Development and validation of chromatographic methods for quantification of peptid structered gastrointestinal system hormones, ghrelin and motilin in human plasma

Ghrelin and motilin are peptide structured gastrointestinal system hormones and belong to the same peptide family (ghrelin/motilin peptide family). These structurally similar molecules are known to play a role in inducing gastrointestinal motility, accelerating gastric emptying, and inducing gastric lavage. Ghrelin is also known to play an important role in the protection of the stomach and intestines as well as the regulation of growth hormone secretion and gut mobility. In this thesis, two High Performance Liquid Chromatography methods were developed and validated for the quantification of ghrelin and motilin in human plasma. Analites were derivatized with fluorescamine for fluorescence detection. Separation was performed on a C8 column (4,6 x 150 mm, 3,5 μm particles) using water and acetonitrile containing trifluoroacetic acid as a mobile phase. Gradient elution was used in both methods. The method develeloped for ghrelin and des-acyl ghrelin determination was linear in the range of 20-800 ng/mL. The quantification limits for ghrelin and des-acyl ghrelin were found 11,32 and 9,81 ng/mL, respectively. The method develeloped for motilin was found linear in the range of 2-200 ng/mL. The quantification limit for motilin was 1,84 ng/mL. Specificity, precision and accuracy of both methods were evaluated and stability of the analyte solutions in different working conditions were investigated. The application of the method developed for motilin was demonstrated by measuring the amounts of motilin in human plasma samples collected from five healthy volunteers.