Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsufonyl chloride.

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55 degrees C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2-5 fmol/injection (S/N=3). Pro-Hyp, Pro-Gly and Pro-Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5-7.9 and 2.4-10.8%, respectively. The recoveries were in the range of 90.8-97.3%. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro and Hyp in normal human serum (n = 10) were 0.64+/-0.35, 0.078+/-0.047, 0.022+/-0.016, 177.0+/-43.0 and 11.1+/-3.5 microM, respectively. The concentrations of Pro-Hyp and Pro-Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.