Determination of A 3,4-diaminopyridine in plasma by liquid chromatography with electrochemical detection using solid-phase extraction

In order to quantify a small amount of a drug, 3,4-diaminopyridine (3,4-DAP), in animal plasma samples, an analytical method was developed. It involved an extraction of 3,4-DAP and phenylephrine, used as internal standard (IS), from plasma with solid-phase extraction (SPE) on C18 cartridges. This analytical method is a hyphenated technique based on high-performance liquid chromatography with electrochemical detection (HPLC-EC) whose purpose is to obtain first a sensitive method and second a satisfying separation between 3,4-DAP and phenylephrine. The analytical method is accurate, specific, and linear between 10 and 500 g of 3,4-DAP per litre. The recovery of 3,4-DAP is estimated at 70.8% with a 95% confidence interval of (66.0 -75.6%). Intermediate precision was evaluated on three quality control samples; the intra-day precision was estimated at 13.5, 9.1, 7.8% and the inter-day precision at 17.9, 8.4, 9.3%. The limit of quantification of the method was evaluated at 10 g l-1. First toxicokinetic parameters determined on dogs plasma samples after one 3,4-DAP oral administration of 1 mg kg-1 were: Cmax=395.7 microg l-1; Tmax =15 min; t1/2=113.6 min; Clearance/F=16.8 ml kg-1 min-1 and Vd/F=2.7 l kg -1.