Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection.

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron-diethylenetriaminepentaacetic acid (DTPA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 microM Fe(III) to be determined with an injection volume of 10 microliters. A mixture of synovial fluid, 20 microM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O2-) generated by a xanthine-xanthine oxidase system and was ultrafiltered through a 30,000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O2- facilitated the release of iron into the synovial fluid. This result suggested that in an inflamed joint with generated O2- and decreased pH, iron may be released into the synovial fluid.