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Supplementary Materials:
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as an example
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Liangpo Liua, Tongwei Xiaa,b, Lihua Guoc, Lanyu Caoc, Benhua Zhaoc, Jie Zhanga,
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Sijun Donga, Heqing Shena, d
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a
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Chinese Academy of Sciences, Xiamen, PR China, 361021
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b
Expressing urine from a gel disposable diaper for biomonitoring using phthalates
Key Laboratory of Urban Environment and Health, Institute of Urban Environment,
School of Environmental Studies, China University of Geosciences, Wuhan, PR
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China, 430074
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c
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d
School of Public Health of Xiamen University, Xiamen, PR China, 361005
Corresponding author e-mail: hqshen@iue.ac.cn
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Deconjugating of the glucuronidated phthalate metabolite:
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After thawing, aliquots of 1mL urine were buffered to pH = 6.5 by sodium acetate
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(1mL, 0.1M NaAc adjusted with 1 M HCl), and spiked with a mixture of isotope
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labeled phthalate monoesters as the internal standards (20 uL, 50ng/mL).
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β-glucuronidase (10 uL, 200 units/mL) was added as deconjugation emzyme and
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4-methylumbelliferyl glucuronide (20 uL, 80 ng/mL) was added as the control
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indicator of the enzyme activity. The samples were incubated at 37 ℃ for 90 min to
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deconjugate the glucuronidated phthalate metabolites and the reaction was stop by
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adding 1mL of 3.6 M phosphoric acid.
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SPE Purification:
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The column was preconditioned with acetonitrile (3 mL) and phosphate buffer (3 mL,
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pH = 2, 0.14 M NaH2PO4 in 0.85% H3PO4) in sequence. The treated urine was loaded
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onto the cartridge at a rate of 1mL/min. Then it was rinsed with 0.1M formic acid (2
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mL) and water (1 mL) and dried with gentle air flow. The analytes were eluted with
1
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acetonitrile (3 mL) followed by ethyl acetate (2 mL) at 1mL/min. The eluate was
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evaporated to dryness under a gentle stream of nitrogen at 45 ℃ and the dried residue
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was resuspended in 200 uL of 60% acetonitrile (0.1% acetic acid) for the further
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analysis.
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HPLC-ESI-MS/MS analysis:
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The target compounds were separated by the LC-20A prominence (Shimadzu, Kyoto,
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Japan) high-performance liquid chromatography (HPLC) equipped with the
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Phenomenex® (Torrance, CA, USA) guard column (Phenyl, 4.0 mm × 2.0 mm
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internal diameter) and separation column (Luna Phenyl-hexyl, 100 mm2.0 mm
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internal diameter, 3.0 um particle size). Mobile phase A was 0.1% acetic acid in water
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and B was 0.1% acetic acid in acetonitrile. Table S1 shows the HPLC gradient
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program. The mobile phase flow rate was set at 0.25 mL/min, and the column
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temperature was set at 30 ℃. The total run time for each injection was 20 min and the
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injection volume was 20 uL.
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The target compounds were ionized by electrospray ionization (ESI) and detected by
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the tandem mass spectrometry (MS/MS) (Applied Biosystems, Concord, Ontario,
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Canada). The mass specific detection was achieved using a MDS Sciex 3200 Q TRAP
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triple quadrupole linear ion trap mass spectrometer equipped with a TurboIonSpray
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electrospray ionization (ESI) source and Analyst Software version 1.5 (Applied
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Biosystems, Concord, Ontario, Canada). ESI-MS/MS was performed at unit mass
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resolution in multiple reaction monitoring (MRM) under the negative ion mode with
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the following parameters: source temperature (TEM) 550 ℃, ion spray voltage (IS)
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-4500 V, curtain gas (CUR) 10 psi (46 kPa of nitrogen), ion source gas 1 (GS1) 40 psi
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(276 kPa of nitrogen), ion source gas 2 (GS2) 40 psi (276 kPa of nitrogen), collision
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gas pressure (nitrogen) medium (corresponds to value of 3 (arbitrary scale of 1–12)),
2
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and dwell time per channel 50 ms. Optimization of the compound-dependent MS/MS
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parameters was performed with phthalate metabolites standards via direct injection
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into the mass spectrometer using a syringe pump (MDS Sciex) at a flow rate of 10
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uL/min. Optimal settings for compound-dependent MS/MS parameters are shown in
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Table S2.
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The Absolute Recovery of SPE Extraction:
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The phthalate metabolite’s absolute recoveries during SPE extraction were calculated
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(Table S3). Considering the background analytes in the urine, we used the labeled
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internal standard to track the SPE recovery instead of the native compounds
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(assuming the labeled internal standards are 100% pure). One of the paired spiking
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samples was added the labeled internal standards before SPE (Concbefore); the other
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was added the labeled internal standards after SPE (Concafter). The SPE recovery was
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calculated as:
SPE recovery = (Concafter/Concbefore)  100%
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Because of the recoveries in the urine expression step are close to hundred percent for
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the analytes, the absolute recoveries of the whole sample preparation procedure
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should be comparable with the reported SPE extraction recoveries for adult urine
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analysis (Liu et al., 2011; Silva et al., 2004). In case of the low SPE recovery, the
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stable isotope-labeled compounds as internal standards had been used to correct the
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possible matrix effects and to improve method accuracy and precision.
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Reference:
1. Liu, L., Bao, H., Liu, F., Zhang, J., and Shen, H. Phthalates exposure of the
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Chinese reproductive age couples and their effect on male semen quality, a
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primary study. Environ Int 2011 (doi:10.1016/j.envint.2011.04.05).
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2. Silva, M.J., Slakman, A.R., Reidy, J.A., Preau Jr, J.L., Herbert, A.R., and Calafat,
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A.M., et al. Analysis of human urine for fifteen phthalate metabolites using
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automated solid-phase extraction. J Chromatogr B 2004: 805: 161-167.
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Table S1. Mobile Phase Gradient Programa
a
Time (min)
0
0.3
1.3
8.0
12
12.5
13.5
14.0
20.0
A (%)
5
5
40
20
20
0
0
5
5
B (%)
95
95
60
80
80
100
100
95
5
A: 0.1% acetic acid in water and B: 0.1% acetic acid in acetonitrile.
Table S2. Transitions and MS/MS conditions for each analyte
Analytes
Parent (Q1)
Daughter (Q3)
Dwell time
DP
EP
CEP
CE
CXP
MMP
179.0
107.0
50.0
-30.0
-7.0
-10.0
-15.0
0.0
MMP-13C4
183.0
110.0
50.0
-33.0
-7.0
-17.7
-14.0
0.0
MEP
193.0
121.0
50.0
-30.0
-7.0
-10.0
-16.0
0.0
MEP- C4
197.0
79.0
50.0
-33.0
-7.0
-18.3
-33.0
0.0
MBP
221.0
77.0
50.0
-30.0
-7.0
-12.0
-20.0
0.0
MBP-13C4
225.0
79.0
50.0
-35.0
-7.0
-19.5
-28.0
0.0
MBzP
255.0
107.0
50.0
-30.0
-7.0
-15.0
-19.0
0.0
MBzP-13C4
259.0
107.0
50.0
-35.0
-7.0
-20.0
-21.0
0.0
MEHP
277.0
134.0
50.0
-30.0
-7.0
-15.0
-22.0
0.0
MEHP-13C4
281.0
137.0
50.0
-45.0
-7.0
-21.8
-22.0
0.0
MEOHP
291.0
121.0
50.0
-40.0
-4.0
-15.0
-35.0
0.0
MEOHP-13C4
295.0
124.0
50.0
-36.0
-7.0
-22.4
-27.0
0.0
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88
89
90
91
92
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DP: declustering potential [V]; EP: entrance potential [V]; CEP: cell entrance potential [V]; CE:
collision energy [V]; CXP: cell exit potential [V].
Table S3. The phthalate metabolite’s absolute recoveries (%) in SPE extraction step (duplicate
results)
Metabolites
30 ng/ml (Mean [SD])
MMP
Concafter
31.6 (1.00)
Concbefore
34.1 (0.04)
MEP
26.9 (0.63)
MBP
29.2 (1.12)
MBzP
90 ng/ml (Mean [SD])
Mean Recovery
92.5
Concafter
99.8 (3.40)
32.1 (0.76)
83.8
27.8 (1.60)
105.0
29.0 (0.14)
32.7 (0.14)
88.7
MEHP
21.0 (0.59)
19.0 (0.64)
MEOHP
32.5 (2.26)
28.7 (1.98)
94
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Concbefore
92.3 ( 3.32)
Mean Recovery
108.2
100.1 (2.19)
87.4 (1.89)
114.5
97.0 (5.28)
101.7 (6.33)
95.4
107 (1.10)
85.1 (0.28)
125.7
110.5
87.0 (2.57)
82.7 (2.62)
105.2
113.2
100.1 (6.86)
93.4 (6.43)
107.2