An immuno polymerase chain reaction screen for the detection of CJC‐1295 and other growth‐hormone‐releasing hormone analogs in equine plasma

Self Cite

CJC‐1295 is a peptide‐based drug that stimulates the production of growth hormone (GH) from the pituitary gland. It incorporates a functional maleimido group at the C‐terminus that allows it to covalently bind plasma proteins such as serum albumin. These CJC‐1295‐protein conjugates have a much greater half‐life compared to the unconjugated peptide and are capable of stimulating GH production for more than six days in humans after a single administration. Conjugated CJC‐1295 is difficult to detect in blood by mass spectrometry due to its low abundance, high molecular weight, and conjugation to a range of different protein substrates. Previously we described a screening procedure for the detection of CJC‐1295 in equine plasma using an immuno‐PCR assay. Here we demonstrate the confirmation of CJC‐1295 in equine plasma by LC−MS/MS after immuno‐affinity capture and tryptic digestion. Using this method, CJC‐1295 was identified down to concentrations as low as 180 pg/mL in 1 mL of equine plasma.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…This experiment was performed with the approval of the University of Melbourne Animal Ethics Committee (Ethics approval No. 1413071.1) …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A method for confirming CJC‐1295 abuse in equine plasma samples by LC–MS/MS

Timms

Ganio

Steel

2019

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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.

Detection of doping peptides and basic drugs in equine urine using liquid chromatography–mass spectrometry

Flores,

Hargrave,

Clifford

et al. 2023

The abuse of prohibited agents including peptides and basic small‐molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone‐releasing peptides, peptide analgesics, beta‐2‐adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography–mass spectrometry (LC–MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide‐based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid‐phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC–MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple‐stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub‐ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.