Abstract
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%.
Highlights
Apixaban is a potent, oral, reversible, direct and highly selective active site inhibitor of Factor-Xa
The accuracy of the assay was defined as the absolute value of the ratio of the calculated mean values of the lowest limit of quantification (LLOQ) QC, LQC, M1QC, MQC and HQC samples to their respective nominal values, expressed in percentage (Fig. 3).Within batch precision ranged from 0.70 % to 3.72 % and the within batch accuracy ranged from 89.24 % to 107.20 %
Intra-day precision ranged from 0.98 % to 3.27 % and the intra-day accuracy ranged from 89.60 % to 94.30 %
Summary
Oral, reversible, direct and highly selective active site inhibitor of Factor-Xa. Apixaban inhibits free and clot-bound Factor-Xa, and prothrombinase activity. Apixaban has no direct effects on platelet aggregation, but indirectly inhibits platelet aggregation induced by thrombin. By inhibiting Factor-Xa, apixaban prevents thrombin generation and thrombus development (Mueck et al., 2011; Mueck et al, 2013; Matchar et al, 2002; Gadisseur et al, 2004; Shenker et al, 2012). Apixaban is extensively metabolized via CYP3A4 and is a substrate for P-glycoprotein (Barrett et al, 2010; Wang et al, 2010; Wang et al, 2006).
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