Transl Clin Pharmacol.  2019 Mar;27(1):33-41. 10.12793/tcp.2019.27.1.33.

Quantification of apixaban in human plasma using ultra performance liquid chromatography coupled with tandem mass spectrometry

• Jeong HC
• Kim TE
• Shin KH

• KMID: 2442244
• DOI: http://doi.org/10.12793/tcp.2019.27.1.33

Abstract

Apixaban, an inhibitor of direct factor Xa, is used for the treatment of venous thromboembolic events or prevention of stroke. Unlike many other anticoagulant agents, it does not need periodic monitoring. However, monitoring is still required to determine the risk of bleeding due to overdose or surgery. Usually, apixaban concentrations are indirectly quantified using an anti-factor Xa assay. However, this method has a relatively narrow analytical concentration range, poor selectivity, and requires an external calibrator. Therefore, the goal of current study was to establish an analytical method for determining plasma levels of apixaban using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To this end, apixaban was separated using 2.5 mM ammonium formate (pH 3.0) (A) and 100% methanol containing 0.1% formic acid (B) using the gradient method with a Thermo hypersil GOLD column. The mass detector condition was optimized using the electrospray ionization (ESI) positive mode for apixaban quantification. The developed method showed sufficient linearity (coefficient of determination [r²â‰¥ 0.997]) at calibration curve ranges. The percentage (%) changes in accuracy, precision, and all stability tests were within 15% of the nominal concentration. Apixaban concentration in plasma from healthy volunteers was quantified using the developed method. The mean maximum plasma concentration (C(max)) was 371.57 ng/mL, and the median time to achieve the C(max) (T(max)) was 4 h after administration of 10 mg apixaban alone. Although the results showed low extraction efficiency (~16%), the reproducibility (% change was within 15% of nominal concentration) was reliable. Therefore, the developed method could be used for clinical pharmacokinetic studies.

Keyword

Anticoagulant; Apixaban; Bioanalytical method; UPLC-MS/MS

MeSH Terms

Ammonium Compounds
Anticoagulants
Calibration
Chromatography, Liquid*
Factor Xa
Healthy Volunteers
Hemorrhage
Humans*
Mass Spectrometry
Methanol
Methods
Plasma*
Stroke
Tandem Mass Spectrometry*
Anticoagulants
Factor Xa
Methanol

Figure

• 

  Figure 1 Mass spectra and chemical structures of (A) apixaban and (B) deuterated internal standard (apixaban-d3) in electrospray ionization (ESI) positive mode. The blue arrows indicate spectral peaks corresponding to each structure.

• 

  Figure 2 Representative chromatogram of (A) double blank human plasma and (B) lower limit of quantification samples.

• 

  Figure 3 Representative chromatogram of (A) apixaban and (B) internal standard in plasma after oral administration of apixaban 10 mg.

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  Figure 4 Mean time-concentration profile after (A) administration of single dose of apixaban 10 mg and (B) co-administration with 20 mg rosuvastatin (crossed square) or 250 mg lapatinib (solid square). Error bars represent the standard deviation (SD).

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