High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites by two-step solid-phase extraction

To provide more accurate measurement of morphine and its metabolites for a study of the genetic differences on morphine response, a method for the ana lysis of morphine and its metabolites is described which has the advantages of increased sensitivity and specificity by using a cleaner extraction. Th e new extraction method involves both the hydrophobic isolation on a carbon cartridge and ion-exchange isolation on ion-exchange resin which has not p reliminary been described for morphine analysis. The combination of these t wo steps successfully purified drugs from human plasma with maximum removal of interfering substance comparing with a conventional C-18 cartridge alon e. The analytes are quantified by high-performance liquid chromatography on a reversed-phase C-18 column employing a mobile phase consisting of 25% ac etonitrile in 0.05 M phosphate buffer (pH 2.1), and 2.5 mM sodium dodecyl s ulfate as the pairing ion with a combination of electrochemical and fluorom etric detections. The recoveries for morphine (M), morphine-3-glucuronide ( M3G), morphine-6-glucuronide (M6G) and hydromorphone after the SPE procedur e were 86+/-7.1%, 82+/-6.9%, 79+/-6.0% and 85+/-6.0%, respectively. Limits of detection for this method are 0.1 ng/ml for M, and 0.18 ng/ml for M3G an d M6G. Limits of quantitation were approximately 0.25 ng/ml for M, and 0.45 ng/ml for M3G and M6G. The present assay was applied to measure M, M3G and M6G content in human plasma to test the applicability and suitability of t his method for clinical and research use. (C) 2000 Elsevier Science B.V. Al l rights reserved.