Supplementary Materials:

Expressing urine from a gel disposable diaper for biomonitoring using phthalates as an example

Liangpo Liua, Tongwei Xiaa,b, Lihua Guoc, Lanyu Caoc, Benhua Zhaoc, Jie Zhanga, Sijun Donga, Heqing Shena, d

aKey Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, PR China, 361021

bSchool of Environmental Studies, China University of Geosciences, Wuhan, PR China, 430074

cSchool of Public Health of Xiamen University, Xiamen, PR China, 361005

dCorresponding author e-mail:

Deconjugating of the glucuronidated phthalate metabolite:

After thawing, aliquots of 1mL urine were buffered to pH = 6.5 by sodium acetate (1mL, 0.1M NaAc adjusted with 1 M HCl), and spiked with a mixture of isotope labeled phthalate monoesters as the internal standards (20 uL, 50ng/mL). β-glucuronidase (10 uL, 200 units/mL) was added as deconjugation emzyme and 4-methylumbelliferyl glucuronide (20 uL, 80 ng/mL) was added as the control indicator of the enzyme activity. The samples were incubated at 37 ℃ for 90 min to deconjugate the glucuronidated phthalate metabolites and the reaction was stop by adding 1mL of 3.6 M phosphoric acid.

SPE Purification:

The column was preconditioned with acetonitrile (3 mL) and phosphate buffer (3 mL, pH = 2, 0.14 M NaH2PO4 in 0.85% H3PO4) in sequence. The treated urine was loaded onto the cartridge at a rate of 1mL/min. Then it was rinsed with 0.1M formic acid (2 mL) and water (1 mL) and dried with gentle air flow. The analytes were eluted with acetonitrile (3 mL) followed by ethyl acetate (2 mL) at 1mL/min. The eluate was evaporated to dryness under a gentle stream of nitrogen at 45 ℃ and the dried residue was resuspended in 200 uL of 60% acetonitrile (0.1% acetic acid) for the further analysis.

HPLC-ESI-MS/MS analysis:

The target compounds were separated by the LC-20A prominence (Shimadzu, Kyoto, Japan) high-performance liquid chromatography (HPLC) equipped with the Phenomenex® (Torrance, CA, USA) guard column (Phenyl, 4.0 mm × 2.0 mm internal diameter) and separation column (Luna Phenyl-hexyl, 100 mmÎ2.0 mm internal diameter, 3.0 um particle size). Mobile phase A was 0.1% acetic acid in water and B was 0.1% acetic acid in acetonitrile. Table S1 shows the HPLC gradient program. The mobile phase flow rate was set at 0.25 mL/min, and the column temperature was set at 30 ℃. The total run time for each injection was 20 min and the injection volume was 20 uL.

The target compounds were ionized by electrospray ionization (ESI) and detected by the tandem mass spectrometry (MS/MS) (Applied Biosystems, Concord, Ontario, Canada). The mass specific detection was achieved using a MDS Sciex 3200 Q TRAP triple quadrupole linear ion trap mass spectrometer equipped with a TurboIonSpray electrospray ionization (ESI) source and Analyst Software version 1.5 (Applied Biosystems, Concord, Ontario, Canada). ESI-MS/MS was performed at unit mass resolution in multiple reaction monitoring (MRM) under the negative ion mode with the following parameters: source temperature (TEM) 550 ℃, ion spray voltage (IS) -4500 V, curtain gas (CUR) 10 psi (46 kPa of nitrogen), ion source gas 1 (GS1) 40 psi (276 kPa of nitrogen), ion source gas 2 (GS2) 40 psi (276 kPa of nitrogen), collision gas pressure (nitrogen) medium (corresponds to value of 3 (arbitrary scale of 1–12)), and dwell time per channel 50 ms. Optimization of the compound-dependent MS/MS parameters was performed with phthalate metabolites standards via direct injection into the mass spectrometer using a syringe pump (MDS Sciex) at a flow rate of 10 uL/min. Optimal settings for compound-dependent MS/MS parameters are shown in Table S2.

The Absolute Recovery of SPE Extraction:

The phthalate metabolite’s absolute recoveries during SPE extraction were calculated (Table S3). Considering the background analytes in the urine, we used the labeled internal standard to track the SPE recovery instead of the native compounds (assuming the labeled internal standards are 100% pure). One of the paired spiking samples was added the labeled internal standards before SPE (Concbefore); the other was added the labeled internal standards after SPE (Concafter). The SPE recovery was calculated as:

SPE recovery = (Concafter/Concbefore) ´ 100%

Because of the recoveries in the urine expression step are close to hundred percent for the analytes, the absolute recoveries of the whole sample preparation procedure should be comparable with the reported SPE extraction recoveries for adult urine analysis (Liu et al., 2011; Silva et al., 2004). In case of the low SPE recovery, the stable isotope-labeled compounds as internal standards had been used to correct the possible matrix effects and to improve method accuracy and precision.

Reference:

1.  Liu, L., Bao, H., Liu, F., Zhang, J., and Shen, H. Phthalates exposure of the Chinese reproductive age couples and their effect on male semen quality, a primary study. Environ Int 2011 (doi:10.1016/j.envint.2011.04.05).

2.  Silva, M.J., Slakman, A.R., Reidy, J.A., Preau Jr, J.L., Herbert, A.R., and Calafat, A.M., et al. Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction. J Chromatogr B 2004: 805: 161-167.

Table S1. Mobile Phase Gradient Programa

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

aA: 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-13C4 / 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

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]) / 90 ng/ml (Mean [SD])
Concafter / Concbefore / Mean Recovery / Concafter / Concbefore / Mean Recovery
MMP / 31.6 (1.00) / 34.1 (0.04) / 92.5 / 99.8 (3.40) / 92.3 ( 3.32) / 108.2
MEP / 26.9 (0.63) / 32.1 (0.76) / 83.8 / 100.1 (2.19) / 87.4 (1.89) / 114.5
MBP / 29.2 (1.12) / 27.8 (1.60) / 105.0 / 97.0 (5.28) / 101.7 (6.33) / 95.4
MBzP / 29.0 (0.14) / 32.7 (0.14) / 88.7 / 107 (1.10) / 85.1 (0.28) / 125.7
MEHP / 21.0 (0.59) / 19.0 (0.64) / 110.5 / 87.0 (2.57) / 82.7 (2.62) / 105.2
MEOHP / 32.5 (2.26) / 28.7 (1.98) / 113.2 / 100.1 (6.86) / 93.4 (6.43) / 107.2

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