An HPLC/RIA method for dynorphin A1-13 and its main metabolites in human blood.
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 ZnSO4 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 micrograms or 1000 micrograms kg-1 Dyn A1-13 over 10 min.
Müller
S
S
University of Florida, College of Pharmacy, Gainesville 32610, USA.
Ho
B
B
Gambus
P
P
Millard
W
W
Hochhaus
G
G
D000097:Acetonitriles; D000701:Analgesics, Opioid / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004399:Dynorphins / Q000097:blood* / Q000493:pharmacokinetics; D006801:Humans; D007262:Infusions, Intravenous; D000432:Methanol; D010446:Peptide Fragments / Q000097:blood* / Q000493:pharmacokinetics; D011863:Radioimmunoassay; D019287:Zinc Sulfate
D000097:Acetonitriles; D000701:Analgesics, Opioid; D010446:Peptide Fragments; C111725:dynorphin A (2-12); C026843:dynorphin (1-13); D004399:Dynorphins; D019287:Zinc Sulfate; C037422:dynorphin (1-12); C036730:dynorphin A, desTyr(1)-desTrp(14)-desAsp(15)-desAsn(16)-desGlu(17)-; D000432:Methanol; C032159:acetonitrile