Determination of hydroxyproline in rat urine by high-performance liquid chromatography with electrogenerated chemiluminescence detection using tris(2,2′-bipyridyl) ruthenium(II)

A high-performance liquid chromatographic (HPLC) method for the determination of total urinary hydroxyproline (Hyp) in rat was developed and validated. Urine samples hydrolyzed with concentrated hydrochloric acid were filtered using a disposable centrifugal filter after the sample solutions had been adjusted to a slightly acidic condition by a buffer solution. Hyp in the fraction could be separated by column-switching HPLC and detected with electrogenerated chemiluminescence detection using tris(2,2′-bipyridyl) ruthenium(II) within an analysis time of 25 min. The calibration curve gave good linearity in the concentration range of 30-1500 nmol/ml, and the lower limit of quantification was 30 nmol/ml. The recovery of Hyp from urine samples was more than 96%. Precision and accuracy including within- and between-run values were less than 6 and ±5%, respectively, in the assay range. We concluded that the present method could be used to measure Hyp in rat urine.