Identification of Di-isononyl Phthalate Metabolites for Exposure Marker Discovery Using In Vitro/In Vivo Metabolism and Signal Mining Strategy with LC-MS Data

Abstract

Di-isononyl phthalate esters (DINPs) are endocrine-disrupting chemicals and have replaced di(2-ethylhexyl) phthalate (DEHP) as the major plasticizer for poly(vinyl chloride) (PVC) products in recent years. Exposure marker discovery of DINPs is crucial, because of their high potential for human exposure and toxicity. Here, we propose an alternative approach for tracing signals derived from stable isotope-labeled precursors with varied labeling ratios to efficiently filter probable metabolite signals. The statistical process, which involves a signal mining algorithm with isotope tracing (SMAIT), has effectively filtered 13 probable DINP metabolite signals out of the 8867 peaks in the LC-MS data obtained from incubated stable isotope-labeled precursors with liver enzymes. Seven of the 13 probable metabolite signals were confirmed as DINP structure-related metabolites by preliminary MS/MS analyses. These 7 structure-related metabolite signals were validated as effective DINP exposure markers, using urine samples collected from DINP-administered rats without time-consuming comprehensive structure identification. We propose that the 7 identified possible DINP metabolite signals of m/z 279.1, 293.1, 305.1, 307.1, 321.1, 365.1, and 375.1 are potential markers for DINP exposure and should be investigated further. The integrated approach described here can efficiently, and systematically, filter probable metabolite signals from a complex LC-MS dataset for toxic exposure marker discovery. It is a relatively low-cost/rapid workflow for exposure marker discovery.

Exposure Marker Discovery of Phthalates Using Mass Spectrometry

“…74,75,82) Using custom synthesized reference standards of speci c oxidized metabolites of DINP, DIDP and, we are now able to assess human exposure to these phthalates. 61,74,75,[82][83][84][85] Table 1 summarizes selected phthalates and their respective metabolites (as biomarkers of exposure) that are currently being investigated in …”

The potential use of diisononyl phthalate metabolites hair as biomarkers to assess long-term exposure demonstrated by a rat model

“…This protocol was based on a protocol developed in our laboratory (Hsu et al, 2011;Lin et al, 2011). Urine (100 lL) was pipetted into 2-mL Eppendorf tubes, and then 25 lL of b-glucuronidase, 10 lL of sulphatase and 100 lL of a 1 lg/mL D 4 -MINP (internal standard) in 100% methanol were added to tube.…”

“…Commercial standards of DINP metabolites were not available, so we used the phase I metabolite MINP, which we had synthesized for a previous study (Hsu et al, 2011) and used it for in vitro liver enzyme incubation to generate DINP metabolites.…”

“…The mass spectrometer settings were described as follows: spray voltage, À4500 V; nebulizer gas (Gas 1), 20 units; auxiliary gas (Gas 2), 10 units; Gas 2 temperature, 300°C; curtain gas, 10 units; collision energy, À25 eV; and dwell time, 100 ms. Precursor/product ions were detected in the negative ion mode and the formation of product ions are listed in Table 1. These parameters were based on the LC-MS/MS method developed in our laboratory that was previously reported (Hsu et al, 2011).…”

“…A previous study in our laboratory used an alternative approach in which the signals derived from stable isotope-labeled precursors with varied labeling ratios were analyzed to detect likely DINP metabolite. With this method, seven metabolite signals were identified as potential markers for DINP exposure (Hsu et al, 2011). DINP metabolites have been previously used as exposure biomarkers (Preau et al, 2010).…”