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ObjectiveTo explore the influence of maternal exposure to chlorpyrifos during pregnancy on neuro-developmental abnormalities of children at age of 2 years for health risk assessment and control of maternal chlorpyrifos exposure. MethodsThe participants of the study were 893 healthy mother-infant pairs enrolled in the Health Baby Birth Cohort in Wuhan municipality, Hubei province from January 2014 through June 2017. Urine samples (n = 2679) were collected from all the surveyed mothers at first, second, and third trimester of pregnancy for detection of 3, 5, 6-trichloro-2-pyridinol (TCPy, a specific metabolite of chlorpyrifos) by high-performance liquid chromatography coupled with tandem mass spectrometry to evaluate internal exposure of chlorpyrifos. The mental development index (MDI) and psychomotor development index (PDI) of surveyed children at age of 2 years were measured using domestically modified Bayley Scales of Infant Development (BSID-CR). The correlation between the children′s neurodevelopmental abnor-malities and maternal chlorpyrifos exposure during pregnancy was analyzed. ResultsThe mean scores of MDI and PDI were 110.0 ± 21.7 and 111.0 ± 17.1 for all the 2-year-old children and 99 (11.1%) and 23 (2.58%) of the children were assessed as having abnormal mental and psychomotor development (with the MDI or PDI score of less than 80), respectively. The median (25th percentile, 75th percentile) of urine specific gravity adjusted concentration (ng/mL) of TCPy for urine samples the mothers in the first, second, and third trimesters were 2.02 (1.30, 3.38), 1.58 (0.98, 2.45), and 1.29 (0.79, 2.05), with the detection rate of 99.4%, 98.8%, and 95.9%, respectively. After adjusting for age at delivery, pre-pregnancy body mass index, maternal education, passive smoking during pregnancy, folic acid supplementation during pregnancy, child sex, parity, breastfeeding duration, and season of urine sample collection, the results of the multifactorial generalized estimating equation analysis indicated that each two-fold increase in maternal urinary TCPy concentration at first trimester was associated with the increased risks of mental and psychomotor developmental abnormalities for all the mothers′ children of 2-year-old, with the odds ratio (OR) and 95% confidence interval (95%CI) of 1.15 (95%CI: 1.01 – 1.32) and 1.34 (95%CI: 1.04 – 1.73); while, for the mother′s boys of 2-year-old, each two-fold increase in maternal urinary TCPy concentration at first trimester was correlated with increased risk of psychomotor developmental abnormality (OR = 1.47, 95%CI: 1.09 – 1.99). ConclusionMaternal exposure to chlorpyrifos during pregnancy, especially in the first trimester, could lead to neurodevelopmental abnormalities in children at age of 2 years, with a more pronounced effect on the neurodevelopment of boys.

Pesticides are widely used in most agricultural areas to protect food crops but adversely affect ecosystems and human beings. Pesticides have attracted great public concern due to their toxic properties and ubiquitous occurrence in the environment. China is one of the largest users and producers of pesticides globally. However, limited data are available on pesticide exposure in humans, which warrants a method for quantification of pesticides in human samples. In the present study, we validated and developed a comprehensive and sensitive method for the quantification of two phenoxyacetic herbicides, two metabolites of organophosphorus pesticides and four metabolites of pyrethroid pesticides in human urine using 96-well plate solid phase extraction (SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). For this purpose, a systematic optimization of the chromatographic separation conditions and MS/MS parameters was conducted. Six solvents were optimized for the extraction and clean-up of human urine samples. The targeted compounds in the human urine samples were well separated within 16 min in one analytical run. A 1 mL aliquot of human urine sample was mixed with 0.5 mL sodium acetate buffer (0.2 mol/L) and hydrolyzed by β -glucuronidase enzyme at 37 ℃ overnight. The eight targeted analytes were extracted and cleaned using an Oasis HLB 96-well solid phase plate and eluted with methanol. The separation of the eight target analytes was performed on a UPLC Acquity BEH C 18 column (150 mm×2.1 mm, 1.7 μm) with gradient elution using 0.1% (v/v) acetic acid in acetonitrile and 0.1% (v/v) acetic acid in water. The analytes were identified using the multiple reaction monitoring (MRM) mode under negative electrospray ionization (ESI - ) and quantified by isotope-labelled analogs. Para-nitrophenol (PNP), 3,5,6-tricholor-2-pyridinol (TCPY) and cis -dichlorovinyl-dimethylcyclopropane carboxylic acid ( cis -DCCA) exhibited good linearities ranging from 0.2 to 100 μg/L, and 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxy benzoic acid (4F-3PBA), 2,4-dicholorphenoxyacetic acid (2,4-D), trans -dichlorovinyl-dimethylcyclopropane carboxylic acid ( trans -DCCA) and 2,4,5-tricholorphenoxyacetic acid (2,4,5-T) showed linearity ranging from 0.1 to 100 μg/L with correlation coefficients all above 0.9993. Method detection limits (MDLs) and method quantification limits (MQLs) of targeted compounds were in the range of 0.02 to 0.07 μg/L and 0.08 to 0.2 μg/L, respectively. The spiked recoveries of target compounds at three levels of 0.5, 5 and 40 μg/L were 91.1% to 110.5%. The inter- and intra-day precisions of targeted analytes were 2.9% to 7.8% and 6.2% to 10%, respectively. This method was applied to the analysis of 214 human urine samples across China. The results showed that all the targeted analytes, except 2,4,5-T, were detected in human urine. The detection rates of TCPY, PNP, 3-PBA, 4F-3PBA, trans -DCCA, cis -DCCA, and 2,4-D were 98.1%, 99.1%, 94.4%, 2.80%, 99.1%, 63.1% and 94.4%, respectively. The median concentration of targeted analytes in a decreasing order were: 2.0 μg/L (TCPY), 1.8 μg/L (PNP), 0.99 μg/L ( trans -DCCA), 0.81 μg/L (3-PBA), 0.44 μg/L ( cis -DCCA), 0.35 μg/L (2,4-D) and below MDLs (4F-3PBA ). For the first time, we developed a method to extract and purify specific biomarkers of pesticides from human samples based on offline 96-well SPE. This method has the advantages of simple operation, high sensitivity, and high accuracy. Moreover, up to 96 human urine samples were analyzed in one batch. It is suitable for the determination of eight specific pesticides and their metabolites in large sample sizes.