P. Spacek et al., ION-EXCHANGE HPLC DETERMINATION OF PYRIDINIUM CROSS-LINKS IN URINE ASMARKERS OF BONE-RESORPTION, Journal of liquid chromatography & related technologies, 20(12), 1997, pp. 1921-1930
Citations number
22
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
The aim of this study was to improve the published method(1) for rapid
and sensitive determination of the urinary pyridinoline as well as de
oxypyridinoline, i.e. the crosslinking elements of bone and cartilage
collagen. This method is based on the HPLC analysis of previously prep
ared urinary hydrolysates. Urine hydrolysate (6 N HCl, 110 degrees C,
16 hours) was purified by selective fractionation using washing with a
mixture of water -acetic acid-butanol (1-1-4 by volume) on small disp
osable columns filled with microgranular cellulose. Liquid chromatogra
phy was based on ion-exchange counteractions and was properly performe
d with the use of a strong cation exchanger as a stationary phase and
with a buffered solution at pH=3.35 as the mobile phase, by keeping th
e oven temperature at 48 degrees C and with the injection of a volume
of 10 mu l. The time desired for HPLC determination alone didn't excee
d 15 min and by using a fluorescence detector (Shimadzu RF 535) set at
297 and 400 nm for excitation and emmision wavelengths, respectively,
it reached a sensitivity of about 200 femtomoles for both the crossli
nks.