Overcoming bioanalytical challenges associated with the separation and quantitation of GSK1278863, a HIF-prolyl hydroxylase inhibitor, and its 14 stereoisomeric metabolites

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1009-1010; pp. 7 - 16
• Main Authors: Licea Perez, Hermes, Knecht, Dana, Evans, Christopher A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.01.2016
• Subjects: Barbiturates - blood; Barbiturates - isolation & purification; Barbiturates - metabolism; Chromatography, High Pressure Liquid - methods; Chromatography, Reverse-Phase - methods; Chromatography, Supercritical Fluid - methods; Cytochrome P-450 Enzyme System - metabolism; Glycine - analogs & derivatives; Glycine - blood; Glycine - isolation & purification; Glycine - metabolism; GSK1278863; High performance fraction collector LCJet system; Humans; Oxidation-Reduction; Prolyl-Hydroxylase Inhibitors - blood; Prolyl-Hydroxylase Inhibitors - isolation & purification; Prolyl-Hydroxylase Inhibitors - metabolism; Reproducibility of Results; Stereoisomeric metabolite separation; Stereoisomerism; Supercritical fluid chromatography (SFC); Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS); Ultra performance convergence chromatography (UPC2); Ultra performance convergence chromatography (UPC2); HIF; UHPLC–MS/MS; SPE; HLQ; UPC2; LLQ; PHI; SFC; High performance fraction collector LCJet system; Supercritical fluid chromatography (SFC); Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS); Stereoisomeric metabolite separation; GSK1278863; Ultra performance convergence chromatography (UPC)
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2015.11.057

•Two methodologies were developed for determination of GSK1278863 and six metabolites.•HPLC–LCJet-SFC analysis for separation of a very complex mixture of stereoisomers.•Identification of which stereoisomeric species circulate in human. GSK1278863 is an investigative drug under investigation for treatment of anemia associated with chronic kidney disease. Its metabolism is primarily metabolized by P450 enzymes where 19 unique metabolic species have been identified. These include multiple products of mono-, di-, and tri-oxygenation. Initially, two separate and complex ultra high performance liquid chromatography (UHPLC) reverse phase methodologies were developed, validated and applied to measure parent and various predominant and circulating metabolites in numerous clinical studies. However, 5 of the 6 oxidative metabolites may exist in different stereoisomeric forms, resulting in 14 separate species; therefore a chiral methodology was required to determine which stereoisomeric forms circulated in human. A variety of conventional approaches were explored, where in the end a supercritical fluid chromatography (SFC) method was required to separate this complex mixture of 14 stereoisomeric metabolites; data from these experiments provided important information on which species circulate in human. The details of these methodologies will be discussed herein.