Development of a high-performance liquid chromatographic assay for digoxin in plasma using post-column fluorogenic derivatization

Digoxin is a cardiac glycoside obtained in purified form from the leaves" of Digitalis lanata. The molecule is composed of a sugar portion and an aglycone (genin) portion. Chemically it is. the-drug of choice for the treatment of congestive heart failure and certain disturbances of cardiac rhythm. The therapeutic significance of digoxin, and particularly the relatively narrow margin between a therapeutic and toxic dose, has warranted the development of a specific and sensitive analytical method for their quantitation in biological samples. The principal method employed by most researchers and hospital laboratories for the assay of digoxin is based on the use of a radioimmunoassay (RIA) procedure. Present reports have shown that the digoxin metabolites, digoxigenin mono-digitoxoside, digoxigenin bis-digitoxoside and digoxigenin cross-react extensively in RIA. Dihydrodigoxin cross-reactivity depends on the RIA kit used. It was also shown that the RIA procedure gave false-positive results in specimens taken from non-digitalized renal patients. Such discrepancies raise questions regarding the "true" plasma digoxin concentrations measured by the current RIA method. Therefore, to ascertain the analytical reliability of available assays in measuring digoxin levels, this thesis reports the development of an HPLC separation of digoxin from its metabolites and some commonly co-administered drugs which, when coupled with post-column derivatization, will quantify the levels of intact digoxin in plasma. Essentially this procedure is based on a novel method developed by Gfeller et al. (9). The post-column detection is based on the reaction of hydrochloric acid with the steroid portion of the cardiac glycosides. Fluorescence of the derivative is further enhanced by the addition of a hydrogen peroxide/ ascorbic acid mixture. The fluorogenic reactants are also employed in the method utilized in the United States Pharmacopeia and hence are considered to be reliable. We have undertaken several modifications to the air segmentation post-column derivatization step developed by Gfeller's group (9). To increase the sensitivity of detection, a non-segmented post-column reactor is used. The glycosides are initially separated on a 15 cm octadecylsilane, 3u packing column, using a combination of methanol, ethanol, isopropanol and water as the mobile phase at a flow rate of 0.3 mL/min. A solution of dehydroascorbic acid and hydrochloric acid are added to the column effluent at 0.23 mL/min to form the fluorophore using a 10 m knitted reactor controlled at a temperature of 79°C. The glycosides react with HC1 and dehydroascorbic acid to form the fluorophore which is monitored by a Waters fluorescence detector equipped with a 360nm excitation filter and a 425 nm emission filter. The quantitation of digoxin in plasma is carried out by the incorporation of an internal standard, digitoxigenin. The method involves the extraction of the compound from plasma with methylene chloride containing 2% propanol. Endogenous substances, commonly co-administered drugs, and metabolites of digoxin do not interfere with the method. The total chromatographic time, including the post-column derivatization step, is about 40 minutes. The detection limit of the method is 0.5 ng/mL of digoxin in plasma. The average extraction recovery is 70% over the therapeutic concentration range. Detector response is linear from 0.5 ng/mL to 3.3 ng/mL of plasma. Using HPLC separation and post-column detection with parallel quantitation by radioimmunoassay, we examined the plasma of digitalized patients. The mean HPLC / RIA ratios are 0.94 1 0.3 (3-D.) (Acute Care Unit of the University Hospital) and 1.0± 0.34 (3.D.) (Vancouver General Hospital). These ratios are comparable to those found by specific HPLC-RIA results reported by other workers. The overall performance demonstrates that this system has the sensitivity, linearity and specificity desired for the determination of drug concentrations in plasma from digitalized patients.Pharmaceutical Sciences, Faculty ofGraduat