Analytical method to determine keratan sulfate in the serum using HPLC.

There is an established method to assess the extent of articular cartilage damage that is based on the determination of keratan sulfate (KS) in synovial fluid by high performance liquid chromatography (HPLC). However, since it is easier to collect serum than synovial fluid, and the serum concentration of KS might reflect the extent of degeneration and injury of the articular cartilage, we aimed at developing a method to determine KS in serum by HPLC. Human serum was spiked with low-sulfated KS or poly-sulfated KS. After digestion of serum protein with a protease, the KS fraction was extracted by anion exchange column chromatography and then desalted. The KS fraction was digested with keratanase II and determined by HPLC. The recovery of KS added to the serum was 98% or more and did not depend on the degree of KS sulfation. A good recovery rate was obtained within a range of 50-100,000 ng/ml. The inter-day assay variation was 6.0% for 10 days. This method enables the determination of serum KS concentration without being influenced by the degree of KS sulfation.