Development and validation of a high-performance liquid chromatographic method for the determination of desmosines in...

The development and the validation of a general strategy for the simple and accurate analysis of desmosines (isodesmosine and desmosine) in tissues coupled with the determination of collagen (as hydroxyproline) is described. The method is based on simplified sample (i.e., lung) pretreatment which involves, in a PTFE screw-capped Pyrex tube, homogenization, collagen extraction with hot 5% trichloroacetic acid and hydrolysis of the elastin-containing residue with 6 M hydrochloric acid, followed by cellulose minicolumn purification of desmosines from the hydrolysates, dansyl chloride pre-column derivatization of the purified desmosines and reversed-phase high-performance liquid chromatographic (HPLC) analysis of the dansyl derivatives using a Spherisorb ODS-2 column, an on-column enrichment sample device and a linear gradient of organic modifier (acetonitrile) in phosphate buffer. The simple sample pretreatment, the optimized chromatographic conditions and the short HPLC analysis time (less than 15 min) allow the accurate and rapid determination of desmosine and isodesmosine, thus permitting the determination of elastin in several kinds of tissues with a minimum of sample manipulation.