Anal Bioanal Chem DOI 10.1007/s00216-014-7908-3

NOTE

Biomonitoring of infant exposure to phenolic endocrine disruptors using urine expressed from disposable gel diapers Liangpo Liu & Tongwei Xia & Xueqin Zhang & Dana Boyd Barr & Ambreen Alamdar & Jie Zhang & Meiping Tian & Qingyu Huang & Heqing Shen

Received: 6 February 2014 / Revised: 9 May 2014 / Accepted: 16 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Infant exposure to endocrine disruptors (EDs) may cause adverse health effects because of their fast growth and development during this life stage. However, collecting urine from infants for exposure assessment using biological monitoring is not an easy task. For this purpose, we evaluated the feasibility of using urine expressed from disposable gel absorbent diapers (GADs) as a matrix for biomonitoring selected phenolic EDs. GADs urine was expressed with the assistance of CaCl2 and was collected using a device fabricated in our laboratory. The analytes were extracted and concentrated using a liquid-liquid method and their hydroxyl groups were modified by dansyl chloride to enhance their chromatography and detection. Finally, the analytes were measured by highperformance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The target chemicals were bisphenol A, triclosan, 17 α-ethynylestradiol, the natural hormone estrone, and 17 βestradiol. The ratio of the CaCl2 to the urine-wetted gel absorbent, variation of the inter-urination volume, and analyte deposition bias in the diaper were assessed. Analyte blank

Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7908-3) contains supplementary material, which is available to authorized users. L. Liu : T. Xia : A. Alamdar : J. Zhang : M. Tian : Q. Huang : H. Shen (*) Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China e-mail: [email protected] X. Zhang (*) Xiamen Maternity and Child Health Care Hospital, Xiamen 361003, China e-mail: [email protected] D. B. Barr Rollins School of Public Health, Emory University, Atlanta, GA 30322, USA

values in the diapers, the sample storage stabilities, and recoveries of the analytes were also evaluated. The results showed that 70–80 % of the urine could be expressed from the diaper with the assistance of CaCl2 and 70.5–124 % of the spiked analytes can be recovered in the expressed urine. The limits of detections (LODs) were 0.02–0.27 ng/mL, well within the range for detection in human populations. Our pilot data suggest that infants are widely exposed to the selected EDs. Keywords Disposable gel absorbent diaper . Biomonitoring . Endocrine disruptors . Infant . Minipuberty

Introduction The ubiquity of human exposure to phenolic endocrine disruptors (EDs) is a widespread concern because of their potential adverse health effects, especially those related to human growth and reproduction. Bisphenol A (BPA) and triclosan (TCS) are two common examples of synthetic environmental EDs [1]. The man-made oral contraceptive 17 αethinylestradiol (EE2) and the endogenous hormones 17 βestradiol (E2) and estrone (E1) can also enter the environment via sewage sludge and wastewater systems. Toxicological and epidemiological evidence suggest that EDs exposure prenatally, perinatally, or in early childhood can result in adverse effects on brain development and neurological welfare. Therefore, assessing infant or childhood exposure to EDs in “critical windows” of development has garnered increasing interest. Biomonitoring is a powerful and often-used tool for assessing exposure to selected EDs [2, 3], but it has been largely confined to adults and toilet-trained children because of the difficulty in obtaining urine samples from diapered children. Therefore, a convenient, validated,

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and low-cost method to obtain infant urine for biomonitoring of phenolic EDs is highly desirable. We previously demonstrated that urine could be successfully expressed from disposable gel absorbent diapers (GAD) and could subsequently use to measure phthalate metabolites [4]. Expanding upon our previous study, the objective of the present study was to evaluate whether GAD-expressed urine could be used to measure selected phenolic EDs including steroid hormones, and, if so, validate the methodology.

Optimization of selected method parameters To optimize the measurement of the selected ED phenols, we evaluated and modified several methodologic parameters including: (1) the ratio of CaCl2 to the wetted absorbent, (2) variation in the urination volume, (3) the possible deposition bias of the analytes in the diaper, (4) the potential background levels of analytes in the diaper materials, (5) analyte stability during sample storage, and (6) analyte recoveries from the spiked absorbent. Ratio of CaCl2 to gel absorbent

Experimental Expressing urine from diaper absorbent Urine absorbed by the GAD was expressed according to our previously reported procedures [4]. To improve the target analyte recoveries, the absorbent was additionally rinsed with 2 mL hexane which was pooled with the previously expressed urine. The expressed urine solution was stored at −80 °C before further handling.

CaCl2 was used to shrink the wetted absorbent thus releasing the urine from the gel sorbent. To test its effectiveness, a medium size commercial diaper (designed for a 7–11 kg baby) was wetted with 250 mL of adult urine and kept at ambient temperature for 30 min. The diapers were cut and the absorbent was removed, mixed, and then split into six aliquots (about 10 g each). The ratios of CaCl2 to absorbent (1:5, 1:10, 1:20, 1:30, 1:50, and 1:70 w/w) were tested in triplicate. After mixing CaCl2 with absorbent, the analyte concentrations in the urine were measured.

Sample preparation and analysis

Urination volume variation

Five phenolic compounds were targeted: BPA, TCS, E1, E2, and EE2. Both the free and the total (free plus conjugated) forms of the analytes in the expressed urine were measured following the previously reported high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method with modifications specific to our target analytes [3]. Details of the materials, sample preparation, and instrumental analysis are presented in the Electronic supplementary materials (ESM).

To ascertain if different volumes of urine absorbed by the same amount of gel absorbent altered the analytic results, five medium-sized diapers were wetted with different volumes of the urine (i.e., 50, 100, 150, 200, and 250 mL in triplicate) and then urine was expressed following the optimized procedures mentioned vide supra using CaCl2 to absorbent of 1:50 (w/w). The analyte concentrations in the expressed urine were measured. The same procedure was used in the following experiments which were all carried out in triplicate. Analyte diffusion in the diaper

Calibration curves and quality control Stock solutions of phenolic standards were prepared at 10 μg/ mL methanol and stored at 4 °C. Subsequent dilutions were carried out with methanol to prepare 0.05, 0.2, 1, 5, 25, 50, and 100 ng/mL working solutions. Two method blanks, two quality control samples (adult human urine spiked with the target compounds at 5 and 50 ng/mL), and two sets of standards were inserted in each analytical batch. Considered to be the valid batch run, the measured analytes with variation ≤20 % were set for the quality control samples. To reduce any likelihood contamination, no plastic vials or other plastic materials were used in the procedure. All glassware was baked for at least 3 h at 500 °C to remove any residual phenols, other interfering chemicals or water.

When a male baby urinates in the center of a diaper, the analytes presumably diffuse evenly together with the urinary aqueous phase. To test this, a diaper was wetted from its center point (diameter approximately 1 cm) with 100 mL of adult urine using a syringe, and then the diaper was cut into five pieces from the central part (along the diaper longitude). The analyte concentrations in the five parts were compared. Analyte blank in the diaper matrix Ensuring the preanalytic and analytic integrity (e.g., preventing contamination) is one of the most important issues in trace analysis. The background analyte levels in diapers from different brands and/or different lots of the same brand were evaluated using water instead of urine. The brand with

Biomonitoring of infant exposure to phenolic endocrine disruptors

the lowest background analyte concentrations was selected to evaluate lot-to-lot differences (N=5 lots).

Analyte stability during overnight sampling procedure Recognizing that overnight diapers may remain on an infant for up to 8 h, we sought to evaluate the stability of the target analytes under these conditions. After wetting diapers with 200 mL of urine, the diapers were wrapped in aluminum foil and incubated at physiologic temperature (37 °C) for 0, 2, 3, 5, and 8 h then the samples were analyzed.

Statistical analysis Linear regression, average, standard deviation (SD), and relative standard deviations (RSDs) were calculated by using the SPSS statistical software, version 13.0 (SPSS Inc., IL, USA). The graphics were generated by using Sigma Plot 10.0 (Systat Software, Inc., Chicago, IL, USA). The analyte concentrations were log-normal (ln) transformed if they were not normally distributed. Spearman rank correlation, Mann-Whitney U tests, and Student-Newman-Keuls tests were performed to compare the significant differences of the intra- and intergroup variations. For data below the LODs, the reported value was set as one half of the LODs. All statistical analyses were twosided, and p values 6 months) Mean (SD)

p valuea

Infant (n=19) (boy) Mean (SD)

Infant (n=19) (girl) Mean (SD)

p valuea

1.00 1.81 0.68 0.90 0.75

0.35–25.3 1.31–8.22 0.22–2.54 0.34–1.06 0.25–1.30

0.85 (0.19) 1.72 (0.26) 0.74 (0.24) 0.96 (0.05) 0.84 (0.21)

1.85 (4.60) 2.10 (1.47) 0.73 (0.42) 0.89 (0.14) 0.75 (0.20)

0.13 0.68 0.21 0.17 0.37

0.84 (0.21) 2.37 (1.73) 0.80 (0.49) 0.89 (0.18) 0.79 (0.23)

2.34 (5.56) 1.61 (0.23) 0.67 (0.22) 0.94 (0.04) 0.75 (0.17)

0.03* 0.02* 0.29 0.49 0.53

1.75 3.78

0.58–76.3 0.97–163

1.25 (1.04) 4.04 (2.38)

5.18 (14.2) 19.8 (44.3)

0.02* 0.14

1.53 (0.77) 28.4 (51.9)

6.76 (17.3) 2.93 (2.17)

0.17 0.02*

1.31 1.03 0.61

0.61–5.50 0.64–1.63 0.21–0.93

1.71 (0.76) 1.03 (0.22) 0.72 (0.11)

1.43 (0.98) 1.06 (0.19) 0.59 (0.16)

0.10 0.41 0.01*

1.65 (1.10) 0.96 (0.16) 0.60 (0.17)

1.36 (0.70) 1.16 (0.17) 0.64 (0.14)

0.34 0.00** 0.58

BPA bisphenol A, TCS triclosan, E1 estrone, E2 17 β-estradiol, EE2 17 α-ethinylestradiol, SD standard deviation a

Tests of differences between groups using Mann-Whitney U tests. No ln transformation for the analysis

expression. No significant differences were observed when either the original urine or the urine expressed from the diaper was analyzed. After rinsing the absorbent with hexane [5], the recovery of TCS was significantly improved.

(4.03 ng/mL). The GM of total E1 (1.31 ng/mL) and E2 (1.03 ng/mL) in infants was significantly lower than that of adults (E1 (5.00 ng/mL) and E2 (2.28 ng/mL), respectively). The latter observation perhaps chiefly reflects the life stage differences of natural hormone production between adults and infants.

Phenolic EDs in infant urine Previous researchers have reported that our target phenols were prevalent in urine samples collected from the adults and older children [2]. However, scarce infant data had been reported. Therefore, the present work serves as the first important step to enable routine biomonitoring of infants to these ED phenols. To demonstrate the viability of our method for testing infant, we conducted a pilot biomonitoring survey of infant urine collected via GADs and analyzed according to our method. Descriptive statistics of infant urine biomarker levels are shown in Table 1. All the target analytes investigated were detected in the infants. The detection rates were similar to our previously reported adult data [3], indicating that infants are commonly exposed to these pollutants in China. TCS possessed the highest geometric mean (GM) concentration followed by total BPA, E1, E2, and EE2, respectively. When comparing infants to adults [3], the profiles of the phenols were different (ESM, Table S4). Infants had significantly higher levels of urinary BPA and EE2 than adults (total BPA 1.75 vs. 0.76 ng/mL with p=0.004; total EE2 0.61 vs. 0.48 ng/mL with p=0.002; MannWhitney U tests). The GM of total TCS in infants (3.78 ng/mL) was slightly lower than those of adults

Conclusion Through a series of single-factor experiments, we concluded that commonly used commercial disposable diapers can be a viable matrix used to monitor infant exposure to phenolic EDs. Our pilot data further suggests that infants are commonly exposed to these EDs in China. The combined analysis of the environmental EDs and steroid hormone metabolites may offer the novel approach to assess pollutant effects on infant hormone surge (i.e., minipuberty). Further, our method may serve as a conduct to promote the inclusion of infants in larger epidemiologic studies.

Acknowledgments We sincerely thank all the participating parents who provided their infants diaper samples. The present work was sponsored by the Xiamen Science and Technology Foundation (3503Z20124025), Fujian Nature Science of Foundation (2013J01063), Ningbo Science and Technology Fund (2013A610189) and CAS/SAFEA International Partnership Program for Creative Research Teams (KZCX2YW-T08). Conflict of interest The authors declare no conflict of interest.

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References 1. Biedermann S, Tschudin P, Grob K (2010) Transfer of bisphenol A from thermal printer paper to the skin. Anal Bioanal Chem 398(1): 571–576 2. Zhang T, Sun H, Kannan K (2013) Blood and urinary bisphenol A concentrations in children, adults, and pregnant women from China: partitioning between blood and urine and maternal and fetal cord blood. Environ Sci Technol 47:4686–4694

3. Xia T, Liu L, Zhang W et al (2012) Determination of phenolic endocrine disrupting chemicals in human urine by high performance liquid chromatography tandem electrospray ionization mass spectrometry. J of Instrum Anal 32(2):211–217 4. Liu L, Xia T, Guo L, Cao L et al (2012) Expressing urine from a gel disposable diaper for biomonitoring using phthalates as an example. J Expo Sci Environ Epidemiol 22:625–631 5. Ye X, Zhou X, Bishop AM et al (2010) Does the composition of urine change when collected from disposable diapers and other absorbent materials quest. J Expo Sci Environ Epidemiol 20(7):644–649

Biomonitoring of infant exposure to phenolic endocrine disruptors using urine expressed from disposable gel diapers.

Infant exposure to endocrine disruptors (EDs) may cause adverse health effects because of their fast growth and development during this life stage. Ho...
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