Determination of p-hydroxybestatin in human serum by high-performance liquid chromatography using fluorescence detection

A high-performance liquid chromatographic method is described for the fluorimetric determination of p-hydroxybestatin (an active metabolite of bestatin) in human serum. p-Hydroxybestatin is formylated in an alkaline medium in the presence of chloroform, and converted to a fluorescent derivative with 1,2-diamino-4,5-dimethoxybenzene. This derivative is then separated on a reversed-phase column (TSK gel ODS-120T) with isocratic elution. The detection limit of p-hydroxybestatin in serum is 15 ng (46 pmol) per ml serum (115 pg in a 100-microliters injection volume). This method is simple and sensitive enough to determine p-hydroxybestatin in serum (200 microliters) from muscular dystrophic patients and from healthy subjects dosed with bestatin.