Development of analytical methods for the quantification of metabolites of lesogaberan in a MIST investigation

Abstract

Analytical methods were developed for the determination of six metabolites of lesogaberan to be used in quantitative determinations of metabolites according to the guidelines of Metabolites in Safety Testing. The γ-amino butyric acid type B receptor agonist lesogaberan and its metabolites are small polar molecules and hydrophilic interaction liquid chromatography was found to be a suitable separation mode. The samples were prepared using protein precipitation and negative electrospray ionization tandem mass spectrometry was used for detection. Initially, exploratory methods for six metabolites were set up for analysis of human plasma samples taken after repeated administration of a high oral dose of lesogaberan. The purpose was to establish which metabolites were present at concentrations significant for further investigation. Four of the six metabolites were then found at clearly detectable concentrations. The analytical methods for these four metabolites were further elaborated and then taken through a qualification procedure, which showed acceptable accuracy (86-114%), precision (<9%) and good linearity in the range 0.03-5 µmol/L. No interferences were seen from endogenous plasma components.

Systemic Exposure to the Metabolites of Lesogaberan in Humans and Animals: A Case Study of Metabolites in Safety Testing

“…The routes to formation of the more surprising defluorinated and guanidino metabolites have previously been discussed (Ekdahl et al, 2013). Bioanalytic methods were subsequently developed and qualified for the determination of metabolite concentration in human and animal plasma samples (Dunér et al, 2013), which is in accordance with the "tiered approach" recommended by the European Bioanalytical Forum (Timmerman et al, 2010): preliminary screening to detect metabolites, followed by the development and use of qualified and/or validated bioanalytic methods to determine absolute parent compound and metabolite exposures.…”

“…All analyses were carried out in accordance with the methods and ethical standards outlined in the relevant study protocol. Metabolite concentrations in each plasma sample were measured using LC-MS, as described previously (Dunér et al, 2013). In the human studies, concentrations of the parent compound were measured by an accredited laboratory (PRA International-Bioanalytical Laboratory B.V., Assen, Netherlands) using LC-MS and in the animal studies as described previously (Fakt et al, 2003).…”

“…1) (Ekdahl et al, 2013). Qualified methods were subsequently developed to determine their plasma concentrations (Dunér et al, 2013). Here, we report the results of investigations conducted to determine exposure to lesogaberan and these six metabolites in humans, rats, and beagle dogs.…”

“…Levels of metabolism in animals in vivo were considerably lower (data on file). This finding was communicated to the FDA, and further experiments were undertaken to identify lesogaberan metabolites and to develop methods for their quantification (Dunér et al, 2013;Ekdahl et al, 2013).…”