UPLC–MS-Based Procedures to Detect Prolyl-Hydroxylase Inhibitors of HIF in Urine

Abstract

This article presents newly developed screening and confirmation analytical procedures to detect the misuse of nine prolyl-hydroxylase inhibitors of the hypoxia-inducible factor: daprodustat, desidustat, FG2216, IOX2, IOX4, JNJ-42041935, molidustat, roxadustat and vadadustat, targeting either the parent drugs and/or their main metabolite(s). For the sample pretreatment, different extraction protocols and technologies were evaluated. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. The chromatographic separation was performed on a C18 column, employing water and acetonitrile, both containing 0.1% formic acid, as mobile phase. Detection was achieved using as analyzer either a triple quadrupole or an Orbitrap, with positive and negative electrospray ionization and different acquisition modes. Validation of the procedures was performed according to the ISO 17025 and World Anti-Doping Agency guidelines. The methods do not show any significant interference at the retention times of the analytes of interest. The extraction efficiency was estimated to be greater than 75% for all analytes and the matrix effect smaller than 35%. Detection capability was determined in the range of 0.25–2.0 for the screening procedure and in the range of 0.5–2.0 ng/mL for the confirmation procedure, that is, in a range of concentration small enough to reveal the abuse of the compounds considered, in case they are used as performance-enhancing agents. The repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the selected ion transitions, considered only in the case of triple quadrupole (CV% < 15), was confirmed to be fit for purpose to ensure the unambiguous identification of all the target analytes in human urine. The applicability of the newly developed methods was verified by the analysis of urine samples containing molidustat, roxadustat or daprodustat. The developed procedures enabled to detect the compounds under investigation and their main metabolites.

Metabolic studies of hypoxia‐inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control

“…IOX4 competes F I G U R E 1 Hypoxia-inducible factor (HIF) stabilisers and drug candidates with and displaces 2-oxoglutarate at the active site of prolylhydroxylase-2. 27,31 When compared with IOX2, lower HIFα induction by IOX4 was observed in the liver, heart and kidney; nevertheless, markedly higher induction was observed in the brain.…”

Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high‐resolution mass spectrometer for the purpose of doping control

“…In light of this, the use of HIF‐PHDs in horses and humans has been strictly prohibited by the International Federation of Horseracing Authorities (IFHA), 17 the Fédération Équestre Internationale (FEI), 18 and in humans by the World Anti‐Doping Agency (WADA) 19 . For the purpose of doping control, a number of analytical methods and metabolism studies for HIF‐PHDs in humans including AKB‐6548, BAY‐348, BAY85–3934, FG‐2216, FG‐4592, and GSK1278863, have been reported by different research groups 10,12,20–26 and mainly by Thevis et al 10,20–24 However, very limited number of reports for IOX4 are available so far in human and horse studies 12,27 . Philip et al recently reported the in vitro metabolic study of IOX4 together with other HIF stabilizers (i.e., IOX2 and IOX3) using equine liver microsomes and the major IOX4 metabolites were identified to be IOX4 glucuronide, O ‐desbutyl IOX4, O ‐desbutyl IOX4 glucuronide, and two forms of mono‐hydroxylated IOX4 27 .…”

“…Philip et al recently reported the in vitro metabolic study of IOX4 together with other HIF stabilizers (i.e., IOX2 and IOX3) using equine liver microsomes and the major IOX4 metabolites were identified to be IOX4 glucuronide, O ‐desbutyl IOX4, O ‐desbutyl IOX4 glucuronide, and two forms of mono‐hydroxylated IOX4 27 . Besides, Mazzarino et al reported an excellent analytical method for the detection of IOX4 in human urine but did not study the metabolic fate of IOX4 12 . In this paper, both in vitro and in vivo metabolic studies of IOX4 have been described.…”

“… 5–11 As a result, it leads to the production of various proteins such as vascular endothelial growth factor (VEGF), glycolytic enzymes, erythropoietin, and its receptor 5–11 . Those HIF signal pathways are regulated by the PHD subfamily (i.e., PHD1–3) 12 . While the HIF‐ β subunit is constitutively expressed, the HIF‐ α subunit is rapidly converted into the inactive form by PHDs together with three cofactors, that is, iron (II), 2‐oxoglutarate (2‐OG), and ascorbic acid, through post‐translational hydroxylation of the proline residues (Pro‐402 and Pro‐564) under normoxic conditions 13–16 .…”

In vitro studies of hypoxia inducible factor‐prolyl hydroxylase inhibitors daprodustat, desidustat, and vadadustat for equine doping control

“…Several studies have already been reported on the detectability and metabolic pathway of HIF stabilizers like roxadustat and FG-2216. 7,8 Mazzarino et al 9 have reported a UPLC-MS-based procedure to detect HIF stabilizers in human urine. Beuck et al 10 developed a liquid chromatography (LC)tandem mass spectrometry (MS/MS)-based analytical assay for the determination of HIF stabilizers.…”