Abstract
A rapid, accurate and sensitive method for the determination of free hydroxyproline and proline in plasma and of total hydroxyproline in urine has been developed. Free imino acids and internal standard are extracted from plasma by trichloroacetic acid precipitation of protein and they are selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of the acid extract with o-phthalaldehyde. The highly fluorescent adducts of imino acids are separated on a Spherisorb ODS 2 reversed-phase column using acetonitrile—0.1 M sodium phosphate buffer, pH 7.2 (9:91, v/v) as mobile phase, followed by fluorometric detection. Total hydroxyproline determination in urine hydrolysates is carried out by reaction of the imino acid with 4-chloro-7-nitrobenzofurazan after clean-up on a Sep-Pak C 18 cartridge of the o-phthalaldehyde-treated sample, high-performance liquid chromatographic separation and fluorometric quantitation of the derivative.
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