Assay of pyridinium crosslinks in serum using narrow-bore ion-paired reversed-phase high-performance liquid chromatography.

The pyridinium crosslinks are important biomarkers of mature hard tissue collagen degradation. This paper describes an isocratic ion-paired reversed-phase high-performance chromatographic assay using narrow-bore columns and high-sensitivity fluorescence detection to enable for the first time the determination of pyridinium crosslinks in both serum and synovial fluid samples. Extracted freeze-dried acid hydrolysates were re-suspended in 20 mM pentafluoropropionic acid (PFPA). Separations were carried out using an Exsil 100 5-microns ODS2 column (100 mm x 2.1 mm I.D.) eluted with 10 mM PFPA in water at 0.15 ml/min and detected using a Jasco 821-FP detector (xenon lamp: excitation 290 nm, emission 400 nm). Fluorescent response was linear from 269 to 8620 fmol for pyridinoline (Pyr) and 85 to 2710 fmol for deoxypyridinoline (dPyr). The limits of detection were 28 and 57 fmol, respectively. The coefficient of variation for extraction and analysis of normal serum was 7.96% for Pyr and 6.30% for dPyr (n = 6). The mean +/- S.D. concentration of Pyr in normal serum was 3.26 +/- 0.83 nM.