UBC Theses and Dissertations
Development of a sensitive, quantitative high-performance liquid chromatographic assay for the measurement of digoxin in patient groups with high levels of digoxin-like immunoreactive substances Embree, Leanne
Digoxin is the most commonly used digitalis glycoside for the treatment of congestive heart failure and certain disturbances of cardiac rhythm. The low therapeutic index observed for digoxin and the clinical significance of digoxin therapy have necessitated the development of sensitive analytical methods for the quantitation of digoxin in biological samples. Digoxin may be analysed by several methods including immunoassays, chromatographic procedures and various biological and chemical methods. Immunoassays, both radioimmunoassay (RIA) and fluorescence polarization immunoassay (FPIA) procedures, are used in the clinical laboratory because of their speed, precision, sensitivity and relatively low cost. However, reaction of the digoxin antibodies used in the immunoassay methods with digoxin metabolites, endogenous compounds such as digoxin-like immunoreactive substances (DLIS), and other drugs that may be co-administered with digoxin continues to be a major problem. The lack of specificity of the immunoassay methods for digoxin has led to difficulties in interpretation of assay values. Attempts to compensate for this lack of specificity have included the use of chromatographic systems as elaborate sample handling methods prior to immunoassay. However, since an immunoassay was used for detection of digoxin in these techniques, the specificity may still be quest ionable. A sensitive and specific assay for digoxin using physico-chemical methods for measurement is therefore needed. A method was developed using pre-column derivatization of digoxin and its metabolites with 3,5-dinitrobenzoyl chloride followed by HPLC analysis with electrochemical detection. A maximum sensitivity of 0.883 ng of 3,5-dinitrobenzoyl digoxin (0.394 ng digoxin) was observed using dual electrode detection in the redox mode. Although resolution between derivatized digoxin and its metabolites was obtained, the low yield of the digoxin derivative and the formation of metabolites when small (ng) samples were derivatized made this method unsuitable for evaluating patient samples. A high-performance liquid chromatographic (HPLC) assay using post-column derivatization of digoxin, which separated digoxin from its metabolites and some commonly coadministered drugs, was developed. Post-column (PC) derivatization of digoxin with concentrated hydrochloric acid and dehydroascorbic acid, followed by fluorescence detection, allowed for quantitation within the therapeutic range of digoxin. Steroids which have been reported to cross-react with digoxin antisera were assayed using the HPLC-PC method developed in this study. The steroid samples either did not elute from the HPLC system or did not produce a fluorescent product under these conditions. Serum samples from digitalized patients were evaluated using both the HPLC-PC and the FPIA methods. When compared to the HPLC procedure, the FPIA assay results gave, on average, higher digoxin levels. This may have been due to the inclusion of digoxin metabolites or endogenous compounds with the FPIA assay. Serum samples from undigitalized patient groups where high DLIS levels have been reported were also evaluated. These included umbilical cord blood samples and samples from hypertensive patients, renal failure patients and hepatic failure patients. Comparison of the HPLC-PC and FPIA methods demonstrated that the HPLC-PC assay gave fewer false positive results than the FPIA. The HPLC-PC assay developed for analysis of digoxin was unaffected by the presence of digoxin metabolites, numerous steroids, co-administered drugs and endogenous compounds, most of which have been reported to give false positive results with the FPIA.
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