Spectrophotometric Methods for Simultaneous Determination of Rivaroxaban and Clopidogrel in Their Binary Mixture

Three rapid, accurate and very simple derivative spectrophotometric methods for RIV and CLP assay in their binary mixture and tablet dosage forms were developed. Method (I) is first derivative spectrophotometric method, derivative amplitudes were measured at the zero crossing wavelength of 289 and 249.5 nm for estimation of RIV and CLP, respectively. The linearity is over the range 2.0 20.0 μg/ml for RIV and 5.0 60.0 μg/ml for CLP with LOD of 0.211 and 0.361 μg mL-1 and LOQ of 0.641 and 1.095 μg mL-1 for RIV and CLP, respectively. Method (II) is ratio derivative spectrophotometric method. The ratio spectra of each drug were derived by dividing its spectra on a constant concentration of the other drug as a divisor. Derivative amplitudes were measured at 256 nm for RIV and at 214.5 nm for CLP over the same linearity range as the first method with LOD of 0.137 and 0.485 μg mL-1 and LOQ of 0.417 and 1.471 μg mL-1 for RIV and CLP, respectively. Method (III) is absorbance ratio method, absorbance of both drugs were recorded at two wavelengths λ1 (232) iso-absorptive point and λ2 (249) λmax of RIV. The final concentrations were obtained by applying the Q equations. The method was linear over the same concentration range as the first method with LOD of 0.272 and 0.485 μg mL-1 and LOQ of 0.826 and 1.471 μg mL-1 for RIV and CLP, respectively. The proposed methods were validated as per ICH guidelines. The proposed methods were successfully applied to both drugs analysis in their laboratory prepared co formulated tablet. Statistical comparison of the obtained results with those of the reference method show good agreement and confirm that there were no significant difference in the accuracy and precision between the proposed and reference one respectively. Figure 1: The structural formulae of the studied drugs. Page 2 of 9 Citation: Sharaf EDM, Ibrahim F, Shalan SH, Abd El-Aziz H (2018) Spectrophotometric Methods for Simultaneous Determination of Rivaroxaban and Clopidogrel in Their Binary Mixture. Pharm Anal Acta 9: 575. doi: 10.4172/2153-2435.1000575 Volume 9 • Issue 1 • 1000575 Pharm Anal Acta, an open access journal ISSN: 2153-2435 the ratio derivative spectra for CLP were recorded in the wavelength range (200-280) nm with Δλ = 2 nm and scaling factor = 1 and ∆λ = 4 nm for the first derivative of the produced ratio spectra with scaling factor = 10. Ultrasonic bath (Model: SS 101 H 230, USA) used for sonication of all prepared solutions. Materials and reagents -Rivaroxaban and Clopidogrel were purchased from Memphis Co. for pharm. & Eva Pharma Company, Cairo, Egypt, respectively. -Laboratory prepared tablet consists of 15 mg RIV, 75 mg CLP, 15 mg maize starch, 15 mg lactose, 7 mg magnesium stearate and 20 mg talc powder. -Methanol was purchased from SigmaAldrich (Germany) as HPLC grade.

The first derivative spectra of both drugs were scanned in the wavelength range (200 -320) nm with ∆λ = 8 nm and applying scaling factor = 10.
The ratio derivative spectra for RIV were recorded in the wavelength range (235-284) nm applying ∆λ = 2 nm and scaling factor = 1 to produce a smooth ratio spectra and ∆λ = 4 nm for the first derivative of ratio spectra appling scaling factor = 10. Meanwhile, the ratio derivative spectra for CLP were recorded in the wavelength range (200-280) nm with Δλ = 2 nm and scaling factor = 1 and ∆λ = 4 nm for the first derivative of the produced ratio spectra with scaling factor = 10.
Ultrasonic bath (Model: SS 101 H 230, USA) used for sonication of all prepared solutions.

Materials and reagents
-Rivaroxaban and Clopidogrel were purchased from Memphis Co. for pharm. & Eva Pharma Company, Cairo, Egypt, respectively.
-Methanol was purchased from Sigma-Aldrich (Germany) as HPLC grade.

Standard solutions
Standard stock solutions of 10.0 mg/100 ml for each of Rivaroxaban and Clopidogrel were prepared separately in methanol. Using methanol for dilution of the stock solutions, working solutions were appropriately prepared. An additional stock solution was prepared using 50 mg/100 ml of Clopidogrel to be used in synthetic mixture preparation. The drugs kept stable for not less than 3 weeks without alteration when stored in the refrigerator.
Calibration graphs establishment D 1 method: A series of 10 ml volumetric flasks was used. Transfer accurately measured volumes of the drug working standard solutions to reach final concentration in the range of 2.0 -20.0 µg/ml for RIV and 5.0-60.0 µg/ml for CLP. Dilute with methanol to the volume and mix well. RIV and CLP zero-order absorption spectra were recorded against blank methanol ( Figure 2). The first derivatives of the zeroorder spectra were recorded. The derivative amplitudes were measured at the zero crossing wavelength of 289 and 249.5 nm for determination of RIV and CLP, respectively (Figures 3 and 4). A plot of derivative absorbance amplitudes was then drawn against the final concentrations. Also, we derived the corresponding regression equations from the obtained data.  volume and then mixed well. The steps stated under "Calibration Graphs Establishment" (a, b, c) were then followed. Graphs of derivative amplitudes against the final concentrations were plotted to obtain the linearity range.
proposed method application for determination of riv and clp in their laboratory prepared co-formulated tablets Ten laboratory prepared tablets were mixed well, then amount equivalent to 15 mg RIV and 75 mg CLP were accurately weighed and transferred into 100 mL volumetric flask and about 80 mL of methanol were added. Sonication of the flask contents for 30 min was applied, and then the flask was completed to the volume with the same solvent and filtered. Aliquot volumes of the filtrate of suitable concentrations in the working concentration ranges for both studied drugs were assayed as illustrated under "Calibration Graphs Establishment". Tablet contents were calculated using calibration graph or corresponding regression equations.

Results and Discussion
RIV UV spectrum showed maxima at 210 and 248 nm and for Ratio derivative spectrophotometry: In case of using ratio derivative spectrophotometry, the first derivative of the ratio spectra (all RIV spectra were divided by CLP spectrum (20.0 μg/mL) ( Figures 5  and 6) and all CLP spectra were divided by RIV spectrum of 5.0 μg/mL) were derived (Figures 7 and 8). After measuring the amplitudes at 256 nm for RIV and at 214.5 nm for CLP, the derivative amplitudes were plotted against the final concentrations to obtain the calibration curves.
Absorbance ratio method: Determination of drugs by absorbance ratio method was also derived, absorbance of both drugs were recorded at two wavelengths λ 1 (232) iso-absorptive point and λ 2 (249) λ max of RIV ( Figure 2). The final concentrations were obtained by applying the Q equations.

Application of the proposed methods (I, II, III) to RIV/CLP binary mixtures assay
Accurate aliquots of RIV and CLP standard solutions in their pharmaceutical ratio (1:5) were transferred into a series of 10 mL volumetric flasks. The solutions were diluted with methanol to the     CLP 222 nm ( Figure 2). Due to this overlap, RIV can be evaluated in presence of CLP in the zero order spectra but the readings were of low sensitivity at 275 nm and CLP cannot be estimated in presence of RIV. Thus, we developed three spectrophotometric methods (derivative and ratio derivative and ratio absorbance) for the quantification of both drugs in the presence of each other.

First derivative spectrophotometric method
The use of derivative spectrophotometric technique make enhancement for the UV absorption spectrum features; the first derivative spectra of both RIV and CLP (Figures 3 and 4) permitted more specific and highly selective determination of each drug at the zero-crossing point of the other. The first derivative amplitudes at 289 nm (zero-crossing of CLP) and at 249.5 nm (zero-crossing of RIV) were selected for assay of RIV and CLP in their binary mixtures, respectively. Figure 5 illustrates the ratio spectra of RIV standards in different concentrations (RIV spectra divided by 20.0 μg/mL of CLP spectrum) while Figure 6 shows their corresponding first derivative spectra. In this figure, the amplitude at 256 nm ( 1 DD 256 ) corresponds to RIV present in the solution, so it can be used for its quantitative assay.

Ratio derivative spectrophotometry
Likewise, Figure 7 and 8 shows the ratio spectra of different concentrations of CLP standards (spectra divided by the spectrum of 5.0 μg/mL RIV solution) as well as their corresponding first derivative spectra, on the basis of which, CLP can be estimated by measuring the amplitude at 214.5 nm ( 1 DD 214.5 ).
The effect of Δλ for obtaining the first derivative of the ratio spectra was tested to obtain the best wavelength interval; Δλ = 8 nm was considered as suitable for both drugs. CLP (20.0 μg /ml) and RIV (5 μg /ml) were selected as the most suitable divisor concentration in relation to signal-to-noise ratio and sensitivity for evaluation of RIV and CLP, respectively.
Absorbance ratio method Figure 2 shows the iso-absorptive point at which both drugs have the same absorbance value and λ max for RIV. Absorbance of each drug was measured twice; at the iso-absorptive point and at λ max for RIV, then calibration for each drug alone was performed and specific absorptivities of both drugs were determined.
Relative concentration of the two drugs in the mixture was calculated by applying the following equations [24].
Where Q M = A 2 (absorbance of the sample at 248.5 nm)/ A 1 (absorbance of the sample at 233.7 nm), Q x = a x2 (absorptivity of RIV at 248.5 nm)/ a x1 (absorptivity of RIV at 233.7 nm) , Q y = a y2 (absorptivity of CLP at 248.5 nm) / a y1 (absorptivity of CLP at 233.7 nm).
A 1 and A 2 are the absorbance of the mixture at 233.7 nm and 248.5 nm, respectively. a x1 and a y1 are the absorptivities of RIV and CLP at 233.7 nm, a x2 and a y2 are the absorptivities of RIV and CLP at 248.5 nm.

Proposed Methods Validation Linearity and range
A plot of the derivative amplitude against the drug concentration in µg/ml showed a linear relationship over the range 2.0-20.0 µg/mL for RIV and 5.0-60.0 µg/mL for CLP. The concentration ranges over which linearity was cited in Tables 1 and 2. The following equations were obtained from data regression analysis: The linearity of absorbance ratio method was performed for each drug twice (at iso-absorptive point and at λ max of RIV).
From data of statistical analysis [24,25] it was found that the   correlation coefficients (r) were of high values indicating high linearity of the proposed methods and the results obeys Beer's law.
Low scattering of the points around the calibration curves was predicted from the small values of the standard deviation of residuals (S y/x ), of intercept (S a ), and of slope (S b ). Owing to small values of percentage relative standard deviation (RSD%) and percentage relative errors (%Er), the proposed method illustrates high accuracy and high precision.

Limit of detection (LOD) and limit of quantitation (LOQ)
Quantitation limit (LOQ) and detection limit (LOD) were determined according to ICH recommendations [26] using the following equation.

LOQ = 10 S a /b LOD = 3.3 S a /b
Where: S a is standard deviation of the intercept of the calibration curve and, b is the slope of the calibration curve. Table 1 shows that: 0.64 and 1.09 µg/mL were LOQ values while 0.21 and 0.36 µg/mL were LOD values for RIV and CLP, respectively for first derivative spectrophotometric method. Table 2 illustrates that: 0.42 and 1.47 µg/mL were LOQ values while 0.14 and 0.48 µg/mL were LOD values for RIV and CLP, respectively for ratio first derivative spectrophotometric method. Table 3 illustrates that: 0.826 and 1.471µg/ mL were LOQ values while 0.272 and 0.485 µg/mL were LOD values for RIV and CLP, respectively for absorbance ratio spectrophotometric method.

Method accuracy
The analysis of the obtained results of the proposed methods for the RIV and CLP were compared with those produced using the reference method [13] for proving the accuracy of our proposed method. The obtained results were analyzed statistically using Student's t-test and variance ratio F-test [25] which illustrated no significant difference between both methods performance regarding accuracy and precision, respectively. Student's t-test and variance ratio F-test are presented in Table 4.
-The precision for proposed methods was determined by (intraday) and (inter-day) assay for both drugs.

Intra-day precision:
The three proposed methods were subjected to intra-day precision through replicate analysis of three concentrations of both tested drugs on three different times within the same day. The obtained results showed small values of % Error and % RSD indicate high accuracy and precision of the proposed methods, respectively. The outcomes are cited in Tables 5-7.
Inter-day precision: Inter-day precision was carried out through replicate analysis of three concentrations of the studied drugs on three successive days. The results are stated in Tables 5-7.      The small values of % Error and % RSD indicate high accuracy and precision of the proposed method, respectively.

Specificity
Common tablet excipients may interfere with the assay of the drugs which affect the specificity of the method. The excipients did not interfere with the results of our proposed methods indicating high specificity of the method. The specificity of the proposed method was investigated by viewing any interference of common tablets excipients such as talc powder, lactose, maize starch, magnesium stearate, calcium hydrogen phosphate and microcrystalline cellulose.

Robustness
The robustness of the method was assessed by evaluating the influence of small variations in the experimental variables on the analytical performance of the method. The small variations in any of the variables did not significantly affect the results; percentage recovery was 99.78 ± 1.02 and 99.95 ± 0.79 for RIV and CLP respectively. This provided an indication of the reliability of the proposed methods during routine work.

Ruggedness
For expressing the stability of the method against extraneous influencing factors such as analyst, laboratory, instrument. The proposed method provided stable results in different laboratories which indicate reliability of the proposed method.

Application of the Proposed Methods to the Determination of RIV/CLP in Laboratory-Prepared Mixtures
Simultaneous evaluation of RIV and CLP in laboratory prepared binary mixtures in their pharmaceutical ratio; 1:5 was performed as an application of the proposed methods. In the laboratory prepared mixtures, the concentrations taken of both drugs were presented in the linearity range. The values of %RSD and %Er were calculated and the results indicate high accuracy of the proposed method as shown in Tables 8-10.

Application of the Proposed Methods to the Determination of the Studied Drugs in Laboratory Prepared Co-formulated Tablets
Our proposed methods were applied for the estimation of both tested drugs in their prepared tablets. Tables 11-13 showed that the results obtained are in good agreement with the results of the comparison one [13]. The obtained data were statistically analyzed using Student's t-test and variance ratio F-test [24] showing no significant difference between the performance of the two methods, so our method is of high accuracy and precision.

Conclusion
RIV and CLP binary mixtures were analyzed using an accurate, sensitive, and rapid three spectrophotometric methods. Also, the studied drugs could be determined in prepared tablets using the proposed spectrophotometric procedures. These proposed methods can be applied in quality control laboratories, science it showed good validation criteria.