Area Under Curve Spectrophotometric Method for Determination of Rivaroxaban in Bulk and Tablet Formulation and Its Validation.

Kasad Pinaz A.*, K.S. Muralikrishna

Shree Dhanvantary Pharmacy College, Department of Quality assurance, Kim, Surat (India)

*Corresponding Author E-mail:- pinazkasad@yahoo.in

ABSTRACT

The present research work discusses the development of an Area under the Curve spectrophotometric method for Rivaroxaban. Simple, sensitive, reproducible, accurate, rapid, simple and cost efficient spectrophotometric method has been developed for the estimation of Rivaroxaban in bulk and dosage form. The optimum conditions for the analysis of the drug were established. The study was conducted in Methanol between the wavelengths of 241nm- 260 nm. The method validations was accomplished through evaluation of analytical parameters of linearity, range, accuracy and precision, limit of detection and limit of quantification and robustness as per ICH guidelines. The developed method was linear (r2= 0.999) in the concentration range of 2-12 μg/ml, precise (Mean %RSD inter day is 0.537 and Mean %RSD intraday is 0.297), accurate (% recovery= 99.31%) and sensitive (LOD and LOQ of 0.059 and 0.179 μg/ml respectively). The proposed method is observed to be suitable for the analysis of Rivaroxaban in bulk and Dosage form for quality control purposes.

KEYWORDS: Rivaroxaban, Oral anticoagulant, AUC method, Validation.

INTRODUCTION:

Anticoagulants are often called blood thinners. They help prevent blood clots from forming and growing and reduce your risk for heart attack, stroke and blockages in your arteries and veins. Rivaroxaban is an oral anticoagulant invented and manufactured by Bayer; in a number of countries it is marketed as Xarelto. ⁽¹⁾ In the United States, it is marketed by Janssen Pharmaceutical. ⁽²⁾ It is the first available orally active direct factor Xa inhibitor. The effects last 8–12 hours, but factor Xa activity does not return to normal within 24 hours so once-daily dosing is possible. There is no specific way to reverse the anticoagulant effect of Rivaroxaban in the event of a major bleeding event, unlike warfarin. Rivaroxaban is an oxazolidinone

derivative optimized for inhibiting both free Factor Xa and Factor Xa bound in the Prothrombinase complex.⁽⁵⁾It is a highly selective direct Factor Xa inhibitor with oral bioavailability and rapid onset of action. Inhibition of Factor Xa interrupts the intrinsic and extrinsic pathway of the blood coagulation cascade, inhibiting both thrombin formation and development of thrombi.

Rivaroxaban does not inhibit thrombin (activated Factor II), and no effects on platelets have been demonstrated.⁽⁶⁾In September 2008, Health Canada and European Commission granted marketing authorization for Rivaroxaban as one 10 mg tablet taken once daily for the prevention of venous thromboembolism (VTE) in patients who have undergone elective total hip replacement or total knee replacement surgery. It was approved by CDSCO on 30 January 2010. In December 2011 Rivaroxaban has been approved by the European Commission for use in two new indications: prevention of stroke and systemic embolism in adult patients with non-valvular atrial fibrillation (AF) with one or more risk factors and treatment of deep vein thrombosis (DVT) and prevention of recurrent DVT and pulmonary embolism (PE) following an acute DVT in adults.

On July 1, 2011, the U.S. Food and Drug Administration (FDA) approved Rivaroxaban for prophylaxis of deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE), in adults undergoing hip and knee replacement surgery.⁽³⁾ On November 4, 2011, the U.S. FDA approved Rivaroxaban for stroke prophylaxis in patients with non-valvular atrial fibrillation.⁽⁴⁾

Figure 1- structural formula of Rivaroxaban

IUPAC name of Rivaroxaban is (S)-5-chloro-N-{[2-oxo-3-[4-(3-oxomorpholin-4-yl) phenyl] oxazolidin-5 yl] methyl} thiophene-2-Carboxamide. It’s Molecular Formula is C₁₉H₁₈ClN₃O₅ and Molecular Mass is 435.882g/mol. Literature survey reveals Colorimetric method^(7-8), RP-HPLC method⁽⁹⁾ and bio analytical method⁽¹⁰⁾ for estimation of Rivaroxaban. The main purpose of the present study was to establish a relatively simple, sensitive, validated and inexpensive spectrophotometric method for the determination of Rivaroxaban in pure form and in pharmaceutical dosage form.

MATERIAL AND METHODS:

Instruments and Reagents

A Shimadzu UV –Visible spectrophotometer- 2450, with 1cm matched quartz cells was used for the absorbance measurements. Sartorious CP225D Analytical balance was used for weighing the samples. D120/1H, Trans-o-sonic- Ultra Sonicator was used for Sonication of solution. All the chemicals used were of analytical grade. Pure Rivaroxaban was procured as a gift sample from Mega Fine Pharma, Mumbai, India.

Preparation of Standard Stock Solution

10 mg of Rivaroxaban was accurately weighed and transferred into a clean, dry 100 mL volumetric flask, dissolved with sufficient volume of methanol, sonicated for 10 min and volume was adjusted to 100 mL with methanol to get a concentration of 100 μg/mL

Method Validation ⁽¹¹⁾

The method validation was carried out as per ICH Q2 (R1) guidelines. The following validation parameters; linearity and range, accuracy and precision, limit of detection (LOD), limit of quantification (LOQ) and robustness were studied.

Linearity and Range

Portions of 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL and 1.2 mL of 100 µg/mL of standard stock solution of Rivaroxaban were transferred into separate 10 mL volumetric flasks and adjusted up to 10 mL with methanol to obtain the concentrations of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, 10 μg/mL and 12 μg/mL respectively. Calibration curve was constructed by plotting Peak Area v/s concentration at the selected wavelength Range. The linearity was calculated by least square regression method.

Accuracy

Accuracy is the percent of analyte recovered by assay from a known added amount. Accuracy of the experiment was established by using recovery studies. For this, standard samples were prepared in triplicate. The spiking was done at three levels 80 %, 100 % and 120 %. Each solution was scanned between 200 nm to 400 nm against methanol as a blank. The spectrum of each was obtained. The amount of Rivaroxaban was calculated at each level and % recoveries were computed.

Precision

I. Repeatability

Solutions of Rivaroxaban containing 6 μg/mL were analyzed six times using developed spectroscopic method and % RSD was calculated.

II. Intraday precision

Solutions of Rivaroxaban containing 2, 6 and 12 μg/mL series were analyzed three times on the same day using developed spectroscopic method and % RSD was calculated.

Ш. Interday Precision

Solutions of Rivaroxaban containing 2, 6 and 12 μg/mL series were analyzed on three different days using developed spectroscopic method and % RSD was calculated.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. Estimation of LOD and LOQ were based on the standard deviation of the response and the slope of the calibration curve.

Equations used for calculation are as follows:

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ is the standard deviation of the absorbance of the sample and, S is the slope of the related calibrations graph.

Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. For the robustness change in the volumetric flask of the different measuring capacity and change in Analyst were done and % RSD was calculated.

RESULTS:

Linearity

The linearity of the response of the drug was verified at 2 to 100 μg/mL concentrations, but linearity was found between 2-12 μg/mL concentrations. The calibration graphs were obtained by plotting the peak area versus the concentration data and were treated by linear regression analysis. The equation of the calibration curve for Rivaroxaban obtained y = 0.113x-0.021.The calibration curve was found linear in the aforementioned concentrations. The correlation coefficient (r²) of determination was 0.999.

Figure 2-Spectrum of Linearity curve for AUC Method.

Figure 3-Linearity of Rivaroxaban for AUC Method.

Table 1-Precision data for AUC Method.

Sr. No	Sample solution concentration (μg/mL)	Mean Absorbance ± SD		% RSD		Mean % RSD
		Intraday	Interday	Intraday	Interday	Intraday	Interday
1	2	0.2106 ± 0.0012	0.213±0.002	0.55	0.94
2	6	0.6516±0.0015	0.652±0.0027	0.23	0.41	0.297	0.537
3	12	1.3566±0.0015	1.3533±0.0035	0.11	0.26

Table 2-Accuracy data for AUC Method.

Sr. No.	Amount of Drug in formulation (μg/mL)	Amount of drug spiked (μg/mL)	Total Amount (μg/mL)	Amount Recovered (μg/mL)	% Recovery	Mean % Recovery
1.	4	3.2 (80%)	7.2	7.132	99.07	99.31 %
2.	4	4 (100%)	8	7.920	99.00
3.	4	4.8 (120%)	8.8	8.7876	99.86

Precision

The relative standard deviation (RSD) values obtained for three concentrations (2 μg/mL, 6 μg/mL and 12 μg/mL) at intraday precision were 0.55, 0.23 and 0.11 respectively (Table 2). The relative standard deviation (RSD) values obtained for three concentrations (2 μg/mL, 6 μg/mL and 12 μg/mL) at inter-day precision on three consecutive days were 0.94, 0.41 and 0.26 respectively (Table 1). All the data are within the acceptance criteria of 1%.

Accuracy (Recovery Study)

The accuracy was calculated as the percentage of the drug recovered from the formulation matrix. The mean percentage recovery obtained was 99.31 % which is presented in (Table 2).

LOD and LOQ

The absorbance of ten solutions (2 µg/mL) was measured at 248.6 nm and calculated according to equation of LOD [3.3 x SD/ slope] and LOQ [10 x SD/ slope]. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.059 μg/mL and 0.179 μg/mL respectively as shown in Table 3.

Table 3-LOD and LOQ data AUC Method

Standard Deviation (SD)	0.002025
Slope	0.113
Limit of Detection(LOD)	0.059
Limit of Quantitation (LOQ)	0.179

Robustness and Ruggedness

These were performed by using different apparatus and analysts. Result were expressed as %RSD and shown in Table 4.

Table 4-Robustness and Ruggedness Data for AUC Method

A) Change in Apparatus (Volumetric Flask)

Volumetric Flask (ml)	Concentration (μg/mL)	Mean	SD	% RSD
10 ml	6	0.6513	0.0011547	0.18
50 ml	6
100 ml	6

B) Change in Analysts

Analyst	Concentration (μg/mL)	Mean	SD	% RSD
Analyst- I	6	0.652	0.0014142	0.22
Analyst- II	6

Determination of Active Ingredients in Tablets

The validated method was applied to the determination of Rivaroxaban in Tablets. Ten tablets were assayed and the results are shown in (Table 5.)

Table 5 - Assay Data for AUC Method.

Sample Label	Claim (mg)	Conc. taken	Conc. found	% Assay
Rivaroxaban	10	6	5.982	99.71 %

Table 6 Summary of Validation Parameter

Sr. No.	Parameters	Result
1.	Linearity (Conc. μg/mL)	2-12 μg/mL
2.	Slope (m)	0.113
3.	Intercept (c)	0.021
4.	Correlation Coefficient	R² = 0.999
5.	Accuracy (%recovery)	99.31 %
6.	Precision (Intraday) (n=3) (%RSD)	0.297
7.	Precision (Interday) (n=3) (%RSD)	0.537
8.	LOD	0.059
9.	LOQ	0.179
10.	Ruggedness Change in Apparatus (Volumetric Flask) (%RSD) Change in Analysts(%RSD)	0.18 0.22

DISCUSSION:

The development of a simple, rapid, sensitive, and accurate analytical method for the routine quantitative determination of samples will reduce unnecessary tedious sample preparations, the cost of materials and labor. Rivaroxaban is a UV absorbing molecule with specific chromophores in the structure that absorb at a particular wavelength and this fact was successfully employed for their quantitative determinations using the AUC spectrophotometric method. The stock and sample solutions of Rivaroxaban were prepared in methanol. The Wavelength of the drug for analysis was determined by taking scans of the drug sample solutions in the entire UV region.

The absorption spectrum of Rivaroxaban was scan in methanol and wavelength selected at the range of 241-260 nm. Calibration curve data was constructed in the range of the concentrations of 2-100μg/ml, but Beer’s law obeyed in concentration range of 2-12 μg/ml. The regression equation was found to be y = 0.113x - 0.021 with correlation coefficient (r2) of 0.999.

Performing replicate analyses of the standard solutions was used to assess the accuracy, precision, and reproducibility of the developed method. The selected concentration within the calibration range was prepared in methanol and analyzed with the relevant calibration curve to determine the intra and inter day variability. The developed method can be successfully applied for assay in tablet dosage forms without any interference. The assay showed that the drug content of this product to be in accordance with the label claim (Table 5).

The repeatability as well as interday precision was found to be within the specified limits (Table 1).The recovery of the analyte of interest from a given matrix can be used as a measure of the accuracy of the method (Table 2).

The obtained results demonstrate the validity and accuracy of the developed method for the determination of drug in tablet. In order to check the accuracy and precision of the developed method and to prove the absence of interference by excipients, recovery studies were carried out after the addition of known amounts of the pure drug to various pre-analyzed formulations of all drugs.

The results reveal that the developed method has an adequate precision and accuracy, and consequently, can be applied to the determination of Rivaroxaban tablet in pharmaceuticals without any interference from the excipients.

CONCLUSION:

The proposed AUC spectrophotometric method was found to be, simple, sensitive, accurate and precise for determination of Rivaroxaban in Bulk and tablet dosage form. Hence it can be conveniently adopted for routine quality analysis of the drug.

REFERENCES:

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Received on 19.06.2013 Accepted on 20.07.2013

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Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 3, Pg 109-113