[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 x 5 mm ID, packed with mu Bondapak C18, 37-50 microns, and an analytical column of 150 mm x 5 mm ID, packed with YWG-C18, 5 microns. 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-triethylamine-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 beta-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 within-day and between-days.