title: Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.
abstract: Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial.
authors.affiliation: Department of Proteomics, Division of Physical-Chemistry, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba.
authors.forename: Jeovanis
authors.initials: J
authors.lastname: Gil
authors.forename: Ania
authors.initials: A
authors.lastname: Cabrales
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Reyes
authors.forename: Vivian
authors.initials: V
authors.lastname: Morera
authors.forename: Lázaro
authors.initials: L
authors.lastname: Betancourt
authors.forename: Aniel
authors.initials: A
authors.lastname: Sánchez
authors.forename: Gerardo
authors.initials: G
authors.lastname: García
authors.forename: Galina
authors.initials: G
authors.lastname: Moya
authors.forename: Gabriel
authors.initials: G
authors.lastname: Padrón
authors.forename: Vladimir
authors.initials: V
authors.lastname: Besada
authors.forename: Luis Javier
authors.initials: LJ
authors.lastname: González
mesh_terms: D000409:Alanine; D002247:Carbon Isotopes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D009842:Oligopeptides / Q000032:analysis* / Q000097:blood; D010940:Plant Proteins; D012015:Reference Standards; D014157:Transcription Factors
chemical_list: D000409:Alanine; D002247:Carbon Isotopes; D009842:Oligopeptides; D010940:Plant Proteins; D014157:Transcription Factors; C083017:R-S protein, Zea mays; C041048:growth hormone releasing hexapeptide