Using a high-resolution mass spectrometry-based metabolomics strategy for comprehensively screening and identifying biomarkers of phthalate exposure: Method development and application.

Di-(2-propylheptyl) phthalate (DPHP) is an alternative plasticizer that can replace other phthalates currently being scrutinized, and its use and production volumes are increasing. This study aimed to develop a high-resolution mass spectrometry (HRMS)-based metabolomics strategy to comprehensively screen urinary biomarkers of DPHP exposure and filter out potentially useful DPHP exposure markers for human exposure assessments. This strategy included three stages: screening of biomarkers, verification of dose-response relationships in laboratory animals, and application in human subjects. The multivariate data analysis method known as orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to screen and find meaningful signals in an MS dataset generated from urine samples collected from DPHP-administered rats. Thirty-six MS signals were verified as exposure marker candidates by assessing dose-response relationships in an animal feeding study. A biotransformation product of DPHP, mono-(2-propyl-7-dihydroxy-heptyl) phthalate, was suggested as a DPHP exposure marker for general human exposure assessments after the human application study and chemical structure identification. Three previously oxidized DPHP biotransformation products might be suitable for human exposure assessments in high-level exposure groups but not in the general population due to their low sensitivity.

Profiling and comparison of toxicant metabolites in hair and urine using a mass spectrometry-based metabolomic data processing method.

Urine and hair are used for assessing human exposure to toxicants. Urine tests can show acute toxicant exposure. Hair analysis can be used to determine chronic toxicant exposure after months to years; however, compared to urine, hair analysis in exposure assessments is much less frequently investigated. Urine and hair are different matrices, and their mechanisms of toxicant metabolite incorporation are different. The toxicant metabolites present in urine and hair may also be different. To clarify this issue, a procedure was developed to identify toxicant metabolites in rat samples using a mass spectrometry-based metabolomic data processing method. Di-(2-propylheptyl) phthalate (DPHP), an industrial plasticizer, was used as the model toxicant. The developed procedure identified not only known DPHP metabolites (mono-(propyl-6-oxo-heptyl) phthalate, mono-(propyl-6-hydroxyheptyl) phthalate, and mono-(propyl-6-carboxyhexyl) phthalate) but also novel metabolites that were structurally related to DPHP in the rat samples, indicating that the developed procedure successfully identified toxicant metabolites in in vivo samples. Among the 62 tentative metabolites identified from the 7th-day urine and the 28th-day hair samples, 33 were detected in only the urine samples, 19 were detected in only the hair samples, and 10 were identified in both the urine and hair samples. A total of 15 out of the 62 metabolites were confirmed as DPHP structure-related metabolites based on MS/MS analysis. Among the 15 DPHP structure-related metabolites, only 2 metabolites were present in both the urine and hair samples. These results suggested that the metabolites identified in urine could not be applied to exposure assessments based on hair analysis.

Exposure marker discovery of di(isononyl)cyclohexane-1,2-dicarboxylate using two mass spectrometry-based metabolite profiling data processing methods.

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is a plasticizer used in polyvinyl chloride (PVC) products, such as toys and food packaging. Because the use of DINCH is on the rise, the risk of human exposure to this chemical may likewise increase. Discovering markers for assessing human chemical exposure is difficult because the metabolism of chemicals within humans is complex. In this study, two mass spectrometry (MS)-based metabolite profiling data processing methods, the mass defect filter (MDF) method and the signal mining algorithm with isotope tracing (SMAIT) method, were used for DINCH metabolite discovery, and 110 and 18 potential DINCH metabolite signal candidates were discovered, respectively, from in vitro DINCH incubation samples. Of these, the 21 signals were validated as tentative exposure marker signals in a rat model. Interestingly, the two methods generated rather different sets of DINCH exposure markers. Five of the 21 tentative exposure marker signals were verified as the probable DINCH structure-related metabolite signals based on their MS/MS product ion profiles. These five signals were detected in at least one human urine sample. Of the five probable DINCH structure-related metabolite signals, two novel signals might be suitable exposure markers that should be further investigated for their application in human DINCH exposure assessments. These observations indicate that the MDF and SMAIT methods may be used to discover a relatively different set of potential DINCH exposure markers.

Identification of urinary biomarkers of exposure to di-(2-propylheptyl) phthalate using high-resolution mass spectrometry and two data-screening approaches.

Di-(2-propylheptyl) phthalate (DPHP) is a plasticizer used in polyvinyl chloride and vinyl chloride copolymer that has been suggested to be a toxicant in rats and may affect human health. Because the use of DPHP is increasing, the general German population is being exposed to DPHP. Toxicant metabolism is important for human toxicant exposure assessments. To date, the knowledge regarding DPHP metabolism has been limited, and only four metabolites have been identified in human urine. Ultra-performance liquid chromatography was coupled with Orbitrap high-resolution mass spectrometry (MS) and two data-screening approaches-the signal mining algorithm with isotope tracing (SMAIT) and the mass defect filter (MDF)-for DPHP metabolite candidate discovery. In total, 13 and 104 metabolite candidates were identified by the two approaches, respectively, in in vitro DPHP incubation samples. Of these candidates, 17 were validated as tentative exposure biomarkers using a rat model, 13 of which have not been reported in the literature. The two approaches generated rather different tentative DPHP exposure biomarkers, indicating that these approaches are complementary for discovering exposure biomarkers. Compared with the four previously reported DPHP metabolites, the three tentative novel biomarkers had higher peak intensity ratios, and two were confirmed as DPHP hydroxyl metabolites based on their MS/MS product ion profiles. These three tentative novel biomarkers should be further investigated for potential application in human exposure assessment.

Clay-catalyzed reactions of coagulant polymers during water chlorination.

The influence of suspended clay/solid particles on organic-coagulant reactions during water chlorination was investigated by analyses of total product formation potential (TPFP) and disinfection by-product (DBP) distribution as a function of exchanged clay cation, coagulant organic polymer, and reaction time. Montmorillonite clays appeared to act as a catalytic center where the reaction between adsorbed polymer and disinfectant (chlorine) was mediated closely by the exchanged clay cation. The transition-metal cations in clays catalyzed more effectively than other cations the reactions between a coagulant polymer and chlorine, forming a large number of volatile DBPs. The relative catalytic effects of clays/solids followed the order Ti-Mont > Fe-Mont > Cu-Mont > Mn-Mont > Ca-Mont > Na-Mont > quartz > talc. The effects of coagulant polymers on TPFP follow the order nonionic polymer > anionic polymer > cationic polymer. The catalytic role of the clay cation was further confirmed by the observed inhibition in DBP formation when strong chelating agents (o-phenanthroline and ethylenediamine) were added to the clay suspension. Moreover, in the presence of clays, total DBPs increased appreciably when either the reaction time or the amount of the added clay or coagulant polymer increased. For volatile DBPs, the formation of halogenated methanes was usually time-dependent, with chloroform and dichloromethane showing the greatest dependence.