An improved high-performance liquid chromatographic assay for the determination of pyridinoline in connective tissues

A high-performance liquid chromatographic (HPLC) method for determination of pyridinoline and its application to connective tissues are described. The chromatographic separation was accomplished by using Inertsil ODS-2 column and a mixture of 0.1 M sodium phosphate and acetonitrile (75:25, v/v) containing sodium dodecyl sulfate (SDS) and ethylenediamine tetraacetic acid (EDTA) as eluent. The chromatogram was developed isocratically and the eluted components were monitored with excitation at 295 nm and emission at 395 nm. The pyridinoline in crude hydrolysate of connective tissues can be determined in 5 min, with as little as 1 pmol of sample. The present method is rapid, simple and can be used for the routine analysis of connective tissues such as cartilage, bone, Achilles tendon and aorta.