Determination of a novel hematoregulatory peptide in dog plasma by reversed-phase high-performance liquid chromatography and an amine-selective o-phthaldialdehyde-thiol post-column reaction with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of SB 107647 (I), a novel synthetic hematoregulatory peptide, in plasma samples of dog and rat. The method involves isolation of I and the internal standard (SB 203285, IS) from plasma by a solid-phase anion-exchange extraction column prior to reversed-phase ion-pair chromatographic separation on an octyl silica column. Following separation, a selective post-column reaction of the epsilon-amino groups of the lysine moieties of the peptide with o-phthaldialdehyde and a thiol under basic conditions was used to generate a highly fluorescent isoindole product, which was subsequently detected on-line with a fluorometer. Optimization of chromatographic conditions resulted in an on-column detection limit of 1 ng. The recovery of I from dog plasma at 20 and 4000 ng/ml was 50.0 +/- 5.94 and 56.6 +/- 1.45% (Mean +/- S.D.), respectively. The limit of quantification for I, for 0.25-ml plasma samples, was 20 ng/ml. Linear response was observed for concentrations of I ranging from 20 to 4000 ng/ml of plasma. The assay was sufficiently sensitive, accurate and precise to support toxicokinetic studies in animal species.