INTRODUCTION:

Rivaroxaban, an oral anticoagulant. It was invented by Bayer and approved from1July 2011.It acts at the crucial points to stop the formation of blood clots¹. It has been reported as a potent, selective and direct inhibitor of factor Xa which prevents thromboembolism during surgical operations^1,2. It is insoluble in water and freely soluble in organic solvents³. It is rapidly absorbed from the gut and produces maximum inhibition of factor Xa within 4 h of oral administration^4,5. Rivaroxaban is usedto reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. It has also been used to lower the risk of developing venous thrombosis postorthopedic surgical procedures⁶.

The drug is available in tablet dosage form. The available tablet strengths are 2.5mg, 10mg, 15mg and 20mg of rivaroxaban. Rivaroxaban is novel oxazolidinone derivative. Its IUPAC name is 5-chloro-N-[[(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl]methyl]thiophene-2-carboxamide⁷.

Fig1. Chemical Structure of Rivaroxaban

Literature survey shows some analytical methods viz. RP-HPLC method development and validation of stability indicating method for determination of rivaroxaban in pharmaceutical formulations⁸, development and validation of stability indicating RP-HPLC method for rivaroxaban and itsimpurities⁹, application of stability indicating HPLC method with UV detector to the analysis of rivaroxaban in bulk and tablet dosage form¹⁰, UV spectroscopy analysis and degradation study of rivaroxaban¹¹, development of a stability-indicating HPLC method and a dissolution test for rivaroxaban dosage Forms¹², Method development and validation of rivaroxaban in pharmaceutical dosage form¹³, A stability-indicating ultra-performance liquid chromatographic method for estimation of related substances and degradants in rivaroxaban active pharmaceuticalingredient¹⁴, UPLC-Q-TOF-MS/MS method for characterization of rivaroxaban¹⁵. HPTLC with MS/TOF for identification, characterization of degradation product of rivaroxaban¹⁶. Most of the reported methods are based on hyphenated techniques, and overall cost of the analysis using these techniques is more as compared to High performance thin layer chromatography.

The stability indicating methods reported show that there is discrepancy in percent degradation values reported in literature. From the information available in literature it was observed that number of methods have been reported already for the study of rivaroxaban. Simple, spectrophotometric methods only give the basic idea about the analytical study of drug. Furthermore various stability studies and characterization studies were developed for evaluation of rivaroxaban by using sophisticated analytical techniques. In spite of this available data there is significant variance observed between percent degradation results and type of forced degradation conditions used. Also, two parameters were used at a time, like reagent and heating process then it may be difficult to conclude that the drop in assay value was due to the likely effect of single stress condition or combination of factors.

Since many articles showed contrasting reports about instability, the main objective of current work was to check the conditions that affect stability of drug by developing stability indicating HPTLC method for rivaroxaban. The simple, easy, high throughput and automation of HPTLC makes it a better choice over conventional analytical tools^17,18. Further, the developed method was validated as per requirements given by International council for harmonisation^19,20.Few papers were also referred for additional details of method development, validation by HPTLC technique^21-30.

MATERIALS AND METHODS:

Chemicals and Reagents:

Rivaroxaban working standard was obtained as a gift sample from Sanofi synthelabo development centre, Verna Goa. All reagents were purchased from LOBA chemie Pvt. Ltd. Mumbai, India.

Instrumentation:

HPTLC system from CAMAG was used for study. Chromatographic separation of the drug was performed on Merck TLC plates precoated with silica gel 60 F₂₅₄ (10cm × 10cm with 250μm layer thickness) from E. MERCK, using a CAMAG Linomat 5 sample applicator (Switzerland). Samples were applied on the plate as a band under nitrogen stream. (6mm of band width) using Camag 100μl sample syringe (Hamilton, Switzerland). A constant application rate of 0.1μl/s was employed. UV- spectral analysis was performed on UV-visible spectrophotometer (Make-JASCO, Model-V730). Photo-stability study was performed in photo-stability chamber (Make-Newtronic, Model- IC DAC version 1.2

Chromatographic Conditions:

Linear ascending development was carried out in 10 ×10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland) by using Ethyl Acetate: Acetonitrile (7:3 v/v) as mobile phase. The mobile phase was saturated in the (CAMAG) twin trough TLC chamber for 30 min before chromatogram development at room temperature. After development, TLC plates were removed and dried. A CAMAG TLC scanner with winCATS software (version 1.4.3) wasused for densitometric evaluation. Deuterium lamp as a source of radiation at the wavelength 249nm was used.

Preparation of Standard Stock solution:

To prepare a standard stock solution of rivaroxaban, 16 mg of the drug was dissolved in 10ml of Dimethyl sulfoxide (DMSO) and sonicated to get a concentration of 1600ppm.

Preparation of Standard and Sample solution:

The Standard stock solution was appropriately diluted using methanol to obtain working stock solution having concentration of 16ppm.

Method Development and Optimization:

Initial trialsinvolved solvents of differing polarity. Different solvent combinations were tried as mobile phase. The optimised method involved ethyl acetate: acetonitrile (7:3v/v). The detection was carried out at wavelength of 249nm for rivaroxaban. The retardation factor of rivaroxaban were found to be 0.75±0.02.

Forced Degradation Studies:

Stress testing establishes intrinsic stability of analyte. Stability testing was carried out according to ICH Q1A (R2) guidelines. The study was carried out by exposing drug to the different stress conditions. The stress conditions were optimized in terms of strength of reagent used and time of exposure to achieve 10-30 percent degradation. The reproducibility of optimised condition is verified. A stressed sample at high concentration was spotted and multi wavelength scanning was done to search for peaks of degradation product. The stress conditions are as follows.

Acid Hydrolysis:

5ml rivaroxaban stock solution (160ppm) was taken, to which 5ml 0.5 N HCl solution was added, and the volume was made up to 50ml. This mixture was heated to 80°C for 4 hours in a refluxing condition. Afterthat solution was cooled at room temperature then volume was made up with methanol andthis solution was applied to the TLC plate and developed.

Alkali Hydrolysis:

1ml stock solution (160ppm) was taken to which 1ml solution of 0.1 N NaOH was added and the volume was made to 10ml, kept at room for 36, 48 and 72hrs. After that, this solution was applied to the TLC plate and developed, observed and scanned.

Neutral Hydrolysis:

Mixed ml of Stock solution (160ppm) was mixed with 1ml of water and the volume made up to 10ml using methanol and this solution was kept at room temperature for 24 hrs. After that, this solution was applied to the TLC plate and developed

Oxidative Degradation:

1ml of Stock solution (160ppm) was mixed with 1ml of 3% w/v H₂O₂ and the volume made up to 10ml using methanol and this solution was kept at room temperature for 4 hrs. After that, this solution was applied to the TLC plate and developed

Thermal Degradation:

For thermal degradation the bulk drug in solid form was exposed to 60°C for 4 and 8 hrs. Then sample was weighed and 16ppm rivaroxaban solution was made from the exposed rivaroxaban sample and after that, this solution was applied to the TLC plate and developed.

Photolytic Degradation:

The bulk drug in solid form was exposed to UV light energy not less than 200 Watt hours/square meter separately and to fluorescent cool white light not less than 1.2million lux hours. After exposure sample wasaccurately weighed. 16mg of drug was transferred to 10ml volumetric flask. The volume was made up with dimethyl sulfoxide (DMSO) to obtain concentration of solution 1600ppm. From this solution 1ml was taken in 10ml volumetric flask and volume was made up with methanol to get concentration of solution 160ppm. and from this solution 1ml was taken in 10ml volumetric flask and volume was made up with methanol to get concentration of working solution 16ppm and then applied on TLC plate.

Method Validation:

The method has been validated according to the guidelines of ICH Q2 (R1) for parameters such as specificity, linearity, range, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness.

RESULTS:

The study was conducted by exposing the drug to different stress conditions for different periods of time like 0.5 Nacid and alkali and duration of exposure 4hrs to 72hrs. We have optimized the stress conditions on the basis of recovery study and reproducibility testing. In Alkali hydrolysis first trial was performed with 0.5 N NaOH and reflux for 4 hrs. It shows 100% degradation. So we kept samples at room temperature by using 0.5 N NaOH for longer period of time for 36, 48, 72hrs. Tocheck the degradation pattern.And in 72 hrs.sample degradation product is seen which shown in Fig. 2 and spectrum scan of rivaroxaban and degradation productis shown in Fig. 3. Order of reaction was calculated for Base hydrolysis by using Graphical method. Fig.4 shows graph for order of reaction, graph shows straight line with negative slope hence it follow First order reaction kinetics³¹. Higher concentration of stressed sample was also spotted to look for degradation product by using multi-wavelength scanning which is shown in Fig. 5 for rivaroxaban. And it state that under all other conditions no degradation product seen at any wavelength. The degradation summary appears in Table 1.

Fig2. Alkali hydrolysis by uing 0.5 N NaOH showing rivaroxaban degradation sample and degradation Product(DP)

Fig3. Spectrum scan of rivaroxaban Std. and degradation product(DP)

Fig4. Graph for order of reaction

Fig5. Multi wavelength scanning of forced condition of rivaroxaban from 239-329nm (Track 2: standard 16ppm, track 4:thermal, oxidative, track 7: UV, track 8:fluro,track 9:neutral,track 10: acid, track11:alkali and track 1,3,5,12:blank track 6

Method Validation:

Linearity:

Different volumes (5-25μl) of rivaroxaban solution (16ppm) were spotted on plate to get the range of 80-400ng/band for rivaroxaban. The simple regression equation method was used to establish the relationship between amounts spotted (ng/band) and peak area. The correlation coefficient was found 0.997 with equation of y = 17.808x+435.31of rivaroxaban.

Specificity:

It is observed that peak purity values for drug peak under stress conditions are within limits. It shows that degradation product do not interfere with drug peak.

Assay:

The amount equivalent to average weight of tablet was taken from blend of synthetic mixture. Blend was prepared by blending100mg of Rivaroxaban with 400 mg of excipients (200mg of Microcrystalline Cellulose MCC and 200mg of Lactose) in mortar pestle by Geometric Mixing. From which 80 mg of blend which is equivalent to 16mg of drug was weighed accurately and dispersed in 10ml dimethyl sulfoxide (DMSO) to get solution (1600ppm). Solution was sonicated, filtered through Whatman filter paper. And this solution was diluted with methanol to achieve 16ppm concentration of rivaroxaban. A 10μl volume of sample solution was applied on TLC plate and peak area was recorded. % recovery was determined from linear equation.Assay of prepared blend was performed and the % drug content was found to be 100.64%.

Accuracy:

Spiking method was used to carry out the recovery studies. Pure drug substance was spiked in blend prepared for assay at three different levels 50%, 100% and 150%. Three replicates of three concentrations were assessed. The results are tabulated in Table 2.

Table 2: Accuracy studies of rivaroxaban

Parameters	Rivaroxaban
% Level	50%	100%	150%
Initial amount(ng/band)	160	160	160
Amount added (ng/band)	80	160	240
% Recovery	99.91	101.09	100.81

Precision:

The method was subjected to intra-day and inter-day precision studies. Intra-day precision was evaluated by applying 6 replicates of 160ng/band of rivaroxaban. Six replicates were also analysed on 3 consecutive days to evaluate the inter-day precision. The method was found to be precise as % RSD was less than 2% and results are given in Table 3.

Table 3: Precision Studies of rivaroxaban

Precision	Concentration	Standard Deviation (S.D.)	% RSD
Intra-day	160 ng/band	47.69	1.22%
Inter-day	160 ng/band	38.81	0.99%

Limit of Detection and Limit of Quantitation:

LOD and LOQ were calculated by formula 3.3 σ/S and 10 σ/S respectively. Where σ is standard deviation of lowest concentration responseor y-intercept.S is the slope of calibration curve. The results are given in Table 4.

Table 4.LOD and LOQ studies of rivaroxaban

Method	LOD (ng/band)	LOQ (ng/band)
By using lowest Concentration	8.53	25.87
By using y-intercept	15.59	47.24

Robustness:

Robustness was tested by doing small and deliberate changes to optimised method like change in mobile phase ratio, saturation time and wavelength. The results are shown in Table 5.

Table 5: Robustness studies of rivaroxaban

Parameters		% RSD
Mobile Phase Composition (± 0.5 ml)	Ethyl acetate: Acetonitrile (6.5:3.5) v/v	1.33
Mobile Phase Composition (± 0.5 ml)	Ethyl acetate: Acetonitrile (7.5:2.5) v/v	1.19
Saturation time (± 5 min)	20 min	0.92
Saturation time (± 5 min)	30 min	1.35
Wavelength (± 1 nm)	249 nm	1.26
Wavelength (± 1 nm)	250 nm	0.99

Validation results proved that the developed method performs well with specificity, linearity, range, accuracy, precision.

DISCUSSION:

The prime objective of present work was to study the stability of rivaroxaban under various stress conditions, using HPTLC, a high throughputtechnique. Because number of research articles found during literature search did not show comparable results for stress degradation.Although rivaroxaban was subjected to acid hydrolysis by using 0.5 N HCl at 80⁰C for 4 hrs.for that no degradation product is observed. But on the other hand it was reported that the one major degradation product was obtained by using 0.1 M HCl at 60⁰C for 3 hrs³. There are few papers that do not reveal degradation percentage⁸. Another article mainly focuses on acid and base hydrolysis and shows degradation percentage for acid was 93.5% degradation by using 0.1 N HCl at 70⁰C for 2 hrs. Andfor base 0.1 N NaOH at room temperature for 1 hr. shows 85.6% degradation³². So our results match for basic condition but not for acid hydrolysis.

We have optimized stability indicating HPTLC method by using simple binary mobile phase that gives good peak parameters. Wavelength chosen was 249 nm based on UV spectrum of drug.

The developed HPTLC method was validated as per ICH guideline and complies with the validation parameters. The samples can be prepared and analysed at room temperature without the special conditions so the method is simple and efficient which can be used for routine stability monitoring study of rivaroxaban. Now, this optimised method can be considered as good alternative for routine analysis of the drug.

CONCLUSION:

The developed method describes simple, sensitive, and specific stability indicating HPTLC method for estimation of rivaroxaban. From the observations it is concluded that rivaroxaban was relatively stable under UV light and cool fluorescent light. The multi wavelength scanning was used to look for the products of degradation.Peak purity was found to be 0.999 for r(s, m) and 0.999 for r(m,e). This indicates that method is specific. The developed method was found to be rapid and cost effective and may be more advantageous for routine quality controlof drug. Further the method was validated according to ICH guideline. It was concluded that the validated method can be used for stability studies of rivaroxaban.

ACKNOWLEDGEMENTS:

Authors are thankful to the Principal and the Management of the AISSMS College of Pharmacy, Pune, Maharashtra, India for providing required facilities for research work. The authors are also grateful to Sanofi Synthelabo Development Centre, Verna Goa, India. for providing rivaroxaban as gift sample.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

REFERENCES:

1. Perzborn E, Roehrig S, Straub A, Kubitza D, Mueck W, Laux V. Rivaroxaban: a new oral factor Xa inhibitor. Arterioscler Thromb Vasc Biol. 2010;30(3):376-81.

2. Roehrig S, Straub A, Pohlmann J, Lampe T, Pernerstorfer J, Schlemmer KH, Reinemer P, Perzborn E. Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor. J Med Chem.2005;48(19):5900-8. doi: 10.1021/jm050101d, PMID 16161994.

3. Abdallah MA, Al-Ghobashy MA, Lotfy HM. Investigation of the profile and kinetics of degradation of Rivaroxaban using HPLC, TLC-densitometry and LC/MS/MS: application to pre- formulation studies. Bull Fac Pharm Cairo Univ.2015;53(1):53-61. doi: 10.1016/j.bfopcu.2015.01.002.

4. Eerenberg ES, Kamphuisen PW, Sijpkens MK, Meijers JC, BullerHR, Levi M. Reversal of Rivaroxaban and dabigatran by pro-thrombin complex concentrate a randomized, placebocontrolled, crossover study in healthy subjects.Circulation. 2011;124(14):1573-9. doi: 10.1161/Circulationaha.111.029017, PMID 21900088.

5. Turpie AGG. New oral anticoagulants in atrial fibrillation.Eur Heart J. 2008;29(2):155-65.

6. Dobesh PP, Fanikos J. Reducing the risk of stroke in patients with nonvalvular atrial fibrillation with direct oral anticoagulants. Is one of these not like the others?J Atr Fibrillation. 2016;9(2):1481. doi: 10.4022/jafib.1481, PMID 27909544.

7. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Rivaroxaban (Accessed on 19 august 2021)

8. Walter ME. Development and validation of a stability-indicating RP-HPLC method for the determination of Rivaroxaban in pharmaceutical formulations. Lat American J Pharm. 2015; 34(8): 1503-10.

9. Girase YN, Srinivasrao V, Soni D. Development and Validation of Stability Indicating RP-HPLC Method for Rivaroxaban and Its Impurities, Symbiosis online. J Biochem. 2018;4(1):1-6.

10. Burla SVS, Peruri VV, Chandra BS. Application of stability indicating HPLC method with UV detector to the analysis of Rivaroxaban in bulk and tablet dosage form. Chem Sci Trans. 2014; 3(4):1546-54.

11. Mandake GR, Patil IS, Patil OA, Nitalikar MM, Mohite SK. UV spectroscopy analysis and degradation study of Rivaroxaban. Asian J Res Pharm Sci. 2018; 8(2): 57-60. doi: 10.5958/2231-5659.2018.00012.7.

12. Souri E, Mottaghi S, Zargarpoor M, Ahmadkhaniha R, Jalalizadeh H. Development of a stability-indicating HPLC method and a dissolution test for Rivaroxaban dosage forms. Acta Chromatographica. 2016; 28(3): 347-61. doi: 10.1556/1326.2016.28.3.05.

13. Mustafa Ç, Tuba R, Engi K, Sacide A. RP-HPLC method development and validation for estimation of Rivaroxaban in pharmaceutical dosage forms, BJPS. Vol.49n(2, Apr/jun). p.34-8.

14. Rajan N, Anver BK. A stability-indicating ultra-performance liquid chromatographic method for estimation of related substances and degradants in Rivaroxaban active pharmaceutical ingredient. J Pharm Res. 2014;8(11):1719-25.

15. Wingert NR, dos Santos No, Nunes MA, Gomes P, Müller EI, Flores ÉM, Steppe M. Characterization of three main degradation products from novel oral anticoagulant Rivaroxaban under stress conditions by UPLC- Q- TOF-MS/MS. J Pharm Biomed Anal. 2016;123:10-5. doi: 10.1016/j.jpba.2016.01.053, PMID 26855380.

16. PatilP, Wawdhane S, Chaudhari P. Identification, Separation and Characterization of Acidic and Alkali Degradation of Rivaroxaban under ICH Recommended stress Condition by HPTLC with MS/TOF, Taylor & Francis Group, TACL. 2017;7(5):706-23.

17. Sethi PD. Quantitative analysis of pharmaceutical formulations. In: HPTLC – High performance thin layerchromatography. 1st ed. Mumbai: CBS Publishers and Distributors. Mumbai; 1996. p.3-5, 10, 18-20, 24, 27-8.

18. StahlE.Thin layer chromatography A laboratory handbook. 2nd ed. India: Springer. India; 2016. p. 52-66.

19. ICH.Q1A (R2) Stability testing of new drug substances and products. International Conference on Harmonization 2003; Geneva.

20. ICH.Q2 (R1) Validation of Analytical Procedures. In: Proceedings of the International Conference on Harmonization 2005; Geneva.

21. Nazira S, Bitar Y, Sarraj M. M.Development and validation of RP-HPLC method for simultaneous estimation of aspirin and Rivaroxaban in synthetic mixture. Res J Pharm Technol. 2020;13(11)(l):5459-65. doi: 10.5958/0974-360X.2020.00953.1.

22. Arous B, Al-Mardini MA, Ghazal H, Al-Lahham F.Stability-indicating method for the determination of Rivaroxaban and its degradation products using LC-MS and TLC. Res J Pharm Technol. 2018; 11(1): 212-20. doi: 10.5958/0974-360X.2018.00040.9.

23. Malik K. C.Karwa M, Jain G.K, Dutt R. Development and Validation of UPLC-MS/MS Method for the determination of Rivaroxaban in human plasma using liquid–liquid extraction. Res J Pharm Technol. 2021; 14(6): 3239-43. doi: 10.52711/0974-360X.2021.00563.

24. Soudi AT, Hussein OG, Eman S. Elzanfaly, Hala E. Zaazaa, Mohamed Abdelkawy. Stability Indicating TLC–Densitometric Method for Determination of Alcaftadine in Presence of its Degradation Products and Dosage form Preservatives. Research J. Pharm. and Tech. 2020 13(11): 5171-5176. doi: 10.5958/0974-360X.2020.00904.X

25. Rawat S, Gupta A. Development of Novel HPTLC Method for Estimation of Qurcetine in Ocimum sanctum. Asian J. Pharm. Tech. 2011 Oct-Dec 1(4): 149-151.

26. Bhusari VK, Dhaneshwar SR. Development and Validation of a Stability-Indicating HPTLC Method for the Estimation of Eszopiclone in Pharmaceutical Dosage Forms. Asian Journal of Research in Pharmaceutical Sciences. 2021 11(3): 219-3. doi: 10.52711/2231-5659.2021.00035

27. Shelar KU, Rao JR, Dhale C. Stability indicating HPTLC method development and validation for the estimation of celecoxib in bulk drug and its Pharmaceutical formulation. Research J. Pharm. and Tech. 2020 13(8): 3661-3665. doi: 10.5958/0974-360X.2020.00647.2

28. Patil PH, Gurupadayya BM, Hamrapurkar PD. Stability Indicating HPTLC Determination of Tadalafil Hydrochloride in Bulk Drug and Pharmaceutical Formulations. Research J. Pharm. and Tech. 2020; 13(6): 2608-2614. doi: 10.5958/0974-360X.2020.00464.3

29. Sinha PK, Jeswani RM, Topgi KS, Damle MC. Stability Indicating RP-HPLC Method for Determination of Potassium Clavulanate in the presence of its Degradation Products. Research J. Pharm. and Tech. 2010 Jan. - Mar 3(1): 141-145.

30. Deshpande K, Ranaware P, Madgulkar AR, Damle MC. Development and Validation of Stability Indicating HPTLC Method for Determination of Repaglinide. Research J. Pharm. and Tech. 2013 Feb. 6(2): 158-161.

31. Bramhankar DM, Jaiswal SB. Biopharmaceutics and pharmacokinetics.2nded. Delhi. Vallah Prakashan:216-7.

32. Badroon T, Sreeramulu J. Development and validation of stability indicating assay by HPLC method for estimation of Rivaroxaban, Journal of Bio-Pharma Research. Vol 8. Issue 5 pp.2582-6, 2019.

Received on 07.09.2021 Modified on 04.01.2022

Research J. Pharm. and Tech 2022; 15(12):5495-5500.

DOI: 10.52711/0974-360X.2022.00927

Sr. No	Parameter	Condition	% Recovery	Peak Purity
Sr. No	Parameter	Condition	% Recovery	r (s,m)	r (m, e)
1	Acid	0.5 N HCl At 80°C for 4 hrs. of reflux.	78.24%	0.999	0.998
2	Base	0.5 N NaOH at Room temperature for 36 hrs.	84.26%	0.999	0.999
		48 hrs.	80.70%	0.999	0.998
		72 hrs.	73.4%	0.999	0.999
3	Neutral	1ml water for 24 hrs.	89.72%	0.999	0.999
3	Oxidation	3% w/v H₂O₂For 4 hrs.	81.44%	0.999	0.999
4	Thermal	60⁰C 4 hrs.	89.74%	0.999	0.998
4	Thermal	60⁰C 8 hrs.	70.28%	0.999	0.998
5	UV	Not less than 200 Watt hrs./square meter	91.06%	0.999	0.999
6	Fluorescence	Not less than 1.2 million lux hrs./square meter	91.68%	0.999	0.999