Development and validation of a bioanalytical LC–MS method for the quantification of GHRP-6 in human plasma

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.

► A quantitative method for GHRP-6 in human plasma was developed and fully validated.
► 13C-labeled Alanine GHRP-6 as internal standard allowed relative quantification.
► Sample cleanup guaranteed no plasma interferences during LC–MS analysis.
► GHRP-6 is highly stable in human plasma.
► Established calibration range permitted clinical samples analysis.