Laboratory Methods

Phthalate laboratory methods were adapted from Kato et al. (2005). Ten phthalate urinary mono-ester metabolites were measured {MEP (monoethyl phthalate), MnBP (mono-n-butyl phthalate), MiBP (monoisobutyl phthalate), MBzP (monobenzyl phthalate), MCHP (monocyclohexyl phthalate), MEHP [mono(2-ethylhexyl) phthalate], MEHHP [mono(2-ethyl-5-hydroxyhexyl) phthalate], MECPP [mono(2-ethyl-5-carboxypentyl) phthalate], MEOHP [mono(2-ethyl-5-oxohexyl) phthalate], MCPP [mono(3-carboxypropyl) phthalate]}, although only MEP, MnBP, and MiBP (metabolites of DEP, DnBP, and DiBP, respectively) were the focus of this study. Urine samples were spiked with a mixture of stable isotope-labeled internal standards (Cambridge Isotopes) and enzymatically digested with glucuronidase at 37°C for 90 min. Five hundred microliters of digested sample solution were injected into an on-line solid-phase extraction (SPE) column and analyzed using a high-performance liquid chromatography/tandem mass spectrometer (HPLC-MS/MS) system (API 5000; AB Sciex). Target analytes were chromatographically separated on a Betasil™ phenyl column in a mobile phase consisting of acetonitrile and 0.1% acetic acid in gradient elution mode (Kato et al. 2005). Ionization of analytes was carried out with an electrospray ionization (ESI) source operating in negative mode. To enhance sensitivity, the mass spectrometer data were acquired using multi-period mode during chromatographic elution time.

The analytical method used to measure environmental phenols in urine has been previously described (Gavin et al. 2014). This method measures four parabens (methyl paraben, ethyl paraben, propyl paraben, and butyl paraben), BP-3, triclosan, and BPA. Urine samples were spiked with stable isotope-labeled internal standards and enzymatically de-conjugated overnight at 37°C. The digested samples were then processed by SPE using C18 cartridges, and the eluents were evaporated and reconstituted with mobile phase immediately before the analysis using a reverse-phase HPLC-MS/MS system (API 5500 QTRAP; AB Sciex). Ionization of the analytes was carried out by atmospheric pressure chemical ionization (APCI).

The r ² of calibration curves for all target analytes for both methods were ≥ 0.99. The limits of detection (LOD) for phthalates were similar to or slightly higher than those used in the NHANES analysis of the general U.S. population (LODs: MEP = 0.5 μg/L, MnBP = 0.9 μg/L, MiBP = 0.4 μg/L) but were sufficiently low to detect analytes in > 97% of the study population. The LODs for phenols were generally lower than those used in NHANES (LODs: methyl and ethyl paraben = 0.5 μg/L, butyl and propyl paraben = 0.2 μg/L, triclosan = 0.2 μg/L, BP-3 = 0.5 μg/L). Randomly selected samples (n = 8 for the phenol panel and n = 11 for the phthalate panel) were analyzed in duplicate, and the relative percent differences (RPD) between duplicate results for all analytes ranged from 0 to 19.6% for samples > LOD. Quality control samples were included in every analytical run, and the recoveries were all within 30% of the respective target values. Precision for each quality control level was good, with coefficients of variation (CV) for all analytes ≤ 15%. Field quality control included collection of 20 field blanks using highly purified water treated as urine samples, including contact with all field collection materials (e.g., urine cups, aliquotting materials, vials). All target analytes in the field blanks were below the respective LODs.

Chemical analyte concentrations were reported in ng/mL of urine. Concentrations below the limit of detection were assigned the value of LOD divided by the square root of 2 (Hornung and Reed 1990).

To account for urinary dilution, urine specific gravity was measured in the field using a handheld refractometer (PAL-10S; Atago USA Inc.). Because NHANES does not report specific gravity–corrected concentrations, we also measured creatinine in the laboratory to facilitate comparison with NHANES. Creatinine was measured using applications of a colorimetric method known as the Jaffe reaction, and based on a method commercially available (BioAssay Systems QuantiChrom Creatinine Assay Kit DICT-500) but using end point reaction measurements and absorbance value collections after 45 min of incubation time at 490 nm.