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 mu l of serum could be loaded onto the extraction cartridges. After washing, the crosslinks were eluted with 400 mu l of 100 mM sodium formate pH 5 and 380 mu l of this was concentrated on a RPB guard column eluted with 100 mM HFBA. The crosslinks were backflushed and separated on a 5-mu m ODS analytical column eluted with 30 mM HFBA with 18% MeCN at I 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 (pM) for pyridinoline (Pyr) and 143 pM 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. (C) 1998 Elsevier Science B.V.