Analysis of encainide and metabolites in plasma and urine by high-performance liquid chromatography

A high performance liquid chromatography (HPLC) method for the quantification of encainide (4-methoxy-2'-[2-(1-methyl-2-piperidinyl)-ethyl]benzanilide hydrochloride) in plasma and urine has been developed and validated. Encainide and two metabolites, the O-demethyl (ODE) and 3-methoxy-O-demethyl (MODE) congeners of the drug, can be quantified simultaneously in plasma and urine by this procedure. The compounds are extracted from buffered plasma (pH 8.5) using N-butyl chloride containing 5% (vol/vol) isopropyl alcohol. The compounds are then separated on a silica column using ethanol:water:methanesulfonic acid, 500/60/0.2 (vol/vol/vol) as the mobile phase and quantified by measuring their UV absorbance at 254 nm. The lower limit of detection for the analytes was 5 ng/ml in plasma and 25 ng/ml in urine. The method was linear from 5 to 5,000 ng/ml for plasma and 25 to 5,000 ng/ml for urine. The intra-assay precision of the method for encainide, ODE, and MODE in plasma and urine ranged from 2 to 8% RSD depending upon concentration. The inter-assay precision of the method was less than 6% for the three analytes per ml of plasma and urine. Absolute recovery of the analytes from plasma ranged from 82 to 92%, while recoveries from urine ranged from 83 to 99%. The analytes were shown to be stable in frozen plasma and urine for up to 52 weeks.