Introduction
Digitoxin and digoxin are cardiac glycosides derived from the digitalis purpurea (Foxglove) plant. They have been in use for centuries for treatment of various heart conditions. Because of their narrow therapeutic range and high toxicity, their levels in patients taking digitoxin or digoxin are monitored.1-2 Immunoassay-based methods for digitoxin or digoxin exist, but are both time consuming and labor intensive. Moreover, the reported immunoassay methods are not selective toward digitoxin or digoxin due to the similarities in their chemical structures, which differ from each other in only one hydroxyl group (Figure 1). The present work was aimed at developing a rapid and selective LC/MS/MS method for the determination of digoxin and digitoxin in biological fluids. In addition, a simple sample cleanup technique for simultaneous removal of proteins and phospholipids using a zirconia-based sorbent, HybridSPE®-PLus, was explored.
Figure 1. Digitoxin and DigoxinExperimental
A 100 μL sample of spiked rat plasma was added to the well of a HybridSPE-PLus 96-well plate followed by 300 μL of 1% formic acid in acetonitrile to precipitate the proteins. The plate was sealed with plastic film and agitated by vibration at 1,000 rpm for 2 minutes on a digital shaker. The plate was transferred to a vacuum manifold and the seal was removed. Vacuum (10 in. Hg) was applied for 4 minutes. The flow-through eluate was analyzed by LC/MS/MS. A Titan™ C18 column packed with 1.9 μm monodisperse, totally porous silica particles was chosen for the UHPLC separation of digitoxin and digoxin. Different mobile phase systems were studied.
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