A cardiotonic steroid multiplex method using ultra-high-performance liquid chromatography-tandem mass spectrometry

Abstract

Background and AimsDigoxin, a cardiotonic steroid (CTS), is commonly prescribed for patients with atrial fibrillation and heart failure. Endogenous CTS have been implicated in cardiovascular disease pathogenesis and can interact with digoxin. We developed an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of eleven CTS. Materials and MethodsIsotopically labelled internal standards were added to samples, followed by protein precipitation and solid-phase extraction. Steroids were separated using an Acquity uPLC chromatography system with a Waters CORTECS T3 column (1.6 m 2.1x50 mm) and quantification performed on a Waters TQ-XS mass spectrometer using electrospray ionisation in positive ion mode. Separation used a methanol/ water elution system containing 0.1% formic acid and post-column infusion of lithium chloride. ResultsRun time was 13.5 minutes. Lower limits of quantification ranged from 0.025 to 0.1 ng/ml. Serum recovery ranged from 40.0-98.6% with matrix effects from -22.9% to 7.6%. Plasma recovery ranged from 27.4-83.9% and matrix effects were -27.6-39.1%. Accuracy and precision at three concentrations were within ideal range ( View larger version (30K): org.highwire.dtl.DTLVardef@180bd42org.highwire.dtl.DTLVardef@109441corg.highwire.dtl.DTLVardef@1568cd9org.highwire.dtl.DTLVardef@1390caf_HPS_FORMAT_FIGEXP M_FIG C_FIG