RP-HPLC Method Development and Validation of Rivaroxaban in Pharmaceutical Tablet Dosage Form
Tushar Bhatkar1*, Pramod Burakle1, Prashant Ajmire2, Rahul Jawarkar1,
Pravin Khatale1, Ekant Taywade1
1Department of Pharmaceutical Chemistry, Dr. Rajendra Gode Institute of Pharmacy, Amravati – 444901.
2Department of Pharmacology, Dr. Rajendra Gode Institute of Pharmacy, Amravati – 444901.
*Corresponding Author E-mail: tusharbhatkar10@gmail.com
ABSTRACT:
For the detection of Rivaroxaban in Pharmaceutical Tablet dosage form, a straightforward, rapid, highly selective, accurate, and exact reverse phase high performance liquid chromatographic (RP-HPLC) approach was designed and validated. The Adamas C8, (150 4.6mm, 5µm) column was used for the development, and the oven temperature used was 30°C. A 60:40% (v/v) combination of acetonitrile and water made up the solvent reservoir mobile phase. With a DAD detector, the flow rate was 1.0 ml per minute, and the wavelength was 253nm. The run time was limited to five minutes. Using EZ Chrome software, the separation was performed using an Agilent 1100 series HPLC. Rivaroxaban's retention period was measured at 3.680 minutes. According to ICH Q2 R1 recommendations, the method was validated in terms of parameters including linearity, accuracy, precision, specificity, limit of detection, limit of quantification, and robustness. With a correlation coefficient of 0.9998, the linearity was in the 10–50µg/ml range. Less than 2% was discovered to be the accuracy's relative standard deviation. The average recovery rate ranged from 100.12% to 101.58%. Limits of quantification and detection were discovered to be 0.3µg/ml and 1.0µg/ml, respectively. The suggested method for determining the dosage of Rivaroxaban in tablet form was successfully established.
KEYWORDS: Rivaroxaban, HPLC, Method validation, Tablets dosage form, Method development, ICH.
INTRODUCTION:
Rivaroxaban (Figure No.1), an oral route of administration oxazolidinone-based anticoagulant, is a potent molecule and selective direct acting selective inhibitor of factor Xa for the prevention and protection against venous thromboembolism in adult patients following total hip or total knee replacement surgery.1 In terms of composition and structure, it is 5-chloro-N-[[(5S)-2-oxo-3- [4-(3- oxomorpholin-4-yl) phenyl]]Thiophene-2-carboxamide, also known as 1, 3-oxazolidin-5-yl] methyl]. The chemical formula is C19H18Cl N3O5S, and the molar mass is 435.882 g/mol.2-3
Figure No. 1: Chemical structure of Rivaroxaban
Based on a literature review, some analytical techniques have been published for determining Rivaroxaban alone or in combination with other medications. Uv IR MS Spectrophotometric4-9, RP-HPLC5-10-17, and HPTLC18 techniques are the ones that were reported. The goal of the current study was to create a precise, rapid, accurate, and fast RP-HPLC method for estimating rivaroxaban in pharmaceutical tablet dosage forms. The analytical method underwent Q2R1 validation in accordance with ICH recommendations.18-20
METHOD AND MATERIALS:
Reagents and Chemicals:
Rivaroxaban pure API sample was acquired from Umedica Labs, Private Limited, Navi Mumbai, India. The Rivaban-10mg tablets (Rivaroxaban 10mg/tablet) commercial formulation The tablet sample, which is produced and distributed by Lupin Limited India, was obtained from a neighbourhood drug store. Acetonitrile that was suitable for HPLC was purchased from Thermosil Fine Chemicals Ltd. in Mumbai, India. Water of the HPLC grade was purchased from Thermosil Fine Chemicals Ltd., Mumbai, India
Instrumentation:
A chromatographic system, Agilent HPLC 1100 series Quaternary pump, 200L injection loop with DAD detector, running on E-Z Chrome application, and a reverse phase Adamas C8 column with 150 x 4.6mm internal diameter, 5µm particle size were used in the development and validation process. Mettler Toledo analytical balance. For weighing purposes, a UV-Visible Spectrophotometer made in India by Systronics (seies1130) was employed.
Chromatographic Prerequisites:
Adamas C8 column (150 x 4.6mm, 5µm particle size) was used to separate rivaroxaban for analysis, and column temperature was kept at 30°C. With a flow rate of 1.0mL/min, the solvent reservoir mobile phase was made up of acetonitrile and water in a 60:40% (v/v) mixture. The DAD detector mobile phase was employed as the diluent, and the detection wavelength was monitored at 251nm. The injection had a 10µL volume. It lasted for five minutes in total. Rivaroxaban's retention period was measured at 3.680 minutes. The optimized chromatographic conditions as show in table no1. and figure no 3.
Table No:1 Optimized chromatographic conditions
|
Mobile Phase Composition |
Acetonitrile: Water 60:40 % (v/v) |
|
Column |
Adamas C8 column (150 x 4.6 mm, 5 µm particle size) |
|
Wavelength |
253nm |
|
Column Oven Temperature |
30°C |
|
Injection volume |
10 µl |
|
Flow Rate |
1.0 mL /min |
|
Run time |
5 min |
Standard stock solution preparation:
10mg of the working standard for rivaroxaban were accurately weighed and then added to a 100mL clean and dry volumetric flask. 40ml of diluent was then added, and the mixture was sonicated for five minutes before being brought up to the desired volume with 60 mL of diluent. It produces a 100µg/ml solution.
Solution Sample Preparation:
10 tablets were precisely weighed and transferred, and the average weight was determined for each tablet. The tablet was put into a 100mL volumetric flask, along with 40mL that was sonicated for 15minutes before the solution was allowed to reach room temperature. The volume was then made up with diluent, and the solution was filtered through a 0.45 nylon syring filter. From this, pipette out 1ml, which you then dilute to 10ml with the same solvent for a Rivaroxaban concentration of 100 µg/ml.
Method Development and Optimization:
Different mobile phases with varying compositions and ratios were tried to establish verified and optimised chromatographic conditions such asymmetry factor, appropriate peak shape, and good theoretical plates. Methanol: acetonitrile, Methanol: Water, and Acetonitrile: Water have all been evaluated for different compositions and varied ratios, flow rates, and ratios in order to choose the right mobile phase composition. The final, developed, and optimised mobile phase, which consisted of an acetonitrile : water (40:60% v/v) mixture with a flow rate of 1.0ml/min, was found to be suitable and proper system suitability parameter results were obtained.19
Validation of Method:
All parameters, including specificity, linearity, accuracy, precision, limit of detection, limit of quantification, and robustness, were included in the validation of the method in accordance with ICH Guidelines Q2 R1.
Specificity:
The capacity to clearly evaluate the analyte in the presence of components that could be anticipated to be present is known as specificity. Determining the impact of excipients, intermediates, contaminants, and other additives that are typically included in the formulation is the specificity of an analytical approach. The test outcomes were compared to those of a typical medication.19 the chromatogram of specificity as show in figure no.4.
Linearity:
The capacity to generate test results that are directly proportional to the concentration of analyte in the sample is known as linearity (within a given range). By visually examining the plot of the signal as a function of analyte concentration, linearity is assessed. The regression line using the least squares approach is used to generate test results if there is a linear relationship.20 For Rivaroxaban, the linearity parameter was calculated at six different concentration levels, ranging from 10 to 60µg/ml The drug's wavelength was 253nm, and the peak area of each chromatogram was recorded using a PDA detector. Rivaroxaban's correlation coefficient was calculated to be 0.9997. The findings demonstrated a strong association between peak area and drug concentration within the specified concentration range.20 Results of Linearity as show in table no. 2 and figure no.2.
Range:
The distance between the analyte concentration in a sample's upper and lower limits is known as the analytical procedure's range.19
Accuracy:
Accuracy of analytical method is ‘measure of how close the experimental value to the true value’ accuracy of the method was determined by standard addition method. A known amount of standard drug is added to the fixed amount of pre-analyzed injection solution. Percent recovery is calculated by comparing the area before and after addition of the standard drug. The standard addition method is performed at 80 %, 100 % and 120 % level. The solutions are analyzed in duplicate at each level as per the proposed method.19 By calculating the recovery of Rivaroxaban using the usual addition approach, the method's accuracy was evaluated. The amount of Rivaroxaban was determined by measuring the peak area ratios and fitting these values to the straight-line equation of the calibration curve after a known amount of the drug was introduced to a pre-quantified sample solution. The recovery investigations were conducted three times at the 80%, 100%, and 120% concentration levels that were indicated. Peak area ratios were measured, and the amount of Rivaroxaban was calculated by fitting these values to the calibration curve's straight-line equation. Percentage recovery and standard deviation of percentage recovery were computed based on the aforementioned conclusion.20 The result of accuracy show in table no. 3.
Precision:
The degree of scatter between measurements obtained from multiple sampling of the same homogeneous sample under the specified conditions. The degree of agreement between measurements. There are three types of accuracy: intermediate precision, reproducibility, and repeatability. Rivaroxaban's intra-day precision study was completed by estimating correspondence responses six times on the same day while concentrating 100%, and its inter-day precision study was completed by estimating correspondence responses six times the following day while concentrating 100%.23 The result of precision show in table no. 4.
Limits of Quantification and Detection:
The lowest concentration of an analyte that produces a measurable response is known as the limit of detection (LOD). The lowest concentration of an analyte that can be measured with a certain level of accuracy and precision is known as the Limit of Quantification (LOQ). Six replicas of the analyte at the lowest concentration are detected and quantified for this study. By injecting progressively lower quantities of the standard solution while utilising the devised HPLC method, the limits of detection (LOD) and quantification (LOQ) of the method were established.19
Robustness:
An analytical procedure's robustness, which measures its ability to be unaffected by little but intentional changes in method parameters, gives a clue as to how reliable it will be in typical conditions. Robustness evaluation depends on the kind of technique being studied and should be taken into account during the development phase. It should demonstrate the accuracy of an analysis in light of purposeful changes to the method parameters. By varying the mobile phase's composition and flow rate, the robustness of the suggested technique is estimated.16
The Adamas C8 column, which has an internal diameter of 150 x 4.6mm and a particle size of 5 µm, was used in the HPLC process, which was developed and optimised with the aim of developing an accurate assay and recovery method for the estimation of Rivaroxaban in pharmaceutical tablet dosage form. The mobile phase consisted of acetonitrile and water in a ratio of 40:60% v/v. The DAD detector was used with a flow rate of 1.0 ml/min and a wavelength of 253nm. The drug peak after elution was well-defined and noticed. Table no.1 displays the outcomes of established and improved chromatographic conditions. Rivaroxaban's retention time, theoretical plate count, and asmmyteric factor were all determined to be 3.420 min., 9522, and 1.01, respectively. The develop method for Rivaroxaban was linear in the 10–60µg/ml range, with a correlation coefficient R2 of 0.9997. It was discovered that Y=31035x, where X is the Rivaroxaban concentration and Y is the corresponding peak area, best describes the relationship between Rivaroxaban concentration and peak area ratio. Table no.2 and Figure no.2 displayed the linearity findings.
Figure No. 2: Standard calibration curve of Rivaroxaban
Figure No. 3: Chromatogram of optimized method
Figure No. 4: Chromatogram of Specificity
Figure No. 5: Chromatogram of rivaroxaban tablet formulation
Table No. 2: Results of Linearity
|
Sr No. |
Concentration |
Area I |
Area II |
Mean |
SD |
%RSD |
|
1 |
10 |
310213 |
310209 |
310211.00 |
2.83 |
0.0009 |
|
2 |
20 |
620427 |
620433 |
620430.00 |
4.24 |
0.0007 |
|
3 |
30 |
920641 |
920637 |
920639.00 |
2.83 |
0.0003 |
|
4 |
40 |
1240855 |
1240849 |
1240852.00 |
4.24 |
0.0003 |
|
5 |
60 |
1861282 |
1861286 |
1861284.00 |
2.83 |
0.0002 |
|
r2=0.999 |
Avg. sd=3.39 |
|||||
The average recovery rate, which was found to be 100.72%, shows that the procedure is reliable. In Table no 3, the accuracy findings were displayed. Rivaroxaban's intra-day precision and inter-day precision were found to have % RSDs of 0.23 and 0.10, respectively. These values were less than 2%, indicating the method's accuracy. Table no. 4. displays the findings of the precision studies. Rivaroxaban's limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.30µg/ml and 1.00µg/ml, respectively, demonstrating the method's sensitivity.
Table No. 3: Results of Accuracy
|
Level of % recovery |
Amount of tablet solution taken (µg/ml) |
Amount of standard solution add (µg/ml) |
Area |
Amount found |
Amount Recovery |
% Recovery |
|
80% |
10 |
8 |
310243 |
18.10 |
8.07 |
100.12 |
|
10 |
8 |
310249 |
18.17 |
8.03 |
100.84 |
|
|
100% |
10 |
10 |
620417 |
20.04 |
10.04 |
100.43 |
|
10 |
10 |
620421 |
19.98 |
9.98 |
99.81 |
|
|
120% |
10 |
12 |
670748 |
21.9100 |
11.91 |
99.30 |
|
10 |
12 |
670737 |
21.9400 |
11.94 |
99.54 |
For the change in flow rate changes from 1ml/min to 1.2ml/min, the robustness parameter was done. Only in flow conditions of less than 2% is the approach robust. The findings are summarised, and it is clear from an analysis of the results above that the procedure was greatly impacted by flow rate change. Therefore, it shows that the approach is reliable even when the flow rate changes by less than 0.2ml/min. Only under low flow conditions is the procedure robust. Mobile phase flow rate was one of the usual variations examined under this parameter. The overall %RSD was determined to be less than 2% for all the changes, proving the robustness of the suggested technique.
Table No. 4: Result of precision
|
Sr. No |
Intra day precision |
Sr. No |
Inter day precision |
||
|
|
Area -I |
Area- II |
|
Area -I |
Area- II |
|
1 |
310221 |
310227 |
1 |
310231 |
310237 |
|
2 |
310217 |
310212 |
2 |
310206 |
310211 |
|
Mean |
310224.00 |
310214.50 |
Mean |
310234 |
310208 |
|
SD |
4.2 |
3.5 |
SD |
4.2 |
3.5 |
|
%RSD |
1.30 |
1.12 |
% RSD |
1.35 |
1.11 |
Table no.5 displayed the summary findings of the system suitability test and validation parameters. For the examination of Rivaroxaban in a commercial tablet formulation, a validated methodology was used. The drug concentration and assay percentage were determined to be 10mg per tablet and 100.60%, respectively. Figure displayed a typical peak chromatogram for the medication Rivaroxaban. It was demonstrated that the excipients employed in the tablet formulation did not interfere by the lack of impurity interfering peaks in the chromatogram of the commercial formulation during the run time.
Table No. 5: Result Summery of Optimized and validation parameter
|
Sr. No |
Parameters |
Results |
|
1 |
Linearity Level |
10-60 (µg/ml) |
|
2 |
Correlation coefficient |
0.9997(r2) |
|
3 |
Retention times |
3.420 min |
|
4 |
Theoretical plates (N) |
9522 |
|
5 |
Tailing factor |
1.01 |
|
6 |
Mean % recovery (%) |
100.72% |
|
7 |
Repeatability |
1.30(% RSD) |
|
8 |
Reproducibility |
1.35(% RSD) |
|
9 |
LOD |
0.3 µg/ml |
|
10 |
LOQ |
1.0 µg/ml |
|
11 |
Robustness |
0.30(% RSD) |
|
12 |
Assay |
100.60 % |
CONCLUSION:
The purpose of this study is to describe a novel HPLC method for determining the dose form of Rivaroxaban in tablets. It was created and verified to be a straightforward, rapid, highly selective, accurate, and exact reverse phase high performance liquid chromatographic (RP-HPLC) technology. The accuracy analysis reveals a high rate of recovery. A study of specificity demonstrates that the approach is free from any interference from the excipients and impurities utilized in the formulation of the tablet. A linearity analysis demonstrates how linear and robust the approach is. Therefore, the method that was developed can be used for regular analysis of Rivaroxaban determination in pharmaceutical formulations.
ACKNOWLEDGEMENTS:
The authors are grateful to the principal and administration of the Dr. Rajendra Gode Institute of Pharmacy in Amravati, India, for giving all the resources necessary to carry out the research.
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Received on 14.06.2023 Modified on 06.11.2023
Accepted on 09.02.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(6):2869-2874.
DOI: 10.52711/0974-360X.2024.00450