INTRODUCTION:

RIV is an anticoagulant and first orally active direct inhibitor of factor X_a approved by FDA in the year2011. It is chemically 5-Chloro-N-({(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl) phenyl]-1, 3-oxazolidin-5yl}-methyl)-2-thiophenecarboxamide with an empirical formula and molecular weight of C₁₉H₁₈ClN₃O₅S and 435.89g/mol respectively. It is an odorless, non hygroscopic and white to yellowish powder which is slightly soluble in organic solvents and is practically insoluble in water and aqueous media. It is marketed by Bayer under the name Xarelto as a tablet (2.5mg, 10mg, 15mg and 20mg)¹.

The literature review revealed that there are few methods for quantitative estimation of RIV using HPLC^2-16, UV spectroscopy^17-20, UPLC^21-22, LC-MS/MS²³ etc.

As the marketed formulations of Rivaroxaban are available in low doses, the present work was an attempt to develop a new sensitive method for the quantitative estimation of Rivaroxaban in Bulk and Pharmaceutical dosage form by HPLC in reverse phase mode.

MATERIALS AND METHODS:

Chemicals- Solvents used for this method are acetonitrile and water which are of HPLC grade and are procured from Merck life science. The entire chemicals i.e. RIVstandard and IS are obtained whose make is from Nifty labs and M/S Granules India limited which were of analytical grade. Marketed RIV tablets were purchased from local pharmacy.

Instrument- The analysis was performed using a gradient high performance liquid chromatography (Schimadzu) equipped with HPLC pump LC-20AT, with LC solutions software, UV-visible detector SPD-20A and Schimadzu-C18 column (250×4.6mm, 5µm).

Preparation of standard stock solution- Stock solution was prepared by transferring 10mg of RIV in 10ml of volumetric flask. It was dissolved in Acetonitrile and made upto the mark, this gives 1000µg/ml solution. Further dilutions were done by serial dilution method for obtaining respective concentrations with acetonitrile.

Preparation of IS solution- Stock solution was prepared by transferring 10mg of Paracetamol in 10ml of volumetric flask. It was dissolved in Acetonitrile and made upto the mark, this gives 1000µg/ml solution. Further dilutions were done by serial dilution method for obtaining a concentration of 1µg/ml with acetonitrile.

Preparations of sample solution- A total number of 20 tablets were weighed and the average weight of tablets was calculated. From the powdered tablets weight equivalent to 10mg was taken and it was dissolved in acetonitrile. The volume was made upto mark with acetonitrile. Further dilutions were done by serial dilution method for obtaining respective concentrations with acetonitrile.

Method development- Different ratios of mobile phases were tried for the separation and resolution. Various method optimization procedures were carried out and compared with system suitability parameters. The choice of wavelength 254nm was considered satisfactory, permitting the detection of the drugs with adequate sensitivity.

Fixed chromatographic conditions- After satisfactory optimizing procedure, acetonitrile and water (60:40%v/v) was used as mobile phase with a flow rate of 1ml/min. The injection volume of sample and standard was 20µl. the detection was carried out using refractive index detector at a wavelength of 254nm. The mobile phase, standard and sample solutions were filtered and degassed using ultrasonicator.

Method validation²⁴:

System Suitability- The system suitability tests were conducted by injecting a standard solution of RIV and IS to ensure the readiness of system before analysis. Under optimum conditions various parameters such as column efficiency (theoretical plates), tailing factor, resolution etc were checked.

Selectivity- Selectivity of a method refers to the extent to which it can determine particular analytes in a solution without interference from other components in the mixture. Selectivity of the method was evaluated by preparing blank and standard solution of drug and IS followed by injecting them into the HPLC system.

Linearity- The linearity of an analytical procedure is its ability (within a given range) to obtain results/response which are directly proportional to the concentration. Linearity of method shall be demonstrated with five different concentration levels. The solutions are prepared at five different concentration levels .The calibration curve is to be drawn by plotting the concentration against the peak area ratios. Correlation coefficient, slope of the curve is reported to show the developed method was linear.

Sensitivity (LOD and LOQ) - The detection limit and quantitation limit can be calculated based on the standarddeviation of the response and the slope.

Accuracy- A standard solution of RIV containing ISwill be prepared at three concentration levelscorresponding to 80%, 100% and 120%. The percentage RSD of three levels will be calculated. It should be within the limits.

Precision- Precision of the method shall be reported by injecting six replicates of standardsolution consecutively under the same analytical conditions. Percentage relativestandard deviation for six replicates should be calculated.

Precision of the method is also determined for both intra-day and inter-day variations. Three different concentrations of standard RIV solutions within the linear range will be analyzed on three consecutive days for inter-day precision and three times (morning, afternoon and evening) within the same day for intra-day precision. The percentage RSD of three levels will be calculated. It should be within the limits.

Recovery studies- Test solutions of RIV shall be prepared at three concentration levelscorresponding to 80%, 100% and 120%. The percentage recovery of three levels will be reported against standard concentrations. Calculation of percentage recoveryis based on the amount of the test solution added vs standard concentration.Percentage recovery value should be not less than 80% and not more than 120%.

Robustness- The robustness of the method is determined to check the reliability of ananalysis with respect to deliberate variations in method parameters. The typicalParameters are given below: Variation in flow rate by ±0.1ml/min, variation in mobile phase ratio by ±0.1% and variation in pH of buffer ±0.05.

RESULTS AND DISCUSSION:

Selection of wavelength- Absorption maximum of RIV was observed at 251nm and absorption maximum of internal standard Paracetamol was observed at 248nm. Thus the wavelength of 254nm was selected as λ_max based on various trails.

Fig-1- Chromatogram of RIV

Fig-2- Chromatogram of IS

Fig-3- Chromatogram of blank solution

Fig-4- Chromatogram of RIV with IS

System suitability- The system suitability parameters are found to be optimal and are as follows-Theoretical plate count- 6455.677, Resolution- 6.488.

Selectivity- There was no interference of peaks obtained in blank solution, at the retention times of analyte peak and IS peak. The chromatograms of blank, drug and IS are shown in fig- the chromatograms of blank, drug and internal standard demonstrate the selectivity results.

Linearity- The working standard solutions of RIV containing IS were prepared for checking the linearity in the concentration range of 100-500ng/ml. Linear regression analysis was performed for checking the linearity of the data obtained. The calibration curve data is shown in table-1 and calibration curve is shown in fig-5

Table-1- Calibration curve data

Concentration (ng/ml)	Peak area ratio
100	0.23505
200	0.29385
300	0.35785
400	0.42155
500	0.49065

Fig-5- Calibration Curve

Sensitivity- The concentration of RIV for determination of LOD was 8.33ng/ml which indicates the sensitivity of method. Similarly LOQ was found 25.25ng/ml which proves that RIV can be estimated at low concentrations.

Accuracy and Precision- The %RSD of RIV during repeatability study was 1.07%. The %RSD of results obtained during inter and intra-day precisions were within the range of 1.16-1.59% and 1.01-1.35% respectively. The %RSD of accuracy of drug substance was found to be within the range of 1.06-1.6%.

Table-2: Values of precision and accuracy of the method

Validation parameter

Concentration (ng/ml)

Mean±Standard deviation

%RSD

Accuracy

(n = 3)

240

300

360

0.3347±0.005356

0.3552±0.003782

0.3925±0.006185

1.6%

1.06%

1.56%

Precision

Repeatability (n = 6)

300

0.353033±0.003771

1.07%

Intra-day precision

(n = 3)

240

300

360

0.3095±0.004167

0.3512±0.003562

0.3990±0.00539

1.34%

1.01%

1.35%

Inter-day precision

(n = 9)

240

300

360

0.3098±0.003592

0.3501±0.005577

0.3989±0.005358

1.16%

1.59%

1.34%

Acceptable Limits (%RSD)-≤2, %RSD- Percentage Relative standard deviation

Table-3: Values of recovery studies of RIV

Concentration of test solution added (ng/ml)	Concentration of standard solution added (ng/ml)	Peak area ratio	Mean	Concentration of solution practically (ng/ml)	Percentage recovery
240	300	0.6360 0.6252 0.6247	0.6286	534.20	98.9%
300	300	0.6872 0.6736 0.6958	0.6855	582.57	97.01%
360	300	0.7352 0.7247 0.7360	0.7320	622.09	94.2%

Acceptable Limits (percentage recovery) -80%-120%

Table-4- Values of robustness of the method

Chromatographic Condition		Concentration	Mean peak Area ratio (n = 3)	Mean±Standard deviation	%RSD
Flow rate	0.9ml/min	300 300 300	0.359233 0.357850 0.361417	0.361417 ± 0.005028	1.39%
	1ml/min
	1.1ml/min
Pump B Concentration	59%	300 300 300	0.354533 0.357850 0.365833	0.359405 ± 0.005808	1.62%
	60%
	61%

Acceptable Limits (%RSD)-≤2

Table-5- Assay results

Brand name of tablets	Label claim of drug	Mean peak area ratio (n = 3) ± Standard deviation	Percentage purity	Amount of drug present in tablet (mg)
Xarelto	2.5mg	0.3361±0.000416	93.32%	2.331
Xarelto	10mg	0.3759±0.012926	115.4%	11.54
Xarelto	15mg	0.3638±0.003516	108.72%	16.30
Xarelto	20mg	0.3573±0.000889	105%	21.02

Acceptable Limits (percentage recovery) -80%-120%

Robustness- In different deliberate varied chromatographic conditions (flow rate and pump B concentration) all analytes were adequately resolved and elution orders remained the same. Results are given in table-4

Assay- The validated HPLC method was applied for determination of RIV in commercially available Xarelto (2.5mg, 10mg, 15mg and 20mg) tablets. 20 tablets were weighed and average weight was calculated from the powder tablets quantity equivalent to 10mg was taken and it was dissolved in acetonitrile and volume was made upto the mark. This solution was further diluted to get a solution having concentration of 300ng/ml. The tablets assay results were given in table-5.

CONCLUSION:

A sensitive RP-HPLC method was developed for the quantitative estimation of Rivaroxaban in bulk and pharmaceutical dosage form. The developed method was optimized prior to validation studies in terms of stationary phase, mobile phase composition and flow rate. The chromatographic peak of RIV was eluted at a retention time of 4.230 mins and that of IS paracetmol was elutes at 2.980 mins. The developed method was subjected to method validation as recommended by ICH guidelines.A simple mobile phase without preparation of any buffer solution or adding ion-pairing agents and a short retention time are advantageous. From the results obtained it is concluded that the developed method was found to be selective, sensitive, linear, accurate, reproducible, robustand applicability of the method can be studied in biological samples..

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Received on 01.10.2020 Modified on 07.04.2022

Research J. Pharm. and Tech 2023; 16(6):2719-2723.

DOI: 10.52711/0974-360X.2023.00447