Determination of total hydroxyproline and proline in human serum and urine by HPLC with fluorescence detection

A method to measure total hydroxyproline (Hyp) and proline (Pro) in human serum and urine by HPLC was developed. Hyp and Pro in acid hydrolysates of serum and urine were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)phenysulfonyl chloride after treatment with o-phthaladehyde and cleanup on Bond Elut C18 column. The derivatives of imino acids were separated on a reversed phase column by gradient elution with acetonitrile and phosphate buffer (1 mmol/l, pH 7) and detected by fluorescence measurement at 315 nm (excitation) and 385 nm (emission). Detection limits for both Hyp and Pro were 10 fmol per injection. The within-day and day-to-day relative standard deviations for Hyp and Pro in serum and urine were less than 3.19%. The recoveries of Hyp and Pro added to serum and urine were about 100%. The present method was applied to determine total Hyp and Pro in serum and urine from normal subjects and patients with chronic renal failure.