An HPLC/RIA method for dynorphin A1-13 and its main metabolites in human blood

Abstract

A selective HPLC/RIA procedure for the determination of dynorphin A1-13 (Dyn A1-13) and its major metabolites in human blood was developed. In order to block peptidase activity, blood samples were transferred into an aliquot of a blocking solution (5% aqueous ZnSO 4 solution-acetonitrile-methanol; 5:3:2, v/v/v). After solid phase extraction, reconstituted aliquots were injected into an isocratic reversed phase HPLC system to separate Dyn A1-13 from its main metabolites (Dyn A2-13, Dyn A1-12 and Dyn A2-12). The isolated and concentrated HPLC-fractions were assayed by RIA using a commercially available antiserum. Intra-day variabilities for quality controls (0.07, 0.25, and 1 ng ml −1) of Dyn A1-13, A2-13, A1-12, A2-12 were between 9 and 41%. Accuracy was between 86 and 132%. Inter-day variability for single quality controls analyzed on five days for Dyn A1-13, A2-13, A1-12, A2-12 was between 4 and 49% for 0.07, 0.25 and 1 ng ml −1 samples, respectively. Accuracy was between 72 and 129%. Five different batches of control blood showed blood levels no different from zero. Considering the complexity of the assay, the method is selective, accurate and reproducible with a limit of detection of 0.07 ng ml −1 for Dyn A1-13, Dyn A2-13, Dyn A1-12 and 0.21 ng ml −1 for Dyn A2-12. The assay was applied to the determination of Dyn A1-13 and its metabolites in blood samples of 2 subjects receiving i.v. infusions of 250 μg or 1000 μg kg −1 Dyn A1-13 over 10 min.