Int J Pharm Pharm Sci, Vol 9, Issue 10, 24-32Original Article
FORMULATION DEVELOPMENT AND STABILITY INDICATING HPLC ASSAY OF TABLETS OF APIXABAN
HEMANT K. JAIN*, VISHAL K. NIKAM
Department of Quality Assurance Techniques, Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune 411041, Maharashtra, India
Email: hemantkjain2001@yahoo.co.in
Received: 29 May 2017 Revised and Accepted: 31 Aug 2017
ABSTRACT
Objective: Cost effective formulation development and stability indicating HPLC method for estimation of apixaban in bulk and tablets dosage form.
Methods: 32 factorial design was applied to formulate the immediate release tablets of apixaban by using direct compression method. The chromatographic separation was performed on Purospher Star RP-18e (5 µm, 250x4, 6 mm)column and a stability indicating assay method was developed by using HPLC. The mobile phase consists of water: acetonitrile (60:40 v/v) was delivered at a flow rate of 1 ml/min and UV detection at 280 nm. The method was validated with forced degradation studies as per ICH guidelines.
Results: Prepared batches were evaluated for all pre-compression parameters and post-compression parameters. Formulation batchF5 prepared by direct compression shows highest dissolution release of 90.97 % over the period of 60 min while disintegration time was found to be 136seconds.The retention time of developed HPLC method was found to be 5.66 min. This method was found to be linear in a concentration range of 5-30 μg/ml of the drug (r2= 0.999). The low value of % RSD in theprecision study indicates reproducibility of the method. The low value of LOD and LOQ suggests the sensitivity of the method. The results of forced degradation studies indicated that the drug was less stable in thermal and photolytic condition and degraded in acidic, basic, oxidative conditions.
Conclusion: On the basis of formulation evaluation, batch F5 was found to be promising formulation suitable for the immediate release of apixaban. Results obtained by validation studies suggested that the developed stability indicating assay method is simple, accurate, specific, sensitive and precise. Thus, this method can be used for routine analysis of apixaban formulation and to check the stability testing.
Keywords:Apixaban,Formulation Development, Tablets, RP-HPLC, Stability indicating assay method, Method validation
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i10.20343
INTRODUCTION
Apixaban(fig. 1) [4] is chemically 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide. Itis a new generation of oral anticoagulant drug that selectively inhibitscoagulationfactor Xa [1].It is used in thromboprophylaxis in patients following total knee replacement surgery with a desiredefficacy and safety profile [2].FDA approved apixaban (Eliquis, Bristol-Myers Squibb/Pfizer) onDecember28, 2012,for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation (AF) [3]. Apixaban is not anofficialdrug in any Pharmacopoeia.Literature survey reveals that only one marketed formulation of apixaban is available which is very costly and cannot afford to poor patients.Therefore, a cost-effective formulation as compared to themarketed formulation is developed. Some methods have been reported for their determination of apixaban byHPLC[4-6] and hyphenated techniques such as UPLC–MS/MS[7-8], LCMS[9-10], GCMS[11],either alone or in combination. This paper presentsformulation development of apixaban as well asdevelopment and validation of stability indicating assay method by using theRP-HPLCtechnique.
Fig.1: Chemical structure of apixaban
MATERIALS AND METHODS
Materials
Commercially available tablets of apixaban (Eliquis®contain 5 mg)were procured from local market and apixaban API was obtained from an approved supplier. All excipients were obtained from Loba Chemie Pvt. Ltd., Mumbai, India. HPLC grade solventsused in this study were obtained from Merck SpecialtiesPvt. Ltd., Mumbai.
Instruments
Apixaban tablets were formulatedon single punch tablet compression machine Mini press 1 (KarnavatiEngineeringlimited).The friability test was performed on Electrolab EF-2 friabilator USP. Disintegration test was performed on Electrolab disintegration tester ED-2L. The dissolution testing was performed on LabindiaDS 8000. The method was performed on Shimadzu LC 2010 CHT, Japan having a quaternary system with automatic injection facility and UV-Visible detection system. The column used was Purospher Star RP-18e (5 µm, 250x4, 6 mm), LC solution software and Shimadzu AY-120 balance was used for this work.
Formulation of tablets
Before formulation and pre-formulation studies (organoleptic properties, solubility, and drug excipient compatibility studies) were carried out. Apixaban tablets were formulated by using 32factorial design as presented in table 1. Drug, binder, super-disintegrant and other excipients were weighed separately for 60 tablets per batch as per proposed formulations. The proposed formulations were coded as F1, F2, F3, F4, F5, F6, F7, F8 and F9. The amounts of drug and excipients are expressed in mg (milligram) unit. Initially, the binder, super-disintegrant and other excipients were passed through sieve no. 40. Then,apixaban (API) was added, mixed properly for 5-10 min and sieved again. Blended mass was taken in the hopper and then die and punch were adjusted to get the desired weight of the tablet (100 mg). Tablets were prepared using flat face round 6.5 mm diameter punch by thedirect compression process.
Table 1: Batches designed by using 32factorial design
| Ingredients | Formulation code (mg/tablet) |
| F1 | |
| Apixaban | 5 |
| Lactose | 49 |
| MicrocrystallineCellulose | 30 |
| CroscarmelloseSodium | 3 |
| Magnesium Stearate | 2 |
| PVPK-30 | 10 |
| SLS | 1 |
Evaluation of powder blend
Angle of repose
The angle of repose (θ) was determined using fixed funnel method. The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the granules. The granules were allowed to flow through the funnel freely onto the surface. The diameter of the granular cone was measured and angle of repose was calculated using the following equation.
Whereh andr are the height and radius of the cone.
Bulk density
Bulk density was determined by pouring a weighed quantity of tablet blend into agraduated cylinder and measuring the height. Bulk density was calculated using the following equation.
Wherem isweight of powder (g),v is bulk volume (cm3),r isradius of cylinder (cm) andh isheight reached by powder in cylinder (cm)
Tapped density
Tapped density is theratio of themass of tablet blend to thetapped volume of tablet blend. Tablet blend was poured intograduated cylinder. Then, thecylinder was allowed to 100 taps under its own weight onto a hard surface. Tapped density was calculated using following equation.
Wherem is weight of powder (g), v is Tapped Volume (cm3),r isradius of cylinder (cm) andh isheight reached by powder in cylinder after tapping (cm)
Hausner’s ratio:
Hausner’s ratio indicates the flow properties of thepowder and measured by the ratio of tapped density to bulk density. Hausner’s ratio was determined by the given formula
Carr’s compressibility index
Compressibility is the ability of thepowder to decrease in volume under pressure using bulk density and tapped density. It is indirectly related to the relative flow rate. Carr’s compressibility index was determined by the following formula
Evaluation of tablets
Appearance
The general appearance of thetabletwas observed for elegance, shape, color, surface textures.
Tablet thickness
The thickness of the tablets was determined by using Vernier calipers. Randomly, 5 tablets selected from each formulation were used for determination of thickness.
Tablet Hardness
The tablet crushing load, which is the force required to break a tablet by compression. The hardness of the 5 tablets (Randomly selected) was determined by diametral compression using Pfizer hardness tester.
Weight variation test
Theweight of 20 tablets was determined individually and average weight was calculated. Weight control test is based on Indian Pharmacopoeia
Tablet friability
The friability of the tablets was measured in an Electrolab EF-2 friabilator (USP).A sample of 10 tablets are dedusted in a drum for a fixed time (100 revolutions) and weighed again. Percentage friability was calculated from the loss in weight as given in equation as below.
% of friability = W0-W1/W0 X 100
Where %f is percentage friability, W0isinitial weight and W1isfinal weightof tablets
Disintegration test
This study was conducted by using disintegration test apparatus on Electrolab disintegration tester ED-2L. The apparatus consists of six plastic tubes which are open at one end and another end is fitted with a rust-proof mesh No. 10. The tubes were suspended in 1-litrephosphate buffer solution (pH 6.8) at a temperature of 37 °C. The plastic tubes were allowed to move up and down at a constant rate through a distance of 53-57 mm. The test was carried out foreach formulation batch(six tablets) and the disintegration times were noted.
Drug content uniformity
10 tablets were weighted and anaverage weight of one tablet was calculated. These tablets were crushed and powder equivalent to one tablet was weighed and dissolved in diluent [water: acetonitrile (60:40v/v)]. This solution was filtered through Whatman filter paper no 41 and further diluted suitably. Then, the solution was injected into HPLC system.
In vitro dissolution study
Dissolution studies of each batch were conducted according to USP apparatus II paddle method with 900 mlphosphate buffer (pH 6.8) at 37 °C and 75 rpm. After5, 10, 20, 30, 45 and 60 min interval samples(10 ml each) were withdrawn from the dissolution medium and replaced with fresh medium to maintain aconstant volume. The samples were filtered through a 0.45μ membrane filter. Then samples were diluted to a suitable concentration with phosphate buffer (pH 6.8). Then, the solution was injected into HPLC system. The cumulative percentage of drug release was calculated [12-15].
Assay of tablets
Twenty tablets were weighed and average weight was calculated. These tablets were crushed and powdered in a glass mortar. The tablet powder equivalent to theaverage weight of apixaban was accurately weighed, transferred to a 50 ml of volumetric flask and diluted up to mark with water: acetonitrile (60:40v/v).The solution was filtered through Whatman filter paper no. 41.This solution was further diluted to obtain 30 μg/ml with diluent and the sample solution was injected into HPLC system. This procedure was repeated in triplicate.
Forced degradation studies
To evaluate stability,apixaban was subjected to force degradation under the condition of acid, base, neutral hydrolysis and oxidation as per international conference on harmonization (ICH) guidelines[16-19].
Acid hydrolysis
100 mg of apixaban was weighed accurately and transferred to 100 ml volumetric flask containing 100 ml of 0.1N hydrochloric acid (HCl). This mixture was refluxed at 80 °C. After 2 h, 5 ml of refluxed sample was withdrawn and neutralized with 5 ml of 0.1 N sodium hydroxide. This solution was further diluted 10 times with mobile phase to obtain a concentration of 100 μg/ml. The chromatogram obtained after 2 h of acid hydrolysis is shown in fig. 6(a).
Alkaline hydrolysis
100 mg of apixaban was weighed accurately and transferred to 100 ml volumetric flask containing 100 ml of 0.1N sodium hydroxide (NaOH). This mixture was refluxed at 80 °C. After 2 h,5 ml of refluxed sample was withdrawn and neutralized with 5 ml of 0.1 N hydrochloric acid. This solution was further diluted 10 times with mobile phase to obtain a concentration of 100 μg/ml. The chromatogram obtained after 2 h of alkali hydrolysis is shown in fig. 6(b).
Oxidative degradation
100 mg of apixaban was weighed accurately and transferred to 100 ml volumetric flask containing 100 ml of 3% hydrogen peroxide (H2O2). This mixture was refluxed at 80 °C. After 2 h, 5 ml of refluxed sample was withdrawn. This solution was further diluted 10 times with mobile phase to obtain a concentration of 100 μg/ml. The chromatogram obtained after 2 hof oxidative degradation is shown in fig. 6(c).
Thermal degradation
100 mg of apixaban IR tablet powder sample was weighed and transferred into 100 ml volumetric flask. The contents were refluxed as such on a water bath previously maintained at 80ᵒ C for 2 h.The sample was allowed to cool to room temperature and then thevolume was made upto the mark with mobile phase and mixed well. The solution was filtered through a0.45µ syringe filter and analyzed. The chromatogram obtained after 2 h of thermal degradation is shown in fig. 6(d).
Photolytic degradation
Photolytic degradation of thedrug was carried out by exposure of about 100 mg of apixaban IR tablet powder sample to UV radiation for 12 h. Then the sample was transferred into 100 ml volumetric flask. The sample was allowed to cool to room temperature and then thevolume was made upto the mark with mobile phase and mixed well. The solution was filtered through a0.45µ syringe filter and analyzed. The chromatogram obtained after 12 hof photolytic degradation is shown in fig. 6(e).
Validation of the method
The developed chromatographic method was validated for system suitability, linearity, range, accuracy, precision, LOD-LOQ and robustness parameters as per ICH guidelines [20-23].
Linearity and range
Working standard solutions were injected in the range of 5-30 μg/ml under the optimized chromatographic conditions and peak areas were calculated at 280 nm. The calibration curve was plotted between areas against concentrations of the drug. Linear regression data, as well as calibration curve, were shown in fig. 7.
Precision
Repeatability study was carried out with six replicates and intermediate precision studies were carried out with three concentrations of apixabanwith three replicates. The values of % relative standard deviation (% RSD) of precision study are shown intable 9.
Accuracy
The accuracy of the method was determined by calculating percent recovery of the drug by standard addition method. Percent recovery of apixabanwas determined at three different level 80%, 100%, and 120% of the target concentration in triplicate. The results of accuracy study are shown intable 10.
Robustness
Robustness of the optimized method was studied by changing flow rate (±0.1 ml/min), change in wavelength (±1 nm) and change in mobile phase composition (±5%) during analysis. The sample was injected in triplicate for every condition and % RSD was calculated for each condition is shown intable 11.
Limit of detection (LOD) and limit of quantitation (LOQ)
Five sets of concentrations were prepared between 5-30 μg/ml and the corresponding areas of these sets were measured. Calibration curves were plotted for each set. The standard deviation of the y-intercept and average slope of the calibration curve was used to calculate LOD and LOQ using following formulae.
Where SD is the standard deviation of y-intercepts of the calibration curves; S is the mean slope of six calibration curves.
Fig.2: IR spectrum of apixaban
RESULTS AND DISCUSSION
Formulation of tablets
Apixaban was found to be soluble in acetonitrile. Drug-excipients interaction studies were performed using FTIR spectrophotometer. The FTIR spectra for the formulation and pure drug are shown in fig. 2 and fig. 3. Characteristics peaks obtained for the pure drug correlated well with that of the formulation peaks.
This indicated that the drug was compatible with the formulation components.
Fig.3: IR spectrum of apixaban formulation
Evaluation of powder blend
The angle of repose of all formulations was between 21.37 ° to 28.14 °, while the result of the Carr's index and Hausner ratio was between 11.71% to 20.02% and 1.03 to 1.25, respectively. Evaluation of powder blend characteristics is presented in table 3. The results indicate that the prepared powder mixtures have acceptable flow properties and compressibility. All pre-compression parameters are found to be within the acceptance criteria.
Table 2: Evaluation of powder blend characteristics
| Parameters | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
| Angle of Repose | 23.05 ° | 22.89 ° | 23.49 ° | 21.37 ° | 25.55 ° | 25.01 ° | 27.07 ° | 27.09 ° | 28.14 ° |
| Bulk Density | 0.606 | 0.571 | 0.588 | 0.606 | 0.588 | 0.588 | 0.588 | 0.571 | 0.588 |
| Tapped Density | 0.689 | 0.714 | 0.666 | 0.714 | 0.689 | 0.714 | 0.666 | 0.714 | 0.689 |
| Hausner’s Ratio | 1.13 | 1.25 | 1.13 | 1.17 | 1.17 | 1.21 | 1.03 | 1.25 | 1.17 |
| % Carr’s Index | 12.04 | 12.02 | 11.71 | 15.12 | 14.65 | 17.64 | 17.97 | 20.02 | 14.65 |
(n=3)
Evaluation of tablets
The evaluation parameters of all formulation batches are presented intable3. The thickness of tablets was observed in the range of 2.4±0.172 to 2.5±0.171 mm, it was found that all prepared tablets had a uniform thickness. The hardness of tablets was in the range of 3.4±0.05 to 3.7±0.10 kg/cm2, which showssufficient mechanical strength. The total weight loss of the prepared tablets due to friability was found in the range of 0.50% to 0.80%, which is within acceptance criteria. The weight variation of prepared tablets was in the range of 98.43±0.61 to 100.82±0.79. The content uniformity of the prepared tablets was in the range of 93.98±1.32%to 95.25±1.12%, which reveals content uniformity. All evaluation parameters were found within the acceptance criteria.
Table 3: Evaluation of formulation batches of tablets
| Parameters | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
Thickness (mm)* |
2.5±0.18 | 2.4±0.17 | 2.5±0.17 | 2.5±0.17 | 2.4±0.17 | 2.4±0.17 | 2.4±0.17 | 2.5±0.17 | 2.5±0.17 |
Weight Variation (mg)* |
98.43±0.61 | 99.06±0.78 | 99.08±0.72 | 99.41±0.93 | 100.11±0.84 | 99.49±0.83 | 100.6±0.81 | 100.82±0.79 | 100.48±0.67 |
Hardness kg/cm2* |
3.4±0.05 | 3.6±0.05 | 3.5±0.15 | 3.5±0.05 | 3.6±0.20 | 3.5±0.05 | 3.7±0.10 | 3.4±0.05 | 3.6±0.20 |
| Friability (%) | 0.50 | 0.60 | 0.80 | 0.60 | 0.80 | 0.60 | 0.60 | 0.60 | 0.50 |
| Disintegration test | 138±1.15 | 137±1.80 | 136±2.11 | 137±1.45 | 136±1.23 | 139±2.10 | 140±2.40 | 145±1.76 | 139±1.32 |
| Content uniformity | 94.62±0.54 | 94.62±1.85 | 94.62±1.68 | 94.30±0.72 | 93.98±1.32 | 94.62±1.87 | 95.25±1.12 | 94.93±0.43 | 95.25±1.85 |
| In vitroDissolution Studies | 36.48 | 40.25 | 29.97 | 38.95 | 90.979 | 69.25 | 62.55 | 44.59 | 67.57 |
(*n=3)data represented as mean±SD
Fig.4: Graphical representation of in vitrodissolution of all batches
From the dissolution study (fig. 4), it is observed that formulation batch F5 shows maximum drug release pattern as compared toother formulations. Therefore,this batchwas selected as optimized formulation batch.
Difference and similarity factor [15]
Results obtained from the dissolution profile were fitted into equations (1) and (2) to determine the difference and similarity factors of the various batches compared to standard. Difference and similarity factors are themodel-independent approach used to estimate the differential factor (f1) and similarity factor (f2) to compare the dissolution profile of optimized formulation (F5) with innovator product. The difference between the reference and test curve at each time point and is a measurement of the relative error between two curves. The FDA suggested that two dissolution profiles were declared similar if thef2 value between 50–100 and f1 was 0–15.
f1= {[Σ t=1n |Rt-Tt|]/[Σ t=1n Rt]} ×100–Equation (1)
f2 = 50+log {[1+(1/n) Σt=1 x n (Rt-Tt) 2]}-0.5 x 100–Equation (2)
Where,
f1 = difference factor, f2 = similarity factor, n = time points
Rt = cumulative percentage dissolved at time t for the reference
Tt = cumulative percentage dissolved at time t for the test.
Observations and calculations of difference and similarity factors are shown in table 4 and table 5, respectively.
Table 4: Observation of % cumulative release of theMarketed and In-house formulation
| S. No. | Time in min | Area of marketed formulation | Area of In-house formulation | % cumulative release of marketed formulation | % cumulative release ofin-house formulation |
| 1 | 5 | 111842 | 117498 | 39.5050 | 41.8812 |
| 2 | 10 | 150023 | 155680 | 55.9835 | 58.3861 |
| 3 | 20 | 192447 | 186791 | 74.4224 | 72.0990 |
| 4 | 30 | 199518 | 202346 | 78.2079 | 79.4224 |
| 5 | 45 | 224972 | 219315 | 89.7492 | 87.4125 |
| 6 | 60 | 244770 | 227800 | 98.0660 | 90.9769 |
Table 5: Calculation of difference and similarity factors
| Time | Rt | Tt | {Rt-Tt} | (Rt-Tt)2 |
| 5 | 39.5050 | 41.8812 | 2.37623762 | 5.6465052 |
| 10 | 55.9835 | 58.3861 | 2.40264026 | 5.7726802 |
| 20 | 74.4224 | 72.0990 | 2.32343234 | 5.3983379 |
| 30 | 78.2079 | 79.4224 | 1.21452145 | 1.4750624 |
| 45 | 89.7492 | 87.4125 | 2.33663366 | 5.4598569 |
| 60 | 98.0660 | 90.9769 | 7.08910891 | 50.255465 |
| sum (Rt-Tt) | 17.742574 | |||
| sum (Rt-Tt)2 | 74.007908 | |||
| sum Rt | 435.93399 | |||
| Similarity factor f2 | 70.0325 | |||
| Difference factor f1 | 4.0700 |
The marketed formulation and in-house formulation shows similarity factor of 70.03 and differential factor of 4.07. The similarity factor (f2) and differential factor (f1) was found within the acceptance criteria.
Optimization of chromatographicconditions
UV spectrum of apixaban showed maximum absorbance was found at 280 nm. Hence, 280 nm was selected for detection wavelength of this drug. Initially, various chromatographic conditions were tried in order to obtain better separation characteristics by changing mobile phase composition and pH. Finally, themobile phase containing water: acetonitrile (60:40 v/v)+0.1 % TEAwas selected at 1 ml/min flow rate. The retention time of apixaban was found to be 5.66 min. The chromatogram of apixabanis shown in fig. 5 and optimized chromatographic conditions are mentioned intable6.
Assay of tablet formulation
The drug content was calculated as an average of three determinations and assay results were shown in table 7. The results were very close to the labeled value of commercial tablets. The value of mean % drug was found to be 99.31% which is within acceptance criteria.
Forced degradation studies
Chromatograms obtained under different stress conditions like acidic, alkaline hydrolysis, oxidative, thermal and photolytic degradation are presented in fig 6(a), 6(b), 6(c), 6(d) and 6(e).
Table 6: Optimized chromatographic conditions
| Parameters | Details |
| Mobile phase | Water: Acetonitrile (60:40) v/v+0.1 % TEA |
| Column | Purospher Star RP-18 end-capped (5 µm) Hibar 250x4,6 |
| Flow rate | 1 ml/min |
| Detection | 280 nm |
| Injection volume | 20 μl |
| Run time | 8 min |
| Retention time | 5.66 min |
| Diluent | water: acetonitrile (60+40) v/v+0.1 % TEA |
Fig.5: Chromatogram of apixaban
Table 7: Results of assay of apixaban
| S.No. | Sample solution concentration (μg/ml) | Actual concentration found | Amount of drug estimated mean±SD* |
| 1 | 30 | 29.82 | |
| 2 | 30 | 29.74 | 99.31±0.16 |
| 3 | 30 | 29.81 |
*The value is represented as a mean±SD of 3 observations.
Fig.6(a)
Fig. 6 (b)
Fig.6 (c)
Fig.6 (d)
Fig.6 (e)
Fig.6: Typically degradation chromatograms of apixaban; (A): in 0.1 HCl at 80 °C after 2h. (B): in 0.1 N NaOH at 80 °C after 2 h. (C): in 3% H2O2 at 80 °C after 2 h. (D): in dry heat at 80 °C after 2 h. (E): exposure to UV radiations for 12 h
The first chromatogram obtained by acid hydrolysis [fig. 6(a)] suggested that 9.15% degradation of the drug was found, when refluxed at 80 °C for 2 h in 0.1 N HCl. The major degradation product formed was at 1.938 min retention time. This study indicates that apixaban was susceptible to acid hydrolysis. Second chromatogram obtained by alkaline hydrolysis [fig. 6(b)] indicated that apixaban was not stable to alkaline hydrolysis when refluxed at 80 °C for 2 h in 0.1 N NaOH. The Value of % degradation was found to be 14.86% and major degradation products appeared at 1.84 and 2.94 min retention time. The third chromatogram obtained by oxidative degradation [fig. 6(c)] suggested that 4.15% degradation was observed when refluxed with 3% H2O2 at 80 °C for 2 h. The major degradation products appeared at 1.45, 1.64 and 2.55 min retention time. Fourth chromatogram obtained by thermal degradation [fig. 6(d)] indicated that apixaban is less degraded when refluxed at 80 °C for 2 h. The minor degradation product was obtained at 1.45 min retention time. Fifth chromatogram obtained by photolytic degradation [fig. 6(e)] indicated that apixaban is less degraded upon exposure to UV radiations for 12 h. The minor degradation product was obtained at 1.45 min retention time.
Validation of the method
The developed chromatographic method was validated for linearity, range, accuracy, precision, LOD-LOQ and robustness parameters as per ICH guidelines.
Linearity and range
The value of correlation coefficient for apixaban (fig.7) demonstrated the good relationship between peak areas and concentrations. Therefore, the developed method was linear in the concentration range of 5-30 μg/ml.
Fig.7: Calibration curve of apixaban in acetonitrile: water (60:40 v/v)
Precision
The repeatability, intra-day precision was calculated as the relative standard deviation of results from three samples, during the same day, and the inter-day precision was studied by comparing on two different days. The percent relative standard deviation (% RSD) was calculated which is within the acceptable criteria of not more than 2were shown in table 8.
Table 8: Repeatability and intermediate precision for apixaban
| Precision | Concentration of drug (μg/ml) | Mean area±SD* | % RSD |
| Repeatability* | 20 | 799859±1636.10 | 0.37 |
| Intra-d | 10 | 377252±6808.76 | 1.80 |
| 20 | 835741±3499.53 | 0.42 | |
| 30 | 121438±2744.61 | 0.23 | |
| Inter-d | 10 | 373366±3209.56 | 0.86 |
| 20 | 801115±8040.09 | 0.10 | |
| 30 | 117499±1936.27 | 0.16 |
*Each value is represented as a mean±SD of n observations. The value of n is 6 for repeatability study and 3 for intraday and interday precision. SD: Standard deviation, %RSD: Percent relative standard deviation
Accuracy
The accuracy was determined by the standard addition method. Amounts of 6; 10; 14 µg/ml of the apixaban standard were added to the sample solution in which 10.0 µg/ml of the drug had been incorporated previously. The final concentrations of the fortified solutions were 16.0, 20.0 and 24.0 µg/ml of apixaban. The recovery experiments were performed in triplicate for each concentration. The value of mean % recovery and % RSD (table9) at each level was found within acceptance criteria that indicate the method is accurate.
Table 9: Accuracy of apixaban
| Levels | Amount taken(μg/ml) | Amount found(μg/ml) | %recovery | % Mean recovery±% RSD |
| 80% | 16.36 | 102.2 | 101.25±0.17 | |
| 16 | 15.72 | 98.25 | ||
| 16.54 | 103.3 | |||
| 100% | 19.36 | 96.8 | 96.1±0.63 | |
| 20 | 19.15 | 95.75 | ||
| 19.15 | 95.75 | |||
| 120% | 27.88 | 92.93 | 93.03±0.13 | |
| 24 | 27.95 | 93.16 | ||
| 27.90 | 93.00 |
*Percent recovery was done in triplicate, % recovery: Percent recovery, %RSD: Percent relative standard deviation
Robustness
Robustness was performed by changing various method parameters like a change in flow rate, the composition of mobile phase and change in detection wavelength. Finally, the effect of these changes was not deliberate. The value of % RSD (table10) was found to be within acceptance criteria which showed the reliability of the method.
Table 10: Robustness study of apixaban
| Parameters | % RSD |
| A: Change in flow rate | 1.98% |
| 0.9 ml/min | |
| 1 ml/min | |
| 1.1 ml/min | |
| B: Change in Mobile Phase | 1.64% |
| Water: ACN (55:45) v/v+0.1% TEA | |
| Water: ACN (60:40) v/v+0.1% TEA | |
| Water: ACN (65:35) v/v+0.1% TEA | |
| C: Change in wavelength | 1.25% |
| 279 nm | |
| 280 nm | |
| 281 nm |
*Each value is represented as % RSD of n observations. The value of n is 3 for change in flow rate, change in wavelength and change in mobile phase composition. %RSD: Percent relative standard deviation
Limit of detection (LOD) and limit of quantitation (LOQ)
The sensitivity of measurement of apixaban by use of proposed methods was estimated in terms of the limit of quantitation (LOQ) and limit of detection(LOD). The values of LOD and LOQ have been found to be 1.020μg/ml and 3.091μg/ml, respectively.These values show that method is sensitive.
CONCLUSION
A cost-effective formulation of apixaban tablets was developed by 32 factorial design. Results of formulation evaluation of prepared between indicate that batch F5 is a promising formulation for theimmediate release of thedrug. Results obtained by validation studies suggested that the developed stability indicating assay method is simple, accurate, specific and precise. Thus, this method can be used for routine analysis of apixaban formulation and to check the stability testing.
ACKNOWLEDGEMENT
Authorsthank Principal, Sinhgad College of Pharmacy, Pune for providing required facility to complete this project.
AUTHOR CONTRIBUTION
Both authors contributed equally to this work
CONFLICT OF INTERESTS
Declared none
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How to cite this article
Hemant K Jain, Vishal K Nikam. Formulation development and stability indicating HPLC assay of tablets of apixaban.Int J Pharm Pharm Sci 2017;9(10):24-32.