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 analysis of morphine and its metabolites is described which has the advantages of increased sensitivity and specificity by using a cleaner extraction. The new extraction method involves both the hydrophobic isolation on a carbon cartridge and ion-exchange isolation on ion-exchange resin which has not preliminary been described for morphine analysis. The combination of these two steps successfully purified drugs from human plasma with maximum removal of interfering substance comparing with a conventional C18 cartridge alone. The analytes are quantified by high-performance liquid chromatography on a reversed-phase C18 column employing a mobile phase consisting of 25% acetonitrile in 0.05 M phosphate buffer (pH 2.1), and 2.5 mM sodium dodecyl sulfate as the pairing ion with a combination of electrochemical and fluorometric detections. The recoveries for morphine (M), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and hydromorphone after the SPE procedure 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 and 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 this method for clinical and research use.