Toxicokinetics of 2-ethylhexyl salicylate (EHS) and its seven metabolites in humans after controlled single dermal exposure to EHS.

The chemical UV filter 2-ethylhexyl salicylate (EHS) is used in various personal-care products. The dermal and oral metabolism of EHS have already been targeted by different studies. However, toxicokinetic data after a single dermal exposure to EHS was missing. In our study, three volunteers were dermally exposed to a commercial EHS-containing sunscreen for 9 h with an application dose of 2 mg sunscreen per cm2 body surface area. The exposure was performed indoors, and sunscreen was applied on about 75% of the total skin area. Complete urine voids were collected over 72 h and eight blood samples were drawn from each subject. Urine samples were analyzed for EHS and seven known metabolites (5OH-EHS, 4OH-EHS, 2OH-EHS, 6OH-EHS, 4oxo-EHS, 5oxo-EHS, and 5cx-EPS) by online-SPE UPLC MS/MS. The peaks of urinary elimination occurred 10-11 h after application. The elimination half-lives (Phase 1) were between 6.6 and 9.7 h. The dominant urinary biomarkers were EHS itself, followed by 5OH-EHS, 5cx-EPS, 5oxo-EHS, and 4OH-EHS. 2OH-EHS, 6OH-EHS, and 4oxo-EHS were detected only in minor amounts. An enhanced analysis of conjugation species revealed marginal amounts of unconjugated metabolites and up to 40% share of sulfate conjugates for 5OH-EHS, 5oxo-EHS, and 5cx-EPS. The results demonstrated a delayed systemic resorption of EHS via the dermal route. Despite an extensive metabolism, the parent compound occurred as main urinary parameter. The delayed dermal resorption as well as the slow elimination of EHS indicate an accumulation up to toxicological relevant doses during daily repeated dermal application to large skin areas.

Preparation and Characterization of Protocatechuic Acid Sulfates.

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) is a phenolic acid present in plants as a secondary metabolite and is also produced in the human organism as a metabolite from the degradation of polyphenols by the intestinal microbiota, particularly of flavonoids. However, PCA, like most polyphenols, is biotransformed in the human body to different conjugates as sulfates, which are found circulating in blood and could be involved in the bioactivity of the original compound. This paper describes a simple process for the preparation of PCA monosulfates with satisfactory yields. Two compounds were obtained that were identified as PCA-3-sulfate and PCA-4-sulfate by mass spectrometry and ¹H and 13C nuclear magnetic resonance using one- and two-dimensional techniques (heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation). Differential MS fragmentation behavior and UV spectra were observed for each compound, which could be used for their identification in samples of unknown composition. The described procedure can be used for the preparation of these polyphenol metabolites in view of their use in in vivo and in vitro studies, as well as standards for their analysis in biological fluids, to contribute to the elucidation of biological effects of dietary polyphenols.

Identification of Trenbolone Metabolites Using Hydrogen Isotope Ratio Mass Spectrometry and Liquid Chromatography/High Accuracy/High Resolution Mass Spectrometry for Doping Control Analysis.

Trenbolone is a synthetic anabolic-androgenic steroid, which has been misused for performance enhancement in sports. The detection of trenbolone doping in routine sports drug testing programs is complex as methods utilizing gas chromatography/mass spectrometry are complicated by unspecific derivatization products and artifacts, and liquid chromatography/mass spectrometry-based assays have shown to allow for comparably high limits-of-detection only. The number of previously reported metabolites in human urine is limited, and most analytical methods rely on targeting epitrenbolone, trenbolone glucuronide, and epitrenbolone glucuronide. In order to probe for the presence of additional trenbolone metabolites and to re-investigate the metabolism, an elimination study was conducted. One single dose of 10 mg of 5-fold deuterated trenbolone was administered to a healthy male volunteer and urine samples were collected for 30 days. For sample processing, published protocols were combined considering unconjugated, glucuronic acid-, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid-phase extractions, liquid-liquid extractions, metabolite de-conjugation, HPLC fractionation, and derivatization. Analytical methods included gas chromatography/thermal conversion/hydrogen isotope ratio mass spectrometry combined with single quadrupole mass spectrometry as well as liquid chromatography/high accuracy/high resolution mass spectrometry of the hydrolyzed and non-hydrolyzed samples. Twenty deuterium-labeled metabolites were identified including glucuronic acid-, sulfo- and potential cysteine-conjugates, and characterized by parallel reaction monitoring experiments yielding corresponding product ion mass spectra. Main metabolites were attributed to trenbolone-diol and potential trenbolone-diketone derivatives excreted as glucuronic acid and sulfo-conjugated analytes with detection windows of 5, respectively 6 days. Further characterization was conducted with pseudo MS3 experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.

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.