Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.

The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH2 , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1-linear is a prodrug of EMP1, which is a performance-enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1-linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1-linear mainly existed in its cyclic form as EMP1, and four N-terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG-NH2 (M1), SCHFGPLTWVCKPQGG-NH2 (M2), WVCKPQGG-NH2 (M3) and VCKPQGG-NH2 (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1-linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1-linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively.

Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing.

The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike-in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.

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.

IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.

Detection of bioactive peptides including gonadotrophin-releasing factors (GnRHs) in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS2 ). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin-releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).

Doping control study of AICAR in post-race urine and plasma samples from horses.

Acadesine, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post-race equine urine and plasma samples including statistical studies of AICAR concentrations determined from 1,470 urine samples collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography-mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma samples between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood samples, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut-off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut-off to urine samples collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd.

Doping control analysis of filgrastim in equine plasma and its application to a co-administration study of filgrastim and recombinant human erythropoietin in the horse.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor regulating granulopoiesis. The recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used for the treatment of granulopenia in humans. Filgrastim is a rhG-CSF analogue and is marketed under various brand names, including Neupogen(®) (Amgen), Imumax(®) (Abbott Laboratories), Neukine(®) (Intas Biopharmaceuticals) and others. It is banned in both human and equine sports owing to its potential for misuse. In order to control the abuse of filgrastim in equine sports, a method to identify unequivocally its prior use in horses is required. This study describes an effective screening method for filgrastim in equine plasma by enzyme-linked immunosorbant assays (ELISA), and a follow-up confirmatory method for the unequivocal identification of filgrastim by analysing its highly specific tryptic peptide (1)MTPLGPASSLPQSFLLK(17). Filgrastim was isolated from equine plasma by immunoaffinity purification. After trypsin digestion, the mixture was analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Filgrastim could be detected and confirmed at 0.2ng/mL in equine plasma. The applicability of the ELISA screening method and the LC/MS/MS confirmation method was demonstrated by analysing post-administration plasma samples collected from horses having been co-administered with epoetin alfa as recombinant human erythropoietin (rhEPO) and filgrastim as rhG-CSF. rhEPO and filgrastim could be detected in plasma samples collected from horses for at least 57 and 101h respectively. To our knowledge, this is the first identification of filgrastim in post-administration samples from horses.

Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography-mass spectrometry.

In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, ß-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses.

High resolution accurate mass screening of prohibited substances in equine plasma using liquid chromatography--Orbitrap mass spectrometry.

A recent trend in the use of high resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged due to significant improvement in high resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution, and mass stability. A number of HRAMS methods have been reported for the detection of multi-drug residues in human or equine urine. As blood has become a common matrix for doping control analysis, especially in equine sports, a sensitive, fast and wide coverage screening method for detecting a large number of drugs in equine blood samples would be desirable. This paper presents the development of a liquid chromatography-high resolution mass spectrometry (LC-HRMS) screening method for equine plasma samples to cover over 320 prohibited substances in a single analytical run. Plasma samples were diluted and processed by solid-phase extraction. The extracts were then analyzed with LC-HRMS in full-scan positive electrospray ionization mode. A mass resolution of 60 000 was employed. Benzyldimethylphenylammonium was used as an internal lock mass. Drug targets were identified by retention time and accurate mass, with a mass tolerance window of ±3 ppm. Over 320 drug targets could be detected in a 13-min run. Validation data including sensitivity, specificity, extraction recovery and precision are presented. As the method employs full-scan mass spectrometry, an unlimited number of drug targets can theoretically be incorporated. Moreover, the HRAMS data acquired can be re-processed retrospectively to search for drugs which have not been targeted at the time of analysis.

Identification of recombinant human relaxin-2 in equine plasma by liquid chromatography-high resolution mass spectrometry.

Relaxin (RLX) is a peptide hormone belonging to the relaxin-like peptide family. Relaxin-2 (RLX-2), a heteromeric polypeptide consisting of an A-chain (24 amino acids) and a B-chain (29 amino acids) linked together by two inter-chain disulfide bonds, is the main circulating RLX hormone in human. Due to its ability to dilate blood vessels surrounding the smooth muscles via induction of nitric oxide resulting in the increase of blood and oxygen supplies to the muscles, it may enhance athletic performance and is therefore banned in horseracing, equestrian competitions, and human sports. In order to control the abuse of rhRLX-2, a definitive method is required to detect and confirm the presence of rhRLX-2 in biological samples. This paper describes, for the first time, the detection and confirmation of rhRLX-2 in equine plasma by liquid chromatography-high resolution mass spectrometry (LC-HRMS) after immunoaffinity extraction. rhRLX-2 could be detected at less than 0.1 ng/ml, and confirmed at less than 0.2 ng/ml in plasma samples.