DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC METHODS FOR THE SIMULTANEOUS ESTIMATION OF RASAGILINE AND RILUZOLE

DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC METHODS FOR THE SIMULTANEOUS ESTIMATION OF RASAGILINE AND RILUZOLE

Ramgopal Dhanwad, Deepali, L. Harini and K. C. Chaluvaraju *

Department of Pharmaceutical Chemistry, Govt. College of Pharmacy, Bengaluru, Karnataka, India.

ABSTRACT: In the present study, an effort has been made to estimate Rasagiline and Riluzole simultaneously in their bulk and physical mixture by UV-Visible spectrophotometric methods. The methods developed are precise, reliable simultaneous equations and area under the curve (AUC) method. The absorption maxima in the simultaneous equation method are 265.20 nm and 263.40 nm, respectively, for rasagiline and riluzole. Similarly, in the area under the curve method, the wavelength selected for estimation is in the range of 260.20 - 270.20 nm and 258.40-268.40 nm, respectively for rasagiline and riluzole.  In the two methods developed, the Beer- Lambartz’s range was in the concentration range of 50 to 250 µg/mL for rasagiline and 4 to 20µg/mL for riluzole.  The percentage recovery was found to be in the range of 100.43% and 100.01% for rasagiline and riluzole. ICH guidelines were followed in the proposed methods for their reproducibility and accuracy. The proposed methods can be adopted industrially and in research laboratories for the routine simultaneous analysis of Rasagaline and Riluzole in their bulk and dosage forms.

Keywords: Rasagiline, Riluzole, Spectroscopic method, Absorption, Simultaneous estimation, Methanol

INTRODUCTION: Rasagiline is chemically (1R)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine (Fig:1) Its molecular formula is C₁₂H₁₃N and molecular weight is 171.23 g/mol. It is soluble in Methanol. It is used in the treatment of Amyotrophic lateral sclerosis (ALS) and belongs to the category of an antiparkinsonian agent ^{1, 2}. Rasagiline is a propargyl amine and an irreversible inhibitor of monoamine oxidase (MAO). MAO, a flavin-containing enzyme, regulates the metabolic degradation of catecholamines and serotonin in the CNS and peripheral tissues. MAO-B is the major form in the human brain and is responsible for the regulation of the metabolic degradation of dopamine and phenylethylamine.

Rasagiline was also shown to be a potent and irreversible inhibitor of MAO-B in platelets ^{3, 4, 5}.

FIG. 1: STRUCTURE OF RASAGILINE

Riluzole is chemically 6-(trifluoromethoxy)-1,3-benzothiazol-2-amine. Its molecular formula is C₈H₅F₃N₂OS, and its molecular weight is 234.198 g/mol⁶. It is slightly soluble in water but completely soluble in methanol ^{7, 8, 9, 10, 11}. It is used in the treatment of amyotrophic lateral sclerosis (ALS) and belongs to the category of anti-convulsant.

FIG. 2: STRUCTURE OF RILUZOLE

It is found that the combination of rasagiline with riluzole is safe and increases survival by about 20% in a dose-dependent manner for the improvement of current neuroprotective treatment strategies of ALS ^{2, 12}. Validated analytical methods are required to characterize these drugs and product composition in all the phases of their formulation developments. On the literature survey, it is revealed that several methods of analyzing rasagiline ^{13, 14, 15,} and riluzole ^{16, 17} by UV-Visible spectrophotometric methods were found. But no analytical method was found for simultaneous estimation of rasagiline and riluzole. Keeping these observations in mind, we have decided to develop newer, simpler, rapid, accurate, and reproducible spectrophotometric methods for simultaneous estimation of rasagiline and riluzole in bulk and pharmaceutical dosage form. A physical mixture was prepared by blending each 50 mg of rasagiline and riluzole. Tablets were prepared from the mixture with other excipients required for tablet formulation as no combined marketed dosage forms are found.

MATERIALS AND METHODS:

Materials: All the chemicals and reagents used for the development of proposed methods to estimate rasagiline and riluzole are of spectroscopic grade. The instrument UV-Visible spectrophotometer (Shimadzu-1800) was used for the analytical method development and validation of rasagiline and riluzole. The present work was carried out at the Department of pharmaceutical chemistry, Govt. College of Pharmacy, Bengaluru. A pure sample of rasagiline and riluzole for the current study was procured from reliable sources. Rasagiline was purchased from Subtle Pharmaceuticals Pvt Ltd., and riluzole was purchased from Trichemie pharma., methanol (HPLC grade) from Merck. Double distilled Millipore water was used for the analysis, and the same was collected from Milli pore Direct Q3.

Methods: The following spectrophotometric methods were selected to analyze the combination of rasagiline and riluzole, namely,

Method A: Simultaneous equation method

Method B: Area under the curve (AUC) method

The instrumental specifications of the UV-Visible spectrophotometer used to perform the above methods are in Table 1.

TABLE 1: INSTRUMENTAL SPECIFICATION

Selection of Solvent for Analysis: The selection of solvents for analysis was carried out by the effect of different solvents on the pure drug and tablet powder. Riluzole was slightly soluble in water. But both the drugs are soluble in methanol and were stable. Hence, methanol was chosen for the preparation of solutions for analysis.Method A: Simultaneous Equation Method:

Selection of Analytical Wavelengths: Standard stock solutions having a concentration of 10 µg/mL was prepared separately, and they were scanned in the wavelength range of 200-400 nm. The maximum (λ_max) absorbance of both the drugs was found to be 265.20 nm for rasagiline and 263.40 nm for riluzole.

Preparation of Standard Stock Solutions and Calibration Curve: The stock solution was prepared by dissolving each 50 mg of accurately weighed rasagiline and riluzole in two different 50 mL standard volumetric flask and the final volume was adjusted to 50 mL with methanol to give the stock solution of 1000 µg/mL (stock A) concentration. From stock A, solution of riluzole 2.5 mL was placed in 25 mL volumetric flask, and volume was adjusted with methanol to give a solution of 100 µg/mL of riluzole (stock B). From rasagiline stock solution A 0.5-2.5 mL and 0.4-2 mL of riluzole stock B were pipetted in to 10 mL of volumetric flasks, volume was made up with methanol to get concentrations of 50-250 µg/mL of rasagiline and 4-20 µg/mL of riluzole respectively. The absorbance of the resulting solution was measured against the wavelength of 265.20 nm and 263.40 nm, respectively, against the blank methanol Fig. 3, 4.

FIG. 3: UV-VISIBLE SPECTRA OF RASAGILINE AT 50-250 µg/mL

FIG. 4: UV- VISIBLE SPECTRA OF RILUZOLE AT 4-20 µg/mL

Determination: Simultaneous equation method can be used to determine the concentration of two drugs in combination if each of the drugs absorbs at the λ_max of the other. The two wavelengths selected for the development of simultaneous equations are 265.20 nm and 263.40 nm. The formula for simultaneous estimation of drugs used is

C_{X =} A₂a_y1-A₁a_y2/ a_x2a_y1-a_x1a_y2     Or

C_X = A₁a_y2-A₂a_y1/a_x1a_y2 –a_x2a_y1

C_y=A₁a_x2-A₂a_x1/a_x2a_y1-a_x1a_y2     Or

C_y= A₂a_x1-A₁a_x2/a_y2a_x1-a_y1a_x2

Where,

C_X = concentration of rasagiline in mixture

C_Y = concentration of riluzole in the mixture

ax₁ and ax₂ are absorptivity of rasagiline at its λ_max and riluzole λ_max

ay₁ and ay₂ are absorptivity of riluzole at its λ_max and rasagiline  λ_max

The absorptivity values are determined by E^1%_1cm. The absorbance values of rasagiline at 265.20 nm and 263.40 nm, riluzole at 265.20 nm, and 263.40 nm are in Table 2 & 3, and absorbance of their mixture is in Table 4.

TABLE 2: ABSORBANCE OF RASAGILINE AT 265.20 nm AND 263.40 nm

Here, ax1 =31.026, ax2 =28.212

TABLE 3: ABSORBANCE OF RILUZOLE AT 265.20 nm AND 263.40 nm

Here a_y1=467.3   a_y2=468.582

TABLE 4: ABSORBANCE OF MIXTURE (RASAGILINE AND RILUZOLE)

Calculation: From the above Table 2 & 3, a_x1 = 31.026, a_x2 = 28.212, a_y1 = 467.3, a_y2 = 468.58 A₁=Absorbance of mixture at λ_max 265.20 nm and A₂=Absorbance of mixture at λ_max 263.40 nm

In mixture 1:

A₁= 0.1784, A₂=0.164 (from Table 4)

a_x1 = 31.026, a_x2 = 28.212, a_y1 = 467.3, a_y2 = 468.58

C_Rasagiline = (0.164 × 467.3 – 0.1784 × 468.58) /

(28.212 × 467.3-31.026 × 468.58)

= 6.9574/1354.6954

= 0.005135mg/mL or 51.357 µg/mL

Similarly, C_Riluzole = (0.1784×28.212-0.164 × 31.026) / 28.212 × 467.3-31.026 × 468.58)

= 0.0552

= 0.00047471mg/mL or 4.747µg/mL

For other mixture the calculations were done similarly as mentioned above

Method B: Area Under Curve Method (AUC):

Selection of Analytical Wavelengths: Standard stock solutions having the concentration of 10 µg/mL was prepared separately, and they were scanned in the wavelength range of 200-400 nm and the maximum (λ_max) absorbance of both the drugs were found to be 265.20 nm for rasagiline and 263.40 nm for riluzole.

Preparation of Standard Stock Solution: Stock solutions of rasagiline and riluzole were prepared as discussed in method A. From rasagiline stock solution  0.5-2.5 mL and 0.4-2 mL of riluzole stock B were pipetted into 10 mL of volumetric flasks, volume was made up with methanol to get concentrations of 50-250 µg/mL of rasagiline and 4-20 µg/mL of riluzole respectively. The absorbance of the resulting solution was measured against the wavelength of 265.20 nm and 263.40 nm, respectively, against the blank methanol. The area under the curve of rasagiline and riluzole are recorded in Fig. 5 & 6.

FIG. 5: AREA UNDER CURVE FOR RASAGILINE            

FIG. 6: AREA UNDER CURVE FOR RILUZOLE

Determination: Area under the curve method is mainly used for the quantification of the drugs in both single and multi-component formulation. It involves calculations of integrated values of absorbance with respect to wavelength in the indicated range.

The formula used to determine the concentration by area under the curve method is

C_{X =} A₂a_y1-A₁a_y2/ a_x2a_y1-a_x1a_y2      or

C_X = A₁a_y2-A₂a_y1/a_x1a_y2 –a_x2a_y1

C_y=A₁a_x2-A₂a_x1/a_x2a_y1-a_x1a_y2      or

C_y= A₂a_x1-A₁a_x2/a_y2a_x1-a_y1a_x2

Where,

C_X= concentration of rasagiline in mixture

C_Y= concentration of riluzole in mixture

A₁ and A₂ are the areas under the curve of the sample mixture at 260.20 nm to 270.20 nm and 258.40 nm to 268.40 nm, respectively.

ax₁ and ax₂ are absorptivity of rasagiline and riluzole at 260.20 nm to 270.20 nm.

ay₁ and ay₂ are absorptivity of  rasagiline and riluzole at 258.40 nm to 268.40 nm

The absorptivity values are determined by E^1%_1cm.

Absorptivity (E^1%_1cm) = (Area under curve /concentration in µg/ml) × 10000

The calibration data and calibration graph of rasagiline at 260.20 nm to 270.20 nm and 258.40 to 268.40 nm are in Tables 5 and 6, Fig. 7. Absorbance of the mixture (rasagiline and riluzole) is in Table 7.

TABLE 5: AREA UNDER CURVE OF RASAGILINE AT 260.20 -270.20 nm AND 258.40-268.40 nm

Here ax₁=30.652, ax₂ =42.466

FIG. 7: CALIBRATION GRAPH OF RILUZOLE

TABLE 7: AREA UNDER CURVE DATA OF RASAGILINE AND RILUZOLE

Calculation:

From the above Table 5 & 6, ax₁ = 30.652, ax2 = 42.466, ay₁ = 254.5, ay2 = 252.25,

A₁ = AUC of mixture at 260.20-270.20 nm, A₂ = AUC of mix at 258.40-268.40 nm

In mixture 1:  A₁= 0.26, A₂ =0.32 (from Table 7)

C_Rasagiline = (0.32 × 254.5 - 0.26 × 252.25) / (42.466 × 254.5 - 30.652 × 252.25)

= 15.855/3075.63

= 0.005155 mg/mL or 51.55 µg/mL

Similarly, C _Riluzole = (0.26 × 42.466 - 0.32 × 30.652) / (42.466 × 254.5 - 30.652 × 252.25)

= 1.24116/3075.63

= 0.0004035mg/mL or 4.035µg/mL

For other mixtures, the calculations were done similarly as mentioned above.

Method validation: The developed method were validated according to their analytical procedures as per ICH guidelines for validation of analytical procedures in order to determine linearity, precision, LOD, LOQ, and accuracy for the analyte.

Linearity: The linearity of an analytical procedure is its ability (within a given range) to obtain test results that are directly proportional to the concentration of analyte in the sample solution.

Preparation of Working Standards of Rasagiline and Riluzole: Working standard stock solutions of rasagiline and riluzole were prepared as discussed in method A. From rasagiline stock solution 0.5-3.5 mL and 0.4-2 mL of riluzole  stock B were pipetted into 10 mL of volumetric flasks, volume was made up with methanol to get concentrations of 50-350 µg/mL of rasagiline and 4-20 µg/mL of riluzole respectively.

The absorbance of the resulting solution was measured against the wavelength of 265.20 nm and 263.40 nm respectively against the blank methanol.

The linearity data and absorbance of rasgiline are in Table 8 and Fig. 8. Similarly, for riluzole in Table 9 and Fig. 9.

TABLE 8: LINEARITY DATA OF RASAGILINE IN METHANOL

FIG. 8: ABSORBANCE LINEARITY OF RASAGILINE

TABLE 9: LINEARITY DATA OF RILUZOLE IN METHANOL

FIG. 9: ABSORBANCE LINEARITY OF RILUZOLE

Precision: Precision studies are carried out to ascertain the reproducibility of the proposed methods. Repeatability was determined by preparing six replicates of the same concentration of the sample, and the absorbance was measured.

A stock solution was prepared by dissolving 50 mg of accurately weighed rasagiline and riluzole into 50 mL volumetric flask, and the final volume was adjusted to 50 mL with methanol to get the stock solution 1000 µg/mL (stock A) concentration. From the resulting solution 2.5 mL of riluzole was placed in 25 mL volumetric flask and volume adjusted with methanol to give a solution of 100 µg/mL of riluzole solution (stock B). From stock solution, A 3.0 mL of rasagiline and 1.2 mL of riluzole of stock B were pipetted into 10 mL volumetric flask, and the volume was made up with methanol to get the concentration of 300 µg/mL of rasagiline and 12 µg/mL of riluzole. The intraday precision study was carried out by preparing drug solutions of the same concentration and analyzing it at three different times in a day Table 10 and 11.

TABLE 10: INTRADAY PRECISION DATA FOR RASAGILINE

 TABLE 11: INTRADAY PRECISION DATA FOR RILUZOLE

To determine interday precision the same procedure was followed for three different days by following the same procedure Table 12 and 13. The results were reported as % RSD.

TABLE 12: INTERDAY PRECISION DATA FOR RASAGILINE

TABLE 13: INTERDAY PRECISION DATA FOR RILUZOLE

Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a true conventional value or as an accepted reference value found. The recovery studies were carried out by adding different amounts (50%, 100%, and 150%) of the pure drug to the pre-analyzed formulation Table 14 and 15.

TABLE 14: ACCURACY DATA FOR RASAGILINE

TABLE 15: ACCURACY DATA FOR RILUZOLE

Ruggedness: Ruggedness expresses the variation within laboratories in analyzing the drugs in different days, by the different analyst, using different equipments, etc. The intermediate precision was performed for rasagiline and riluzole by a different analyst on a different day.

Preparation of Standard Solutions of Mixtures: The working standard solutions of rasagiline and riluzole were prepared as per the procedure mentioned in the precision method.

Determination: The resulting mixtures of samples are subjected to UV analysis by different analysts, and the obtained absorbance was recorded, and the % RSD of replicates was calculated. The results obtained were presented in Tables 16 and 17.

TABLE 16: RUGGEDNESS DATA FOR RASAGILINE AND RILUZOLE BY ANALYST 1

TABLE 17: RUGGEDNESS DATA FOR RASAGILINE AND RILUZOLE BY ANALYST 2

Limit of Detection and Limit of Quantification: Limit of detection (LOD) and limit of quantification (LOQ) were determined based on the  standard deviation of response and the slope and were calculated by using the equation

LOD = 3 × s/S and LOQ = 10 × s/S

Where s is the standard deviation of intercept and S is the slope of the line

RESULTS: Rasagiline and Riluzole were individually analyzed by UV-Visible spectrophoto-metric method using the solvent methanol.  Optical characteristics such as λ_max, E^1%_1cm, slope intercept, correlation coefficient, linearity and range, LOD and LOQ were observed as in Table 18 and 19.

TABLE 18: RASAGILINE CALIBRATION DATA

The mixture of rasagiline and riluzole was analyzed by UV-Visible spectroscopic method using simultaneous equation method and area under the curve method (AUC). These methods were validated according to the ICH guidelines. The observed results of the developed and validated methods in the present study suggest that these methods can be adopted for routine analysis of drugs simultaneously.

TABLE 19: RILUZOLE CALIBRATION DATA

CONCLUSION: A physical mixture was prepared in the laboratories of Govt. College of Pharmacy as no combined dosage forms of rasagiline and riluzole are available in the market. As spectroscopic methods are highly powerful and convenient methods of analysis, in the present study, two different such methods were developed and validated for routine analysis of rasagiline and riluzole in their bulk and physical mixtures. In the simultaneous equation method, different wavelengths are selected to calculate their concentrations in both bulk and in combination.

The simultaneous equation method is extended to calculate the concentration as the area under the curve method at a wavelength of ±5 nm λ_max of rasagiline and riluzole. These developed methods are economical, accurate, and precise. From the observations of both methods developed and validated, they may be used for routine analysis of rasagiline and riluzole simultaneously at the industrial level in their dosage forms.

ACKNOWLEDGEMENT: Authors are thankful to the Principal, Govt. College of Pharmacy Bangalore for his constant support and encouragement in our research work and for providing laboratory facilities to complete the present research work. The authors are also thankful to Rajiv Gandhi University of Health Sciences, Karnataka, for their financial support.

CONFLICTS OF INTEREST: Authors have no conflicts of interest to declare.

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    How to cite this article:

    Dhanwad R, Deepali, Harini L and Chaluvaraju KC: Development and validation of spectroscopic methods for the simultaneous estimation of rasagiline and riluzole. Int J Pharm Sci & Res 2021; 12(11): 6028-36. doi: 10.13040/IJPSR.0975-8232.12(11).6028-36.

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