Doping control analysis of TB-500, a synthetic version of an active region of thymosin β4, in equine urine and plasma by liquid chromatography–mass spectrometry

CJC‐1295 is a 30 amino acid peptide‐based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance‐enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC‐1295 remains active in the bloodstream for significantly longer than non‐conjugated peptide‐based drugs that are rapidly excreted. Conjugation of CJC‐1295 to plasma proteins prevents its detection by top‐down mass‐spectrometry‐based peptide screening protocols as it effectively becomes a macromolecular protein with an undefined molecular weight. Using a pair of monoclonal antibodies raised against the CJC‐1295 peptide, we present an immuno‐polymerase chain reaction (I‐PCR) assay that is capable of detecting the CJC‐1295‐protein conjugate at concentrations down to 0.8 pg/mL. Detection of endogenous equine GHRH necessitated a screening threshold for CJC‐1295 in equine plasma of 50 pg/mL. The effectiveness of the assay for controlling the illicit use of CJC‐1295 was confirmed in equine blood samples after administration in thoroughbred race horses.

Section: Resultssupporting

confidence: 70%

“…The rapid clearance of mGRF 1-29 from plasma is similar to that observed in other peptide-based drugs administered to horses. 18,19 FIGURE 5 Background levels of CJC-1295 screening in equine plasma. To determine the range for background signals in plasma, 974 equine plasma samples were tested in the I-PCR CJC-1295 assay.…”

Section: Equine Administration Studiesmentioning

confidence: 99%

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

Timms

Ganio

Forbes

et al. 2018

“…These levels remained elevated after administration for up to 6 days and 14 days, respectively . The long biological half‐life of CJC‐1295 makes it an attractive analytical target compared to most other peptide based‐drugs which have a biological half‐life of 2 hours or less …”

Section: Discussionmentioning

confidence: 99%

“…3 The long biological half-life of CJC-1295 makes it an attractive analytical target compared to most other peptide baseddrugs which have a biological half-life of 2 hours or less. 12,13 While sold as a peptide-based drug, the ability of CJC-1295 to covalently bind plasma proteins means the drug behaves like a protein-based doping agent, rather than a peptide, with increased stability and reduced renal excretion. By the same logic, CJC-1295 needs to be analyzed by methods developed for protein-based doping agents, rather than those typically used to detect peptide-based drugs.…”

Section: Discussionmentioning

confidence: 99%

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

Timms

Ganio

Steel

2019

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.

“…14 The considerable attention to small bioactive peptides has led to the constant development and modification of methods for their detection. Thus, some methods for anti-doping analysis of GnRH, 15 its 3 synthetic analogues (buserelin, triptorelin, leuprolide), 16 and other peptides, such as GHRPs [16][17][18][19][20][21] and TB-500 22 in urine were developed. As was shown in various in vivo 17,[23][24][25] and in vitro 20,24,[26][27][28] studies, prohibited substances metabolites identification is of considerable importance, so some of these methods include not only the parent substance but also target metabolites.…”

Section: Introductionmentioning

confidence: 99%

Determination of GnRH and its synthetic analogues' abuse in doping control: Small bioactive peptide UPLC–MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable biomarkers

Zvereva¹,

Dudko²,

Dikunets³

2017

Gonadotropin-releasing hormone (GnRH) and its small peptide synthetic analogues are included in Section S2 of the World Anti-Doping Agency (WADA) Prohibited List as they stimulate pituitary luteinizing hormone (LH) and testicular testosterone (T) secretion. Both the following approaches can be applied for determination of abuse of these peptides: direct identification of intact compounds and their metabolites in athletes' biofluids and evaluation of LH and T concentrations as mediate markers of drug intake. To develop an effective concept for GnRH and its analogues determination in anti-doping control, in vitro and in vivo studies were conducted. A new method was applied to the evaluation of the slow-release profile of buserelin, goserelin, and leuprolide biodegradable microspheres after the intramuscular injection in male volunteers. Eight metabolites of 10 GnRH analogues were identified after incubation with human kidney microsomes, most of them were leuprolide degradation products. Obtained data were added into ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for GnRH analogues determination. The detection time windows for administered peptides and their metabolites in urine samples were evaluated with 2 sample preparation techniques: dilute-and-shoot and solid-phase extraction. To support the second hypothesis, the measurement of LH and the main parameters of the steroid profile were performed in urine samples. Just 1 compound among those investigated resulted in the LH concentration dropping to non-physiological levels. Thus, for doping-control purposes, monitoring of hormone levels fluctuations could be applied only together with longitudinal passport steroid profile data.