Gas-liquid chromatography-mass spectroscopy determination of clopamide in plasma

A gas-liquid chromatography-mass spectroscopy (GLC-MS) method for the determination of clopamide (1) in human plasma was developed to evaluate the pharmacokinetics and bioavailability of 1 in humans. The method is specific, sensitive, and rapid and allows routine analysis as required for extensive pharmacokinetic studies. The assay procedure involves addition of furosemide (2) as an internal standard to plasma, separation on Sep Pack-C18 cartridges, elution by ether: methanol (1:1), evaporation, and subsequent derivatization with trimethylanilinium hydroxide in methanol (Methelute). Then, 5 microL of the reaction mixture is injected into a GLC-MS system which consists of a 1% SE-30 on Gas Chrom Q (100-120 mesh) glass column. The MS information was obtained under the following conditions: ionization beam energy 70 eV, ion source 200 degrees; and m/e 372 for single ion monitoring. The retention times for 1 and 2 were 0.6 and 1.0 min, respectively. The limit of detection is 10 ng/mL of 1 in plasma and the calibration curve was shown to be linear between 10 and 500 ng/mL of 1. After repeated analysis of spiked plasma samples, the coefficient of variation ranged from 5.1 to 8.3%. The recovery from the extraction procedure was 92 +/- 2.2%. Spiked samples frozen at -20 degrees C were stable for at least 8 weeks. The method has been successfully used in a pharmacokinetic study with po dosing of 5 mg of 1 to eight healthy volunteers. Peak plasma concentrations of 197 +/- 56 ng/mL were observed after 1.1 +/- 0.34 h. No measurable concentration of 1 beyond 12 h after dosing was observed.