A mixed-mode chromatographic separation method for the analysis of dialkyl phosphates

While reversed-phase (RP) liquid chromatography can separate a wide range of analytes, for strongly acidic compounds such as environmentally relevant dialkyl phosphates (DAPs), this remains a challenge because they have low affinity for standard RP columns or they exhibit inferior peak shapes. Mixed-mode chromatographic (MMC) columns, which contain both RP and ion-exchange functionalities, can address these resolution problems. However, using current MMC separation approaches, analyte peaks are relatively broad as compared to conventional RP chromatography. Herein we present an enhanced MMC-based UHPLC/ESI-MS method for the analysis of DAPs. In contrast to commonly available MMC-based methods, we applied the MMC Luna® Omega PS C18 column that was conditioned by 0.1% formic acid and equilibrated with the initial mobile phase before sample injection. This conditioning step tremendously improved the retention and separation of the DAPs, especially for those with high water solubility and shorter carbon chain lengths. Using water/methanol (95 v/5 v) and ammonium acetate in methanol as the mobile phases, nine DAPs could be baseline resolved with very sharp peaks, including the shorter-chain dimethyl phosphate, diethyl phosphate and bis(2-chloroethyl) phosphate. Other columns were examined to facilitate method optimization, and to identify stationary phases with the ability to separate DAPs as well as to elucidate the retention and separation mechanisms. With this novel UHPLC and post-column dication ion-pairing ESI-MS/MS method, instrumental detection limits as low as 0.01 ng/mL level were achieved. Representing other strongly acidic analytes, the short-chain perfluoroalkyl acid, perfluorobutyl sulfonic acid could also be analyzed with this method

<i>In Vitro</i> Metabolic Formation of Perfluoroalkyl Sulfonamides from Copolymer Surfactants of Pre- and Post-2002 Scotchgard Fabric Protector Products

Currently there is a scientific debate on whether fluorinated polymers (or copolymers) are a source, as a result of their degradation and subsequent formation, of perfluorinated carboxylic acids (PFCAs) and perfluorinated alkanesulfonates (PFSAs). The present study investigated whether commercially available fluorinated surfactants, such as Scotchgard fabric protector (3M Company), can be metabolically degraded, using a model microsomal <i>in vitro</i> assay (Wistar-Han rats liver microsomes), and with concomitant formation of PFCAs, PFASs, and/or their precursors. The results showed that the main <i>in vitro</i> metabolite from the pre-2002 product was perfluorooctane sulfonamide (FOSA), and coincident with the detection of the major fabric protector components, which contains the <i>N</i>-ethyl-perfluorooctanesulfonyl chemical moiety (C₈F₁₇SO₂N­(C₂H₅)−); the main <i>in vitro</i> metabolite of the post-2002 product was perfluorobutane sulfonamide (FBSA), which was coincident with the detection of the major fabric protector components, and contains the <i>N</i>-methyl-perfluorobutanesulfonyl chemical moiety (C₄F₉SO₂N­(CH₃)−). FOSA or FBSA metabolite concentrations increased over the 0–60 min microsomal incubation period. However, concentrations of their small molecule precursors such as alkylated FOSAs or FBSAs were not detectable

In vitro metabolic activation of triphenyl phosphate leading to the formation of glutathione conjugates by rat liver microsomes

The present study investigated the metabolism of the flame retardant and plasticizer chemical, triphenyl phosphate (TPHP), in a rat liver microsome-based in vitro assay with glutathione (GSH) in order to elucidate metabolic pathways leading to formation of conjugates. A highly sensitive and efficient method was developed for the detection and characterization of GSH reactive metabolites using LC-Q-TOF-MS/MS both in the negative and positive electrospray ionization modes. Seven GSH conjugates formed as a result of microsomal incubation, which were identified as S-conjugates based on MS/MS spectra, and confirmed by subsequent time-dependent incubation assays. With the exception of hydrolysis reactions leading to formation of a diester metabolite, diphenyl phosphate (DPHP), the results demonstrated that Phase I epoxidation on phenyl ring of TPHP leading to mono- and di-hydroxylated TPHP metabolites, which can further conjugate with GSH. Depending on hydroxylated TPHP formation, an o-hydroquinone intermediate formed in vitro via Phase I metabolism, and the o-benzoquinone form reacted with GSH and also formed GSH conjugates. The present study showed that via hydroxylated TPHP Phase I formation that GSH conjugates are important Phase II metabolites for TPHP metabolism in vitro. Some GSH conjugates may be valuable candidate biomarkers for monitoring TPHP exposure in biota