Background: A sensitive method specific for ghrelins is needed for investigations of this gastrointestinal peptide. Our aim was to develop and validate a quantitative mass spectrometry (MS) method to measure ghrelin and desacyl-ghrelin simultaneously.
Methods: After deproteinization by precipitation, we performed reversed-phase separation with a rapid 2-column online extraction design coupled to a quadrupole mass spectrometer for electrospray ionization MS detection. Chromatography was performed on a C(18) monolithic column, with ammonium acetate buffer/methanol as the mobile phase and a chromatographic run time of 6 min/sample. The 4-fold-charged ions were used for multiple reaction monitoring experiments.
Results: The method was linear with injections of 0.01-10 ng. Limits of detection and quantification were 0.02 and 0.07 microg/L for ghrelin, respectively, and 0.03 and 0.35 microg/L for desacyl-ghrelin. Intra- and interday imprecision (CVs) were 9%-4% and 12%-6% at concentrations of 0.33-5.93 microg/L for ghrelin, respectively, and 16%-6% and 15%-8% at concentrations of 1.12-10.02 microg/L for desacyl-ghrelin. The mean (SD) recoveries in plasma of added ghrelin and desacyl-ghrelin were 95.8% (12%) and 101% (1.2%), respectively. Using kinetic modeling, we determined the mean (SD) periods of half-change (t(1/2)) of ghrelin to be 156 (16) min in EDTA plasma and 49 (1) min in Li-heparin plasma. Bland-Altman analysis showed that the median differences between EIA and liquid chromatography-tandem mass spectrometry (MS/MS) for desacyl-ghrelin were -40% for plasma/serum samples and 85% for cell supernatants and for ghrelin were 6% for enriched plasma samples and 44% for cell supernatants.
Conclusion: Our HPLC-MS/MS procedure has excellent selectivity and sufficient limit of quantification to allow the monitoring of concentration-time profiles in biological matrices.