Rapid determination of total hydroxyproline (HYP) in human urine by HPLC analysis of the phenylisothiocyonate (PITC)-derivative

A recent development in the preparation for amino acid analysis is the use of phenylisothiocyanate (PITC) as a precolumn derivatizing agent prior to analysis to form a stable derivative. These derivatives can then be separated by reversed phase high pressure liquid chromatography (HPLC). Because of interest in the accurate measurement of urinary hydroxyproline (HYP), PITC was used for derivatization followed by HPLC analysis. Using an automated computer-controlled sampler system, up to 75 samples can be analyzed each 24 h. Technical details, specificity, and reliability of this method are provided. Duplicate measurements of HYP had a coefficient of variation (CV) of 5.5% and with a recovery of HYP in spiked urine samples of 94 to 104%. A sample frozen and thawed 10 times showed no change in the concentration of HYP. When total HYP was measured in 230 healthy women between 20 and 39 years of age, it was found to be 236.62 ± 146.41 μmoles/L. These samples were researched in 3.5 days using this technique.