Supporting Information

Parabens in human urine from several Asian countries, Greece, and the

United States

Masato Honda1, Robinson Morgan1, Kurunthachalam Kannan1,2,3*

1 Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201-0509, United States

2 Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, New York 12201-0509, United States

3 Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

*Corresponding author: Kurunthachalam Kannan

Wadsworth Center, Rm. D547

New York State Dept. of Health

Empire State Plaza, Albany, NY 12201-0509

Tel: 1-518-474-0015

Fax: 1-518-473-2895

E-mail: kurunthachalam.kannan@health.ny.gov

For submission to: Chemosphere

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Tables: 7

Figure: 1

Table S1. Sample information.

Table S2. Conditions of instrumental analysis.

Table S3. Information of analytical characteristics.

Table S4. Urinary concentrations (ng/mL) of parabens in nine countries.

Table S5. Results of Steel-Dwass test for urinary concentrations of parabens among nine countries.

Table S6. Age related differences in urinary parabens concentration.

Table S7. Results of Steel-Dwass test for urinary concentrations of parabens among six age-group.

Fig. S1. Correlation between urinary concentration of MeP, EtP and PrP.

Sample pretreatment procedure

Parabens were extracted from urine by liquid-liquid extraction method. 0.5mL of spot urine sample was

transferred into a 15 mL polypropylene (PP) tube and 0.3 mL of 1 M ammonium acetate buffer

containing 100 unit/mL of ß-glucuronidase (pH not adjusted) and 5 ng of internal standard mixture

(containing 13C6-MeP, 13C6-EtP, 13C6-PrP, 13C6-BuP, D4-HeP and D4-BzP) were added. After vortexing,

samples were incubated overnight at 37°C. After incubation, 3 mL of ethyl acetate was added, and shaken

in an orbital shaker for 60 min. The organic and aqueous layers were separated by centrifugation at 3400

g for 5 min. The upper layer (organic extract) was transferred into a new 15 mL PP tube. The extraction

was repeated twice with 3 mL aliquots of ethyl acetate. 1 mL of ultrapure water was then added to the

combined extracts, which were shaken for 30 min, and centrifuged at 3400 g for 5 min. The upper layer

was pipet transferred into a new 15 mL PP tube, which was evaporated to near dryness under a gentle

stream of nitrogen. The extract was reconstituted with 0.5 mL of methanol and transferred into a liquid

chromatograph (LC) vial for instrumental analysis.

Calibration curve

A 12 point standard calibration curve within the range of 0.02 - 100 ng/mL (0.02, 0.05, 0.10, 0.20, 0.50,

1.00, 2.00, 5.00, 10.0, 20.0, 50.0 and 100 ng/mL) was made in methanol. To minimize the effect of

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instrumental ion saturation, quadratic fitting and weighting with “1/x” was used to calculate the

regression equation of the calibration curve (Analyst 1.6 Software; AB SCIEX). At each point of the

calibration curve, a 20% error from back calculated concentration was set as the acceptable limit.

Table S1. Urine sample information. NA: not available.

Table S2. Instrumental conditions for parabens analysis.

Table S3. Limit of quantification (LOQ), procedural background, spiked recovery (1.25 ng of native

compounds spiked into urine samples), and calibration range of analysis.

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Table S4. Urinary concentrations (ng/mL) of parabens in nine countries. KM: Kaplan-Meier; DR: detection rate; Min: minimum concentration;

Max: maximum concentration; 75th: 75th percentile; 95th: 95th percentile; nd: not detected. Sample number: China (n = 47), India (n = 41), Japan (n

= 36), Kuwait (n = 40), Korea (n = 26), Saudi Arabia (n = 30), Vietnam (n = 19), Greece (n = 30), the U.S. (n= 31).

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Table S5. Results of Steel-Dwass test between urinary concentrations of MeP, EtP and PrP among nine countries studied. Bold and underlined:

significance level is p < 0.05.

Table S6. Age related differences in urinary parabens concentration (ng/mL). *a: sample number.

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Table S7. Results of Steel-Dwass test between urinary concentrations of MeP, EtP, PrP and total parabens among six age-group in male and female.

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Fig. S1 Correlation between log transformed urinary concentration of MeP, EtP and PrP among nine countries (base: 10, n = 300).

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