Development and validation of a rapid reversed-phase liquid chromatography method for CnAMP1 peptide quantification in human intestinal cell lines
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Plant foods are rich sources of biologically active peptides that may have a role in the prevention of diseases. Coconut water is a valuable beverage due to its nutrient composition and the presence of bioactive compounds, such as the peptide CnAMP1. It is unknown if CnAMP1 can be absorbed into intestinal cells. We, therefore, aimed to develop and validate a simple reversed-phase liquid chromatographic method to quantify the peptide in Caco-2 and LS180 cell lysates. CnAMP1 standards (1–200 µmol/L) and spiked cell lysates were injected onto a Reprosil-Pur 120 C18-AQ column (4.6 × 250 mm) using acetonitrile:water:trifluoroacetic acid (14.0:85.9:0.1, by volume) as mobile phase in isocratic mode at flow rate of 1 mL/min. The method achieved rapid separation (total run time of 6 min), with linear response, good sensitivity (limit of detection, 8.2 ng; lower limit of quantification, 30.6 ng) and no interfering peaks. Best recoveries (84–96%), accuracies (7.6–14.8%) and precision (1.5–8%) were found for LS180 cell lysates spiked with medium (50 µmol/L) and high (100 µmol/L) amounts of the peptide. Uptake assays detected no peptides in the cell lysates; however, after the first 15-min incubation CnAMP1 underwent partial hydrolysis upon incubation with LS180 cells (29%) and extensive hydrolysis with Caco-2 cells (93%).
KeywordsCnAMP1 peptide Reversed-phase liquid chromatography Absorption Caco-2 cells LS180 cells
This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian Ministry of Education. The authors would like to thank Prof Dr Tatiane Santi-Gadelha for her support on proof reading the article.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
All authors listed have contributed to the conception, design, gathering, analysis or interpretation of data and have contributed to the writing and intellectual content of the article. All authors gave informed consent to the submission of this manuscript.
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