A Rapid Quantification of Hydroxyproline in Leather using High-Performance Liquid Chromatography - Fluorescence Detection (HPLC-FLD) Method

Abstract

In the area of leather development research, determining collagen content through measurement of 4-Hydroxyproline (Hyp) has been major challenging task due to the interference of various leather-processing chemicals. To overcome this problem, for the first time we have used a High-Performance Liquid Chromatography coupled with Fluorescence Detection (HPLC-FLD) method for accurate determination of Hyp in leather samples through chemical derivatization of Hyp with 7-chloro 4 nitro benzofuran (NBD-Cl) reagent. The HPLC-FLD analysis was performed on a PICO TAG column with an isocratic mobile phase (80 % of 0.1M, pH 7.2 sodium acetate and 20% of acetonitrile, v/v) at a flow rate of 0.7mL/min. The detection was carried out at an excitation wavelength of 465 nm and its emission at 535nm. The retention time of Hyp was found to be ~3.5 minutes and the total run time was about 10 minutes. The method validation indicated that this analytical method is precise (3-12%RSD), accurate (90-100%), the limit of detection 0.01μg/ml, the limit of quantification - 0.03 μg/ml and linear (R2 -0.9995) over the concentration range of 0.1 -2.0 μg/ml. The obtained result indicated that the assay linear range was acceptable for repeated analysis and suitable for the complete range of hydroxyproline levels present in leather samples. Compared to the traditional method (IUC 17:1980), this analytical method demonstrates higher simplicity, specificity, reproducibility, and it could be useful for certifying leather products as well as inspecting international trade in leather and hides.