Abstract:

Phthalates (PAEs) are endocrine-disrupting chemicals that are widely present in everyday life and enter the human body through various pathways. The release of PAEs into the environment through pathways that include leaching, evaporation, abrasion, and the use of personal care products exposes humans to PAEs via ingestion, inhalation, and dermal absorption. Pregnant women, as a particularly vulnerable population, risk adverse newborn growth and development when exposed to PAEs. While the concentrations of PAEs in urine reflect recent exposure levels in humans, urinary levels of phthalate metabolites (mPAEs) are commonly used as biomarkers of internal exposure owing to the relatively short biological half-lives of PAEs (<24 h). In this study, we developed a solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for simultaneously detecting eight mPAEs in the urine of pregnant women. Urine samples were enzymatically hydrolyzed with β-glucosidase and then purified using the Bond Elut Plexa SPE column, with subsequent elution, concentration, and redissolved performed prior to HPLC-MS/MS. Separation was achieved using an Agilent Eclipse Plus C18 column (100 mm×3 mm, 3.5 μm), with gradient elution performed using 0.1% acetic acid aqueous solution and 0.1% acetic acid acetonitrile as mobile phases. Multiple reaction monitoring (MRM) mode was used for detection, with quantification performed using the internal-standard method. Good linearities were obtained in the range of 0.1-200 ng/mL for the eight mPAEs, with limits of detection (LODs) and quantification (LOQs) of 0.015-0.048 and 0.050-0.160 ng/mL, respectively. The eight mPAEs exhibited recoveries of 80.2%-99.7% at three spiked levels (1, 10, and 50 ng/mL). This method was subsequently used to analyze the eight mPAEs levels in urine samples of 497 pregnant women from the Ezhou Maternity and Child Health Care Hospital. The participants exhibited widespread exposure to PAEs, with monobutyl phthalate (MBP) showing the highest median level of 104.46 ng/mL, and monobenzyl phthalate (MBzP) showing the lowest (0.22 ng/mL). In addition, this study assessed neonatal birth outcomes. Linear regression modeling revealed that gestational age decreased by 0.11 weeks (95% confidence interval (CI): -0.18--0.03) for every natural-log (ln) increase in the level of monoethyl phthalate (MEP) in urine samples of pregnant woman. Moreover, the birth weight decreased by 39.28 g (95% CI: -76.48--2.09) and 39.62 g (95% CI: -73.73--5.52), for every ln increase in mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono(2-ethylhexyl) phthalate (MECPP) levels, respectively. The developed method is characterized by its simplicity, low LODs, high accuracy, and precision. This study provides clear evidence that PAE exposure during pregnancy negatively affects newborn growth and development by measuring the levels of eight mPAEs in the urine of pregnant women and linking these findings to neonatal outcomes. Further large-scale cohort studies are needed to validate these findings, along with mechanistic studies using animal models or in-vitro systems that elucidate the biological pathways through which mPAEs contribute to adverse birth outcomes.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), phthalate metabolites (mPAEs), urine samples of pregnant women, birth outcomes