Analytical Sciences, Talk
Quantification of ghrelin and des-acyl ghrelin in human plasma by using cubic selected reaction monitoring LC-MS
Jonathan Sidibé1, Emmanuel Varesio1, Gérard Hopfgartner1
1University of Geneva
Ghrelin is a gastrointestinal hormone peptide which stimulates appetite and growth. Two forms of the peptide were observed, the acyl-ghrelin with an octanoyl modification on the third serine residue and the des-acyl ghrelin lacking this modification. Quantification using ELISA test may over-estimates ghrelin concentration because the immunoassay may not be selective enough to differentiate ghrelin from desacyl-ghrelin or from a common precursor the prepro-ghrelin. LC-MS analysis is particularly adapted to overcome this selectivity issue especially when using the selected reaction monitoring (SRM) mode for quantitative analysis. Peptides such as ghrelin, are generally ionized, in electrospray, in several charge states (4+ to 8+) depending on the mobile phase conditions. Beside the loss in sensitivity the charge state distribution may changes over the concentrations range and sample background which affects the limit of quantification, the accuracy, the precision and the dynamic range of the assay. To overcome these limitations a method based on the sum of multiple charged states and their corresponding fragments was developed for the quantitative analysis of ghrelin and des-acyl ghrelin in human plasma.

However, limited selectivity of the SRM transitions has been observed in human plasma samples at low concentrations (100 pg/mL). Thus, a LC-MS/MS/MS method based on a triple quadrupole linear ion trap was developed, where second generation product ions from multiple charge states peptides are selected and summed (LC-SRM3/MS). The LC-SRM3/MS method was found to be linear from 50-75 to 2500 pg/mL for the ghrelins using a 0.5 mL plasma sample. The accuracies and precisions at LOQ for des-acyl ghrelin (50 pg/mL) and ghrelin (75 pg/mL) were found to be better than 91% and 2%, respectively. A partial protein precipitation method combined with a column-switching LC analysis was developed for sample clean-up and analytes separation. Blood and plasma stabilization was found to be essential for good assay performance. Additionally, because ghrelin and des-acyl ghrelin are endogenous analytes bovine plasma was used as surrogate matrix where the sequences in amino acids of the ghrelin peptides are different.

Compared to the LC-SRM/MS method the addition of an additional MS step did significantly improve the selectivity and therefore the sensitivity. The LC-SRM3/MS method could be successfully applied for the quantification of ghrelin and des-acyl ghrelin in human plasma samples using a generic sample preparation procedure.