Non-targeted Screening Of Biomarkers Of Phthalates Exposure In Human Urine Samples Using HPLC-HRMS/MS

Phthalates are a group of chemicals commonly used as plasticizers in a variety of daily consumer products.With the widespread use of phthalates in numerous everyday products,these compounds are ubiquitous in the environment and have become one of the most common groups of synthetic chemicals to which humans are exposed.Many studies have demonstrated that phthalates are considered as a kind of environmental endocrine disruptors and can affect human development and reproductive system.Human biomonitoring is an important tool for detecting environmental contaminants in the human body.Currently,the targeted liquid chromatography-tandem low resolution mass spectrometry（LC-LRMS/MS）is a commonly used approach for human biomonitoring of phthalates.However,the targeted LC-LRMS/MS is limited to measure the known biomarkers with available reference standards and cannot analyze the other contaminant present in the sample.To meet the desire to make a more comprehensive and systematical exposure assessment,it is of great importance to discovery new potential unknown biomarkers for phthalate exposure biomonitoring.and as many known and potentially unknown phthalate metabolites in human samples.As the advances in liquid chromatography and mass spectrometry,which makes the detection and identification of unknown compounds using liquid chromatography-tandem high mass spectrometry become possible and have gained much attention in human biomonitoring.Herein,the aim of this study is to develop a non-targeted screening method for rapidly screening and identification of the new phthalate exposure biomarkers using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry（HPLC-QTOF MS/MS）.（1）The fragmentation patterns of the 23 phthalate metabolites were investigated using collision-induced dissociation experiments at 10,20,and 40 e V with HPLC-QTOF MS/MS.Except observed the reported fragmentation pattern that undergoes a cleavage of bondαto carbonyl,the study also found a new fragment pattern of the side chain that is undergoing a charge-immigration from the C₁ of the R group to the benzene ring followed by loss of a molecule of CO.Based on this fragmentation pattern,the fragmentation pattern of a radical product ion at m/z134.0373 observed for phthalate metabolites was first proposed.Athough the fragmentation patterns of PAEs primary and secondary metabolites are different,from insight of the MS/MS spectra,three common specific product ions at m/z 121.0297,m/z147.0088,and m/z 165.0193 corresponding to C₇H₅O₂^-,C₈H₃O₃^-,and C₈H₅O₄^-,respectively were observed for all the phthalate metabolites.The discovery of the three common product ions provides a new strategy to quickly screen out those chromatogram peaks of phthalate metabolites in the samples.（2）The molecular and formula characteristics of phthalate metabolites were investigated and the reslut shows that the number of nominal ions are odd,mass defect<0.2,molecular composition is C≥8、H≥6,O≥4 and degree of unsaturation≥6。It provides a stragety for picking up molecular ions of the potential unknown PAEs metabolites in mass spectra.A high efficiency core-shell column allows for a fast separation of 23 PAEs metabolites in 24 min,which provides an optimum condition for separation of potential PAEs in samples.The 23 known phthalate metabolites were used to validate these two developed methods.Both of the established non-targeted screening methods have good sensitivity.（3）The correlation of the retention time and the 23 known phthalate metabolite structures was explored.After the correlation analysis for eight descriptor,a linear regression equation RT=5.9461log Kow-1.8407（R²=0.8649）was obtained for retention time prediction of phthalate metabolite.The degree of equation fit is much higher than other log Kow prediction model and the RT accuracy is higher.All the compounds are eluted with RT accuracy within±5 min and 87.0%of the selected compounds are eluted with RT accuracy within±3 min.This RT prediction model provides a useful tool to reduce the number of candidate structures in the non-targeted screening.（4）Following the suspect screening workflow,five hits were quickly identified as the metabolites of phthalates from the 2097 hits in 150 human urine samples,corresponding to mono-（4-methyl-7-carboxyheptyl）phthalate,mono-（carboxy-iso-butyl）phthalate,mono-（oxo-iso-nonyl）phthalate,mono-（hydroxy-iso-decyl）phthalate and mono-（carboxy-iso-decyl）phthalate on the suspect list.Following the unknown screening workflow,three peaks were identified as new phthalate metabolites from 777hits in 150 human urine samples,corresponding to mono-（4-hydroxy-2-butoxyethyl）phthalate,mono-（hydroxymethyl-phenyl）phthalate and mono-（hydroxyundecyl）phthalate.The two substances mono-（4-hydroxy-2-butoxyethyl）phthalate and mono-（hydroxymethyl-phenyl）phthalate identified in this study have never been reported before.Then the correlations of the eight tentatively identified compounds between the normal and infertile group were analyzed,the results showed that there was a statistically significant difference between the normal and infertile group for the suspect mono-（oxo-iso-nonyl）phthalate.This study is the first attempt to discover phthalate exposure biomarkers in human urine using non-targeted HPLC-HRMS/MS.The results of this study suggest that non-targeted screening is a useful method for discovering human phthalate exposure biomarkers and the compounds identified in this study may be appropriate biomarkers for assessing human exposure to phthalates and association with health outcomes.As the diversity of phthalate used,it is of great importance to establish the non-targeted methods for phthalate metabolites screening in human biomonitoring.