Automated solid-phase extraction with high-performance liquid chromatography analysis of pyridinium crosslinks

An automated solid-phase extraction procedure for free and total pyridinium crosslinks in urine, with on-line HPLC analysis, is described. The pyridinium crosslinks either following hydrolysis in 3 M HCl or free in urine were extracted using a Gilson Aspec system onto extraction cartridges containing an octasilane/cation-exchanger sorbent. After washing, the crosslinks were eluted with 400 microl 100 mM sodium formate, pH 5, and 50 microl was automatically injected onto the analytical HPLC column. Detection was by fluorescence (Ex 295 nm, Em 400 nm). Recoveries were 95-100% with a limit of quantitation (s/n = 5) of 1.97 nmol/liter (nM) for pyridinoline and 2.79 nM for deoxypyridinoline. The interassay coefficient of variation was 6.5% for pyridinoline and 8.2% for deoxypyridinoline. The throughput of the system was up to 70 samples per day. The method correlated well with the established manual cellulose extraction but was substantially simpler and more reliable. A good correlation with an immunoassay for deoxypyridinoline was also obtained.