SOME ASPECTS OF SIMULTANEOUS DETERMINATION OF PYRIDINOLINES AND DESMOSINES AS COLLAGEN AND ELASTIN RESORPTION MARKERS BY MEANS OF REVERSE PHASE HPLC, UTILIZING FLUORESCENCE AND/OR UV-ABSORPTION MONITORING

Abstract

Isocratic reverse phase HPLC method was elaborated and optimized for simultaneous determination of pyridinoline (PD), deoxypyridinoline (DPD) and isodesmosine (IDES) in urine and in tissues within approximately 16 minutes. Mobile phase is 0.02 M heptafluorobutyric acid (HFBA) with 0.01 M (NH2)2SO4 and 8 - 12 % acetonitrile (ACN), at pH 1.55, flow rate 0.5 mL/min. and temperature of 40°C. The addition of ammonium sulphate affects positively the quality of chromatographic separation. The glass column 150 × 3 mm is filled with 7 μm spherical silica C18. The concentrations of determined substances are monitored by detector of fluorescence at λEX/λEM = 297/400 nm for both PD and DPD, and at λEX/λEM = 275/320 nm for IDES. Both PD and DPD are measurable with the same sensitivity limit of 200 femtomols; in the case of IDES the determination sensitivity is approximately 5x lower (1 pmol). Since IDES concentration in urine is considerably lower, it means that approximately 25 mL of the sample must be processed and the time of preparatory work is, therefore, considerably increased. In this system, desmosine (DES) is practically not detectable by fluorescence. It was verified by comparison of detection sensitivity in fluorescence and UV-absorption measurements that fluorescence is substantially more sensitive (with the detectors used by us) for the determination of PD, DPD, and IDES. If it is necessary to quantify DES as well, UV-absorption must be measured, and therefore the urine must be concentrated at least 5 times more. HPLC separation itself is very effective, on baseline, and with high number of theoretical plates of the column for all relevant substances. HPLC assays of tissue samples are of higher quality and more transparent than those of urine.