Measurement of pentosidine in human plasma protein by a single-column high-performance liquid chromatography method with fluorescence detection.

A rapid and sensitive single-column high-performance liquid chromatography method and application for the detection of protein bound pentosidine is described. Pentosidine, a cross-link between arginine and lysine, is a well-characterized advanced glycation endproduct. In order to detect protein-bound pentosidine, plasma proteins were hydrolysed in 6N HCl. Detection of pentosidine is done based on its own fluorescence characteristics using fluorimetric detection (E(x)=325 nm, E(m)=385 nm). Separation is done, with a run-to-run time of 30 min, on a C(18) Allspehere ODS-II column with a citric acid acetonitrile buffer. This detection enables sensitive and specific determination of protein bound pentosidine in plasma with a detection limit of 2.2 nmol/l or 0.02 pmol/mg protein (signal-to-noise: 6). The intra-assay coefficient variation is 6.5% at a plasma pentosidine concentration of 0.47 pmol/mg protein and 2.0% at a concentration of 1.27 pmol/mg protein. The inter-assay coefficient variation is 3.1% at a plasma pentosidine concentration of 0.43 pmol/mg protein and 1.6% at a concentration of 1.40 pmol/mg protein. Linearity is tested in 4 different plasma samples and showed linearity (0-200 nmol/l, r(2)>0.99). Recovery of pentosidine in 4 different plasma samples at different concentration levels is 102+/-10% (mean+/-SD). Using this method protein bound pentosidine concentration is investigated in healthy controls (n=24, age 67+/-9 years) and patients with end stage renal disease (n=24, age 65+/-10 years). Higher plasma concentrations of protein bound pentosidine are measured in the patient group as compared with the control group 3.05 (2.03-3.92)pmol/mg protein and 0.21 (0.19-0.33)pmol/mg protein, respectively (median (interquartile range), p<0.00001). These results are consistent with previously reported results.