.

Quality control (QC), quality assurance (QA)

Procedural blanks and matrix spikes were analyzed for every batch of 18 samples in

analysis of blood BPA and urinary BPA and BPA-conjugates. BPA-d16 was used as a surrogate

to estimate the BPA recovery throughout whole extraction process. All equipment was rinsed

with acetone and hexane before use to avoid sample contamination. During analysis of

human plasma samples, laboratory blanks and spiked matrices were analyzed per every batch

of 18 samples to check for instrumental drift, matrix interference and contamination of

solvent and glassware. Estimations of the method detection limit (MDL) and method

quantification limit (MQL) were carried out using a lower spike concentration, and it was

determined by the consecutive analysis of a series of n spiked samples according to the

following equations:

MDL = t × σ

where σ is the standard deviation of the data and t is the compensation factor from the

Student’s t-Table with n – 1 degrees of freedom at a confidence interval of 95%.

MQL = 10 × σ

where σ is the standard deviation of the data

In the analysis of BPA in human plasma and urine samples, BPA was found in the blank

samples with the mean level of 0.002 ± 0.005 ng mL-1 and its MDL was 0.021 ng mL-1.
Recovery of BPA-d16 in human plasma samples was 89 – 101% while the matrix-spiked

recovery was 85 – 105%.

 1.4x108 4.0x108
-
[M-H]
3.5x108 [M-H]-
1.2x108

Relative intensity ( a. u. )
Relative intensity ( a. u. )
3.0x108
1.0x108 398 400 402 404 406 408 410 412 804 806 808 810 812 814 576 578 580 582 584
- 2.5x108
8.0x107 [2M-H]
2.0x108
7
6.0x10
1.5x108
4.0x107 1.0x108
398 400 402 404 406 408 410 412 804 806 808 810 812 814 576 578 580 582 584
2.0x107 5.0x107

0.0 0.0
200 400 600 800 1000 200 400 600 800 1000
m/ z m/ z

804 806 808 810 812 814

 5.0x107
3.0x107

4.0x107 2.5x107

Relative intensity ( a. u. )
-

Relative intensity ( a. u. )
[M-K]
304 306 308 310 312 314
2.0x107 418 420 422 424 426 428 430 432 434
3.0x107
[M-K]
-
1.5x107
2.0x107
1.0x107

1.0x107 304 306 308 310 312 314 5.0x106 418 420 422 424 426 428 430 432 434

0.0
0.0
200 400 600 800 1000 200 300 400 500 600 700 800
m/ z m/ z

Figure S1. ESI-MS of the Bisphenol A phase II conjugates: Bisphenol A mono-glucuronide (upper left); Bisphenol A di-glucuronide (upper
right); Bisphenol A mono-sulfate (lower left) and Bisphenol A di-sulfate (lower right). The corresponding insets show the experimental (solid
lines) and calculated (red bars) isotopic patterns of the peaks corresponded to: Bisphenol A mono-glucuronide (upper left) [M-H]- and [2M-H]- at
207 208 209 210 211 212 213 214 215 216 217

m/z of 403 and 807 amu, respectively; Bisphenol A di-glucuronide (upper right) [M-H]- at m/z of 589 amu; Bisphenol A mono-sulfate (lower
left) [M-K]- at m/z of 307 amu; and Bisphenol A di-sulfate (lower right) [M-K]- at m/z of 425 amu.
Figure S2. 1H NMR of Bisphenol A mono-glucuronide
Figure S3. 1H NMR of Bisphenol A di-glucuronide

Figure S4. 1H NMR of Bisphenol A mono-sulfate

Figure S4. 1H NMR of Bisphenol A di-sulfate
Table S1. LC-MS/MS transitions and MS parameters of BPA-conjugates (upper) and GC-MS/MS
transitions and MS parameters of BPA analysis (lower) adopted in this study.

MRM Transitions Entrance

Declustering
MRM Collision
Collision Energy
Collision
Analytes
Analytes Transitions Potential Cell
Potential (V) Energy (V)
(m/z) (m/z) (V) (V) Potential (V)

403  227 -50 -5 -36.5 -2

357  73 30
BPA mono-
403  113
BPA di-TMS derivatives -50 -5 -30 -2
glucuronide
357  191 20
403  59 -50 -5 -55 -0.5

579  403
d16-BPA di-TMS derivatives -90 73
368 -4 -35 24 -3
(surrogate standard)579  113
BPA di-glucuronide  197
-80
368 -5 -42 16 -0.5

d10-Pyrene 289  59 -30 -2 -35 -0.5

212  208 36
(internal stranded) 307  227 -50 -5 -30 -2
BPA mono-sulfate
307  80 -55 -5 -45 -1.5

387  307 -55 -3 -26 -2.5

BPA di-sulfate
193  80 -35 -2 -25 -1
d16-BPA
243  223 -40 -4 -28 -2
(internal standard)
 Table S2. Proposed fragmentation assignments of mass spectra of BPA analyzed by GC-MS/MS.
Compound Transition (m / z), Collision energy (V) & Assignments
Bisphenol A 357  73, 357  191
30V 20V
[M – C15H14O2 – Si – C3H9]+ [M – C7H8O – Si – C3H9]+
m / z = 357 m / z = 73 m / z = 357 m / z = 191
CH2
C C C
Si
Si Si Si Si Si
O O O O O

d16-Bisphenol A 368  73 368  197

24V 16V
[M –C15D14O2 – Si – C3H9]+ [M – C15H14O2 – Si – C3H9]+
m / z = 368 m / z = 73 m / z = 368 m / z = 197
D CD3 D D CD3 D D CD2
D C D D C D D C
Si
Si Si Si Si Si
O O O O O D
D D D D
D D D D D

d10-pyrene (internal standard) 212  208

36V
[M – D2]∙+
m / z = 212 m / z = 208
D D
D D D D

D D D D

D D D D
D D D D
 Table S3. Proposed fragmentation assignments of mass spectra of BPA phase II conjugates analyzed by LC-MS/MS.
Compound Transition (m / z) & Assignments
BPA mono-glucuronide 403  227 [M – C6H10O6 – H]- 403  113 [M – C15H15O2 – CO2 – H2O – H]- 403  59[M –C15H15O2 – CO2 – H2O –CO –
m / z = 227
OOC
O HO
OOC
O C2H2– H]-
HO HO O HO
HO O OH
OH -
O OOC
O O
OH HO
OH HO O O
m / z = 113 OH O

OH m / z = 59

BPA di-glucuronide 579  403[M –C6H10O6 – H]- 579  113[M – C15H14O2 – C6H9O6 – CO2 – 289  59 [M – C15H14O2 – C6H8O6 – CO2 –
m / z = 403
OOC
O
H2O – H] -
H2O –CO – C2H2– H]-
HO
HO O
OH OOC OOC
O O
HO HO
O HO O HO O
HO OH OOC OH
OH O O
OH HO O-
O O HO O
COOH HO O HO
OH m / z = 113 OH m / z = 59
OH OH
O O
COOH COO

BPA mono-sulfate 307  227[M – SO3 – H]- 307  80[M – C15H11O2Br3 – SO3 – H]-
m / z = 227
O3S O

O OH SO3
O3S
m / z = 80
OH

BPA di-sulfate 387  307[M – SO3 - H]- 193  80 [M – C15H14O2 – SO3 - H]-
m / z = 307
O3S O
O3S O
O SO3
SO3
O m / z = 80
SO3H

D16-Bisphenol A 241  223[M – CD3 – D]-

 D D
D D3C D CD3
CD3D D
C
DO D DO D

D DD D DD
O O
D D

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5. R. W. Bonsnes, H. H. Taussky, On the colorimetric determination of creatinine by the Jaffe reaction, J. Biol. Chem. 158 (1945) 581–591.