Development of a high-performance liquid chromatographic-post-column fluorogenic assay for digoxin in serum.
A quantitative, sensitive and specific assay for digoxin was developed using a high-performance liquid chromatographic (HPLC) system with post-column (PC) fluorogenic derivatization. Separation of digoxin from its metabolites was accomplished using a 15 cm X 4.6 mm I.D., 3-microns octadecylsilyl HPLC column and an optimum mobile phase of methanol-ethanol-isopropanol-dehydroascorbic acid (52:3:1:45, v/v). Concentrated hydrochloric acid, used as the PC derivatization reagent, was delivered by hexane displacement from a polyvinyl chloride pressure vessel. Construction of the pressure vessel is described. The mixture of HPLC effluent and PC reagent was passed into a 20-m knitted reactor (PTFE tubing) maintained at 79.0 +/- 0.2 degrees C. The resultant fluorophores were monitored by a fluorescence detector equipped with a 360-nm excitation filter and a 425-nm emission filter. Specificity of this HPLC-PC assay for digoxin in the presence of its metabolites was demonstrated. Also, numerous steroids evaluated did not produce fluorescence under these conditions. An extraction procedure for evaluating digoxin in serum without interference from endogenous compounds was also developed. Detector response to digoxin was linear from 0.5 to 3.3 ng extracted from serum.
Embree
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L
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
McErlane
K M
KM
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D004077:Digoxin / Q000097:blood*; D006801:Humans; D013050:Spectrometry, Fluorescence
D004077:Digoxin