A Validated LC-MS/MS Method for the Estimation of Apixaban in Human Plasma

A high performance liquid chromatography mass spectrometric method for the estimation of apixaban in human plasma has been developed and validated using apixaban 13CD3 as an internal standard (IS). The extraction of analyte and IS was accomplished by liquid-liquid extraction technique. The method has been validated over a concentration range of 1.00 ng mL-1 to 301.52 ng mL-1. Chromatographic separations was achieved using Thermo Beta basic-8, 100 mm x 4.6 mm, 5μ, column eluted at flow rate of 1.0 mL minute-1 with 1:1 splitted post column with mobile phase Acetonitrile: Ammonium formate buffer pH 4.2 (70:30 v/v).The overall run time of method was about 3.0 min. with elution times of apixaban and its internal standard apixaban 13CD3 at around 1.2 min. The multiple reaction monitoring transitions were set at 460.2 > 443.2 (m/z) and 464.2>447.4 (m/z) for apixaban and apixaban 13CD3 respectively. The calibration curves were linear (r2≥0.99) over the range of 1.0-301.52 ng mL-1 with lower limit of quantitation validated at 1.0 ng mL-1. Extraction recoveries were >98 % for both apixaban and its stable labeled IS apixaban 13CD3. The within run and between run precisions were within 0.70%-6.98%, while accuracy ranged from 89.2 to 107.2%.