Quantification of hydroxyproline in small amounts of skin tissue using isocratic high performance liquid chromatography with NBD-F as fluorogenic reagent.

In order to determine the collagen content of small amounts of skin tissue, we developed a new, simple and highly sensitive method of measuring the quantity of hydroxyproline (Hyp) using isocratic high performance liquid chromatography (HPLC) with a fluorogenic agent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F). The recovery rate of Hyp and reproducibility of the assay were high, and the test was sensitive enough to detect Hyp in less than 1 mg of skin tissue. This method is clinically useful for ensuring accurate diagnosis and for monitoring specific skin conditions using small human skin samples collected in biopsies.