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

Journal of Chromatography. a
I T James, D Perrett

Abstract

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 microliters of serum could be loaded onto the extraction cartridges. After washing, the crosslinks were eluted with 400 microliters of 100 mM sodium formate pH 5 and 380 microliters of this was concentrated on a RPB guard column eluted with 100 mM HFBA. The crosslinks were backflushed and separated on a 5-micron ODS analytical column eluted with 30 mM 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 (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.

Citations

Nov 5, 1999·Journal of Clinical Densitometry : the Official Journal of the International Society for Clinical Densitometry·M J Seibel, H W Woitge
Apr 5, 2001·Rheumatic Diseases Clinics of North America·H W Woitge, M J Seibel
May 3, 2003·The British Journal of Nutrition·Silvia ValtueñaFrancesco Branca
Aug 15, 2000·Journal of Chromatography. a·D T Rossi, N Zhang

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