Research Paper

Development and Validation of a HPTLC Method for

Rivaroxaban in Human Plasma for a Pharmacokinetic
Study
A. H. SHUKLA, P. J. SHAH, P. P. DEDHIYA*, B. A. VYAS AND S. A. SHAH
Maliba Pharmacy College, Maliba Campus, Gopal Vidyanagar, Bardoli, Gujarat, India-394 350

Shukla et al.: Validated HPTLC method for determination of Rivaroxaban in human plasma

The present study is concerned with the development and validation of a bioanalytical method for
estimation of rivaroxaban in human plasma using high performance thin layer chromatography. The
chromatographic separation was achieved on pre-coated silica gel 60F254 thin layer chromatography plate
using toluene:ethylacetate:methanol (6:3:1, % v/v/v) as a mobile phase. Detection was carried out at 284 nm.
A compact spot was obtained with an Rf value of 0.44±0.02. The linearity was found over the concentration
range of 25-125 ng/band in human plasma. The relative standard deviation for repeatability of sample
application and sample measurement was 0.60 and 1.65 %, respectively. The relative standard deviation
for intraday and interday precisions was in the range of 1.37 to 1.85 % and 1.03 to 2.77 %, respectively.
The limit of quantitation was 8.00 ng/band and the limit of detection was 2.64 ng/band. Percent recovery
of rivaroxaban was in the range of 66.95-69.03%. This method was applied to a pharmacokinetic study
of rivaroxaban. The Cmax and tmax for test and marketed formulation were found to be 63.83 and 47.3 ng/
ml and 3 and 4 h respectively. The area under the curve0-t for test and marketed formulation were found
to be 290 and 219.0 ng/ml.h, respectively. The t1/2 for test and marketed formulation was found to be 6.87
and 6.64 h.

Key words: Rivaroxaban, method development, validation, high-performance thin-layer chromatography,

pharmacokinetic study, human plasma

Rivaroxaban, 5-chloro-N-[[(5S)-2-oxo-3-[4- MATERIALS AND METHOD

(3-oxomorpholin-4-yl)phenyl]-1,3oxazolidin-5-yl]
methyl]thiophene-2-carboxamide (fig. 1), is the first Rivaroxaban was received as a gift sample from Symed
orally bioavailable oxazolidinone derivative and direct Labs Ltd., (Hyderabad, India). Chromatography was
inhibitor of the coagulation factor Xa with anticoagulant performed on aluminum plates pre-coated with silica gel
activity, which has been available for prophylaxis and 60 F254 supplied by LobaChemie Pvt., Ltd., (Mumbai,
treatment of deep vein thrombosis[1-3]. Researchers have India). Other chemicals used were toluene, methanol,
developed various analytical methods for estimation of ethyl acetate, acetonitrile of Analytical Reagent grade
rivaroxaban in its pharmaceutical dosage forms, which (Rankem, Thane, India). Human plasma was obtained
included high performance liquid chromatography, from Sardar Smarak Hospital, Bardoli and the dosage
ultra-performance liquid chromatographic method form used was Xarelto film-coated tablets 10 mg,
and liquid chromatography-mass spectrometry Bayer Zydus Pharma. Chromatographic analysis was
(LC-MS)[4-18]. Unlike high-performance thin-layer accomplished using Linomat V semiautomatic sample
chromatography (HPTLC), the above reported methods
are complex, time consuming and expensive techniques This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
for estimation of drugs in biological samples. Therefore, allows others to remix, tweak, and build upon the work non-commercially,
this paper aimed to develop bioanalytical method for as long as the author is credited and the new creations are licensed under
the identical terms
estimation of rivaroxaban in human plasma and to
apply this method in a pharmacokinetic study. Accepted 10 February 2020
Revised 24 January 2020
Received 05 December 2019
*Address for correspondence
E-mail: [email protected] Indian J Pharm Sci 2020;82(2):315-320

March-April 2020 Indian Journal of Pharmaceutical Sciences 315

 with 500 µl ml of drug free plasma. Spiked plasma
samples were extracted using extraction method as
described above. Twenty five microlitres of the sample
was spotted on a TLC plate and the plate was developed
using the chromatographic conditions described above.

Method validation:

Fig. 1: Structure of rivaroxaban The developed bioanalytical method was validated in

terms of specificity, linearity, precision, accuracy, limit
applicator, Hamilton syringe (100 μl), Camag TLC of detection and limit of quantification. Specificity of
Scanner IV, Camag (Muttenz, Switzerland) twin- the method was ascertained by analysing the standard
trough developing chamber (10×10 cm), ultraviolet sample and rivaroxaban extracted from plasma.
(UV) cabinet with dual wavelength UV lamps. Electro The spot of rivaroxaban extracted from plasma was
analytical balance used was of Shimadzu, AUX-220. confirmed by comparing its Rf and UV spectra of the
spot with standard rivaroxaban. The peak purity of
Preparation of standard solutions:
both the spots was assessed by comparing the spectra
Standard stock solution was prepared by dissolving acquired at three different positions on the band, i.e.
10 mg of rivaroxaban in acetonitrile and making it up at peak start (s), peak apex (m) and peak end (e).
to 10 ml with the same. Further dilutions were made Linearity for rivaroxaban in plasma was ascertained
to prepare 50 and 100 μg/ml solutions with the same from regression coefficient by plotting graph of peak
solvent. area versus respective concentration (n=5) by analyzing
solutions corresponding to 25 to 125 ng/band. Method
Selection of mobile phase and optimized precision was ascertained by performing repeatability
chromatographic conditions: of measurement, repeatability of sample application
and intra-day and inter-day precisions. The accuracy of
Number of trials was carried out by using different
the method was determined in terms of the extraction
solvents like toluene, methanol, ethyl acetate,
efficiency of the method by comparing the area of
chloroform, triethylamine and glacial acetic acid
rivaroxaban extracted from plasma and rivaroxaban
individually and in combinations. Bioanalytical method
standard. The limit of detection and limit of quantitation
for estimation of rivaroxaban was developed using
of the developed method were calculated from the
following chromatographic conditions. Stationary phase
standard deviation of the intercepts and mean slope of
used was aluminum plate precoated with silica gel 60F254 the calibration curves of rivaroxaban using the formula
(10×10 cm), mobile phase was toluene: LOD = 3σ/S and LOQ = 10σ/S, where, σ is the standard
ethylacetate:methanol (6:3:1, % v/v/v), chamber deviation of the intercepts of 5 calibration curves and S
saturation time was kept at 30 min, temperature was is the mean slope of 5 calibration curve.
24±2º, migration distance was 75 mm and detection
wavelength was 284 nm. Pharmacokinetic study:

Extraction of rivaroxaban from plasma: Healthy, Adult Wistar female rats (200–230 g) were
obtained from the Central Animal House, Maliba
Five hundred microlitres of plasma was transferred Pharmacy College. The animals were housed at
into a centrifuge tube and spiked with fixed aliquots of temperature 27±2º, humidity 55 %, 12-h light/12-h
working standard solution of rivaroxaban and vortexed, dark cycle and had free access to food and drinking
3 ml of acetonitrile was added and sonicated for water up to the time of experimentation in normal
10 min. The above solution was centrifuged for 15 min acrylic cages. The animals were acclimatized to the
at 1000 rpm. The supernatant was transferred to a 5-ml laboratory environment 1 week before the experiment.
volumetric flask volume made up with acetonitrile and The experiments protocol described in the present
filtered through a 0.22 µm syringe filter. study was approved by the Institutional Animal Ethics
Committee (IAEC, Protocol No. MPC/IAEC/06 /2018)
Preparation of calibration curve:
and conducted according to the Committee for the
Aliquot 100, 200, 300, 400, and 500 μl of the working Purpose of Control and Supervision of Experiments on
standard solution (50 μg/ml) of rivaroxaban were spiked Animals (CPCSEA) guidelines. Five tablets of Xeralto
316 Indian Journal of Pharmaceutical Sciences March-April 2020
 www.ijpsonline.com
of the strength 10 mg were crushed and 8 ml of 1% of 0.44±0.02. (fig. 2). Chromatogram of blank plasma
CMC was added to prepare a suspension. The prepared was run to check any interference. Comparison of the
suspension was administered to rats by oral route. spectra scanned at s, m and e positions of individual
bands showed a high degree of correlation (>0.9994),
Blood collection and preparation of plasma sample which confirmed purity of the corresponding bands.
for Pharmacokinetic studies:
Calibration curve of rivaroxaban from plasma was
After administration of rivaroxaban to the rats, blood obtained by plotting peak area versus concentration
was collected by retro-orbital technique at 0, 1, 2, 3, over the range of 25 to 125 ng/band. The calibration
4, 5, 6, 12 and 24 h time intervals. One ml of blood curve was found to be linear with a correlation
was drawn into vacutainer tubes containing 1.8 mg of coefficient of 0.9954. 3D chromatogram is shown in
EDTA. The vacutainer tubes were inverted 10 times to fig. 3. Calibration data is shown in Table 1. RSD for
mix blood and the anticoagulant, centrifuged at 2000 to repeatability of measurement and repeatability of
3000 rpm for 15 min and the supernatant was separated. sample application was found to be 0.60 and 1.65 %,
Protein precipitation technique was used to prepare respectively, which ensured precision of spotter device.
plasma sample for analysis. Aliquots of 0.5 ml was RSD for intra-day and inter-day precision was found in
taken from plasma samples collected at 0, 1, 2, 3, 4, 5, the range of 1.37-1.85 and 1.03-2.77 %, respectively.
6, 12 and 24 h. To this, 3ml of acetonitrile was added These results indicated that the method is reproducible
and vortexed, the sample was sonicated for 10 min and could be used for estimating rivaroxaban in plasma.
and centrifuged for 15 min at 1000 rpm. Supernatant
The minimum detectable concentration in terms of
was transferred to a 5-ml volumetric flask diluted to LOD and the minimum quantifiable concentration
the mark with acetonitrile, filtered through a 0.22 µl in terms of LOQ were found to be 2.64 and
syringe filter and 25 µl of the filtrate was applied to the 8.00 ng/band, respectively, which indicated good
TLC plate. sensitivity of the method. The accuracy of the method
RESULTS AND DISCUSSION in terms of extraction efficiency was determined for
rivaroxaban from plasma and % recovery was found
Numerous initial trials were carried out with toluene, to be in the range of 66.95 to 69.03 % (Table 2).
methanol, ethyl acetate, chloroform and modifiers glacial Robustness of the method was determined by changing
acetic acid and triethylamine to determine their effect on mobile phase composition, mobile phase volume,
drug migration. After these trials, various combinations saturation time, wavelength and scanning speed. The
of solvents, toluene:methanol:triethylamine, % RSD was found in the range of 1.32-1.67, which
chloroform:methanol:glacial acetic acid,
toluene:methanol, toluene:ethyl acetate:methanol TABLE: 1 CALIBRATION DATA FOR RIVAROXA-
were tried in various proportions. Better results were BAN IN PLASMA
obtained in toluene:ethyl acetate:methanol in the ratio Amount (ng/band) Peak area (Mean±SD, n=5) % RSD
of 6:3:1, where sharp peak was observed with an Rf of 25 617.0±15.2 2.46
50 1025.9±23.9 2.32
0.44.
75 1439.6± 26.0 1.81
Comparison of chromatograms of standard rivaroxaban 100 1776.3±21.6 1.21
and rivaroxaban from plasma showed identical Rf values 125 2080.3±37.2 1.78

Fig. 2: Densitogram of rivaroxaban

Densitograms of rivaroxaban a) standard and b) from plasma
March-April 2020 Indian Journal of Pharmaceutical Sciences 317
ensured robustness of the method (Table 3). The overall using above obtained regression line Eqn.. Data for
validation summary is given in Table 4. pharmacokinetic study is depicted in Tables 5 and
6, which was calculated through PK solver (excel
Pharmacokinetic studies of the test (solid dispersion
add in). Comparison of plasma concentration versus
based tablet) and a marketed formulation (Xarelto
time profile of test and marketed formulation is shown in
tablet) were conducted in female Wistar rats after
fig. 4. The values of t1/2 for test and marketed formulation
oral administration and sampling was done via retro-
was found to be 6.87 and 6.64 h, respectively, which
orbital vein. The plasma concentration was calculated
showed no significant difference between the t1/2 values
for samples collected at different time intervals
suggesting similar time for elimination of rivaroxaban
from both the formulations.
The Cmax value for test and marketed formulation
were found to be 63.83 and 47.3 ng/ml, respectively.
The statistical analysis showed significant difference
between these Cmax values P<0.05. The Cmax of the
test formulation is 1.35 times higher than that of the
marketed formulation. This suggested better release

Fig. 3: 3D chromatogram of calibration range of

rivaroxaban from plasma

TABLE 2: RECOVERY DATA OF RIVAROXABAN

FROM PLASMA
Standard From plasma
Amount
Peak area±SD Peak area±SD % Recovery
(ng/band)
(n=5) (n=5)
25 893.8±6.7 617.0±15.2 69.03
50 1506.2±6.6 1025.9±23.9 68.11 Fig. 4: Overlay of plasma concentration versus time
75 2132.4±11.7 1439.6±26.0 67.51 profile of rivaroxaban
100 2649.6±6.8 1776.3±21.6 67.04
Overlay of plasma concentration versus time profile of
125 3107.7±12.0 2080.9±37.2 66.95
rivaroxaban from (l) Xarelto tablet and (l) test
formulation
TABLE 3: ROBUSTNESS DATA FOR RIVAROXABAN
Robustness Mean Peak area
Parameters Peak area % RSD
condition ±SD
7:2.5:0.5, v/v/v 1403.5
5:3.5:1.5, v/v/v 1450.4
Mobile phase composition
6:3.5:0.5, v/v/v 1447.7 1429.68±23.99 1.67
(toluene:ethyl acetate:methanol)
6:2.5:1.5, v/v/v 1403.6
6:3:1, v/v/v 1443.2
9 ml 1410.9
Mobile phase volume 10 ml 1438.8 1435.47±23.08 1.61
11 ml 1456.7
20 min 1440.3
Saturation time 30 min 1445.7 1454.93±20.84 1.43
40 min 1478.8
281 nm 1432.8
Wavelength 284 nm 1436.9 1423.87±19.13 1.34
287 nm 1401.9
10 mm/sec 1436.8
Scanning speed 20 mm/sec 1434.3 1446.60±19.17 1.32
40 mm/sec 1468.7

318 Indian Journal of Pharmaceutical Sciences March-April 2020

 www.ijpsonline.com
TABLE 4: METHOD VALIDATION SUMMARY FOR In conclusion, the present work involved development
RIVAROXABAN IN PLASMA and validation of a HPTLC method for the estimation
Parameters Result of rivaroxaban in plasma. The developed method was
Linearity range 25-125 ng/band found to be specific, precise, accurate and robust. The
Regression equation Y= 14.71x+284.52
method was applied to a pharmacokinetic study of
Regression coefficient (R2) 0.9954
rivaroxaban. The results indicated better release, faster
Precision (RSD)
Repeatability of measurement (n=7) 0.60
onset of action and better bioavailability of the test
Repeatability of sample application formulation compared to the marketed formulation.
1.65 Although the present method showed considerable
(n=7)
recovery of rivaroxaban from plasma, other extraction
Intra-day precision (n=3) 1.37-1.85
methods such as solid phase extraction could have been
Inter-day precision (n=3) 1.03-2.77
Limit of detection (LOD) 2.64 ng/band tried for obtaining a greater recovery of rivaroxaban.
Limit of quantification (LOQ) 8.00 ng/band The present study could also be extended to estimation
Recovery (n=3) 66.95-69.03% of rivaroxaban in urine.
Robustness Robust
Acknowledgements:
TABLE 5: IN-VIVO DRUG RELEASE PROFILE OF
The authors thank Symed Labs limited, Hyderabad,
RIVAROXABAN India for providing rivaroxaban as a gift sample and
Time
Test formulation Marketed formulation Maliba Pharmacy College for providing the required
(h)
Mean concentration±SD Mean Concentration±SD facilities to carry out this research work.
(ng/ml, n=3) (ng/ml, n=3)
0 0 0
REFERENCES
1 9.81±1.26 13.17±1.71 1. Chandra B, Vankayalapati H, Mittapalli R, Anaparthi S.
Development and validation of UV spectrophotometric
2 21.83±4.21 27.21±2.98
method for the determination of Rivaroxaban. Der Pharma
3 63.83±4.19 29.0±1.36
Chemica. 2013;5(4):1-5.
4 36.63±3.20 47.3±2.76
2. Wolfgang M, Jan S, Dagmar K, and Michael B. Clinical
5 33.2±1.30 19.66±1.66 pharmacokinetic and pharmacodynamic profile of
6 14.8±1.96 10.22±1.10 Rivaroxaban. Clin Pharmacokinet 2014;53(1):1-16.
12 8.13±1.16 5.46±1.86 3. Timothy B. Experimental and clinical pharmacology, new
24 0 0 Oral Anticoagulant Drugs-Mechanisms of Action. Aust Prescr
2010;33(2):38-41.
TABLE 6: DATA OF PHARMACOKINETIC STUDY 4. Attimarad M, Ahmed KK, Aldhubaib BE, Harsha S. High-
FOR RIVAROXABAN performance thin layer chromatography: A powerful analytical
Parameters Test formulation Marketed formulation technique in pharmaceutical drug discovery. Pharm Methods
Cmax (n=3) 63.83±2.47 ng/ml 47.3±1.92 ng/ml 2011;2(2):71-5.
tmax 3h 4h 5. Mohamed A, Medhat A, Hayam M. Investigation of the profile
AUC 290.7 ng/ml.h 219.9 ng/ml.h and kinetics of degradation of Rivaroxaban using HPLC, TLC-
Half-life 6.87 h 6.64 h densitometry and LC/MS/MS: application to pre-Formulation
studies. Bull Fac Pharm Cairo Univ 2015;53(1):53-61.
of rivaroxaban from the test formulation. The Tmax of 6. Kasad PA, Muralikrishna K. Area under curve
Spectrophotometric method for determination of Rivaroxaban
the test formulation occurred at 3 h as compared to
in bulk and tablet formulation and its validation. Asian J Pharm
4 h for the marketed formulation. These values indicated Sci 2013; 3(3):109-13.
a rapid onset of action of the test formulation. Based 7. Shivashankar V, Gandhimanthi M, Ravi TK. Development of
on the Cmax and Tmax values it could be concluded that validated RP-HPLC method for estimation of Rivaroxaban
the test formulation exhibited greater and faster onset in pharmaceutical formulation. Int J Pharm Anal Res
of absorption. The AUC0-t for the test and the marketed 2015;4(4):406-10.
8. Mustafa C, Tuba R, Engin K, Sacide A. RP-HPLC method
formulation was found to be 290.7 and 219.90 ng/ml.h,
development and validation for estimation of Rivaroxaban in
respectively. The AUC of test formulation was pharmaceutical dosage forms. Braz J Pharm Sci 2013;49:2.
1.32 times higher as compared to the marketed 9. Basima A, Mohammad A, Francois K, Manal D, Fida A,
formulation indicating better rivaroxaban bioavailability Alyamama A. Development and validation of a liquid
from the test formulation compared to the marketed chromatography method for the analysis of Rivaroxaban and
determination of its production related impurities. Pharm
formulation. Chem J 2018;52:483-90.

March-April 2020 Indian Journal of Pharmaceutical Sciences 319

10. Burla S, Peruri V, Chandra B. Application of stability performance liquid chromatography tandem mass spectrometry
indicating HPLC method with UV detector to the analysis of (HPLC-MS/MS). Plos One 2017;12(2):1-14.
Rivaroxaban in bulk and tablet dosage form. Chem Sci Trans 15. Rajan N, AnverBasha K. A stability-indicating ultra-
2014;3(4):1546-54. performance liquid chromatographic method for estimation
11. Nageswara R, Ramakrishna K. Development and validation of of related substances and degradants in Rivaroxaban active
a stability indicating LC-PDA-MS/MS method for separation, pharmaceutical ingredient. J Pharm Res 2014;8(11):1719-25.
identification and characterization of process related and 16. Patil P, Wawdhane S, Chaudhari P. Identification, separation
stress degradation products of Rivaroxaban. RSC Adv and characterization of acidic and alkali degradation of
2014;4(44):231-55.
Rivaroxaban under ICH recommended stress condition by
12. Rohde G. Determination of Rivaroxaban – A novel, oral, direct HPTLC with MS/TOF. Anal Chem Lett 2017;7(5):706-23.
factor Xa inhibitor – In human plasma by high-performance
17. Vaghela D, Patel P. High performance thin layer
liquid chromatography–tandem mass spectrometry. J
Chromatogr B 2008;872:43-50. chromatographic method with desitometry analysis for
determination of Rivaroxaban form its tablet dosage form. Int
13. Yadav S, Dubey N. Development and validation of
bioloanalytical method for estimation of Rivaroxaban using J Pharm Pharm Sci 2014;6(6)383-6.
HPLC-PDA in human blood plasma. J Drug Deliv Ther 18. Lories I, Mostafa A, Girges. High performance liquid
2017;7(7):123-5. chromatography, TLC densitometry, first-derivative and
14. Derogis P, Sanches L, Aranda V, Colombini M, Mangueira first-derivative ratio spectrophotometry for determination of
C, Katz M, et al. Determination of Rivaroxaban in patient’s Rivaroxaban and its alkaline degradates in bulk powder and its
plasma samples by anti-xa chromogenic test associated to high tablets. J Chromatogr Sep Tech 2013;4:1-6.

320 Indian Journal of Pharmaceutical Sciences March-April 2020