Metabolites profiling of R14 in rat urine by ultra performance liquid chromatography–quadrupole time-of-flight mass spectrometry
Introduction
NOX Inhibitor VII (R14) is a cell-permeable, selenium-to-sulfur substituted Ebselen derivative, with similar NOX2 inhibitory activity as Ebselen, but more potent and NOX2-selective than Ebselen in cell-based assays and exhibits no glutathione peroxidase activity. R14 has recently been identified as a novel agent targeting triple-negative breast cancer and has undergone preclinical drug development. In the present study, the metabolites profiling of R14 in rat urine was elucidated after a single oral dose of R14 to male Sprague-Dawley rats.
Methods
Rat urine samples were collected during 0-24 h after a single oral dose of R14 (50 mg/kg) to the rats (n=3). The samples were analyzed on an X500B QTOF mass spectrometer (SCIEX, USA) equipped with a Synergi Fusion-RP column (4 µm, 150 × 2 mm, 80 Å). Water containing 0.1% formic acid (FA) and methanol were used as gradient mobile phase A and B. The temperature and spray voltage were set at 500°C, and 5000 v, respectively. The curtain gas, collision gas (CAD), nebulizer gas (gas 1) and heater gas (gas 2) were set at 30, 10, 55 and 60 psi, respectively. TOF MS and MS/MS data were monitored using the Information-dependent-acquisition (IDA) in positive mode by SCIEX OS software 1.6.1. The ion chromatograms (EIC) of metabolites were extracted with ±0.002 Dalton. The chemical formula of metabolites was confirmed by TOF MS matching with 10 ppm mass tolerance and 20% isotope intensity tolerance using formula finder. The structures of the metabolites were proposed based on the TOF MS/MS fragments.
Results
The carbonyl group of R14 was easily hydrogenated and yielded a metabolite with a hydroxyl group (M1). R14 itself and M1 further underwent multiple phase I metabolisms, and these metabolites might further produce phase II metabolites (i.e., glucuronides and sulfates). A total of 40 metabolites of R14 were identified in the rat urine. M2 and M33 were identified as two major metabolites. M2 was the glucuronide of M1. M33 was a methylation and bi-hydroxylation metabolic product of M1.
Conclusion
R14 underwent extensive metabolism in vivo. Methylation, hydroxylation, glucuronidation and sulfation were major metabolic pathways. Further studies are warranted to understand full biotransformation processes of R14 in in vitro and in vivo models.