Automated high-performance liquid chromatographic determination of hydroxylysylpyridinoline and lysylpyridinoline in urine using a column-switching method.
An on-line urine clean-up system was developed for the simultaneous determination of free and total pyridinoline, hydroxylysyl-pyridinoline (HP) and lysylpyridinoline (LP) by high-performance liquid chromatography (HPLC) using a column-switching technique. The method is based on a combination of gel permeation chromatography (GPC) and ion-pair reversed-phase HPLC. In the GPC column, pyridinoline is preseparated from endogenous urinary substances with 0.03 M heptafluorobutyric acid (HFBA) as the mobile phase. After column switching, the eluate fraction containing pyridinoline is further separated by ion-pair chromatography using an octadecylsilica (ODS) column with 0.03 M HFBA-acetonitrile (81:19) as the mobile phase. The detection limits were 36 and 44 pmol/ml for free and total HP, respectively, and 44 pmol/ml for both free and total LP at a signal-to-noise ratio of 3. The coefficients of variation for free and total pyridinoline were 1.5 and 3.5%, respectively. The determination of one sample including the clean-up is completed within 25 min. This system is precise and is useful for the determination of pyridinoline in large amounts of urine. The usefulness of pyridinoline as a biomedical marker for bone resorption was also examined.
Yoshimura
Y
Y
Drug Safety Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Ohnishi
K
K
Hamamura
M
M
Oda
T
T
Sohda
T
T
D000596:Amino Acids / Q000652:urine*; D000818:Animals; D001331:Automation; D001847:Bone Diseases / Q000652:urine; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D051381:Rats; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; C015484:pyridinoline; C036020:deoxypyridinoline