The effects of glycerol ingestion (GEH) on hydration and subsequent cycle ergometer submaximal load exercise were examined in well conditioned subjects. We hypothesized that GEH would reduce physiologic strain and increase endurance. The purpose of Study I (n = 11) was to determine if pre-exercise GEH (1.2 gm/kg glycerol in 26 ml/kg solution) compared to pre-exercise placebo hydration (PH) (26 ml/kg of aspartame flavored water) lowered heart rate (HR), lowered rectal temperature (Tc), and prolonged endurance time (ET) during submaximal load cycle ergometry. The purpose of Study II (n = 7) was to determine if the same pre-exercise regimen followed by carbohydrate oral replacement solution (ORS) during exercise also lowered HR, Tc, and prolonged ET. Both studies were double-blind, randomized, crossover trials, performed at an ambient temperature of 23.5-24.5 degrees C, and humidity of 25-27%. Mean HR was lower by 2.8 +/- 0.4 beats/min (p = 0.05) after GEH in Study I and by 4.4 +/- 1.1 beats/min (p = 0.01) in Study II. Endurance time was prolonged after GEH in Study I (93.8 +/- 14 min vs. 77.4 +/- 9 min, p = 0.049) and in Study II (123.4 +/- 17 min vs. 99.0 +/- 11 min, p = 0.03). Rectal temperature did not differ between hydration regimens in both Study I and Study II. Thus, pre-exercise glycerol-enhanced hyperhydration lowers HR and prolongs ET even when combined with ORS during exercise. The regimens tested in this study could potentially be adapted for endurance activities.

The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-phase extraction combined with a novel derivatization method to improve the chromatography of basic peptides. The method was validated with respect to linearity, repeatability, intermediate precision, specificity, limits of detection, limits of confirmation, ion suppression, and stability. As proof of principle, all eight GHRPs or their metabolites could be detected in urine collected from rats after intravenous administration.

The Dermorphins are a family of peptides that act as potent agonists of the opioid μ receptor. Originally identified as a seven amino acid peptide on the skin of the South American Phyllomedusa frog, peptide chemists have since developed a large number of Dermorphin variants, many with superior opioid activity to the original peptide. Dermorphins are unique among the peptide opioid agonists as they appear to have a limited ability to cross the blood brain barrier, producing effects on both the central and peripheral nervous systems. It is this ability of Dermorphins to provide central anaesthesia after intravenous or subcutaneous administration that allows their use as analogues of the opioid class of drugs. Recently, illicit use of the Dermorphin peptide in the racing industry has shown the need for an analytical method to control the use of these peptides. We present a high-throughput liquid chromatography-tandem mass spectrometry screen for 17 Dermorphin peptides in equine urine and plasma with limits of detection down to 5 pg/mL. The peptide extraction technique is also suitable for use in human urine.

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.

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening.

No abstract

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α‐neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled “Conotoxin”, was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α‐neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one‐step affinity purification method was developed for the detection of α‐neurotoxins in plasma.

Gonadotropin-releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high-resolution mass spectrometry (LC-HRMS) and data-independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnRH and analogues in plasma, combined with the analysis of urine from administration studies, reveals a common pattern of peptide catabolites that can be used to guide the design of MS-based screens for this class of drugs. This culminated in the successful detection of the peptide in two out-of-competition canine urine samples.