The phthalates of interest for this study are commonly described in the literature (Benjamin et al., 2017 (link); Radke et al., 2020 (link)) and include metabolites of the parent compounds di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), and diethyl phthalate (DEP). Thus, the measured metabolites for this study were mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), mono-2-carboxymethylhexyl phthalate (MCMHP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), mono-ethylhexyl phthalate (MEP), mono-isobutyl phthalate (MiBP).
The metabolites measured in this study include 1-napthol, 2-naphthol, 2- and 3-hydroxyfluorene, 1-hydroxypyrene, and 1-, 2-, 3-, and 4-hydroxyphenanthrene. PAHs like these that are composed four rings or less (classified as low molecular weight PAHs) are more readily excreted in the urine (Yang et al., 2021 (link)), and thus were the target metabolites for this study.
Participants were instructed to collect the first urine in the morning at the end of the 24-h sample period in a provided plastic urine collection container (previously tested to be BPA and phthalate-free). Field blanks were collected alongside of urine samples for quality assurance purposes to assess potential field contamination. Urine samples were collected from all participants when the technicians returned to pick up air monitoring equipment. Samples were transported to the field laboratory and were processed within 8 h of collection. Samples were stored in the laboratory's freezer at −20 °C until shipment to Emory University within 6 months of collection (Atlanta, GA, USA).
All urine samples were randomized using a Fisher-Yates shuffling algorithm prior to analysis to reduce any potential batch effects. A 0.5-mL aliquot of urine was spiked with isotopically labeled analogues of the target phthalates and phenols and then was subjected to an enzyme hydrolysis to liberate glucuronide-bound conjugates. The hydrolysate was extracted using an ABS Elut-NEXUS solid phase extraction column, eluting with acetonitrile and ethyl acetate. The extract was concentrated to dryness and reconstituted in mobile phase for analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) using two separate injections and acquisition methods.
Analyte concentrations were calculated using isotope dilution calibration. Two quality control materials (one high and one low) and one blank sample were analyzed concurrently with each set of 28 unknown samples. Further quality assurance measures were included in the sample analyses including the analysis of NIST SRM 3672 and 3673 (one of each per 50 samples), and bi-annual participation in the German External Quality Assessment Scheme (G-EQUAS). Specific gravity was measured using a refractometer.
All metabolite concentrations were adjusted for measured creatinine concentrations to account for variability in the volume of urine and the concentrations of endogenous and exogenous chemicals from void to void (Barr et al., 2005 (link)).
The metabolites measured in this study include 1-napthol, 2-naphthol, 2- and 3-hydroxyfluorene, 1-hydroxypyrene, and 1-, 2-, 3-, and 4-hydroxyphenanthrene. PAHs like these that are composed four rings or less (classified as low molecular weight PAHs) are more readily excreted in the urine (Yang et al., 2021 (link)), and thus were the target metabolites for this study.
Participants were instructed to collect the first urine in the morning at the end of the 24-h sample period in a provided plastic urine collection container (previously tested to be BPA and phthalate-free). Field blanks were collected alongside of urine samples for quality assurance purposes to assess potential field contamination. Urine samples were collected from all participants when the technicians returned to pick up air monitoring equipment. Samples were transported to the field laboratory and were processed within 8 h of collection. Samples were stored in the laboratory's freezer at −20 °C until shipment to Emory University within 6 months of collection (Atlanta, GA, USA).
All urine samples were randomized using a Fisher-Yates shuffling algorithm prior to analysis to reduce any potential batch effects. A 0.5-mL aliquot of urine was spiked with isotopically labeled analogues of the target phthalates and phenols and then was subjected to an enzyme hydrolysis to liberate glucuronide-bound conjugates. The hydrolysate was extracted using an ABS Elut-NEXUS solid phase extraction column, eluting with acetonitrile and ethyl acetate. The extract was concentrated to dryness and reconstituted in mobile phase for analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) using two separate injections and acquisition methods.
Analyte concentrations were calculated using isotope dilution calibration. Two quality control materials (one high and one low) and one blank sample were analyzed concurrently with each set of 28 unknown samples. Further quality assurance measures were included in the sample analyses including the analysis of NIST SRM 3672 and 3673 (one of each per 50 samples), and bi-annual participation in the German External Quality Assessment Scheme (G-EQUAS). Specific gravity was measured using a refractometer.
All metabolite concentrations were adjusted for measured creatinine concentrations to account for variability in the volume of urine and the concentrations of endogenous and exogenous chemicals from void to void (Barr et al., 2005 (link)).
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