Development of a Sensitive HPLC Method with Fluorescence Detection for the Determination of Motilin in Human Plasma

Abstract

Background: Motilin is a peptide-structured gastrointestinal system hormone. In this study, a sensitive HPLC-fluorescence detection method was developed and validated for the quantification of motilin in human plasma. Materials & methods: Optimization processes were carried out with the experimental design methodology. Analyses were performed on a C8 column (4.6 × 150 mm, 3.5 μm particles) using water and acetonitrile containing trifluoroacetic acid as the mobile phase. Results & conclusion: The method was linear from 2 to 200 ng/ml of motilin. The assay variability was less than 5%. The limit of quantification was found to be 1.84 ng/ml. The applicability of the developed method was successfully demonstrated by quantifying the levels of motilin in human plasma samples.

Motilin fluctuations in healthy volunteers determined by liquid chromatography mass spectrometry

“…Despite these differences in bioanalytical approaches, the concentrations generated by RIA and LC-MS/MS are similar for fasted individuals – in the 100-1000 pg/mL concentration range. The LC-MS/MS method was capable of measuring much lower motilin concentrations than achievable by LC-fluorescence after derivatisation with a fluorescent tag ( 18 ), which only measured levels of around 7 ng/mL in a single timepoint of unknown fed/fasted state. In addition to the selectivity of the LC-MS/MS approach, the ability to detect multiple peptides in a single sample makes it an advantageous analytical method from the perspective of sample volume, as a single plasma sample of 100 μL was used to measure all the peptides in parallel.…”

“…One published LC-MS/MS method for measuring motilin concentrations used rat plasma spiked with porcine motilin as the reference standard ( 16 ) but had selectivity and sensitivity problems with a detection limit of 10 ng mL -1 , considerably above the reported motilin levels measured in humans which are in the region of 0.2-0.5 ng mL -1 ( 13 , 17 ). A more recent study used LC-fluorescence to detect motilin in human plasma, but the reliability of this methodology is questionable ( 18 ) as borne out by their conclusion that endogenous motilin concentrations are in the region of 6-8 ng mL -1 , substantially higher than reported by RIA.…”