Reversed-phase liquid chromatography method for measurement of procainamide and three metabolites in serum and urine: percent of dose excreted as deethyl metabolites.

We report a reversed-phase high-performance liquid chromatography method for the determination of procainamide (PA) and three of its metabolites, n-acetylprocainamide (NAPA), deethylprocainamide (DEPA), and deethyl-n-acetylprocainamide (DENAPA), in serum and urine. (p-Amino)-n-(2-dipropylaminoethyl)-benzamide was the internal standard. A phenyl column (1.0-mL/min flow rate) and a mobile phase consisting of 0.075 M acetate buffer (pH 4.3):acetonitrile (20:3) resulted in a total chromatography time of 21.6 min. The optimum detector wavelength was 270 nm. Maximum linear concentrations were 37.8, 34.2, 20.0, and 16.3 mg/L for DEPA, DENAPA, PA, and NAPA, respectively. Minimum detectable concentrations were 0.05 mg/L or less for all four compounds. One-tenth milliliter of sample was extracted into methylene chloride:2-propyl alcohol (9:1). Extraction efficiencies were independent of concentration or biological fluid for each compound. Standard curves were linear and best-fit by dividing the curve into two portions and/or using weighted linear regression. Within-day and day-to-day precison were excellent. No interfering substances were observed in the serum or urine of normal subjects with the exception of caffeine, which was resolved by alteration of the mobile phase. Advantages of this method include a small sample volume, low minimum detectable concentrations, and an order of elution which enhances the detectability of the deethyl metabolites. Urinary excretion of DEPA and DENAPA accounted for an average of only 0.58 and 0.53%, respectively, of PA doses administered intravenously to six normal volunteers.