Optimization of the determination of 4-aminopyridine in human serum and urine by column liquid chromatography

Two convenient reversed-phase column liquid chromatographic procedures are described for the determination of 4-aminopyridine in human serum and urine. A 0.5-ml aliquot of serum after the addition of a 0.5-ml solution of 4-(aminomethyl)pyridine in 0.1 M Na2HPO4 as the internal standard is passed through a 1-ml BondElut C18 silica extraction column. The column is selectively washed to remove acidic, neutral and weakly basic compounds. The desired compounds are eluted with a 0.3-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 10-microliters aliquot of the eluate is injected onto a 150 x 4.6 mm I.D. column packed with 5-microns C18 silica particles that is eluted at ambient temperature with a mobile phase containing octanesulfonic acid as the ion-pairing agent. The peaks are monitored at 263 nm. A. 0.25-ml aliquot of urine or 0.5 ml of serum is mixed with N-propionylprocainamide as the internal standard and subjected to benzoylation by Schotten Baumann reaction. The reaction mixture is adjusted to pH 5.5-6 and extracted with a BondElut C18 extraction column. An aliquot of the eluate is chromatographed at ambient temperature with a mobile phase containing tetramethylammonium perchlorate. The peaks are monitored at 278 nm.