Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

Abstract

A liquid chromatography-tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05-2.0 ng/ml depending on the target analyte), specificity, recovery [[60 %, coefficient of variation (CV) \15 % except for TB500 17-23 fragment, AOD9604, and ARA290 for which recovery was \50 %), ion suppression/enhancement (\35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25°C, 2 weeks (4°C), 2 months (-20°C)], and repeatability of retention times (CV \0.1 %) and relative abundances of the selected ion transitions (CV \15 %).The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.

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

“…Our results show that GHRP-1, GHRP-6, hexarelin, and ipamorelin could not be detected using the standard elution conditions but could be detected using other conditions ( Figure 1); this was in agreement with a study conducted by Mazzarino et al. 13 We found that the standard elution condition sometimes eluted all substances within critical sample pH condition (about pH 5). Thus, when using the standard elution conditions, we should control the pH of the urine sample to be analyzed.…”

“…Therefore, almost all antidoping laboratories have reported the use of these elution conditions when analyzing GHRPs. However, Mazzarino et al 13 recently reported that some GHRPs and their metabolites could not be detected under these elution conditions. Here we compared the standard elution conditions (5% formic acid in methanol) and Mazzarino's condition (2% ammonia and 1.2% formic acid in methanol).…”

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).…”

Multianalyte LC–MS-Based Methods in Doping Control: What are The Implications for Doping Athletes?

“…Recently, multianalyte screening procedures have also been developed to detect small peptides by means of mixed-mode weak cation exchange SPE followed by LC-ESI-MS-based techniques [15][16][17]; while peptide hormones with medium-molecular weight (e.g., insulins, tesamorelin, sermorelin, CJC-1293, CJC-1295, synacthen and IGF1 analogs) were proposed to be screened for by methods based on immunoaffinity purification followed by LC-ESI-MS/MS [18].…”