Establishment And Application Of Method For Detecting Phthalate Metabolites In Human Urine

Objective:To develop and validate a HPLC-MS/MS method for determination of 13 phthalate metabolites in human urine, and to study exposure levels and influencing factors of phthalates of two years old infants.Methods:(1) A method for the determination of 13 phthalate metabolites including MMP, MEP, MCHP, MiBP, MBP, MBzP, MEHP, MEHHP, MEOHP, MECPP, MOP, MCPP, MNP, in human urine, using solid-phase extraction(SPE) and isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry(MS), has been developed.(2) The questionnaires and spot urine samples were collected from 344 two years old infants in a county of Jiangsu Province from June 2011 to January 2012. The infants’general information and phthalate exposure relevant behaviors and habits were asked.(3) 13 phthalate metabolites in urine were determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).(4) As the skewed distribution of all detectable metabolites, geometric mean (GM), the median,25th and 75th percentiles were used to describe the results. Correlations between metabolites were evaluated using Spearman’s (rank) correlation coefficient analysis. The significance of the differences among individual characteristics for each metabolite levels was estimated by nonparametric tests, i.e., Kruskal-Wallis and Mann-Whitney U-test. Non-parametric tests and multiple linear regression were used to assess the influence factors of infants’phthalate exposure.Results:(1) We developed a sensitive analytical method, based on SPE followed by isotope dilution-HPLC-ESI-MS/MS, to quantitatively measure 13 phthalate metabolites in human urine. Good linear relationships were obtained in the range of 0.5μ/Lto 200μg/L with the correlation coefficients above 0.999. The limits of detection were between 0.1μg/L and 0.2μg/L. The recoveries were in the range of 94.94% to 117.81% and relative standard deviations were lower than 10%.(3) Phthalate metabolites in urine were detected of all the 344 two years old infants. Except MCHP, MOP and MiNP, the detection frequencies of the other 10 phthalate metabolites were as follows:MMP,100.0%; MEHHP,100.0%; MECPP, 100.0%; MEP,99.7%; MiBP,99.7%; MEOHP,99.7%; MBP,99.4%; MCPP,96.8%; MEHP,89.8%; MBzP,22.7%.The median levels were as follows:MMP,15.73μ/L; MEP,13.99μg/L; MBP,39.30μg/L; MiBP,29.23μg/L; MCPP,2.90μg/L; MEHP,2.88μg/L; MEHHP, 11.93μg/L; MEOHP,8.70μg/L; MECPP,50.10μg/L.(4) The consumption of commodity and food containing phthalates had statistic effect on concentrations of urinary phthalate metabolites in infants (p<0.05). The consumption of commodity and food containing phthalates might increase the infants’ phthalates exposure.Conclusion:A sensitive analytical method, based on SPE followed by isotope dilution-HPLC-ESI-MS/MS, to quantitatively measure 13 phthalate metabolites in human urine has been developed and validated.The study showed that there was a widespread exposure to phthalates among the 2 years old infants. The consumption of commodity and food containing phthalates might increase the infants’phthalates exposure.