INTRODUCTION:

Figure 1: Chemical Structure of Safinamide Mesylate.

INTRODUCTION:

Safinamide mesylate is an orally administered drug used to treat idiopathic parkinsonism. It is marketed worldwide under the brand name xadago and in india as xafinact. It is a reversible and selective MAO inhibitor. Safinamide is used with another medication (Levodopa/ Carbidopa) to treat symptoms of Parkinson’s disease. It can help improves symptoms such as shakiness, stiffness and difficulty in moving. It can also help reduce the amount of “off” time (periods of slow movement or stiffness). Safinamide belongs to a class of drugs known as MAO inhibitors. It works by increasing the levels of certain natural substances in the brain. It is used between 3-4 stage in Parkinson.^1,4,5

IUPAC name: N₂-{4-[3-flurobenzyl) oxy] benzyl}-L-alaninamide, methanesulfonic acid

Molecular formula: C₁₇H₁₉FN₂O_2.CH₄SO₃

Molecular weight: 398.45g/mol

Solubility: Freely soluble in water, methanol and dimethyl sulphoxide, Sparingly soluble in ethanol and insoluble in ethyl acetate

Protein binding: 88-90%

Bioavailability: 95%

Half-life: 22hour

Pka: 15.76 146

Drug approval: by USFDA in March 2017 and in India by CDSCO in 2019.^[2,3]

On literature survey it was found that not much work has been done on this idiopathic anti-parkinsonism drug Safinamide mesylate and have found that HPTLC method was used commonly but not HPLC method.

In view of need for suitable method for routine analysis of anti-parkinsonism drug, attempt is made to develop simple, precise and accurate analytical method for its estimation. Analytical validation is one of the important steps of process validation. Without proven measurement system, it is impossible to confirm whether the manufacturing process has done what purpose to do and hence there is need to validate new method developed.^6,7,8

MATERIALS AND METHODS:

1. Chemicals:

Table 1: Lists of chemicals used.

Serial no	Name	Manufacturer
1	Acetonitrile HPLC grade	Merck Life science private limited
2	Ammonium acetate	Merck specialties private limited
3	Glacial acetic acid HPLC grade	Merck life science private limited

2. Apparatus:

Table 2: Lists of apparatus used.

Serial no	Name	Manufacturer
1	Water purification system	Direct-Q MILLIPORE France
2	HPLC (LC Compact 1120)	Agilent technologies
3	C₁₈ Column 5µm (250X4.6 mm)	XBridge
4	Analytical weighing balance	Shimadzu-AUX 220
5	UV-Visible spectrophotometer	Shimadzu-UV 1700
6	Vacuum pump	SUPER FIT
7	Ultrasonication Bath	Ana lab scientific instruments private limited
8	Filtration kit	TARSONS
9	Nylon membrane filter 0.22µm	Millipore (India) private limited

3. Software:

EZ Chrome Elite software-dual channel was used for acquisition, evaluation and storage of chromatographic data.

4. Drug sample:

Commercially available Safinamide Mesylate tablets (50mg) were procured from local pharmacy, manufactured by Sun Pharma Laboratories Ltd, villi: Kokihi Mirza Palash Bari road PO Palash Bari Dist. Kamrul, Assam - 781128.

5. Active Pharmaceutical ingredients:

Safinamide Mesylate was obtained as gift sample from Alkem Pharmaceuticals, Mumbai, India.

6. Preparation of mobile phase:

A mixture of 10mm Ammonium acetate buffer pH 5.8 and Acetonitrile in the ratio of 55:45 (V/V%) was used as the mobile phase. Ammonium acetate buffer pH 5.8 was prepared by dissolving 0.7708g of Ammonium acetate in 1000ml of water, adjusting the pH 5.8 with glacial acetic acid, and degassed in ultrasonic water for 15min and vacuum filtered through 0.22µm filter.^9.10,11

7. Preparation of Standard stock solution:

A standard stock-solution of concentration 1mg/ml was prepared using the mobile phase as a diluent by taking 50mg of Safinamide Mesylate in 50ml of volumetric flask. The solution is degassed in ultrasonic water for 10min, and vacuum filtered through 0.22µm filter.^12,13

8. Preparation of Working standard solution:

A working standard solution of concentration 10µg/ml was prepared from the above stock solution using the mobile phase as diluent.

9. Preparation of Sample stock solution:

A sample stock solution of concentration 1mg/ml was prepared using the mobile phase as a diluent by taking an amount of sample equivalent to 50mg of Safinamide mesylate in 50ml of volumetric flask. The solution is degassed in ultrasonic bath for 15min, and vacuum filtered through 0.22µm filter.

10. Chromatographic conditions:

XBridge^TM C₁₈ column 5µ (250X4.6mm) was used for the analysis. The flow rate was set at 1ml/min with a run time of 8min. The injection volume was 20µl. The detector was set at a wavelength of 226nm.

RESULT:

1. Chromatogram of blank:

Figure 2: Chromatogram of blank (Ammonium acetate: Acetonitrile).

2. Chromatogram of Safinamide Mesylate (10µg/ml):

Figure 3: Chromatogram of Safinamide mesylate (10µg/ml).

Table 3: System suitability testing (Safinamide mesylate 10µg/ml)

Theoretical Plates (USP)	Capacity factor	Asymmetry (Tailing factor)	S/N (6σ)
5376	0.01667	1.24958	10.483180

3. Linearity:

The linearity of the method was determined by the analysis of analyte concentration across a range of 10µg/ml-60µg/ml of Safinamide mesylate and area was plotted graphically as a function of analyte concentration.

Figure 4: Chromatogram of Safinamide mesylate (10-60µg/ml).

Figure 5: Linearity graph for Safinamide mesylate (10-60µg/ml).

Table 4: Linearity at a concentration range of 10-60µg/ml.

Serial No	Concentration (µg/ml)	Peak area
1	10	6822784
2	20	11019225
3	30	17651769
4	40	24278139
5	50	30726096
6	60	36796413

4. Precision:

Precision of the analytical method was studied by analysis of multiple sampling of homogenous sample. Method reproducibility was demonstrated by repeatability and intermediate precision measurements of peak area and peak symmetry parameters. The repeatability (within-day in triplicates) and intermediates precision (for 2 days) were carried out at single concentration level. Six injections were made and the obtained results within and between the days of trials were expressed as %RSD.

Figure 6: Chromatogram showing interday precision (day 1).

Figure 7: Chromatogram showing interday precision (day 2).

Table 5: Interday Precision.

Serial No	Sample	Peak Area
		Day 1	Day 2
1	Sample 1	1895431	1819322
2	Sample 2	1873133	1807783
3	Sample 3	1833789	1816164
4	Sample 4	1835309	1845596
5	Sample 5	1815629	1811949
6	Sample 6	1805185	1817234
	Average	1843179	1819675
	SD	34583.55	13352.3
	% RSD	1.88%	0.73%

Figure 8: Chromatogram showing intraday precision (morning).

Figure 9: Chromatogram showing intraday precision (afternoon).

Table 6: Intraday precision.

Serial number	Sample	Peak area
		Morning	Afternoon
1	Sample 1	1960797	1866475
2	Sample 2	1978287	1891082
3	Sample 3	1967379	1821702
4	Sample 4	1967237	1868131
5	Sample 5	1908387	1827929
6	Sample 6	1906055	1849784
	Average	1948024	1854184
	SD	321100.7	263466.9
	% RSD	1.65%	1.42%

5. Accuracy:

The recovery of Safinamide mesylate was used to find the accuracy of the developed method. The accuracy of the method was determined by calculating recoveries of Safinamide mesylate by the standard addition method. In pre-quantified sample solution (30µg/ml), a known number of standard solutions of Safinamide mesylate (80%, 100% and 120%) were added. The quantity of Safinamide mesylate measured using a calibration curve.

Figure 10: Chromatogram for Accuracy (80%).

Figure 11: Chromatogram for Accuracy (100%).

Figure 12: Chromatogram for Accuracy (120%).

6. Robustness:

The robustness of the method was studied by deliberate changes in the method like alteration of flow rate and wavelength of detection.

Figure 13: Chromatogram for decreased flow rate (0.9ml/min).

Figure 14: Chromatogram for decreased flow rate (1.1ml/min).

Figure 15: Chromatogram for decreased Wavelength of detection (225nm).

Figure 16: Chromatogram for increased Wavelength of detection (227nm).

Table 8: Data for robustness.

Serial no	Parameter	Conditions	Retention time (min)	Peak area
1	Flow rate	0.9ml	4.397	12151313
		1ml	3.863	6822784
		1.1ml	3.577	13922451
2	Detection wavelength	225nm	3.950	17561217
		226nm	3.863	6822784
		227nm	3.943	18176519

7. Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ were calculated according to ICH recommendations where the approach is based on the single-to-noise ratio. A signal to noise ratio 3:1 and 10:1 was considered for calculating LOD and LOQ respectively.

Table 9: Data for LOD and LOQ.

LOD	2.85 µg/ml
LOQ	9.5 µg/ml

DISCUSSION:

The system suitability test was applied to the chromatograms taken under optimum conditions to check various parameters such as theoretical plates (5376), capacity factor (0.01667), asymmetry (1.24958) and signal to noise ratio (10.483180). Suitable tests results were achieved for the proposed method. All these results indicate the suitability of the instrument for the developed method.

For study of precision six replicates of the standard solution was injected into the HPLC system in inter day and intraday intervals. The %RSD values of day 1 and day 2 for inter day intervals were found to be 1.88% and 0.73% respectively. While the %RSD values of morning and afternoon sessions for intervals were found to be 1.42% and 1.425 respectively. Therefore, the %RSD values for precision studies are within the accepted limits of 2%.

Linearity was performed using standard solutions in the concentration range of 10-60µg/ml. Calibration curve was constructed for the standards by plotting the concentrations versus peak area and evaluated by linear regression analysis. The correlation coefficient (R²) was found to be 0.99, which is within the accepted limits.

Accuracy was performed by spiking a pre-quantified sample with standard at 80%, 100% and 120%. The solutions were prepared in triplicates and analyzed through the developed method. The mean recovery values of obtained for 3 trials were 102%, 99% and 98% respectively, which indicates that there is an extremely less interference coming from matrix components.

For robustness a change of ±0.1ml/min in the optimized flow rate of 1ml/min of the method was done, resulting in the change of retention time from 3.86min to 4.39min and 3.5min respectively for each deliberate change in flow rate. Similarly, a change of ±1nm in the optimized detection wavelength of 226nm of the method was done, resulting in the change of retention time from 3.86min to 3.95min and 3.94min respectively for each deliberate change.

Considering the accepted limits for signal to noise ratio of 3:1 and 10:1 for calculating LOD and LOQ and it was found to be 2.85µg/ml and 9.5µg/ml.

CONCLUSION:

The proposed method showed acceptable accuracy, precision, linear concentration range and robustness. The results of analysis proved that the method is suitable for the determination of Safinamide mesylate in bulk and tablet dosage form without any interference from its excipients and this method can be used for the routine determination of Safinamide mesylate in bulk drug and in pharmaceutical dosage form.

REFERENCES:

1. https://www.webmed.com>safinamide mesylate.

2. www.google.com

3. https://www.drugbank.ca>safinamide mesylate.

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Received on 26.11.2021 Modified on 13.03.2022

Asian J. Pharm. Ana. 2022; 12(3):173-178.

DOI: 10.52711/2231-5675.2022.00029

Serial No	Spike level	Amount of drug in pre-quantified sample (µg/ml)	Conc. Of standard addition (µg/ml)	Average peak area	Conc. Found (µg/ml)	Mean % recovery
1	80%	30	24	15318437	24.5	102%
2	100%	30	30	1747619	29.7	99%
3	120%	30	36	8186588	35.5	98%