Liquid chromatography–tandem mass spectrometry to determine the stability of collagen pentapeptide (KTTKS) in rat skin

The objective of this study was to develop a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine the stability of collagen pentapeptide (KTTKS), which is a subfragment of collagen and has been proved to promote the extracellular release of collagen in skin fibroblast, in rat skin. The chromatographic condition was optimized on an Acclaim C-18 column (2.1 mm × 150 mm, 3 μm) under isocratic elution using a mobile phase consisting of deionized water and acetonitrile (87:13, v/v) mixture containing 5 mM pentafluoropropionic acid as an ion-pairing reagent. The quantitation of KTTKS was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The calibration curve showed good linearity in the concentration range of 0.05–10.0 μg/mL (r2 > 0.999). The intra- and inter-day precisions were 0.8–6.5% and 2.4–5.8%, respectively, and the intra- and inter-day accuracies were 96.3–102.7% and 92.8–98.5%, respectively. The developed LC–MS/MS method was successfully applied to investigate the degradation rate and sites of KTTKS in rat skin homogenate. KTTKS was found to be very susceptible to the peptide bond cleavage by aminopeptidases present in the skin.