Validation of an analytical method for the quantification of a marker compound and determination of its biological activities in skate skin collagen peptides

Abstract

In this study, we developed and validated a high-performance liquid chromatography/photodiode array method for the quantification of glycine-proline-hydroxyproline (GPH) as a marker compound in skate skin collagen peptides (SCPs). The accurate molecular mass and amino acid sequence of this marker were determined using a QTOF mass spectrometer. Chromatographic separations were conducted using a 95:5 isocratic mobile phase of 0.1% trifluoroacetic acid (A) and acetonitrile/methanol (1:4 v/v, B) at a flow rate of 0.3 mL/min; the separated compounds were detected at 214 nm using a Jupiter® 4 μm Proteo 90Å column. The method was validated for specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision. It was found to be linear in the range of 1.0-50.0 μg/mL, with a good correlation coefficient (R2=0.9999) and excellent specificity. The LOD and LOQ were 0.07 and 0.22 μg/mL, respectively. A recovery study determined the accuracy of the method, with an average recovery of 103.76%, 100.35% and 103.97% of the marker at 10, 15 and 25 μg/mL, respectively, and a precision study showed a relative standard deviation of less than 2%. Additionally, the DPPH and ABTS radical scavenging activities of the SCPs increased in a concentration-dependent manner, and UV-protective effect was confirmed by human keratinocyte (HaCaT cell) viability. Our study thus provides the foundation for developing functional foods or cosmetics using SCPs.