Characterization of the metabolites of H3B‐6545 in vitro and in vivo by using ultra‐high performance liquid chromatography combined with electrospray ionization linear ion trap‐orbitrap tandem mass spectrometry

Abstract

H3B‐6545 is a selective ERα covalent antagonist, which has been demonstrated to be effective in anti‐tumor. To fully understand its mechanism of action, it is necessary to investigate the in vitro and in vivo metabolic profiles. For in vitro metabolism, H3B‐6545 (50 μM) was incubated with the hepatocytes of rat and human for 2 h. For in vivo metabolism H3B‐6545 was orally administered to rats at a single dose of 10 mg/kg, and plasma, urine and fecal samples were then collected. All samples were analyzed by using ultra‐high performance liquid chromatography combined with linear ion trap‐orbitrap tandem mass spectrometry (UHPLC‐LTQ‐Orbitrap‐MS) operated in positive ion mode. The structures of the metabolites were elucidated by comparing their MS and MS2 spectra with those of parent drug. A total of 11 metabolites, including a GSH adduct, were detected and structurally identified. M2, M7 and M8 were further unambiguously identified by using reference standards. Among these metabolites, M1, M5, M7 and M10 were newly found and reported for the first time. The metabolic pathways of H3B‐6545 included deamination (M8 and M9), dealkylation (M2, M3 and M10), N‐hydroxylation (M6), hydroxylation (M1 and M4), formation of amide derivatives (M5 and M7) and GSH conjugation (G1).

Identification and characterization of the metabolites of sinomenine using liquid chromatography combined with benchtop Orbitrap mass spectrometry and nuclear magnetic resonance spectroscopy

“…In addition, M10 showed prolonged chromatographic retention under the acidic mobile phase system, which may be due to the reduced alkalinity. This finding also suggested that M10 was sinomenine N ‐oxide 20,21 . To further confirm the structure of the metabolite, the incubation sample was pretreated with TiCl 3 .…”

“…Like M10, M12 showed prolonged chromatographic retention under the acidic mobile phase system, which may be due to the reduced alkalinity. 20,21 This finding also suggested that N-hydroxylation occurred at nitrogen atom. N-desmethyl-sinomenine (<10%).…”

“…The ion at m/z 60.0452 suggested that oxygenation may occur at the nitrogen atom. Like M10, M12 showed prolonged chromatographic retention under the acidic mobile phase system, which may be due to the reduced alkalinity 20,21 . This finding also suggested that N ‐hydroxylation occurred at nitrogen atom.…”

“…This finding also suggested that M10 was sinomenine Noxide. 20,21 To further confirm the structure of the metabolite, the incubation sample was pretreated with TiCl 3 . After reaction, this metabolite was found to be reduced by TiCl 3 .…”