LC-MS/MS Method for Simultaneous Analysis of Growth Hormone-Releasing Peptides and Secretagogues in Human Urine

Abstract

: Growth hormone (GH)-releasing peptides (GHRPs) and GH secretagogues (GHSs) are listed in the World Anti-Doping Agency (WADA) Prohibited List. In the present study, we developed and validated a method for the simultaneous analysis of seven GHRPs (alexamorelin, GHRP-1, -2, -4, -5, -6, and hexarelin) and three GHSs (anamorelin, ibutamoren, and ipamorelin) in human urine. Method validation was performed at minimum required performance levels specified by WADA technical documents (2 ng/mL) for all substances, and the method was validated with regard to selectivity (no interference), linearity (R2 > 0.9986), matrix effects (50.0%-141.2%), recovery (10.4%-100.8%), and intra-(2.8%-16.5%) and inter-day (7.0%-22.6%) precisions. The limits of detection for screening and confirmation were 0.05-0.5 ng/mL and 0.05-1 ng/mL, respectively.

Development of a multi‐functional concurrent assay using weak cation‐exchange solid‐phase extraction (WCX‐SPE) and reconstitution with a diluted sample aliquot for anti‐doping analysis

“…To evaluate the sensitivity of the developed method, we compared it with the previous in‐house methodologies . Even if the assays were performed under different chromatographic and mass spectrometric conditions, we were still able to appraise the sensitivity of our novel method with some degree of certainty.…”

Equine metabolism of the growth hormone secretagogue MK‐0677 in vitro and in urine and plasma following oral administration

“…The metabolism of MK‐0677 in humans has been investigated not only in vitro using pooled human liver microsomes but also in vivo following an oral administration of 10 mg of MK‐0677 to a healthy male volunteer 17 . Analytical methods have also been developed for the detection of MK‐0677 in human doping samples 18–20 . More recently, the equine metabolism has been investigated in vitro using equine liver microsomes, with a total of 20 phase I and 12 phase II metabolites that were detected 21 .…”

“…17 Analytical methods have also been developed for the detection of MK-0677 in human doping samples. [18][19][20] More recently, the equine metabolism has been investigated in vitro using equine liver microsomes, with a total of 20 phase I and 12 phase II metabolites that were detected. 21 Knowledge of species-specific metabolism profiles allows the refinement of screening targets in doping control.…”

A rapid non‐target screening method for determining prohibited substances in human urine using liquid chromatography/high‐resolution tandem mass spectrometry

“…Urine samples were prepared by modifying the previously described method . Briefly, each urine sample (2 mL) was spiked with 10 μL of ISTD solution.…”

“…In addition, peptide drugs are also being added to the prohibition list. Therefore, many analytical methods for analyzing peptide drugs have been introduced in the field of drug analysis . Depending on the variety of drugs, various instrumentation and experimental methods are required for doping tests …”