Rapid determination of desmosine and isodesmosine in tissue hydrolysates by isocratic high performance liquid chromatography and precolumn derivatization

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Abstract
A rapid and sensitive isocratic high performance liquid chromatographic method has been developed for the single and specific determination of low concentrations of desmosine (Des) and isodesmosine (Ide), the major specific crosslink aminoacids in elastin. Samples of isolated elastin or whole tissue were hydrolysed in 6N HCl, and the hydrolysates were prefractionated on cellulose CF1. Des, Ide,γ-glutamyl-glutamic acid as internal standard were dansylated and derivatives were extracted from reaction mixture by ethylacetate. Their separation on a Lichrosphere 100-NH2 column, using methanol-water as mobile phase containing acetic acid and 0.25 M sodium acetate, final pH 6.5, was followed by fluorescence detection (340–510 nm). The overall reproducibility was 5.9% for Des and 5.0% for Ide. The limits of detection were 2.2 pmol and 2.5 pmol, respectively. The method was successfully applied for the determination of Des and Ide in normal pig aortas.