ANALYSIS OF ENCAINIDE IN PLASMA BY RADIOIMMUNOASSAY AND HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

A radioimmunoassay for encainide (4-methoxy-2''-[2-(1-methyl-2-piperidyl)ethyl]benzanilide hydrochloride), a new antiarrhythmic agent, was developed by immunization of rabbits with an O-carboxymethyl analog of encainide conjugated to bovine serum albumin. A radioactive tracer containing 125I was prepared by chloramine-T iodination of the tyramine amide of the O-carboxymethyl analog. Separation of bound from free tracer in the assay is accomplished using a 2nd antibody. The assay is capable of detecting 20 pg encainide directly or 0.2 ng encainide/ml plasma or urine. Interassay variability of the slope, .gamma.-intercept and 50% bound points on the standard curve had coefficients of variation (CV) of 6.9, 7.8, and 6.0%, respectively. Mean recoveries of 94.4% with a CV of 12.0% were obtained after extraction and analysis of standards from plasma at concentrations of 20, 200 and 2000 ng/ml. No interference with the assay was observed from any components in normal, hemolyzed or turbid [human] plasma or from urine. The extent of cross-reactivity of possible metabolites of encainide was evaluated, and the assay for unchanged encainide was validated by high pressure liquid chromatography (HPLC). An HPLC procedure for the quantification of encainide in plasma is described. Encainide and 3 putative metabolites, the O-demethyl, N-demethyl and N,O-bis-demethyl congeners of encainide, can be quantified simultaneously by this procedure. All 4 compounds are extracted from buffered plasma (pH 8.5) with n-butyl chloride containing 5% isopropyl alcohol (vol/vol). The compounds are then separated by HPLC on a .mu.Porasil column and quantified by measuring their UV absorbance at 254 nm. Each compound can be quantified over a concentration range of 10-1000 ng/ml using 1 ml plasma samples.