Samples were prepared by SPE extraction with Oasis HLB columns. Separation was achieved with an ACQUITY UPLC HSS T3 column (2.1 × 100; 1.8 μm), at 35°C. The mobile phase consisted of acetonitrile (70%) and ammonium formate 5 mM (30%). Total run time was 1.5 min operating at isocratic mode with a flow rate of 0.3 mL/min. Mass spectrometry detection was performed by positive mode electrospray, on the ammonium adducts with two transitions for each analyte and one for the IS (d(3)-digoxin). The method proved to be specific and linear over the range (0.3-10) ng/mL. This technique also showed high sensitivity with a 0.09 ng/mL LOD and a 0.28 ng/mL LOQ, for both substances. Percentage recovery ranged from 83 to 100% for digoxin and from 62 to 94% for metildigoxin. The intra- and interday precision CV were ≤10%.
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