High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

Abstract

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenyl-methylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-μm, 250 × 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile—water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile—methanol—tetrahydrofuran— water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (λ max  264) was about 5 ng/ml and by fluorescence detection (λ excitation  270 nm, λ emission  300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 ± 4.2% from serum and 102.0 ± 2.62% from urine.