A Sensitive and Specific Procedure for Quantitation of ADR-529 in Biological Fluids by High-Performance Liquid Chromatography (HPLC) with Column Switching and Amperometric Detection |
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Authors: | Lewis Richard C. Phillips Barbara A. Baldwin John R. Rossi David T. Narang Prem K. |
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Affiliation: | (1) Bioanalytical Research Laboratory, Department of Pharmacokinetics/Dynamics, Adria Laboratories, Inc., Columbus, Ohio, 43216;(2) Department of Pharmacokinetics/Dynamics, Adria Laboratories, P.O. Box 16526, Columbus, Ohio, 43216 |
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Abstract: | An HPLC method using electrochemical detection (ED) has been validated for the determination of ADR-529 in plasma and urine using ICRF-192 as an internal standard (IS). Prior to storage and quantitation, both plasma and urine samples require acid stabilization. Acidified plasma samples were prepared for HPLC using a two column solid-phase extraction (SPE). An aliquot of buffered plasma (i.e., pH 6-7) was first deproteinated and desalted on a C-18 SPE column. The analytes were then eluted onto a C-8 SPE column where retention and selective cleanup were achieved in the cation-exchange mode via silanol interactions. Acidified urine samples were diluted in acetonitrile prior to injection. The HPLC system for plasma and urine samples employed two narrow-bore silica columns used in the weak cation-exchange mode and separated by a switching valve. To prohibit late-eluting peaks from passivating the glassy carbon working electrode, a heart-cut containing ADR-529 and the IS was vented from the first silica column to the second using an automated switching valve. Amperometric detection at an oxidation potential of +1050 mV vs a Ag/AgNO3 reference electrode was used. Linearity was validated between 5 and 500 µg/ml in plasma and between 2 and 100 µg/ml in urine. Imprecision and percentage bias were typically <10% for both plasma and urine controls throughout their respective dynamic ranges. The absolute recoveries for ADR-529 and the IS from plasma were >95%. This method is being successfully applied to the pharmacokinetic/dynamic evaluation of ADR-529 in animals and humans. |
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Keywords: | column switching high-performance liquid chromatography electrochemical detection cardioprotectant (ADR-529) sensitive validation pharmacokinetics |
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