High-performance liquid chromatographic assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-l-arginine (ST 789) in plasma by cyclization with benzoin and fluorimetric detection

Abstract This paper describes a new highly sensitive assay for N 2 -[5-(hypoxanthin-9-yl)pentyloxycarbonyl]- l -arginine, an immunomodulatory agent, required for clinical pharmacokinetic investigation. A pre-column derivatization by cyclization with benzoin in aqueous medium produces the fluorescent 2-substituted amino-4,5-diphenylimidazole fluorescing at 450 nm (excitation wavelength 310 nm). l -Arginine-acetyl- l -carnitinamide chloride (ST 857, II), another arginine derivative, was used as an internal standard. A C 18 DB column (5 μm, 250 mm × 4.6 mm I.D.) and a 45:55 (v/v) mixture of 0.05 M ammonium phosphate at pH 7.2 and methanol as mobile phase were used. Linearity was ascertained in the range 5–100 ng. Extraction recovery from plasma proved to be higher than 90% in the range 5–50 ng/ml. Intra-day precision, expressed as coefficient of variation, was in the range 4.7–6.0%. The limit of quantification proved to be 5 ng/ml and the limit of detection 2 ng/ml at a signal-to-noise ratio of 5. The method is specific.