Simultaneous determination of serum and urinary hydroxyproline and proline by liquid chromatography using two fluorescent labeling reagents

A method for the simultaneous determination of free serum and total urinary hydroxyproline (Hyp) and proline (Pro) concentrations by liquid chromatography (LC) using two fluorescent labeling reagents was developed. Serum Hyp and Pro, after treatment with o-phthalaldehyde, were derivatized with 4-(2-phthalimidinyl)phenylsulfonyl chloride (Phisyl-Cl). Imino acids in hydrolysed urine were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride (DPS-Cl) after treatment with o-phthalaldehyde and cleanup on a Bond Elut C18 column. The two reaction mixtures were combined and mixed with dichloromethane to extract the excess of the reagents, and then the aqueous layer (10 μl) was subjected to LC. The Phisyl and DPS derivatives of imino acids were separated on a reversed-phase column by gradient elution with phosphate buffer (5 mM, pH 2.5) and acetonitrile at 35°C and detected by fluorescence measurement at 300 nm (excitation) and 400 nm (emission). The detection limits (s:n=3) for Hyp and Pro were 30 and 40 fmol/injection, respectively, for Phisyl derivatives and 5 fmol/injection for both DPS derivatives. The within-day and day-to-day relative standard deviations for Hyp and Pro in serum and urine were <2.8%. The recoveries of Hyp and Pro added to serum and urine were 97.6–103.6%.