COLUMN SWITCHING HPLC METHOD FOR DETERMINATION OF DEXTRORPHAN, AN ACTIVE METABOLITE OF DEXTROMETHORPHAN, IN PLASMA

An HPLC method for the determination of dextrorphan, an active metabolite of dextromethorphan, in plasma was established using column switching technique. The column switching system was equipped with a per-column of 30 mm×5 mm ID, packed with μ Bondapak C₁₈, 37～50 μn, and an analytical column of 150 mm×5 mm ID, packed with YWG-C₁₈, 5 μm. A 0.2 % acetic acid solution was used as the pretreating mobile phase to wash out impurities from the per-column. The analytical mobile phase consisted of acetonitrile—water—acetic acid—triethyl-amine—dichloromethane (17:82:1:0.05:0.025). The plasma samples were directly injected into the HPLC system after enzymatic hydrolysis of dextrorphan glucuronide ester conjugate to free form with β-glucuronidase. The dextrorphan was monitored with a fluorescence detector at 290 nm (excitation) and 315 nm (emission). The method was linear within the plasma concentration range of 20～640 ng/ml (r=0.9987), and the detection limit was 4 ng/ml. The mean recoveries of the method averaged 103.8%. The relative standard deviations of the assay were less than 10% for both withinday and between-days.