AUTOMATED SOLID-PHASE EXTRACTION WITH HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ANALYSIS OF PYRIDINIUM CROSS-LINKS

An automated solid-phase extraction procedure for free and total pyrid inium crosslinks in urine, with online HPLC analysis, is described. Th e pyridinium crosslinks either following hydrolysis in 3 M HCl or free in urine were extracted using a Gilson Aspec system onto extraction c artridges containing an octasilane/cation-exchanger sorbent. After was hing, the crosslinks were eluted with 400 mu l 100 mM sodium formate, pH 5, and 50 mu l was automatically injected onto the analytical HPLC column. Detection was by fluorescence (Ex 295 nm, Em 400 nm), Recoveri es were 95-100% with a limit of quantitation (s/n = 5) of 1.97 nmol/li ter (nM) for pyridinoline and 2.79 nM for deoxypyridinoline. The inter assay coefficient of variation was 6.5% for pyridinoline and 8.2% for deoxypyridinoline. The throughput of the system was up to 70 samples p er day. The method correlated well with the established manual cellulo se extraction but was substantially simpler and more reliable. A good correlation with an immunoassay for deoxypyridinoline was also obtaine d. (C) 1996 Academic Press, Inc.