Identification of gonadotropin‐releasing hormone metabolites in greyhound urine

Choi

Drug Testing and Analysis

et al. 2020

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).

Section: Methods Applicabilitymentioning

confidence: 99%

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

Choi

Drug Testing and Analysis

et al. 2020

Drug Testing and Analysis

“…87 Proper collection protocols must be followed to ensure substitution or contamination cannot occur, notwithstanding substitution with human urine can easily be detected. 87 Urine was used as biological matrix to target gonadotropin-releasing hormone detection by LC-MS 348 ; boldenone, danazol, ethylestrenol, mesterolone, methandriol, nandrolone, and norethandrolone by GC-MS 344 ; methyltestosterone by GC-MS 343 ; SARM by LC-MS 49 ; chlorpropamide by fluoroimmunoassays 349 ; flunixin by ELISA 341 ; carprofen and main metabolites by GC-MS 342 ; and phenylbutazone and lignocaine by GC-MS, in this case using both urine and blood. 143 The tail was considered the best site for hair collection.…”

Section: Greyhounds (Canis Familiaris)mentioning

confidence: 99%

Doping detection in animals: A review of analytical methodologies published from 1990 to 2019

Moreira

Carmo

Pinho

et al. 2021

Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti‐doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices published from 1990 to 2019 were considered. A total of 317 studies were included, of which 298 were related to horses, demonstrating significant advances toward the development of doping detection methods for equine sports. However, analytical methods for the detection of doping agents in sports involving other species are lacking. Due to enhanced accuracy and specificity, chromatographic analysis coupled to mass spectrometry detection is preferred over immunoassays. Regarding biological matrices, blood and urine remain the first choice, although alternative biological matrices, such as hair and feces, have been considered. With the increasing number and type of drugs used as doping agents, the analytes addressed in the published papers are diverse. It is very important to continue to detect and quantify these drugs, recognizing those that are most frequently used, in order to punish the abusers, protect animals' health, and ensure a healthier and genuine competition.

“…The measurement of GnRH in urine may circumvent this problem. In greyhound dogs and humans, GnRH metabolites have been detected in urine, but intact GnRH in urine has only been detected after [but not prior to] administration of GnRH, using mass spectrometry [ 77 , 78 ]. Similarly, NOX 1255 (a GnRH spiegelmer) was unable to detect GnRH in the urine of healthy horses, but it detected GnRH one hour after GnRH administration [ 79 ].…”

Section: Gnrh Aptamersmentioning

confidence: 99%

Using Aptamers as a Novel Method for Determining GnRH/LH Pulsatility

Izzi‐Engbeaya

Abbara

Cass

et al. 2020

IJMS

Aptamers are a novel technology enabling the continuous measurement of analytes in blood and other body compartments, without the need for repeated sampling and the associated reagent costs of traditional antibody-based methodologies. Aptamers are short single-stranded synthetic RNA or DNA that recognise and bind to specific targets. The conformational changes that can occur upon aptamer–ligand binding are transformed into chemical, fluorescent, colour changes and other readouts. Aptamers have been developed to detect and measure a variety of targets in vitro and in vivo. Gonadotropin-releasing hormone (GnRH) is a pulsatile hypothalamic hormone that is essential for normal fertility but difficult to measure in the peripheral circulation. However, pulsatile GnRH release results in pulsatile luteinizing hormone (LH) release from the pituitary gland. As such, LH pulsatility is the clinical gold standard method to determine GnRH pulsatility in humans. Aptamers have recently been shown to successfully bind to and measure GnRH and LH, and this review will focus on this specific area. However, due to the adaptability of aptamers, and their suitability for incorporation into portable devices, aptamer-based technology is likely to be used more widely in the future.