Assay and Organic Impurity Profiling of Apixaban using an Ascentis Express C18 column and UV Detection

Sonal Shinde, Application Scientist

Introduction

Apixaban is a selective, orally bioavailable, and reversible inhibitor of free and clot-bound factor Xa (which catalyzes the conversion of prothrombin to thrombin). Apixaban is sold under the tradename Eliquis, and is typically prescribed to lower the risk of stroke and embolism in people with nonvalvular atrial fibrillation. It is also used to prevent deep vein thrombosis (DVT) which may lead to pulmonary embolism (PE) in knee or hip replacement surgery patients. Apixaban was developed in a joint venture by Pfizer and Bristol-Myers Squibb, and it was approved 2012 in Europe, and 2014 in the U.S.

At present there are no pharmacopeial monographs available with either an assay and/or organic impurity profiling method for Apixaban. We have therefore developed and validated a new HPLC method for said purposes using a column with C18 solid core particles and UV detection. The limit of detection (LOD) is 0.33 ppm and the limit of quantitation (LOQ) is 1.0 ppm at 235 nm for Apixaban using HPLC-UV detection.
Apixaban structure
Apixaban

 

Experimental conditions
column: Ascentis® Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles (53829-U)
buffer: dissolve 2.04 g of potassium dihydrogen phosphate in 1500 mL Milli-Q® water (10 mM). Adjust to pH 5.0 with triethylamine.
mobile phase: [A] buffer:methanol (90:10) (v/v) [B] buffer:acetonitrile:methanol (20:20:60) (v/v/v)
Gradient: Time (min) A(%) B(%)
     0 65 35
   18 55 45
   20 55 45
   22 50 50
   25 40 60
   30 30 70
   35 30 70
   40 65 35
   50 65 35
flow rate: 1 mL/min
pressure: 132-147 bar (1914-2131 psi)
column temp.: 40 °C
detector: UV @ 235 nm (analytical flow cell; 10 μL)
injection: 3 μL
diluent: mix water and acetonitrile 50:50 (v/v)
stock standard solution: 0.5 mg/mL of Apixaban standard in diluent.
standard solution: dilute 5 mL of stock standard solution to 100 mL to obtain 25 ppm standard solution. (Figure 1)
SST solution: weigh 2.5 mg of each impurity in 20 mL volumetric flask. Dilute to volume with diluent. Add 1 mL of this solution into a 50 mL volumetric flask and dilute to volume with standard stock solution. (Figure 2)
sample solution: 0.5 mg/mL of Apixaban sample in diluent (Figure 3)

 

Figure 1. Chromatographic data - blank & standard solution.

Chromatographic data - blank & standard solution

 

Figure 2. Chromatographic data - system suitability test (SST) solution.

Chromatographic data - system suitability test (SST)
solution

 

Peak Compound Retention Time (min) RRT Resolution Tailing Factor
1 Apixaban 16.0 1.0 - 1.2
2 Impurity A 24.8 1.5 40.3 1.0
3 Impurity D 27.0 1.7 13.6 1.1

 

Figure 3. Chromatographic data - sample solution.

Chromatographic data - sample solution

 

Peak Compound Retention Time (min) Resolution Tailing Factor
1 Apixaban 16.0 50327 1.2

 

Validation and verification

1. Specificity

Inject standard solution and determine the retention time of desired analyte in the presence of other components like impurities and excipient.

 

  Compound Retention Time (min)
1 Apixaban 16.0
2 Impurity A 24.8
3 Impurity B 27.0

2. Standard repeatability (25 ppm)

Sample Apixaban (area)
STD 1 165029
STD 2 165045
STD 3 165194
STD 4 165159
STD 5 165032
   
Mean 165091.8
Standard deviation 78.5
RSD (%) 0.1

3. Linearity, LOD & LOQ

Concentration (μg/mL) Apixaban (area)
0.25     2015
0.5     3527
1.25     8605
2.5   16392
6.25   41517
12.5   81937
20 132946
25 164362
30 196291
37.5 247176
   
LOQ (S/N ratio 10) 1.0 ppm
LOD (S/N ratio 3) 0.33 ppm

 

Figure 4. Apixaban calibration curve data (0-37.5 ppm).

Apixaban calibration curve data

Materials