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Simultaneous Quantification of Ghrelin and Desacyl-Ghrelin by Liquid Chromatography–Tandem Mass Spectrometry in Plasma, Serum, and Cell Supernatants

¹ Department of Pediatrics, Friedrich-Alexander-University, Erlangen, Germany.

^aAddress correspondence to this author at: Department of Pediatrics, Friedrich-Alexander-University, Erlangen, Loschgestr. 15, 91054 Erlangen, Germany. Fax 49-9131-8533714; e-mail manfred.rauh{at}kinder.imed.uni-erlangen.de.

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 C₁₈ 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 µg/L for ghrelin, respectively, and 0.03 and 0.35 µg/L for desacyl-ghrelin. Intra- and interday imprecision (CVs) were 9%–4% and 12%–6% at concentrations of 0.33–5.93 µg/L for ghrelin, respectively, and 16%–6% and 15%–8% at concentrations of 1.12–10.02 µg/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.

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