A Review of New Analytical Methods for Quantitative Determination and Validation of Apixaban

Abstract

The development and validation of analytical methods form an ongoing, interconnected component of research and development, as well as quality assurance and quality control. These activities play a key role in risk management and equivalence testing because they help define product-specific acceptance criteria and ensure trustworthy analytical results. Validation studies determine whether a method is suitable for its intended analytical purpose. A comprehensive survey of existing literature shows that Apixaban can be evaluated using various analytical approaches, including UV spectroscopy, RP-HPLC, and HPTLC, either alone or in combination with other drugs. Validation parameters—such as accuracy, precision, robustness, and others—have been rigorously assessed following ICH guidelines. Owing to their simplicity, sensitivity, and reproducibility, these analytical techniques are applicable to both bulk drug and tablet formulations of Apixaban. The review also discusses the advantages and limitations of the available analytical methods for Apixaban. This extensive overview will be highly beneficial for researchers conducting studies involving Apixaban.

Keywords

Introduction

Apixaban is a new generation of oral anticoagulant drug that selectively inhibits coagulation factor Xa. It {is chemically 1-(4-methoxyphenyl)-7-oxo-6-[4 (2-oxopiperidin-1-yl) phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4c]pyridine-3-carboxamide. It is used in thromboprophylaxis in patients following total knee replacement surgery with a desired efficacy and safety profile¹. Apixaban belongs to the anti-coagulant category of drug which acts by directly inhibiting the Factor Xa involved in the conversion of prothrombin to thrombin in the coagulation cascade. Thus helps in inhibiting the clot formation. Apixaban is used in the treatment of venous thrombosis and reduce the risk of stroke and systemic embolism².

ANALYTICAL METHODS REPORTED ON APIXABAN

1. Kunala Anusha et al., (2018) have developed two sensitive zero order (D0) and first order derivative (D1) UV spectroscopic methods have been developed and validated for the determination of Apixaban in pharmaceutical formulations using phosphate buffer pH 5.0 (Method A), and phosphate buffer pH 7.0 (Method B). The drug showed maximum absorbance at 276 nm in zero order and the amplitude was measured in the range of 264 nm - 305 nm in first order derivative. Beer-Lambert’s law was obeyed in the range of 1 – 60 µg/mL. Accuracy of the methods were established by standard addition and the recovery was found to be in the range of 98.3 - 101.1 % (% RSD < 0.66, zero order), 99.06 -100.4 % (% RSD < 1.75). The developed methods were successfully applied for the assay of Apixaban in tablets and there was no interference from the excipients. The developed methods were found to be sensitive as observed from the optical characteristics. These methods were validated as per ICH guidelines and can be suitably used for the analysis of Apixaban in API and tablets.
2. B. Mahendra et al., (2019) aim to Develop UV spectrophotometry method for the estimation of Apixaban in its dosage forms. Analysed the marketed formulations for their reliability and accuracy and performed the recovery studies for the developed UV spectrophotometric method. The developed method was validate for its accuracy precision reproducibility. On the basis of results the UV spectrophotometric method developed for the determination of Apixaban is found to be precise, accurate and cost effective. Hence this method can be used for routine analysis of Apixaban in bulk and pharmaceutical dosage forms.
3. Narender Boggula et al., (2018) to validate the apixaban content in bulk and pharmaceutical dosage formulation and validate it as per ICH guidelines. A simple, rapid, precise and highly selective spectrophotometric method was developed for estimation of apixaban in tablet dosage form by Area Under Curve method. Area Under Curve method, involves the measurement of absorbances of apixaban at the wavelength of 269 nm-289 nm. Methanol was used as solvent. Linearity was observed in the concentration range of 5–25 µg/ml for apixaban. The accuracy of the method was confirmed by recovery studies of tablet dosage forms and was found to be 100% for Apixaban. The method showed good reproducibility and recovery with % RSD less than 0.988%. The LOD of apixaban was found to be 0.335 µg/ml and LOQ of apixaban was found to be 1.015 µg/ml. Thus the proposed method was found to be rapid, specific, precise, accurate and cost effective quality control tool for the routine analysis of Apixaban in bulk and tablet dosage form. Drug stability studies have been determined for the formulation under specified conditions and it was found stable.
4. A G Radhika et al., (2018) A simple, rapid, precise and highly selective spectrophotometric method was developed for estimation of Apixaban in tablet dosage form by Zero Order Derivative and Area Under Curve. Zero order derivative and Area Under Curve methods involve the measurement of absorbances of Apixaban at the wavelength of 279 nm and 269-289 nm respectively. Methanol was used as solvent. Linearity was observed in the concentration range of 5-25 µg/ml for Apixaban. The accuracy of the Zero order derivative method and AUC were confirmed by recovery studies of tablet dosage forms and were found to be 100% and 100% for Apixaban respectively. The Zero order derivative method and AUC showed good reproducibility and recovery with % RSD less than 0.790% and 0.988%. The LOD for Zero order derivative method and AUC of Apixaban were found to be 0.23 µg/ml and 0.335 µg/ml and LOQ for Zero order derivative method and AUC of Apixaban were found to be 0.713 µg/ml and 1.015 µg/ml respectively. Thus, the proposed method was found to be rapid, specific, precise, accurate and cost-effective quality control tool for the routine analysis of Apixaban in bulk and tablet dosage form. Drug stability studies have been determined for the formulation under specified conditions and it was found stable.
5. Swarup Suresh Prabhune et al., (2014) have developed a simple, robust, and stability-indicating reversed-phase high-performance liquid chromatographic (HPLC) method for the analysis of apixaban and its related substances has been successfully developed. Chromatography was performed on a 250 mm × 4.6 mm, 5 µm C18 column with a gradient mixture of a phosphate buffer–methanol 60:40 (v/v) at 1.0 mL min−1. Ultraviolet detection of apixaban was at 220 nm. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting apixaban solution to photolytic, acidic, basic, oxidative, and thermal degra dation. The peaks from the degradation products did not interfere with that from apixaban. The method was used to quantify the related substances in apixaban in the bulk drug and can be used for routine quality control purposes.
6. Rajendra Singh Rajput et al., (2022)  have developed A simple, consistent and sensitive stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of Apixaban in pharmaceutical dosage form by using Zorbax C18 (150 mm x 4.6 mm, 5μm particle size) with a mobile phase consisting of Ammonium Formate Buffer: Acetonitrile in a ratio of 65:35% v/v and successfully validated as per the international council for harmonization (ICH) guideline. The method was found to be simple, robust, precise, sensitive, accurate; specific and stress degradation studies were performed with acidic, alkaline, oxidative, thermal, humidity and photolytic stress conditions as per ICH guidelines.
7. Amit Gosar et al., (2020) The purpose of this research study is to develop a novel, simple, precise, accurate and economical method for determination of Apixaban API isomers. Apixaban API has three isomer as ortho, meta and para. In reverse phase and normal phase chromatography it was very difficult to separate these isomer; hence new chiral technique was adopted. This chromatographic method was developed on chiralpak IA column (250×4.6×5µm) with isocritic technique. The detection of isomeric impurities were observed at wavelength 290nm. This analytical method was validated as per ICH guideline and regression analysis showed R value (correlation coefficient) > 0.999 for Apixaban API 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.
8. Israa Al-Ani  et al., (2021) to develop a sensitive, specific, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method and validate it for the quantification of process related and degradation impurities of apixaban; an anticoagulant drug. The chromatographic separation was achieved on a Sigma-Aldrich’s Ascentis Express® C18 (4.6 mm × 100 mm, 2.7 µ) HPLC column with a runtime of 40 min. Mobile phase-A and mobile phase-B were phosphate buffer and acetonitrile, respectively. The column oven temperature was set at 35 °C, and the photodiode array detector was set at 225 nm. The newly developed method was utilized to detect nine process-related impurities (Imp-1 to Imp-9) in a test sample of Apixaban. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic, and thermal conditions to demonstrate the stability-indicating nature of the developed RP-HPLC method. The developed method was validated as per ICH guidelines and found to be specific, precise, sensitive, and robust. In conclusion, the RP-HPLC method was successfully developed and validated then effectively applied to analyze both; Apixaban drug substance and product.
9. Erten Akbel et al., (2023) In this study, two different chromato graphic methods were developed for the determination of APX in pharmaceutical products. In the first method, an Agilent C18 column (250 mm × 4.6mm, 5μm) wasused, and the temperature was kept constant at 30°C. The mobile phase was chosen to be 0.1% trifluoroacetic acid solution and acetonitrile (65:35, v/v), and isocratic elution was applied. The flow rate of the mobile phase was found to be 1.0mL·min−1 and the injection volume was 20µL. The detection was carried out at a wavelength of 276nm using a UV detector. In the second method, ethanol was used as an organic modifier. The only difference between these methods was the organic modifier. All other conditions of the methods were the same. Both chromatographic methods were vali dated in accordance with ICH guidelines for various para meters such as selectivity, linearity, accuracy, precision, detection and quantification limit, and robustness.
10. A N Shaikh et al., (2022) to develop and validate a simple, precise and sensitive method for estimation of Apixaban in bulk drug and its marketed formulation using the RP-HPLC method. The separation was achieved on C18 column (4.6 id x 250mm; 5µm) using mobile phase (Water: ACN) in the ratio of 40:60(v/v) with a run time of 7 minutes and wavelength for estimation of Apixaban was taken as 280 nm. Literature survey reveals that there are very few HPLC, UV methods available, Hence RP-HPLC method for estimation of Apixaban was developed. Various parameters likeLinearity, Accuracy, Repeatability, Precision, Robustness, LOD and LOQ, % Recoverywas validated as per ICH guideline.
11. Natalia Olegario dos Santos et al., (2022) to develop and validate an in vitro method to assess the dissolution profile of apixaban immediate-release (5 mg) coated tablets. Several conditions were tested in this study until the most suitable one was reached. The selected method included the following parameters: 0.01 M hydrochloric acid (pH 2.3, 500 mL), USP paddle apparatus, 75 rpm, 37 °C, with seven sampling points and a total test time of 90 minutes. Quantitative analysis was performed by high performance liquid chromatography using a previously validated method. The dissolution method was validated following the official guidelines, demonstrating specificity, linearity, precision, accuracy, and robustness. This study enabled the development of an adequate, effective, and reliable method, which contributes to the evaluation of apixaban release in new products and the quality control of formulations containing this drug.
12. Rashmi Shukla et al., (2024) The presented work highlights the use of the quality by design methodology in accordance with International Council on Harmonization (ICH) recommendations for developing an reverse phase high performance liquid chromatographic (RP-HPLC) method for apixaban (APX) and clopidogrel (CLP). An efficient approach based on a factorial design incorporating the essential method parameters of the RP-HPLC method such as flow rate, pH, and proportion of organic phase, is presented. The optimum conditions for analysis were derived by using Design Expert software 10.0 Version, i.e., Hypersil ODS C18 column (5.0 μ, 25 cm × 4.6 mm), methanol and 0.05M potassium dihydrogen phosphate buffer (63.5:36.5%v/v, pH 3) as mobile phase, and 0.8 ml/minute as the flow rate. The derived condition gave excellent resolution between APX and CLP with Rt of 4.89 and 14.35 minutes, respectively, with the best possible system suitability parameters. The developed method presented excellent linearity in the range of 1.25–3.75 µg/ml for APX and 37.5–112.5 µg/ml for CLP at 245 nm. The optimized method was further validated in accordance with ICH guidelines on analytical method validation. Finally, the approach was successful in determining APX and CLP from a binary combination.
13. Mrinalini C. Damle et al., (2019) to develop and validate simple, sensitive stability indicating HPTLC (High performance thin layer chromatography) method for apixaban. Methods: The chromatographic separation was performed on aluminium plates precoated with silica gel 60 F254 methanol (3:6:1 v/v/v) as mobile phase followed by densitometric scanning at 279 nm. using toluene: ethyl acetate. The chromatographic condition shows sharp peak of apixaban at Rf value of 0.38±0.03. Stress testing was carried out according to international conference on harmonization (ICH)Q1A (R2) guidelines and the method was validated as per ICH Q2(R1) guidelines. The calibration curve was found to be linear in the concentration range of 100-500 ng/band for apixaban. The limit of detection and quantification was found to be 11.66ng/bandand35.33ng/band, respectively.
14. Jennifer Lagoutte-Renosi et al., (2018) have developed a method for simultaneous determination of dabigatran, rivaroxaban and apixaban in human plasma using high performance liquid chromatography coupled with a mass spectrometry assay and applied it to 26 patient samples. Our method presents a total run time of 5 minutes and extends from 25 to 1000 µg/L for apixaban and dabigatran; and from 5 to 1000 µg/L for rivaroxaban. Intra- and inter-assay accuracy were between -22.3 and 25.4 %; and -23.7 and 3.8%, respectively. Precision at low and high concentrations were below 17.5 %. Frozen samples were stable up to 3 months. No significant cross contamination was observed. In conclusion, our assay can be used during clinical studies and in daily routine practice for the management of specific clinical situations at reasonable cost..
15. Ramzia I. El-Bagary et al., (2016) the development of simple and valid spectrofluorimetric methods for the determination of Apixaban at λ ex/ λ em= 284/ 450 nm and tirofiban HCl at λ ex/ λ em= 227/ 300 nm in aqueous media. Different experimental parameters affecting fluorescence intensities were carefully studied and optimized. The fluorescence intensity-concentration plots were linear over the ranges of 0.2-6 µg ml-1for apixaban and 0.2-5 µg ml-1for tirofiban HCl. The limits of detection were 0.017 and 0.019 µg ml-1and quantification limits were 0.057 and 0.066 µg ml-1for apixaban and tirofiban HCl, respectively. The fluorescence quantum yield of apixaban and tirofiban were calculated with values of 0.43 and 0.49. Method validation was evaluated for linearity, specificity, accuracy, precision and robustness as per ICH guidelines. The proposed spectrofluorimetric methods were successfully applied for the determination of apixaban in Eliquis tablets and tirofiban HCl in Aggrastat intravenous infusion. Tolerance ratio was tested to study the effect of foreign interferences from dosage forms excipients. Using Student's t and F tests, revealed no statistically difference between the developed spectrofluorimetric methods and the comparison methods regarding the accuracy and precision, so can be contributed to the analysis of apixaban and tirofiban HCl in QC laboratories as an alternative method.

RESULTS AND DISCUSSION:

A variety of analytical methods have been developed and validated for the determination of Apixaban in bulk drug and pharmaceutical formulations, ranging from simple UV spectrophotometric techniques to advanced chromatographic and spectrofluorimetric methods. UV-based zero-order, derivative, and AUC approaches proved accurate, precise, and economical for routine quality control. Stability-indicating RP-HPLC methods provided high specificity by effectively separating Apixaban from degradation products under stress conditions. Specialized chromatographic techniques, including chiral and impurity-focused HPLC, successfully detected isomeric and process-related impurities. Additional methods such as HPTLC offered fast screening options, while LC–MS/MS enabled sensitive quantification in biological samples for clinical use. Spectrofluorimetric analysis demonstrated very high sensitivity, and validated dissolution testing supported formulation evaluation. Overall, these studies confirm that Apixaban can be reliably analyzed using diverse, ICH-compliant methods depending on sensitivity needs, matrix complexity, and analytical objectives.

CONCLUSION

A wide range of validated analytical methods—including UV spectrophotometry, derivative and AUC techniques, RP-HPLC, stability-indicating HPLC, chiral chromatography, HPTLC, LC–MS/MS, spectrofluorimetry, and dissolution testing—have been developed for Apixaban. UV methods offer simple and cost-effective routine analysis, while HPLC techniques provide high specificity and stability-indicating capability for impurities and degradation products. Chiral and impurity-profiling methods ensure quality of the API, and LC–MS/MS enables sensitive detection in biological samples. Spectrofluorimetry offers excellent sensitivity, and dissolution testing supports formulation evaluation. Overall, all methods meet ICH validation standards and reliably quantify Apixaban across diverse applications.

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