Equine Urine Screening for IOX Compounds Using Solid Phase Extraction and Triple Quadrupole LC-MS

A. Background IOX compounds belong to the family of hypoxia inducible factor stabilizers and oxygen enhancers. “The hypoxia inducible factor pathway is the major signaling pathway responsible for cellular oxygen sensing and adaptation to a hypoxia (Günter, Ruiz-Serrano, Pickel, Wenger, & Scholz, 2017).” The hypoxia inducible factor is a transcription factor, meaning it is a protein that binds to DNA and affects the expression of genes (Khan Academy, 2020). This gives the hypoxia inducible factor pathway the ability to control the expression of several downstream genes responsible for homeostasis (Ziello, Jovin, & Huang, 2007). During normoxia, the hypoxia inducible factor proteins are constantly degraded. “In the α-subunit there is an oxygen degradation (ODD) domain, which is hydroxylated by proline-hydroxylase-2 (PHD2), rendering the subunit vulnerable (Ziello et al., 2007).” The hydroxylation is carried out by enzymes known as also known as 2-oxoglutarate dependent oxygenases. These hydroxylase enzymes are dependent upon molecular oxygen and 2-oxoglutarate as substrates to function, making them cellular oxygen sensors responsible for the regulation of the pathway (Günter et al., 2017; Pagé, Chan, Giaccia, Levine, & Richard, 2008; Yang, Sun, Wang, & Jiao, 2013). The proline hydroxylation allows the hypoxia inducible factor to bind with the von Hippel-Lindau protein. The von Hippel-Lindau is a ubiquitination ligase complex that marks the hydroxylated hypoxia inducible factor for proteasomal degradation (Haase, 2013; Pagé et al., 2008; Ziello et al., 2007). In a hypoxic state, the lack of oxygen means there is a lack of substrate for the prolyl-hydroxylase enzymes to interact with, prohibiting the degradation of the hypoxia inducible factor (Pagé et al., 2008). When stabilization occurs, the hypoxia inducible factor is free to bind to its specific genes and induce their transcription. These genes are responsible for stimulating angiogenesis and erythropoiesis. (Rabinowitz, Barrett, Rosen, & Venkatesan, 2010; Strowitzki, Cummins, & Taylor, 2019). Red blood cells contain hemoglobin, otherwise known as an oxygen binding protein. Therefore, through hypoxia, the formation of red blood cells is stimulated and an increase in oxygen levels occurs. There are several therapeutic drugs in development to take advantage of the pathway. These therapeutic compounds are hypoxia inducible factor-prolyl hydroxylase domain protein inhibitors, or HIF prolyl hydroxylase inhibitors (HIF PHIs). The purpose of developing hypoxia inducible factor prolyl hydroxylase domain inhibitors is to help patients with anemia from chronic kidney disease, anemia from chemotherapy, or anemia from a chronic disease by increasing their erythropoietin levels (Haase, 2017; Kansagra et al., 2018). As a result of the oxygen enhancing capacity of these new therapeutic compounds, it follows that they have the abuse potential as many of the other erythropoietin stimulating agents that came before. The concept of blood doping began with humans in the Olympics receiving transfusions of their own blood before competing and has since continued to advance with the creation of new blood doping substances. Human sports typically set the precedent for the newest compounds and trends used for doping purposes. Eventually, these ideas and drugs find their way into non-human sports where there are even more possibilities of what substances can be used for doping purposes and what substances can be banned. B. Statement of the Issue Since IOX compounds have been developed for the purpose of increasing erythropoietin levels in anemic patients, it is obvious why they have been chosen as doping agents within the industry of horseracing. In January of 2020, the presence of IOX-2 in two standard bred horses was first documented by the New York Equine Drug Testing Program (Shoemaker, 2020). While this has been the first known case of IOX compounds detected in racehorses, other oxygen enhancing and blood doping compounds have already been banned by the International Federation of Horseracing Authorities (IFHA), as well as the World Anti-Doping Agency (WADA) for human sports. C. Significance of the Issue IOX compounds are just one type of the many substances making their way into human sports and horseracing as new doping agents. The list of prohibited and banned substances is ever-increasing, making it harder for human and equine toxicology laboratories to constantly be developing new detection methods. For example, UIC AFTL did not have a validated method for the detection of IOX compounds in equine urine prior to the completion of this project. D. Purpose of Study The purpose of this study is to establish and validate a reproducible and reliable method for extracting and detecting IOX-1, IOX-2, FG-2216, and IOX-4 from equine urine using solid phase extraction and liquid-chromatography-tandem mass spectrometry. The validation of the method followed the guidelines provided by the UIC AFTL Standard Operating Procedure ‘Validation Requirements for Methods Using Instrumental Analysis’ #AFTL GE005-04. E. Significance of the Study Limited studies and research exist on these novel compounds and the more information that is available, the easier it is for different laboratories to quickly validate and integrate these compounds into their methods. It is important for more information on these compounds to be available to assist laboratories in staying up to date and being prepared to handle the detection with newly developed compounds that have a possibility of doping use.