Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography

Abstract

1. In this study, hydrophilic interaction liquid chromatography (HILIC), radiochemical activity monitoring and linear trap quadrupole orbitrap mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were used to identify the metabolites of a highly polar novel γ-aminobutyric acid type-B receptor agonist, lesogaberan, in rats.

2. Urine was collected from three male Wistar rats for 24 h after dosing with ¹⁴C-labelled lesogaberan (170 mg/kg, 10 MBq/kg); plasma samples were taken 2 and 24 h after dosing. Pooled samples were separated by HILIC and eluents were analysed by radiochemical activity monitoring, MS and MS/MS.

3. Only the parent compound was detected in plasma, but six metabolites (M1–M6) were detected in urine. Analysis of MS and MS/MS data and comparison with synthetic reference standards enabled the identification of the structure of each metabolite. M1 was identified as the N-acetylated species [(2R)-3-acetamido-2-fluoropropyl]-phosphinic acid, and M6 as [(2R)-3-amino-2-fluoropropyl]-phosphonic acid. Metabolites M2 and M5 were the alcohol and carboxylic acid species 3-hydroxypropyl-phosphinic acid and 3-hydroxyphosphonoyl-propanoic acid, respectively, both of which had lost the fluorine atom present in the parent compound. M3 was the corresponding carboxylic acid species retaining the fluorine atom, (2R)-2-fluoro-3-hydroxyphosphonoyl-propanoic acid. Finally M4 was identified as [(2R)-2-fluoro-3-guanidino-propyl]-phosphinic acid.

Keywords::

• Mass spectrometry
• drug evaluation
• preclinical
• metabolites
• Gastroesophageal reflux

Acknowledgement

We thank Xueqing Li for her expert assistance in performing the HPLC, radiochemical activity monitoring, MS and MS/MS analysis, and Jonas Brånalt, Tomas Gustafsson, Sverker von Unge and Maria Ölwegård-Halvarsson for making the synthetic reference compounds.

Declaration of interest

Anja Ekdahl and Ann Aurell Holmberg are employees of AstraZeneca. Neal Castagnoli Jr. has received consulting fees from AstraZeneca DMPK Center of Excellence, Global DMPK, Mölndal, Sweden. Writing support was provided by Dr Stephen Sweet of Oxford PharmaGenesis™ Ltd, and was funded by AstraZeneca R&D, Mölndal, Sweden.