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-methoxyphenylsulfonyl chloride
H. Inoue et al., 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-methoxyphenylsulfonyl chloride, J CHROMAT B, 757(2), 2001, pp. 369-373
A highly sensitive HPLC method for the determination of prolyl dipeptides,
Pro and Hyp in serum was developed. After deproteinization of serum and pre
treatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-
dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degreesC
for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp,
were separated on tandem reversed-phase columns by a gradient elution at 5
5 degreesC and detected by fluorescence measured at 318 nm (excitation) and
392 nm (emission). The: detection limits for prolyl dipeptides were 2-5 fm
ol/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 deviat
ions were 1.5-7.9 and 2.4-10.8%, respectively. The recoveries were in the r
ange of 90.8-97.3%. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro an
d Hyp in normal human serum (n = 10) were 0.64 +/-0.35, 0.078 +/-0.047, 0.0
22 +/-0.016, 177.0 +/- 43.0 and 11.1 +/-3.5 muM, respectively. The concentr
ations 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. (C) 2001 Elsevier Science B.V. All right
s reserved.