2.2. Phthalate Measurements

Urinary samples were collected twice from each woman during the second (mean: 23.0 weeks) and third trimester clinical visits (mean: 31.9 weeks). Specific gravity (SG) was measured with a handheld refractometer at the time of urine collection. Samples were collected in sterile, phthalate-free specimen cups, transferred to cryovials, and stored at −80 °C in the study repository (UTHSC Department of Pathology) until shipment for analysis. The phthalate metabolites present at detectable concentrations in at least 70% of the measurements at both time points were included in this analysis, including mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP), mono-methyl phthalate (MMP), mono-n-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), mono-carboxy-isononyl phthalate (MCNP), mono-carboxy-isooctyl phthalate (MCOP), mono-(3-carboxypropyl) phthalate (MCPP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(4-hydroxypentyl) phthalate (MHPP) and mono(2-carboxymethylhexyl) phthalate (MCMHP). Analysis of phthalate metabolites involved enzymatic deconjugation, automated online solid phase extraction, separation with high performance liquid chromatography (HPLC), and detection by isotope-dilution tandem mass spectrometry. Process and instrument blanks were included for quality assurance. More details of the analytic method have been described elsewhere [38]. The limits of detection (LODs) were between 0.01 and 0.30 ng/mL. Measurements <LOD were replaced by LOD divided by the square root of 2 [39]. Metabolite concentrations were adjusted for specific gravity to account for urine dilution using the following Formula (1):

where P is the measured urinary phthalate concentration, SG is the specific gravity for each participant, and SGmedian is the median SG over all samples. We further calculated the summed molar concentration across all DEHP metabolites (∑DEHP) as ∑DEHP = {[MEHP × (1/278.348)] + [MEHHP × (1/294.347)] + [MEOHP × (1/292.331)] + [MECPP × (1/308.330)] + [MCMHP × (1/308.33)]}. All the metabolites and ∑DEHP were natural log transformed, to achieve an approximately normal distribution, for analyses.