Development and Validation of an New High Performance Liquid Chromatography Method for Determination of Apixaban Isomers

The purpose of this research study is to develop a novel, simple, precise, accurate and economical method for determination of Apixaban API isomers. This chromatographic method was developed on chiralpak IA column(250 x 4.6 x 5µm) with isocritic technique. The detection of impurities was observed at wavelength 290nm. This analytical method was validated and regression analysis showed R value (correlation coefficient) > 0.999 for Apixaban and its isomeric impurities. A solution of Apixaban in dichloromethane was found stable up to 48 hrs. The degradation study was done within the given guidelines prescribed by ICH. The method is validated for linearity, accuracy and precision.


Introduction
Apixaban is chemically known as 1-(4-methoxyphenyl)-7oxo-6-[4-(2-oxopiperidin-1-yl) phenyl]-4, 5, 6, 7-tetrahydro-1H-pyrazolo [3, 4-c] pyridine-3-carboxamide is a Anticoagulant drug, a direct inhibitor of factor X, also used in the prevention of venous thrombo embolism.Apixaban is recommended by the National Institute for Health and Clinical Excellence for the prevention of stroke and systemic embolism in people with non-valvular atrial fibrillation and at least one of the following risk factors: prior stroke or transient ischemic attack, age 75 years or older, diabetes mellitus, or symptomatic heart failure [1].

Objectives of the Study
The literature survey reveals that few analytical methods have been reported for Apixaban and its related compounds including spectroscopic methods, high performance liquid chromatography (HPLC) methods [7][8][9][10][11].Our objective is to develop and validate new normal phase HPLC method for determination of Apixaban and its isomer.The proposed NP-HPLC method utilizes economical solvent system having advantage like better retention time, peak sharp and symmetric nature.In reverse phase Chromatographic these impurities are not well separated from the product hence new chiral technique was developed.This method was validated according to ICH Guidelines.

Instrumentation
Waters, Alliance 2695 series HPLC system comprising a Quaternary pump, an auto-sampler, a thermostat column compartment, a solvent cabinet with gasser along with photo diode array (PDA) 2998 and ultraviolet (UV) 2487 detectors were used for separation and detection.Data acquisition and calculations were carried out using Waters Empower3 software (Milford).Sartorius (Germany) analytical balance was used for weighing material.

Preparation of Blank
Transfer 5 ml Dichloromethane into 10 ml volumetric flask and make up with diluent.

System Suitability Solution
Transfer about 5 mg of Apixaban working standard, Apixaban ortho impurity and Apixaban meta impurity into 10 mL volumetric flask, add 5 ml dichloromethane and dissolve in and make upto the mark with diluent.

Reference Solution (a)
Transfer about 60 mg of Apixaban working standard into 10 mL volumetric flask, dissolve in about 5 mL of dichloromethane and make upto the mark with diluent.
Transfer 1 mL of above solution to 100 mL volumetric flask and make upto the mark with diluent.Further transfer 1 mL of above solution into 10 mL volumetric flask and make upto the mark with diluent.

Test Solution
Transfer about 60 mg of Apixaban sample into 10 mL volumetric flask, dissolve in about 5 mL of dichloromethane and make upto the mark with diluent.Reference solution (a) 5 5 Test solution 2

Procedure
Equilibrate the HPLC system with the initial composition until a steady baseline is obtained.Inject Blank, System suitability solution and reference solution (a).Ensure that all the system suitability parameters meet the requirements.Inject test solution as per injection sequence and record the chromatograms.Make blank correction if necessary.

Acceptance Criteria
Resolution: The resolution between the peaks due to meta impurity and Apixaban in the chromatograms obtained with system suitability solution should not be less than 1.5.
%RSD: The percent relative standard deviation of three replicates for the peak due to Apixaban in the chromatograms obtained with reference solution (a) should not be more than 5.0.

Calculation
Calculate impurity content by formula given below:

Analytical Method Validation
The developed method is subjected to analytical method validation, which is conducted according to International Council for harmonization (ICH) guidelines.The parameters with which analytical method is validated are specificity, limit of detection and limit of quantification, linearity, accuracy, precision [4][5][6].

Specificity
Specificity is capability of the method to measure the analyte response in presence of impurities.The typical chromatograms of blank Solution, System suitability solution, Reference Solution (a), and Impurities Spike Solution are given from figure 1 to figure 4 respectively.The results indicate that all impurities are well separated under the current chromatographic conditions.There was no interference of peak from blank solution and samples solution within retention time of impurities obtained.Peak purity for Apixaban and its impurities were passing.For retention time of each impurity and its peak purity refer table no.5

Limit of Detection and Limit of Quantification
Series of standard solution of Apixaban and its impurities were prepared and injected in concentration ranging from 50% to 500%.Limit of detection (LOD) and Limit of quantification (LOQ) was calculated based on residual standard deviation of regression line and slope.Both calculated LOD and LOQ were well within limit.The LOQ is below 0.057% for impurities and Apixaban API.For details of LOQ and LOD refer Table.

Linearity
Series of linearity solution of Apixaban and its impurities were prepared from 50% to 500% of test concentration.Linearity curves were drawn by plotting the peak areas of Apixaban and its impurities against its corresponding concentration of linearity solution.Observed regression coefficient was greater than 0.998 and % y-intercept was less than 5.0%

Accuracy
Accuracy of method is calculated and established by carrying out recovery studies of impurities.The test solution was spiked with impurities solution at specific limit level concentration 100%, 150%, 300%.Each spiked test solution was analyzed for recovery study of impurities.Recovery established is between 100% to 105%.

Precision
System precision was carried out by analyzing six injections of reference solution (a) of Apixaban API at limit level concentration.Relative standard deviation for peak area of Apixaban was calculated and found to be 0.33%.

Robustness
Robustness was studied by making small but deliberate changes in optimized method conditions and evaluating the effect on resolution between ortho impurity, meta impurity and Apixaban.The mobile phase flow rate was changed by±0.1 units from 1.0 to 0.9 and 1.1 ml/min.

Solution Stability
The solution stability of Apixaban and its impurities in related substances method was evaluated by leaving a spiked solution in tightly capped volumetric flask at room temperature for 48 hrs and analysis of all its impurities content was done at interval of 12 hrs.

Result and Discussion
Since most of the impurities are non-polar in nature, various chiral columns were screened.The parameters like tailing factor and theoretical plates were recorded during the study.From obtained data Chiralpak IA (250 x 4.6 x 5µm) was found suitable for analysis.
All impurities were prepared at 100 ppm and their UVvisible spectra was acquired.The Apixaban and all its impurities has good and satisfactory response at 290nm.Hence detection at 290nm was selected for methoddevelopment.
Retention time were confirmed by injecting working standards of Apixaban, Meta and ortho impurities.For System Suitability, the resolution between Apixaban and Meta imp should be not less then 1.5.Relative Standard Deviation for five replicate injections peak due to Apixaban should not be more then 5%.

Conclusion
The analytical method validation for Apixaban by normal Phase HPLC was carried out by performing the parameters such as specificity, limit of detection and limit of quantification, linearity, accuracy, precision, robustness and solution stability.All the data has been compiled and found to be satisfactory.Hence, method developed for normal Phase HPLC can be suitably used for analysis of Apixaban isomer in pharmaceutic industries.

4. 1 .
Chromatographic Condition and Measurement Procedure 4.1.1.Mobile Phase Mix 65 volume of n-Hexane, 15 volume of Toluene, 10 volume of Methanol and 10 volume of Ethanol and degas by sonication for 2 mins.
peak area for respective impurity in test solution.AR = Average peak area of Apixaban in reference solution (a).WS = Weight in mg of Apixaban working standard taken for reference solution (a) preparation.WT = Weight in mg of Apixaban sample taken for test solution preparation.P = Potency of Apixaban working standard (%).

Table 1 .
Chemical name of Apixaban and its isomers.

Table 4 .
Peak name with Retention Time and Relative Response Factor.

Table 5 .
Peak purity and RT Ratio for Apixaban and its impurities.

Table 7 .
Linearity table and its R 2 values Apixaban.

Table 8 .
Linearity table and its R 2 values of Meta Impurity.
Figure 6.Linearity graph of Meta Impurity.

Table 9 .
Linearity table and its R 2 values of Ortho Impurity.