INTRODUCTION:

Parkinson’s disease is common neurodegenerative disorder typified by loss of dopaminergic neurons from the basal ganglia, and by a characteristic clinical syndrome with cardinal physical signs of resting tremor, bradykinesia and rigidity(1). Globally, it is estimated 6.3 million people have the disease. Many anti-parkinsonism drugs are available in market as a monotherapy or as an adjunct therapy (2).

Rasagiline is an effective drug in market used for treatment of Parkinsonism. It is a highly potent, selective and irreversible second generation mono amino oxidase inhibitor (3). It is selective for MAO type B over type A by a factor of fourteen which are being developed for treatment of parkinson’s disease. MAO-B inhibitors cause an increase in extracellular levels of dopamine in the striatum. Rasagiline also possesses neuroprotective properties those are independent of its MAO inhibitory activity (4, 5). Rasagiline, an MAO-B inhibitor similar to selegiline but without amphetamine metabolites, has similar effects as selegiline on enhancing L-dopa effects, as well as modest beneficial effects in monotherapy (6).

Chemically Rasagiline is (R)-N-2 Propynyl-1-indanamine (7) (Fig 1). Its molecular weight is 171.24gm/mol (CAS No. : 136236-51-6) and it is freely soluble in water, ethanol and sparingly soluble in isopropanol (8). The dissociation constant of Rasagiline base is 7.12 at 25°C (9). It is a chiral compound with one asymmetric carbon atom in a five member ring with an absolute R-configuration which is produced as single enantiomer (10)

Fig. 1: Chemical Structure of Rasagiline

In the literature review few analytical methods for the determination of Rasagiline in biological ﬂuids were found. Other method used was a gas chromatography–mass spectrometry procedure in electron impact mode (11). Analytical method for the quantification of Rasagiline mesylate by a reverse phase liquid chromatography method in biodegradable PLGA microspheres (12), in tablet dosage forms(13) and stability indicating method for determination of Rasagiline mesylate(14) were also reported. It was revealed that none of the RP-HPLC method is reported for the determination of Rasagiline using an internal standard in tablet dosage form.

Hence in present work attempt has been made for the development and validation of simple, rapid, sensitive, and precise HPLC method, using an internal standard. UV detection was performed at 210 nm.

EXPERIMENTAL:

Reagents and Chemicals

Rasagiline mesylate was kindly provided by APOTEX Research Private Limited (India). Caffeine (used as an internal standard) was provided by Juggat Pharma (India). Acetonitrile, Isopropyl alcohol, o-phosphoric acid and Water used were of HPLC grade. Tablets of 1, 0.5 mg Rasagiline (Rasalect 1, 0.5 Sun Pharmaceutical Ind. Ltd. and Relgin 1 Intas Pharmaceuticals Ltd.) were procured from local pharmacy.

Instrumentation

The HPLC system used was Shimadzu LC-20AT pump, Rheodyne injector (20μl), SPD-20A UV detection and the system was controlled through Spinchrome software. Analytical column used for this method was Gracesmart RP18 (250X4.6mm, 5μm). Sartorius digital Balance, Digisun 7007 pH meter, RC Systems sonicator and vacuum pump were used in the experiment.

Chromatographic Conditions

The composition of the mobile phase used was isopropyl alcohol: acetonitrile: water (20:10:70, v/v) (adjusted to pH 2.5 with o-phosphoric acid). The mobile phase was vacuum-filtered through 0.45m nylon Millipore membranes (Millipore, USA), and degassed by ultrasonication for 10min before use. The mobile phase flow rate was set at 0.8 ml min−1. After equilibration with the solvent to obtain a stable baseline, aliquots of samples (20µl) were injected through Rheodyne injector in the column. The total run time was 10 min. The absorbance of the eluent was monitored at 210 nm with a detection sensitivity of 0.100 aufs. Caffeine (10µgml−1) was used as an internal standard.

Preparation of Standards and Sample Solutions

Standard stock solutions of Rasagiline (1000µgml−1) and Caffeine (IS) (100µgml−1) were prepared in HPLC grade water. These solutions were kept and stored under refrigeration (4.0±0.5 ◦C). Working standard solutions were freshly prepared daily by appropriate dilution of the stock solutions with mobile phase. Sample solutions were prepared by weighing 30 tablets accurately and finely powdered. The powder equivalent to 10 mg of Rasagiline was taken in a 50 ml volumetric flask, about 40 ml of HPLC grade water was added and kept in Ultrasonic bath for 10 minutes then made up to volume 50 ml. The resulting solution was vacuum-filtered through 0.45m nylon Millipore membranes (Millipore, USA). From the above solution 2 ml was transferred in to 10ml volumetric flask along with 1ml of Caffeine solution (100 μgml−1) made up to the volume with mobile phase (40μgml−1 Rasagiline and 10μgml−1 Caffeine). Results of assay of marketed formulations are shown in Table 1.

METHOD VALIDATION

Method validation was carried out under the guidelines of International Conference on Harmonization (ICH) (15, 16). The assay was validated with respect to linearity, precision, accuracy, sensitivity and robustness.

Linearity

Calibration curves were obtained from injecting the six sets of eight serial different drug concentrations (1, 5, 10, 20, 40, 80, 100 and 200 μgml−1 of Rasagiline). The curves were generated by plotting the peak area ratios between Rasagiline and Caffeine against Rasagiline concentration. Linearity was evaluated by linear regression using ANOVA.

Precision

The precision of the method was determined by repeatability (intra-day) and intermediate precision (inter-day) and was expressed as relative standard deviation (R.S.D.). Repeatability was determined by performing nine determinations from triplicate injections of three different concentrations of Rasagiline (10, 40 and 100μgml−1) on the same day at different time intervals and on three different days for inter-day precision.

Accuracy/Recovery

In this study, accuracy was determined based on the recovery (percentage) of known amounts of standard Rasagiline added in the assay samples. This was performed by analyzing Rasagiline at three different concentration levels 50%, 100% and 150% to the assay sample, with a constant concentration of 10μgml−1 of internal standard. Samples were prepared in triplicate. The accuracy of the assay was determined by comparing the found concentration with the added concentration.

Sensitivity

Sensitivity of the method was determined by means of the detection limit (LOD) and quantification limit (LOQ). The LOD and LOQ were measured based on the method described by the International Conference on Harmonization. Calculations for LOD and LOQ were based on the standard deviation of the calibration curve (σ) and the slope of curve (S), using the equation LOD= 3.3×σ/S and the equation LOQ= 10×σ/S.

Robustness

Robustness of the method was evaluated by the analysis of Rasagiline solution under different experimental conditions such as pH of the mobile phase and flow rate. The flow rate and pH were varied by 3% and their effects on the retention time (tR), tailing factor (T), resolution of the peaks (R) and repeatability were studied.

RESULTS AND DISCUSSION:

Optimization of the chromatographic method

The chromatographic conditions were adjusted to provide the best performance of the assay. For system optimization the important parameters such as type and concentration of organic solvents, pH and mobile phase flow rate were investigated.

Effect of pH

Different pH values of the mobile phase were checked to establish the optimum separation and highest analytical sensitivity for Rasagiline and Caffeine. The pH values tested were from 2.5 to 5.0. Finally, the best results were obtained at pH 2.5±0.2 by using 1% o-phosphoric acid. The choice of this pH for the mobile phase is justified by the excellent symmetry of the peaks and the adequate retention times of Rasagiline and Caffeine.

Effect of mobile phase composition

Different mobile phase composition like acetonitrile: methanol: water, acetonitrile : isopropyl alcohol : water, acetonitrile : water were tried first on RPTLC plates for separation of Rasagiline and Caffeine. Acetonitrile: isopropyl alcohol: water and acetonitrile : water were further tried on HPLC system. It was observed that the acetonitrile : isopropyl alcohol : water system gave a better resolution and peak symmetry than the water: acetonitrile system.

Different proportions of acetonitrile : water (10 : 90, 15 : 85, 20 : 80, 30 : 70 v/v) and acetonitrile : isopropyl alcohol : water (10:10:80, 10:20:70 and 20:10:70 v/v/v) were tried before the final chromatographic conditions were selected. As the percentage of acetonitrile and isopropyl alcohol changed, Rasagiline retention time was varied in the range of 2 min to 5.5min. Finally acetonitrile: isopropyl alcohol: water (10:20:70 v/v/v) (adjusted to pH 2.5±0.2 with 1% o-phosphoric acid) was chosen as a mobile phase. As a result, the standard solutions of Rasagiline and Caffeine showed symmetric and well-defined peaks, with an average retention time for Rasagiline at 3.2 min and 4.23 min for the Caffeine. Resolution between peaks was 4.60. Tailing factor was 1.83 for Rasagiline and 1.62 for Caffeine.

Effect of Flow Rate

Different mobile phase flow rates (0.8, 1.0 and 1.2ml min−1) were investigated. The optimum flow rate for which the column plate number (N) was maximum, with the best resolution between all components and with a short run time (<15min) was found to be 0.8 ml min−1.

Table 2: Summary of precision determined during method validation

Concentration (μgml−1)	intra-day (n^a = 3) R.S.D.^b (%)	inter-day (n^a = 3) R.S.D.^b (%)
10	2.69	2.66
40	1.85	1.41
100	2.70	2.79

a : No. of analyzed sample, b : Relative standard deviation

Internal standard

Different compounds were tested as an IS for the chromatographic procedure. Among them, Caffeine eluted before 10 min of the analysis and has a better symmetry and resolution with respect to Rasagiline. Therefore, Caffeine has been chosen as an IS.

The overlay chromatogram of blank and Rasagiline with Caffeine with finalized chromatographic conditions is shown in the Fig 2.

METHOD VALIDATION

Precision

The R.S.D. of repeatability (intra-day) and intermediate precision (inter-day) ranged between 1.41% and 2.79%. These values show a low variability between the values obtained for each concentration. These values are shown in Table 2.

Accuracy

The results of the accuracy studies are shown in Table 3. Recovery ranged is between 95.16% and 102.24 % with R.S.D. less than 3%. The values obtained show a suitable accuracy for the analytical method.

Linearity

The standard calibration curve was linear over the concentration range 1–200μgml−1(Fig 3). The correlation coefficient obtained after linear regression analysis was 0.99935. The equation of the calibration curve based on the peak ratio of Rasagiline and IS with respect to Rasagiline concentration was y= 0.04242x + 0.02846 (Fig. 3). The standard error was 0.07982. According to statistical analysis by ANOVA, the curve was linear with p<0.005(Table 4).

Fig. 2: Overlay chromatogram of blank and Rasagiline (tR 3.20 min.) with Caffeine (tR 4.23 min)

Table 3: Accuracy of the method determined according to ICH Q2 guidelines.

Brand Name	Concentration (μgml−1)		Recovery (%)^a (Mean ± SD^b)	R.S.D.^c (%) (n^d=3)
Brand Name	Amount added	Amount found (Mean ± SD^b)	Recovery (%)^a (Mean ± SD^b)	R.S.D.^c (%) (n^d=3)
Rasalect 1 (rasagiline 1mg)	20	19.61 ± 0.38	101.36 ± 0.47	0.46
	40	40.27 ± 0.70	99.52 ± 0.17	0.17
	60	61.08 ± 1.05	99.89 ± 0.84	0.84
Rasalect 0.5 (rasagiline 0.5mg)	20	19.44 ± 0.03	97.24 ± 0.14	0.14
	40	39.32 ± 1.04	98.32 ± 2.62	2.67
	60	57.75 ± 0.69	96.26 ± 1.14	1.19
Relgin 1 (rasagiline 1mg)	20	20.01 ± 0.38	100.07 ± 1.96	1.96
	40	39.23 ± 0.82	98.08 ± 2.04	2.08
	60	60.04 ± 0.77	100.08 ± 1.29	1.29

a : (Found concentration/added concentration) ×100, b : Standard deviation, c : Relative standard deviation, d : No. of analyzed sample

Table: 4: Statistical analysis of linearity.

Multiple regression analysis
Parameter	Coefficient	Standard Error	t-statistic	p-value	Lower 95%	Upper 95%
Intercept	0.02845	0.03785	0.75174	0.48064	-0.06417	0.12107
Slope	0.04242	0.00044	95.8609	0.00000	0.04134	0.04350
ANOVA
Parameter	Sum of square	Degree of freedom	Mean of square	F	p-value	F critical
Regression	351.5398	7	50.2199	2562.289	0.0000	0.921968
Residual	0.783986	40	0.0196
Total	352.3238	47
Multiple R		0.99967
R square		0.99934
Adjusted R square		0.99923
Standard Error		0.07982

Table 5: Results of Robustness of the Method.

Parameter

Value

R^a

T^b

tR^c (min) Rasagiline

Amount found (%)

R.S.D.^d (%)

2.42

2.50

2.58

5.682

4.608

4.920

1.91

1.83

1.85

3.160

3.200

3.147

99.73

100.00

100.44

4.4

2.9

4.5

Flow rate (mlmin−1)

0.776

0.800

0.824

5.712

4.608

5.037

1.85

1.83

1.76

3.933

3.200

3.727

103.1

100.0

97.74

0.9

2.9

3.9

a : Ressolution, b : Tailing factor, c : Retention time, d : Relative standard deviation

System suitability

System suitability was performed to confirm that the equipment was adequate for the analysis to be performed. The test was carried out by making six replicate injections of a standard solution containing 10.0μgml−1 of Rasagiline and 10.0μgml−1 of Caffeine (IS), and analyzing each solute for their peak area, theoretical plates (N), resolution (R) and tailing factor (T). The results of system suitability in comparison with the required limits are shown in Table 6. The proposed method fulfils these requirements within the accepted limits.

Table 6: System suitability results of the proposed method.

Analyte	R^a	N^b	T^c	RSD(%) ^e tR^d peak area
Rasagiline	4.60	3395	1.838	0.26 2.3
Caffeine (IS)	4.60	5352	1.628	0.22 2.8
Required limits	R >2	N > 2000	T < 2	R.S.D. < 5%

a : Resolution, b : No. of theoretical plate, c : Tailing factor, d : Retention time

e : Relative standard deviation(%)

CONCLUSION:

In the present research work to achieve highest precision in quantitative estimation of Rasagiline in pharmaceutical dosage form, a reverse phase liquid chromatography method for Rasagiline using IS was developed and validated. The method was validated in terms of linearity, precision, accuracy, detection limit, quantification limit and robustness. It involves a simple procedure for the preparation of the samples and shorter run times for analytical procedure (less than 15 min). A low percent of organic solvent (acetonitrile 10% and isopropyl alcohol 20%) was used in the composition of the mobile phase. Hence the present HPLC method can be considered as simple, rapid, suitable and easy to apply for routine analysis of Rasagiline in pharmaceutical dosage form.

ACKNOWLEDGEMENTS:

The authors shall remain grateful to APOTEX Research Private Limited, Bangalore, India and Juggat Pharma, Bangalore, India for providing Rasagiline Mesylate and Caffeine in the form of ‘Gift sample’ respectively and Nargund College of pharmacy, Bangalore for providing lab and research facilities to complete this project.

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Received on 01.03.2012 Modified on 16.03.2012

Asian J. Research Chem. 5(4): April 2012; Page 515-519

Brand Name	Amount found(mg)	Mean ± SD^a	Amount found (%)	Mean ± SD^a	R.S.D.^b (%) (n=3)
Rasalect 1 (Rasagiline 1mg)	0.96 1.02 1.03	1.003±0.037	96 102 103	100.33±3.78	3.77
Rasalect0.5 (Rasagiline 0.5mg)	0.515 0.516 0.501	0.510±0.008	103.09 103.38 100.35	102.27±1.67	1.634
Relgin 1 (Rasagiline 1mg)	0.952 1.013 0.964	0.964±0.032	95.21 101.36 96.49	96.49 ± 3.24	3.30