In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

A mathematical method for calibrating the signal drift in liquid chromatography - mass spectrometry analysis.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) has become the most versatile analytical tool for profiling small-molecule compounds and increasingly been applied in many fields. Nevertheless, LC-MS based quantification still face some challenges, such as signal drift in LC-MS, which may affect the validity of the obtained data and lead to misinterpretation of biological results. Here, we established a calibration method known as "RIM" to compensate the signal drift of LC-MS. To this end, a mixture of d4-2-dimethylaminoethylamine (d4-DMED)-coded normal fatty acids (C5-C23) was used as calibrants to construct RIM calibration. With the addition of calibrants, not only the MS signal drift, but also the mass accuracy and LC retention time can be calibrated, thereby improving the reliability of quantitative data. The effectiveness of RIM was carefully validated using a human serum extract spiked with 34 standards and then RIM was applied for rat brain untargeted metabolome research. In addition, to expand the functionality and flexibility of RIM for data handling, we generated a MATLAB-based RIM program, which implements the above concepts and allows automatic data process. Taken together, the proposed RIM method has potential application in large-scale quantitative study of complex samples.