Quantitation of Indomethacin in Serum by HPLC Using Fluorescence Detection

Abstract

An improved, sensitive and accurate HPLC procedure using fluorescence detection for quantitation of indomethacin in serum has been developed. After addition of an equal volume of phosphate buffer, pH 6.6 to serum along with the internal standard, the samples were extracted with methylene chloride. Prior to chromatography, the extracted indomethacin was deacylated to its fluorescent product (DBI) in 0.01 N-NaOH. The mobile phase consisted of methanol and pH 4 acetate buffer (3:7 V/V) and the separation was achieved on a C18 reversed phase column. The retention times of DBI and the internal standard were 7.5 and 16.0 min. respectively. The fluorometric excitation and emission wavelengths were 278 and 358 nm, respectively. The sensitivity of the assay was 1 ng/ml of serum and the CV at this concentration was 2.46%. The standard plot was linear (r > 0.999) for indomethacin concentrations between 1 and 500 ng/ml. The inter-and intra-day studies showed high reproducibility (CV = 2.8%, F = 0.89, p > 0.05). The method was used to determine the complete serum level vs. time profiles of indomethacin in animals.