Analysis by high-performance gel permeation chromatography of hyaluronic acid in animal skins and rabbit synovial fluid.

The simultaneous analysis of the molecular weight and concentration of hyaluronic acid in biological samples using high-performance liquid chromatography with two gel permeation columns is described. The elution volumes of various molecular weights of hyaluronic acids were linearily related to the logarithms of their molecular weights up to 600,000. The concentration of hyaluronic acid could be determined in the range from 20 to 100 micrograms/ml, i.e., from 4 to 20 micrograms per 200 microliter injected. The method was applied to the analysis of several animal skin extracts and rabbit synovial fluid. Skin extracts from mouse, rat, guinea-pig and rabbit could be chromatographed without prior isolation and purification. Hyaluronic acids in skin were separated clearly from chondroitin sulphates and their concentrations were determined. The molecular weights were estimated simultaneously to be more than 10(6). Rabbit synovial fluids from intact joints and saline- and carrageenin-treated joints could be chromatographed directly. The chromatograms showed that the concentration of hyaluronic acid in carrageenin-treated synovial fluid is lower than that in saline-treated fluid and the molecular weight distribution is broader. This technique enabled the rapid analysis of hyaluronic acid present at low levels in biological samples.