Pentosidine assay

Pentosidine was determined by reversed phase HPLC combined with sensitive fluorescent detection, as published previously [21]. Briefly, serum samples were mixed with equal amounts of 35 % HCl and hydrolyzed for 16 h at 105 °C. The bone samples were treated as above. In the next step, the hydrolysate was purified and pre-concentrated using solid phase extraction; the hydrolysate was mixed with a suspension of cellulose and combined with a solution containing n-butyl alcohol and acetic acid. This mixture was then applied to a purification column filled with cellulose. The PEN-containing fraction was then desorbed using 0.05 mol/l HCl, and the extract was evaporated. The residue was reconstituted in a solution containing 0.02 mol/l heptafluorobutyric acid, 0.01 mol/l ammonium sulphate and acetonitrile (ergo mobile phase), and this was applied to a HPLC system (Shimadzu, LC-10ADvp, Kyoto, Japan) equipped with a C18 reversed phase column (Separon SGX C18, 150 x 3 mm). We monitored the emission signal at 385 nm upon excitation at 335 nm. Synthetic PEN was used as a standard. The concentration of PEN was expressed in nmol/l.

The peak area reproducibility involving complete analytical procedure (including sample hydrolysis, drying, reconstitution, and HPLC analysis) in actual samples was 95.6 %. Reproducibility of the HPLC determination itself was 98.8 %. Based on our PEN measurements, limit of detection was 1.76 nmol/l and limit of quantitation 5.87 nmol/l.