Determination of urinary hydroxyproline in rabbits of hepatic fibrosis after administration by high performance liquid chromatography with pre-column derivatization

A method was developed for the determination of hydroxyproline (Hyp) in urine by reversed phase high performance liquid chromatography (RP-HPLC), and the variance in concentration of urinary hydroxyproline in rabbit models of schistosomiasis hepatic fibrosis before and after oral administration of the HDS (a compound recipe of Chinese medicine) was measured. After derivatization by 9-fluorenylmethyl chloroformate (FMOC), a baseline separation of hydroxyproline and 3, 4-dehydro-L-proline (the internal standard) was achieved on a Zorbax SB-C-18 column by gradient elution with methanol and sodium acetate buffer. The detection was carried out with diode array detector( DAD) by absorbance at 262 nm. The relationship between the peak area and the concentration had a good linearity (correlation coefficient was 0.9999) in the range of 5.0-200 mu mol/L. While the signal-noise ratio was 3, the detection limits for hydroxyproline, was 2.0 mu mol/L. The recovery of urinary hydroxyproline was 96.5%-99.0%. The method is simple, accurate, rapid and suitable for the analysis of great mass of samples.