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Supplemen a y in o ma ion
De e mina ion o he u ina y concen a ions o six bisphenols
in public se an s by online solid-phase ex ac ion-liquid
ch oma og aphy- andem mass spec ome y
And ea Es é ez-Dan a*, Rosa io Rodil, José Beni o Quin ana, Rosa Mon es*
Aqua ic One Heal h Resea ch Cen e (ARCUS) & Depa men o Analy ical Chemis y,
Nu i ion and Food Chemis y. R. Cons an ino Candei a S/N, IIAA building,
Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain
Analy ical & Bioanaly ical Chemis y
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Tex S1. Enzyma ic hyd olysis op imiza ion
P.3
Tex S2. Analy ical me hodology o c ea inine de e mina ion in u ine.
P.4
Table S1. Chemical s uc u e o a ge bisphenols
P.5
Table S2. Summa y o sociodemog aphic cha ac e is ics and c ea inine le els o
he s udied popula ion.
P.6
Table S3. Linea i y pa ame e s, in e cep and slope es ima es and s anda d e o
P.7
Table S4. Compa ison o he pe o mance o he me hod p oposed in his wo k
wi h o he online SPE-LC-MS/MS me hods published in he li e a u e.
P. 8-9
Table S5. Compa ison o he u ina y concen a ions wi h o he ecen published
s udies.
P.10-11
Fig. S1 S anda dized pa e o cha s o (a) BPA, (b) BPS and (c) BPF, ob ained
du ing enzyma ic deconjuga ion DOE op imiza ion.
P.12
Fig. S2 Es ima ed esponse su ace o (a) BPF and (b) BPS, ob ained du ing
enzyma ic deconjuga ion DOE op imiza ion.
P.13
Fig. S3 Rela i e esponse in u ine o he a ge compounds employing di e en
concen a ions o NH4F, as modi ie o bo h LC mobile phases (n=2).
P.14
Fig. S4 Ch oma og am o a 100 ng mL-1 spiked u ine sample unde inal
condi ions.
P.15
Fig. S5 E ec o online SPE aqueous phase modi ie s (B1) in he peak shape and
signal in ensi y o BPS in u ine
P.16
Fig. S6 S abili y o bisphenol in u ine s o ed a oom empe a u e. RSD (n=3) <
10%
P.17
Fig. S7 Compa ison o c ea inine co ec ed concen a ions (µg g-1) acco ding o
obacco use: (a) BPF and (b) BPS
P.18
Fig. S8 Compa ison o c ea inine co ec ed concen a ions (µg g-1) acco ding o he
esidence en i onmen : (a) BPF and (b) BPS
P.19
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Tex S1. Enzyma ic hyd olysis op imiza ion
Design o expe imen s (DOE)
A cen al composi e design (2^2 + s a ) wi h 4 cen e poin s was c ea ed o make an e icien
op imiza ion o he enzyme concen a ion (expe imen al domain: 250- 850 uni s) and incuba ion
ime (expe imen al domain: 1.5 – 4.5 h) a iables. The ob ained expe imen s we e:
Expe imen 1. Enzyme concen a ion = 550 uni s, incuba ion ime = 0.88 h
Expe imen 2. Enzyme concen a ion = 250 uni s, incuba ion ime = 1.5 h
Expe imen 3. Enzyme concen a ion = 850 uni s, incuba ion ime = 1.5 h
Expe imen 4. Enzyme concen a ion = 126 uni s, incuba ion ime = 3 h
Expe imen 5. Enzyme concen a ion = 550 uni s, incuba ion ime = 3 h
Expe imen 6. Enzyme concen a ion = 550 uni s, incuba ion ime = 3 h
Expe imen 7. Enzyme concen a ion = 550 uni s, incuba ion ime = 3 h
Expe imen 8. Enzyme concen a ion = 550 uni s, incuba ion ime = 3 h
Expe imen 9. Enzyme concen a ion = 974 uni s, incuba ion ime = 3 h
Expe imen 10. Enzyme concen a ion = 250 uni s, incuba ion ime = 4.5 h
Expe imen 11. Enzyme concen a ion = 850 uni s, incuba ion ime = 4.5 h
Expe imen 12. Enzyme concen a ion = 550 uni s, incuba ion ime = 5.12 h
Sample p epa a ion and injec ion
375 µL o il e ed u ine was adjus ed a pH 5 wi h sodium ace a e bu e and spiked wi h 100 ng
mL-1 o a ailable sul a e and glucu onide me aboli es mix u e (BPA-S, BPA-DS, BPS-S, BPF-S, BPA-
G, BPA-DG, BPS-G and BPF-G) + 20 ng mL-1 o ISs BPA-d6 and BPS-d8. Then, each expe imen was
andomly pe o med acco ding o he condi ions speci ied in he DOE able and injec ed in o he
online SPE-LC-MS/MS sys em ollowing he op imized p o ocol.
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Tex S2. Analy ical me hodology o c ea inine de e mina ion in u ine.
Sample p epa a ion
U ine samples we e il e ed h ough 0.45 µm PVDF sy inge-d i en il e s. Then, each aliquo was
dilu ed 10,000 imes in ul apu e wa e , spiked wi h he in e nal s anda d (c ea inine-d3) a 10
ng mL-1 and ans e ed o an inse o injec ion in he LC-MS/MS sys em.
Liquid ch oma og aphy- andem mass spec ome y pa ame e s
Ins umen al analyses we e pe o med wi h a Wa e s Acqui y UPLC® H class sys em (Mil o d,
MA, USA) equipped wi h a qua e na y sol en pump, a he mos a ed LC column compa men ,
and a sample manage . The UPLC® sys em was in e aced o a iple quad upole mass
spec ome e Xe o TQD om Wa e s.
The ch oma og aphic sepa a ion was pe o med a 30 ºC on a Luna C18 column (50 x 2.0 mm,
I.D., 3 µm pa icle size) om Phenomenex. A dual eluen sys em consis ing o (A) 0.1% o mic
acid in ul apu e wa e and (B) 0.1% o mic acid in MeOH was used a a low a e o 0.3 mL min-
1 in isoc a ic mode (50:50) o 3 minu es. Injec ion olume was se a 3 µL.
The in e ace be ween he UPLC® sys em and he Xe o TQD mass spec ome e was an
elec osp ay ioniza ion (ESI) sou ce ope a ing in posi i e mode a a ixed capilla y ol age o 3
kV and a empe a u e o 150 ºC. Ni ogen, p o ided by a ni ogen gene a o om Peak Scien i ic
Spain (Ba celona, Spain), was used as desol a ion gas a 600 L h-1 and 450 ºC (desol a ion
empe a u e), and as cone gas a 10 L h-1. Analyses we e pe o med by MS/MS in Selec ed
Reac ion Moni o ing (SRM) mode, whe e: c ea inine SRM ansi ions 114 > 44 (quan i ica ion),
114 > 86 (quali ica ion) and c ea inine-d3 SRM ansi ions 117 > 47 (quan i ica ion), 117 > 89
(quali ica ion).
Valida ion
Calib a ion cu es we e p epa ed in ul apu e wa e and anged om he me hod quan i ica ion
limi (MQL) o 250 ng mL-1, wi h IS le el o 10 ng mL-1, being he MQL 0.02 ng mL-1 and he
ob ained R2 was 0.9995. Me hod epea abili y was e alua ed a 1 and 50 ng mL-1 and he RSD
we e 7 and 5 %, espec i ely o 5 consecu i e injec ions. Me hod accu acy was e alua ed
h ough spiking 6 di e en eal u ine samples a 2 mg mL-1 (which co esponds o 200 mg dL-1
in sample and 200 ng mL-1 in he dilu ed u ine) and he ob ained alues we e 102 ± 6 %.
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Table S1. Chemical s uc u e o a ge bisphenols
Name
S uc u e
Bisphenol A (BPA)
Bisphenol AF (BPAF)
Bisphenol B (BPB)
Bisphenol E (BPE)
Bisphenol F (BPF)
Bisphenol S (BPS)
HO OH
S
HO OH
O O
OHHO
HO OH
CF3
HO OH
F3C
HO OH
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Table S2. Summa y o sociodemog aphic cha ac e is ics and c ea inine le els o he s udied popula ion ( ull da a p esen ed in ZENODO eposi o y
(h ps://doi.o g/10.5281/zenodo.10477935)
Sociodemog aphic cha ac e is ics
Gende %
Tobacco use %
Residence loca ion %
Age
C ea inine (g L-1)
Female
Male
Yes
No
U ban
Subu ban
Ru al
Mean
Median
SD
Mean
Median
SD
67.6
32.4
16.1
83.9
56.1
33.8
10.1
51.3
52
7.08
0.94
0.82
0.64
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Table S3. Linea i y pa ame e s, in e cep and slope es ima es and s anda d e o .
In e cep
es ima e
In e cep
s anda d e o
Slope
es ima e
Slope s anda d
e o
S a da d e o o
es ima e
BPS
3.74E-04
7.87E-04
3.80E-03
7.83E-05
3.03E-03
BPF
1.43E-03
1.27E-03
7.82E-03
1.31E-04
5.19E-03
BPE
-8.49E-04
1.24E-03
1.38E-02
1.30E-04
5.20E-03
BPA
2.85E-03
6.08E-04
4.42E-03
6.42E-05
2.56E-03
BPAF
-1.94E-03
9.84E-02
3.94E-01
1.04E-02
4.10E-01
BPB
-3.84E-03
1.87E-03
2.07E-02
1.98E-04
7.87E-03
Calcula ions pe o med using 3 independen calib a ion cu es wi h S a g aphics Cen u ion 19 so wa e
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Table S4. Compa ison o he pe o mance o he me hod p oposed in his wo k wi h o he online SPE-LC-MS/MS me hods published in he li e a u e.
Sample p epa a ion
Sepa a ion and de ec ion
Re e ence
Ta ge
Bisphenol
P e- ea men
Ex ac ion
LC-MS/MS
%R
MQL
(ng mL-1)
This s udy
BPA, BPAF,
BPB, BPE,
BPF and
BPS
200 µL o u ine
Fil a ion h ough 0.45 µm PVDF sy inge-d i en il e s
Addi ion o IS + 1mM sodium ace a e bu e a pH 5 + 700 uni s o ß-
glucu onidase + 392 µL o ul apu e wa e
Incuba ion o 5 h a 37 °C
Online SPE on S a a-X 25 µm ca idges
Mobile phases: 15 mM o sodium ace a e
bu e a pH 5 in ul apu e wa e - MeOH
Inj. Vol.: 500 µL
LC-(ESI-)-MS/MS on QqQ (SRM)
Luna C18 (150 x 2 mm I.D., 3 µm)
Mobile phase: 2 mM o NH4F in
ul apu e wa e – 2 mM o NH4F in
MeOH
92-112 %
(excep
BPAF 11-
40%)
0.049-2.2
Ye e al.,
2005
BPA
100 µL o u ine
Addi ion o IS + 50 µL o enzyme/ ammonium ace a e Incuba ion
o e nigh a 37 °C
Online SPE on LiCh osphe e RP-18 ADS 25 µm
column
Mobile phases: ul apu e wa e - MeOH
Inj. Vol.: 1000 µL
LC-(APCI-)-MS/MS on QqQ (MRM)
Ch omoli h Pe o mance RP-18 (100 x
4.6 mm I.D.)
Mobile phases: ul apu e wa e - MeOH
100 %
1.3
Koch e
al., 2012
BPA
300 µL o u ine
Addi ion o IS + 1 mM ammonium ace a e bu e a pH 5 + 6 µL o ß-
glucu onidase
Incuba ion o 4 h a 37 °C
Online SPE on LiCh osphe e RP-8 ADS 25 µm
column
Mobile phases: ul apu e wa e - Ace oni ile
Inj. Vol.: 100 µL
HPLC-(ESI-)-MS/MS on QTRAP (MRM)
Wa e s A lan is T3 analy ical column
(150 x 3 mm I.D., 3 µm)
Mobile phases: ul apu e wa e -
Ace oni ile
96.8%
0.1
Zhou e .
al., 2014
BPA, BPF
and BPS
100 µL o u ine
Addi ion o IS + dilu ion o 1 mL wi h 0.1 M o mic acid + 50 µL o ß-
glucu onidase/sul a ase
Incuba ion o 4 h a 37 °C
S op solu ion: 750 µL 0.1 M o mic acid in ul apu e wa e
Online SPE on LiCh osphe e RP-18 ADS 25 µm
column
Mobile phases: ul apu e wa e - MeOH
Inj. Vol.: 350 µL
LC-(APCI-)-MS/MS on QqQ (MRM)
Ch omoli h High Resolu ion RP-18e (100
x 4.6 mm I.D.)
Mobile phases: ul apu e wa e - MeOH
77-106%
0.03-0.1
Page 41 o 52 Analy ical & Bioanaly ical Chemis y
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He e nan
e al.,
2016
BPA, BPAF,
BPB, BPF
and BPS
50 µL o u ine
Addi ion o IS + 25 µL o ß-glucu onidase (200 uni s) + 440 µL
ul apu e wa e
Incuba ion o 90 min a 37 °C
S op solu ion: 400 µL o 0.5 % o mic acid in ul apu e wa e .
Online SPE on S a a-X 25 µm ca idges
Mobile phase: 0.05 % o ace ic acid in
ul apu e wa e : 0.05 % o ace ic acid in
MeOH
Inj. Vol.: 500 µL
LC-(ESI-)-MS/MS on QTRAP (MRM)
Syne gi MAX-RP column (150 x 3 mm
I.D., 4 µm)
Mobile phase: 0.05% o ace ic acid in
ul apu e wa e : 0.05 % o ace ic acid in
MeOH
101-110%
0.005-0.39
Jo e al.,
2020
BPA, BPF
and BPS
100 µL o u ine
Addi ion o IS + 100 µL o ß-glucu onidase/sul a ase (1000 uni s)
Incuba ion o 24h a 37 °C
S op solu ion: 80 µL 1 M o mic acid + 670 µL ul apu e wa e
Online SPE on MAYI-ODS column 50 µm
Mobile phases: ul apu e wa e - MeOH
Inj. Vol.: 100 µL
LC-(APCI-)-MS/MS on QqQ (MRM)
ACE 5 C18-pen a luo ophenyl column
(150 x 2.1 mm I.D., 5 µm)
Mobile phases: ul apu e wa e - MeOH
99.4-108%
0.13-0.24
Re e ences:
1. Ye X, Kuklenyik Z, Needham LL, Cala a AM. Au oma ed on-line column-swi ching HPLC-MS/MS me hod wi h peak ocusing o he de e mina ion o nine en i onmen al phenols in u ine.
Analy ical Chemis y 2005; 77: 5407-5413.
2. Koch HM, Kolossa-Geh ing M, Sch ö e -Ke mani C, Ange e J, B üning T. Bisphenol A in 24 h u ine and plasma samples o he Ge man En i onmen al Specimen Bank om 1995 o 2009: a
e ospec i e exposu e e alua ion. J Expo Sci En i on Epidemiol 2012; 22: 610-6.
3. Zhou X, K ame JP, Cala a AM, Ye X. Au oma ed on-line column-swi ching high pe o mance liquid ch oma og aphy iso ope dilu ion andem mass spec ome y me hod o he
quan i ica ion o bisphenol A, bisphenol F, bisphenol S, and 11 o he phenols in u ine. Jou nal o Ch oma og aphy B 2014; 944: 152-156.
4. He e nan AL, Thompson K, Eaglesham G, Vijayasa a hy S, Muelle JF, Sly PD, e al. Rapid, au oma ed online SPE-LC-QTRAP-MS/MS me hod o he simul aneous analysis o 14 ph hala e
me aboli es and 5 bisphenol analogues in human u ine. Talan a 2016; 151: 224-233.
5. Jo MJ, Pa k J-H, An K-A, Choi H, Kang Y-s, Hwang M. Quan i ica ion o bisphenols in Ko ean u ine using online solid-phase ex ac ion-high-pe o mance liquid ch oma og aphy- andem mass
spec ome y. En i onmen al Toxicology and Pha macology 2020; 80: 103491.
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Fig. S5 E ec o online SPE aqueous phase modi ie s (B1) in he peak shape and signal in ensi y
o BPS in u ine
3
x10
0
0.5
1
1.5
213.191
1
2
x10
0
2
4
6
8
13.154
1
3
x10
0
1
2
3
413.348
1
4
x10
0
0.5
1
2.5
13.245
1
4
x10
0
0.5
1
1.5 13.275
1
Time (min)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
0.1% o mic acid
0.1% ace ic acid
5mM o sodium ace a e bu e a pH 5
15mM o sodium ace a e bu e a pH 5
25mM o sodium ace a e bu e a pH 5
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Fig. S6 S abili y o bisphenol in u ine s o ed a oom empe a u e. RSD (n=3) < 10%
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Fig. S7 Compa ison o c ea inine co ec ed concen a ions (µg g-1) acco ding o obacco use: (a)
BPF and (b) BPS
(a)
(b)
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Fig. S8 Compa ison o c ea inine co ec ed concen a ions (µg g-1) acco ding o he esidence
en i onmen : (a) BPF and (b) BPS
(a)
(b)
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