RESUMO
This article describes the development and validation of a radioreceptor assay for the determination of morphine and morphine-6-beta-glucuronide (M6G) in serum. The assay is based on competitive inhibition of the mu-opioid-selective radiolabeled ligand [3H]-DAMGO by opioid ligands (e.g. M6G) for binding to the striatal opioid receptor. The assay has been validated according to the Washington Conference Report on Analytical Method Validation. The radioreceptor assay can be performed in serum without prior pre-treatment of the sample. Direct addition of the sample results in no significant loss in maximal binding sites, and therefore, no loss in sensitivity. The assay proves to be selective for a multitude of opioid agonists and antagonists (e.g. morphine IC50 = 4.1 nM and M6G IC50 = 12.8 nM). Moreover, morphine-3-glucuronide (M3G) displays a low affinity (IC50 = 1100 nM) for the mu-opioid receptor and according to the literature demonstrates no analgesic activity. This makes discrimination, in relation to the analgesic effect, of the two metabolites of morphine possible. The assay is fast (assay time <4h, analysis 5 min/sample), easy and the sensitivity (limit of detection (LOD) = 1.6 nM M6G-equivalents) is such that very potent agonists, like morphine and M6G, can be measured at the desired serum levels. The assay is accurate (<18%), but precision is limited if measured over several days (>35%). The assay is most accurate and precise if measured over a range from 3.5 to 40 nM M6G-equivalents. Based on the limited inter-assay precision, we propose to use this receptor assay mainly as a screening tool for neonates treated with morphine.
