A high‐throughput LC‐MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography

Abstract

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.

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) – sample preparation, mass spectrometric detection, and data review

“…[1][2][3] In general, the combination of direct urine injection, two-dimensional liquid chromatography, DMSO-assisted ESI, high resolution mass spectrometric detection and a tailored reporter template provides a high-throughput initial testing assay for peptidic and peptiderelated analytes. [1][2][3] In general, the combination of direct urine injection, two-dimensional liquid chromatography, DMSO-assisted ESI, high resolution mass spectrometric detection and a tailored reporter template provides a high-throughput initial testing assay for peptidic and peptiderelated analytes.…”

“…While the first analytical methods were based on sophisticated and laborious sample preparation procedures [1][2][3] [eg, solid-phase extraction (SPE)], more recent publications demonstrated the fitness-for-purpose of simplified approaches 4,5 (eg, dilute-and-inject approaches). While the first analytical methods were based on sophisticated and laborious sample preparation procedures [1][2][3] [eg, solid-phase extraction (SPE)], more recent publications demonstrated the fitness-for-purpose of simplified approaches 4,5 (eg, dilute-and-inject approaches).…”

Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP‐1, GHRP‐2, GHRP‐6, Hexarelin, and Ipamorelin

“…It has been demonstrated that GHRPs are rapidly eliminated from blood after intravenous administration . Recently some methods for their effective detection in urine were developed . The routine screening for GHRPs was successfully implemented for the analyses of more than 1000 official sample during XXII Winter Olympic Games in Sochi in 2014 .…”

Detection of GHRP‐2 and GHRP‐6 in urine samples from athletes

“…They mimic the biological action of the endogenous hormone ghrelin to induce the secretion of growth hormone (GH) from the pituitary. [5,6] Since use of GHRP-2 is only approved in Japan, few human administration studies have been reported. GHRP-2, GHRP-6, ipamorelin and hexarelin have been detected in vials confiscated from athletes in Germany, Belgium, and Australia.…”