INTRODUCTION:

Parkinson’s disease is a neurodegenerative disorder. The dopamine generating cells die in the part of mid brain called substantia nigra which leads to Parkinson’s disease. Rasagiline (Figure 1), [(R) - N- (prop-2-ynyl)-2,3-dihydro-1H-inden-1-amine] is a monoamine oxidase. Earlier it was used as a monotherapy agent in Parkinson disease. Chemical Formula is C₁₃H₁₇NO₃S and molecular weight is 267.34g/mol^1,2. Rasagiline is quickly absorbed by the GIT and it readily crosses the BBB. The drug Rasagiline is not official in any of the pharmacopoeias. The literature surveys revealed various analytical methods like UV and Visible spectrophotometry^3-6, HPLC^7-13, HPTLC¹⁴ and LC-MS/MS^15,16methods available for the estimation of the drug in formulations and in human plasma.

Received on 13.07.2019 Modified on 10.09.2019

Research J. Pharm. and Tech 2020; 13(2):843-849.

DOI: 10.5958/0974-360X.2020.00159.6

Image result for rasagiline

Figure 1 Chemical strucutre of Rasagiline

The suggested technique is based totally on the direct evaluation of Rasagiline with a great degree of accuracy and precision. This technique is simple, with less cost and it can be applied to bulk drug and its formulations. Multivariate standardization method shows the shift of common single species analysis from one dependant variable to 'm' dependant variables. e.g. wavelengths or sensors, which can be concurrently comprised within the calibration model^17-20.

Under the optimized circumstances, the applied analytical technique gives great resolving power, rapidity, sensitivity and low price for quantitative and regular analysis of an investing admixture.

If the absorbance of an analyte (X) Rasagiline in this case, is scanned at 5 wavelengths set (λ = 211, 213, 215, 217 and 219 nm), the subsequent equation can be written for any selected wavelength.

A_λ211 = a X C_x + k₁ …………………..……………… (1)

A_λ213 = b X C_x + k₂…………………..……………… (2)

A_λ215 = c X C_x + k₃ …………………..……………… (3)

A_λ217 = d X C_x + k₄ …………………..………………(4)

A_λ219= e X C_x + k₅ …………………..……………… (5)

Where, A_λrepresents the absorbance of the analyte; a, b, c, d, e are the slopes of the linear regression functions of the analyte; k_1,k_2,k_3,k_4,k₅ are the intercepts of the linear regression functions at the five selected wavelengths and C_xrepresents the concentration of the analyte. The above five equation systems (1-5) can be summarized as

A_T = a x C_x+ b x C_x+ c x C_x+ d x C_x+ e x C_x+ K_T…...(6)

The above equation can be further simplified to

A_T = C_x (a+b+c+d+e) + K_T………………………….. (7)

Where A_T and K_T represents the sum of the absorbance obtained and the sum of intercepts of regression equations at five wavelength set respectively. The concentration of the analyte X in a solution can be computed by using the formula.

A_T- K_T

C_ᵡ= ––––––––––––

(a+b+c+d+e)

MATERIALS AND METHODS:

Chemicals and solvents:

· Distilled H₂O

· Analytical grade rasagiline was obtained as a gift sample from Ideal analytical and Research Institute production, Pondicherry. The marketed tablet formulation used was Azilect, Teva Pharmaceuticals, India, (Label claim - 10milligram rasagiline), procured from the local market.

Instrumentation:

· Perkin-Elmer UV-Visible double beam spectrophotometer Lambda 25

· Ultra Sonicator

· Analytical balance

Method development:

Solvent selection:

Rasagiline was freely soluble in distilled H₂O. Hence it was used as a solvent to dissolve the standard drug and sample.

Preparation of standard stock solution:

The standard stock solution of Rasagiline was prepared by dissolving 100 mg of the reference drug in 50mL of distilled water and then finally made upto the mark in a 100 mL standard flask with the same solvent. Different concentrations (8-12µg/mL) of solution were prepared from this standard stock solution.

Determination of λ_max:

About 1mL of the stock solution was pipetted into a 100 mL standard flask and diluted to the mark with distilled H₂O to obtain a concentration of 10µg/mL. This solution was measured in the UV region from 200 - 400nm. The λmax was obtained as 215nm (Figure 2). The linear curve was obtained when the absorbance was plotted against the concentration (Table 1). The solutions were scanned across the range surrounding 215 nm i.e., 211, 213, 215, 217, 219 nm to enhance the correlation and to reduce the instrumental fluctuations.

Preparation of sample solution:

Accurately weigh and powder 20 tablets of Rasagiline and weight equivalent to 10mg was measured and dissolved in 10mL of distilled water to obtain 1mg/mL solution. This solution was filtered and used for further analysis.

Method Validation²¹:

According to ICH Q2B guidelines the technique was validated for sensitivity, accuracy, precision and linearity.

Linearity:

The different concentrations over the range of 8-12 µg/mL were prepared from the standard stock solution of Rasagiline. In order to minimize the instrumental fluctuations and to improve the correlation these solution were scanned over a range of wavelength surrounding its absorbance maxima. i.e. 211, 213, 215, 217, 219nm. The absorbances were recorded and the standardization graphs were obtained by plotting the concentration vs absorbance. (Figure 3, Table 1).

Table 1: Multivariate UV calibration obtained at five wavelengths

_{Conc (µg/ml)}	_Absorbance
_{Conc (µg/ml)}	_{211 nm}	_{213 nm}	_{215 nm}	_{217 nm}	_{219 nm}
₈	_0.2549	_0.2598	_0.2648	_0.2655	_0.2348
₉	_0.2868	_0.2942	_0.2956	_0.2969	_0.2648
₁₀	_0.3188	_0.3256	_0.3285	_0.3288	_0.2898
₁₁	_0.3478	_0.3589	_0.3613	_0.3596	_0.3187
₁₂	_0.3818	_0.3893	_0.3942	_0.3875	_0.3477

By calculating the detection limit and quantification limit using the below formulae, the sensitivity of the method was determined.

LOD = 3.3 σ/S ……………………………………… (8)

LOQ = 10 σ /S............................................................. (9)

Where, σ is the standard deviation (SD) of the lowest concentration and

S is the slope of the standard curve.

Precision:

To assess the intraday and interday precision, 10µg/mL solution was scanned six times in a short interval of time in one day for intraday precision and on six different days for interday precision (Table 3 and 4).

Accuracy:

The recovery study of the suggested technique was resolved by standard addition method at 80%, 100% and 120%. The stock solution of standard and the sample was prepared. Pipette out 0.5mL of standard into a three standard flask and add the sample 0.3, 0.5, 0.7mL into the above volumetric flasks and make up to the volume of 10mL with distilled water. The solutions were measured under UV spectrometer and the percentage recovery was calculated.

Assay:

The absorbance of extracted tablet solution was recorded at 215nm and the amount present in the formulation was calculated as in Table.

RESULTS AND DISCUSSION:

The λmax of Rasagiline was obtained at 215 nm with distilled H₂O as the solvent.

The technique was linear in the selected concentration range over 8-12µg/mL. The data from the linear regression analysis shows good linear relationship with R²=0.9999 for all the calibration plots. The equation for the linear regression is Y=0.0325x+0.0044. For precision, the % relative standard deviation was found to be 0.0409 and 0.0228. The LOD and LOQ obtained is 0.0760µg/mL and 0.2304µg/mL respectively. Therefore according to ICH guidelines each of the validation parameters were obtained to be within the limits.

Linearity:

The linearity was recorded at 211, 213, 215, 217 and 219nm in the concentration range 8-12µg/mL and it was shown in the Figure 3 and their calibration curves and residual plots were shown in Figures 4 to 8 & 9-13 respectively. At each of the wavelength the fewer values of standard deviation shows the technique was precise and the LOD and LOQ were calculated and reported in Table 2.

Table 2: Linearity data showing LOD and LOQ at all five wavelengths

Wavelength (nm)	Regression equation	R²	SD	LOD (_µg/mL)	LOQ (_µg/mL)	% RSD
211	Y=0.0315x+0.0032	0.9996	0.0011	0.1248	0.3784	0.3748
213	Y=0.0324x+0.0019	0.9996	0.0011	0.1176	0.3565	0.3548
215	Y=0.0325x+0.0044	0.9998	0.0007	0.0760	0.2304	0.2277
217	Y=0.0307x+0.0210	0.9994	0.0013	0.1500	0.4545	0.4259
219	Y=0.028x+0.01150	0.9993	0.0013	0.1546	0.4685	0.4505

$Description: C:\Users\SATYAM\Desktop\Thesis Book\New folder (2)\PROJECT\GOVIND\RASAGILINE\PRECISION 1.PNG$

Figure 2 UV spectrum of Rasagiline

Figure 3 UV spectrum showing linearity of Rasagiline at 215 nm

Figure 4 calibration curve at 211 nm Figure 9 Residual plot at 211 nm

Figure 5 Calibration curve at 213 nm Figure 10 Residual plot at 213 nm

Figure 6 Calibration curve at 215nm Figure 11 Residual plot at 215 nm

Figure 7 calibration curve at 217 nm Figure 12 Residual plot at 217 nm

Figure 8 Calibration curve at 219 nm Figure 13 Residual plot at 219 nm

(a) (b)

Figure 14 UV spectra showing intraday precision (a) Morning (b) Evening

(a) (b)

Figure 15 UV spectra showing interday precision (a) Day 1 (b) Day 6

Table 5: Recovery Studies

Wavelength (nm)	Amount present (_µg/mL)	Amount added (_µg/mL)	Absorbance	Amount recovered (_µg/mL)	%Recovery
211	5	3	0.2910	7.9825	99.78
		5	0.3069	9.9839	99.83
		7	0.3780	11.8756	98.96
213	5	3	0.3010	7.8979	98.72
		5	0.3260	10.1258	101.25
		7	0.3804	11.9895	99.91
215	5	3	0.3040	7.9892	99.86
		5	0.3370	10.2310	102.31
		7	0.3999	12.0571	100.47
217	5	3	0.2210	7.9836	99.79
		5	0.3050	9.9597	99.59
		7	0.3760	11.9896	99.91
219	5	3	0.1463	7.9546	99.43
		5	0.3020	9.9951	99.95
		7	0.3580	12.1502	101.25

$Description: C:\Users\seetharaman\Desktop\peoject\GOVIND\RASAGILINE\ACCURACY 2.PNG$

Figure 16 UV Spectrum showing accuracy of Rasagiline

Table 6: Assay of Rasagiline

Label claim (mg)	Amount estimated (mg)	% Assay
10	9.99	99.9
10	9.98	99.8
10	10.01	100.1
Average		99.93
SD		0.1528
% RSD		0.1529

Assay:

The absorbance of the tablet formulation was documented at 215nm. The amount and assay percentage was found to be 9.99 mg and 99.93 % w/w with a % RSD value of as in Table 6.

CONCLUSION:

The newly developed method was evidenced to be accurate, specific, sensitive and reproducible for the evaluation of Rasagiline in its pharmaceutical formulation by multilinear regression technique. The method was validated using various validation parameters and are obtained to be within the limits according to ICH guidelines. This method is a simple working procedure as comparable to the expensive techniques like HPLC, and therefore, it can be employed for regular analysis of Rasagiline in bulk drug and pharmaceutical formulations.

LIST OF SYMBOLS/ABBREVIATIONS:

nm = Nanometer

μg/mL = Microgram per millilitre

ICH = International Conference on Harmonization

UV = Ultraviolet

g/mol = Gram per Mole

BBB = Blood Brain Barrier

GIT = Gastro Intestinal Tract

HPLC = High Performance Liquid Chromatography

HPTLC=High Performance Thin Layer Chromatography

LC/MS = Liquid chromatography-Mass spectrometry

M = Molar

CONFLICTS OF INTEREST:

The authors report NO conflict of interest on the study.

ACKNOWLEDGEMENT:

The authors are thankful to the Chancellor, SRM Institute of Science and Technology, and the management of SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur for carrying out the this research work.

REFERENCES:

1. Barnett-Cowan M, Dyde RT, Foxe SH, Moro E, Hutchison WD, Harris LR. Multisensory determinants of orientation perception in Parkinson's disease. Neuroscience. 167; 2010: 1138–50.

2. Sean C Sweetman, Martindale, The complete Drug Reference, 36^th ed, Pharmaceutical Press, 2009: 815.

3. Patel Pinal R Mulgund Sugandha V. Development and Validation of a Spectrophotometric method for estimation of Rasagiline Mesylate in bulk and tablet dosage Form. International Journal of Drug Development and Research. 5(3); 2013: 174-177.

4. Rama R, Preeti K. UV spectrophotometric method for the determination of Rasagiline mesylate in bulk and pharmaceutical formulations. International Journal of Pharmaceutical Sciences Review and Research. 5(1); 2010: 5-7.

5. Devala Rao G, Kathirvel S, Satyanarayana SV. Simple spectrophotometric methods for the determination of Rasagiline mesylate in pharmaceutical dosage form. Journal of Pharmacy Research. 4(1); 2011: 61-62.

6. Chennaiah M, Veeraiah T, Charan Singh T, Venkateshwarlu G. Extractive spectrophotometry methods for determination of Rasagiline mesylate in pharmaceutical formulations using acidic triphenyl methane dyes. Journal of the Chilean Chemical Society. 56(4); 2011: 926-929.

7. Ravi PR, Aditya N, Cherian L, Patil S. LC Method for Determination of Rasagiline Mesylate in Different Plasma Matrices and its Application to Oral Pharmacokinetic Study in Rabbits. Journal of Chromatographic Science, 51(1); 2013: 1–7

8. Fernandez M, Barcia E, Negro S. Development and validation of a reverse phase liquid chromatography method for the quantification of Rasagiline Mesylate in biodegradable PLGA microspheres. Journal of Pharmaceutical and Biomedical Analysis. 49(5); 2009: 1185-1191.

9. Sundara Murthy P, Balaji V, Rajasekhara R, Raju I. Validated and stability indicating dissolution test with RP-HPLC analysis for Rasagiline Mesylate in tablet dosage form. International Journal of Pharmaceutical Sciences Review and Research. 10(1); 2011: 18-23.

10. Kumar RJ, Jeyabaskaran M, Kumari PM. Validated RP –HPLC method for the estimation of Rasagiline in pure and tablet dosage form. Journal of Pharmacy Research. 5; 2011: 1376-1377.

11. Lakshmi MV, Rao JVLNS, Rao AL. Development and validation of RP-HPLC method for the estimation of Rasagiline tablet dosage forms. Rasayan Journal of Chemistry. 3; 2010: 621-624.

12. Reddy KU, Sriramulu J, Reddy UM, Reddy YP. Stability indicating RP-HPLC method for the determination of Rasagiline in pharmaceutical products. Int J Sci Inn Disc. 1; 2011: 19-32.

13. Kumar RN, Rao GN, Naidu PY. Stability indicating RP-LC method for determination of Rasagiline mesylate in bulk and pharmaceutical dosage forms. International Journal of Applied Biology and Pharmaceutical Technology. 1; 2010: 247-59.

14. Singaram Kathirvel, Suggala Venkata Satyanarayana, and Garikapati Devalarao. Development and Validation of a Stability indicating HPTLC Method for Analysis of Rasagiline Mesylate in the Bulk Drug and Tablet Dosage Form. Chromatography Research International. Volume 2012, Article ID 273604, http://dx.doi.org/10.1155/2012/2736042012.

15. Ma J, Chen X, Duan X, Deng P, Wang H, Zhong D. Validated LC-MS/MS method for quantitative determination of Rasagiline in human plasma and its application to a pharmacokinetic study. Journal of Chromatogr B. 873; 2008: 203-208.

16. Song M, Wang L, Zhao H, Hang T, Wen A, Yang L et al. Rapid and sensitive liquid chromatography-tandem mass spectrometry: assay development, validation and application to a human pharmacokinetic study. Journal of Chromatogr B. 875; 2008: 515-21.

17. Arayne MS, Najma Sultana, Zuberi MH, Siddiqui FA. Spectrophotometric quantification of metformin in bulk drug and pharmaceutical formulations using Multivariate technique. Indian Journal of Pharmaceutical Sciences.71; 2009: 331-5.

18. Kokilambigai KS, Seetharaman R, Kavitha J, Sowndaravel P, Lakshmi KS. Multivariate Calibration technique for the spectrophotometric quantification of Zaleplon in bulk and pharmaceutical formulations. Journal of Pharmaceutical Sciences and Research. 9(6); 2017: 824 - 829.

19. Sai Susmitha A, Kokilambigai KS, Lakshmi KS. Spectrophotometric quantification of Telmisartan employing multivariate calibration technique in bulk and pharmaceutical formulations. Research Journal of Pharmacy and Technology. 12(4); 2019: 1799-1805.

20. Madhan S, Kavitha J, Lakshmi KS. Multivariate calibration technique for the spectrophotometric quantification of Ivermectin in Pharmaceutical Formulation. Asian Journal of Pharmaceutical and Clinical Research. 12(2); 2016: 444-451.

21. Q2R1 ICH guidelines for analytical method development. Available at: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2 _R1__Guideline.pdf.