Automated on-line solid-phase extraction and high-performance liquid chromatographic analysis of total and free pyridinium crosslinks in serum

An automated on-line solid-phase extraction procedure for free and total pyridinium crosslinks in serum, with HPLC analysis, is described. The pyridinium crosslinks either following hydrolysis in 3 M HCl or free in neat serum were extracted using a Gilson Aspec XLi system onto extraction cartridges containing an octylsilane/cation exchanger sorbent. Up to 525 μl of serum could be loaded onto the extraction cartridges. After washing, the crosslinks were eluted with 400 μl of 100 m M sodium formate pH 5 and 380 μl of this was concentrated on a RPB guard column eluted with 100 m M HFBA. The crosslinks were backflushed and separated on a 5-μm ODS analytical column eluted with 30 m M HFBA with 18% MeCN at 1 ml/min and detected by their native fluorescence (excitation 295 nm, emission 400 nm). The use of a high sensitivity fluorescence detector was essential. Recoveries were 95–100% with a limit of detection ( S/ N=2) of 109 pmol/l (p M) for pyridinoline (Pyr) and 143 p M for deoxypyridinoline (dPyr). The inter-assay R.S.D. was 9% for pyridinoline and 10.8% for deoxypyridinoline. The throughput of the system was up to 50 samples per day.