Phthalate metabolite measurements

First trimester urine was collected in sterile and phthalate-free specimen cups during initial recruitment visits, transferred to cryovials, and stored in freezers at <–80°C. We measured specific gravity using a handheld refractometer at the time of urine collection, which was calibrated with deionized water before each measurement. Phthalate metabolite concentrations were analyzed at two different sites. Samples from the mothers of girls were analyzed at the Environmental Health Laboratory at UW per a modified version of the Centers for Disease Control and Prevention (CDC) method 6306.03. Glucuronidated phthalate monoesters underwent enzymatic deconjugation, followed by online solid-phase extraction coupled with reversed HPLC-electrospray ionization-MS/MS to quantify the simple monoesters in urine (45). Samples from the mothers of boys were analyzed by the Division of Laboratory Sciences, National Center for Environmental Health, CDC. At the CDC, urine samples were analyzed using a modified method described in Silva et al. (46) that involved the enzymatic deconjugation of the phthalate metabolites from their glucuronidated form, automated online solid-phase extraction, separation with HPLC and detection by isotope dilution MS/MS. Process and instrument blanks as well as field blanks were run in each laboratory for quality assurance of analytical and sampling procedures. For the field blank collection, deionized water was purchased, poured into phthalate-free urine cups, and transferred with disposable pipettes to 5-mL cryovials. These blanks were then interspersed with subject samples to be shipped to laboratories.

The limit of detection (LOD) of metabolites was between 0.2 and 2.0 ng/mL for the UW samples and 0.2 and 0.6 ng/mL for the CDC samples, and all blanks were below the LOD. For participant concentrations below the LOD, a value equal to each sample’s specific LOD divided by the square root of 2 was used (47). All urinary phthalate metabolite levels were adjusted for dilution using specific gravity measurements and logarithmically transformed to normalize distributions. To calculate the molar sum of the DEHP metabolites, mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-hydroxy-hexyl phthalate (MEHHP), mono-2-ethyl-5-oxy-hexyl phthalate (MEOHP), and mono-2-ethyl-5-carboxypentyl phthalate (MECPP) were divided by their molecular weights and added: ∑DEHP metabolites = ([(MEHP/278) + (MEHHP/294) + (MEOHP/292) + (MECPP/308)] × 1000). Other metabolites measured included monoethyl phthalate (MEP), monoisobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), monobutyl phthalate (MBP), mono(carboxynonyl) phthalate (MCNP), and mono(carboxy-isooctyl) phthalate (MCOP).