Determination of Apixaban Levels in Human Plasma by a High-Throughput Liquid Chromatographic Tandem Mass Spectrometry Assay

A high-throughput liquid chromatography method with detection by tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantification of apixaban in human plasma. The separation was performed on a Gemini-NX column under isocratic conditions using a 33: 67 (v/v) mixture of acetonitrile and 1 mM ammonium formate in water at 40 degrees C with a flow rate of 0.5 mL/min. The detection of apixaban was performed in multiple reaction monitoring mode (m/z 417.2 from m/z 460.2) with electrospray positive ionization. A single-step protein precipitation with methanol was used for plasma sample preparation. The method was validated with respect to selectivity, linearity (r > 0.994), intra-day and inter-day precision (CV < 14.4 %) and accuracy (bias < 9.5 %) over the range of 9.70 - 970.00 ng/mL plasma. The lower limit of quantification (LLOQ) was 9.70 ng/mL and the recovery was between 97.4 - 104.5 %. The method is fast, efficient, requires the processing of a small volume of plasma (50 mu L), a short run-time (1 min) for chromatographic analysis, and a simple and rapid preparation of samples. It is very well suited for clinical therapeutic drug monitoring and pharmacokinetic studies.