INTRODUCTION:

Safinamide Mesylate is a third-generation reversible MAO-B inhibitor, is a recently developed medicine to treat Parkinson's disease (PD). Parkinson's disease (PD) is the second most common chronic progressive neurological condition among the elderly, after Alzheimer’s disease. Parkinson's disease is a progressive neurodegenerative disorder affecting about 1 person every 1000 in the fifth decade and 19 every 1000 above 80 years to every person¹. Safinamide has been associated with a low rate of serum enzyme elevations during treatment, but has not been linked to instances of clinically apparent acute liver injury. Safinamide is a unique molecule with multiple mechanisms of action and a very high therapeutic index. Safinamide has been described as a multimodal medication, combining dopaminergic and nondopaminergic activities, although it remains unclear how the nondopaminergic properties contribute to its overall effect. The drug was approved in February 2015 and in the US on March 21, 2017².

Safinamide Mesylate is an alpha- aminoamide (chemical formula C₁₇H₁₉FN₂O₂ CH₄O₃S) with multiple mechanisms of action. Safinamide Mesylate (SAF)(S1), also known as (S)-2-(4 ((3-Fluorobenzyl) oxy) benzyl) amino) propanamide methane sulfonate, which also blocks sodium voltage sensitive channels and modulates stimulated release of glutamate³.

Chemistry:

The IUPAC name of Safinamide is ((2S)-2-[[4-[(3-fluorophenyl) methoxy] phenyl] methylamino] propanamide. The Chemical Structure of Safinamide is shown in fig-1

Fig. 1: Structure of Safinamide Mesylate⁴.

Materials and Instruments:

Double beam UV – Visible Spectrophotometer
Model	UV 550
Make	Jasco
Software	Spectra manager

Table No 1: List of Instruments used

HPLC System HPLC Quaternary Gradient System
Model No.	1260Infinity II
Make	Agilent
Column	Phenomenex
Software	Openlab EZ Chrome

Table No 2: List of Reagents Used

Sr. No.	Chemicals/ Reagents/ Solvents	Supplier	Grade
1	Acetonitrile	Merck	HPLC grade
2	Water	Siddhi Lab	HPLC grade
3	Trifluro acetic acid	Thermofisher Scientific	Analytical grade

HPLC Method Development:

Preparation of Standard Solution:

· Weighed accurately 26.35mg Safinamide mesylate (Equivalent to 20mg of Safinamide) and transferred into 20ml volumetric flask

· Added 15ml of Water and sonicated to dissolve the standard completely and diluted up to the mark with Water (1000 PPM).

· Further diluted 0.5mL to 25mL with Water. (20 PPM)

Preparation of standard stock solution for Chromatographic development:

· Weighed 26.35mg of Safinamide mesylate (equivalent to 20mg Safinamide).

· Dissolved in 15mL water in a 20mL volumetric flask.

· Volume made up to the mark with water.

· Final concentration: 1000PPM.

· Further diluted 2ml of stock solution to 20mL with mobile phase (100PPM).

Preparation of System suitability test (Safinamide standard solution):

· Weighed about 32.94mg of Safinamide mesylate (Equivalent to 25mg of Safinamide) and transferred in 50mL volumetric flask, added 35mL of Water, sonicate to dissolve it, made volume up to the mark with Water.

· Pipette out 1.5ml from standard stock solution and transferred into 25ml volumetric flask and made volume up to the mark with mobile phase (30µ/mL= working concentration), chromatograms were recorded.

Analysis of marketed Test sample:

Marketed test sample Having Name Xafinact 50mg tablets are selected for analysis and for doing validation.

Sample Prepared in duplicate. Summary of sample preparation as follows:

Table No 3: Summary of Marketed Test samples Preparation

Sample	Sample (mg)	Diluted to (mL)	Volume taken	Diluted to (mL)
Sample 1	80.7	50	1.5	25
Sample 2	81.4	50	1.5	25

So, Placebo prepared at lab level by using formula as follows:

Table No 4: Formula for Placebo preparation

Sr. No.	Ingredients	Role	Qty (mg)
1	Lactose	Filler	80
2	Starch	Binder	5
3	Magnesium stearate	Lubricant	5
4	Talc	Glidant	5
5	Crospovidone	Disintegrants	5
Total			100 mg

Total 10 gm of placebo prepared.

Sample preparation of Marketed test sample:

Weighed 20 tablets transferred in mortar pestle and crushed to fine powder. Mixed the contents with butter paper uniformly. Weighed the powder material equivalent to 25mg of Safinamide (81.20mg of powder material) and transferred to clean and dried 50mL of volumetric flask. Added 35 mL of Water, sonicated for 10 minutes with intermittent shaking. After 10 minutes allow to cool the solution to room temperature and made volume up to the mark with Water. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5mL of initial filtrate. Further diluted 1.5ml of filtered stock solution to 25ml with mobile phase (30mcg of Safinamide), injected the resultant solution and chromatograms were recorded and results are recorded.

Sample Prepared in duplicate. Summary of sample preparation as follows:

Table No 5: Summary of Marketed Test samples Preparation

Sample	Sample (mg)	Diluted to (mL)	Volume taken	Diluted to (mL)
Sample 1	80.7	50	1.5	25
Sample 2	81.4	50	1.5	25

Formula for % Assay calculation:

% Assay= (Safinamide Spl area)/(Safinamide Std avg area) X (Safinamide STD wt (mg))/50 X 1.5/25 X 50/ (Tablet sample weight (mg)) X 25/1.5 X (Avg wt of tablet (mg))/(Label claim of Safinamide) X Factor X 100

Validation of Rp-Hplc Method:^5-10

1) Filtration Study:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample.

This study was conducted with Safinamide Test sample (Tablet solution).

Filtration study carried out with unfiltered and filtered test solution. During filtration activity 0.45µm PVDF and 0.45µm Nylon syringe filters used by discarding 5 mL of aliquot sample.

2) Stability of Analytical Solution:

Stability study was conducted for standard and test sample solution. Stability study was performed at normal laboratory conditions.

The solution was stored at normal illuminated laboratory conditions and analyzed after 12 hours and 24hours.

Standard and Test solution stability study was performed by calculating the difference between results of test solution at each stability time point to that of initial.

3) Specificity:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Following solution shall be prepared and injected to prove the specificity nature of the method.

1. Blank (Mobile phase as a diluent)

2. Placebo

Placebo Sample solution preparation:

Weighed 48.26mg of placebo material (Which is equivalent to 25mg of Safinamide) and transferred to clean and dried 50mL of volumetric flask. Added 35mL of Water, sonicated for 10minutes with intermittent shaking. After 10minutes allow to cool the solution to room temperature and made volume up to the mark with Water. Filtered the solution through suitable 0.45µ PVDF syringe filter discarding 3-5mL of initial filtrate. Further dilute 1.5ml of filtered stock solution to 25ml with mobile phase, injected the resultant solution and chromatograms were recorded.

4) Linearity and Range:

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

Linearity Safinamide stock solution:

Weighed 32.94mg of Safinamide mesylate (Equivalent to 25mg of Safinamide) and dissolved in 25mL with Water. Further diluted 5ml of stock solution to 50mL with Water. (100 PPM).

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

As per ICH Q2R1 guidelines LOD and LOQ was determined by using the approach Based on the Calibration Curve in which residual standard deviation of a regression line was calculated and determined the LOD and LOQ by using following formula:

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line

S = Slope of regression line

6) Accuracy (% Recovery):

The accuracy of the analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value of the value found, Accuracy will be conducted in the range from 50 % to 150% of working concentration. Solution of each accuracy level was prepared in triplicate. Calculated % Recovery for each sample, Mean %recovery for each level and overall recovery and also calculated %RSD for each level and % RSD for overall recovery.

7) Precision:

Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous test under the prescribed conditions. Precision is of two types, Repeatability and Intermediate precision. It is performed on tablet test sample.

1. Repeatibility

2. Intermediate precision.

8) Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

RESULT AND DISCUSSION:

Selection of analytical wavelength:

Fig. No. 2 UV spectrum of Water as a blank

Optimized Chromatographic Conditions:

Table no 6: Optimized Chromatographic Conditions

Parameter	Description
Standard solution	Safinamide 100 PPM
Column	Agilent poroshell C18
Mode	Isocratic
Column Dimension	(150 mm X 4.6 mm i.d.) 5μm
Detector	U.V. Detector
Wavelength	226 nm
Flow Rate	1.0 ml/min
Mobile phase	Acetonitrile: 0.1% TFAA in Water (70:30)
Injection Volume	20 μl
Retention time	2.63

Fig. No. 3 Typical chromatogram of optimized trial

Validations of the RP-HPLC method:

The optimized method for estimating Safinamide Mesylate was validated for the following parameters using ICH Q2(R1) guidelines

1) Filtration Study:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on Tablet test sample.

Results of Filter study:

Table No 7: Results of Filter study

Sample description	Area	% Absolute difference
Unfiltered	5876923	NA
0.45 µ PVDF filter	5840645	0.62
0.45 µ Nylon filter	5828369	0.83

2) Solution Stability:

Stability study was conducted for Standard as well as Test Sample. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed at initial, after 12 hours and 24 hours.

Table No 8: Results of Solution stability.

Sample solution
Time point	Area	% Absolute difference
Initial	5886312	NA
12 Hours	5843650	0.72
24 Hours	5833005	0.91
Standard solution
Time point	Area	% Absolute difference
Initial	5976047	NA
12 Hours	5943625	0.54
24 Hours	5929690	0.78

3) SPECIFICITY:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Blank and Placebo solution prepared and injected to check interference at R.T. of Safinamide.

Results of Specificity.

Table No 9: Results of Specificity

Description	Observation
Blank	No interference at R.T. of Safinamide due to blank
Placebo	No interference at R.T. of Safinamide due to placebo

Linearity and Range:

Linearity of an analytical method is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range.

Fig. No 4: Calibration curve of Safinamide

Data of linearity of Safinamide:

Table No 10: Data Linearity for Safinamide

Sr No.	Parameter	Result value	Acceptance criteria
1	Beer's linearity range	15.0 - 45.0 µg/mL	NA
2	Correlation coefficient (R²)	0.99998	NLT 0.98
3	Intercept	-79691.800	To be report
4	Slope	200842.920	To be report
5	% RSD for area at each level	NA	NMT 2.0

The respective linear equation for Safinamide was:

Y = M X + C

Y = 200842.920 X + -79691.800

Where, X = concentration of Analyte in µg/mL

Y = is area of peak.

M = Slope

C= Intercept

Conclusion:

From the calibration curve it was concluded that the Safinamide shows linear response in the range of 15.0 -45.0μg/ml. The Regression value was found well within the limit.

4) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

σ = 15769.38 (Residual standard deviation of a regression line)

s = 200842.920

Detection limit (LOD):

LOD = 3.3 σ/ S

LOD = 3.3 x 15769.38 / 200842.920

LOD = 0.259µg/mL

Quantitation limit (LOQ):

LOQ = 10 σ/ S

LOQ = 10 x 15769.38 / 200842.920

LOQ = 0.785 µg/mL

5) ACCURACY (RECOVERY):

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method is determined by applying the method to analyzed samples to which known amounts of analyte have been added.

Chromatograms:

Fig No 5: Typical chromatogram of Accuracy 50%.

Fig No 6: Typical chromatogram of Accuracy 100%.

Fig No 7: Typical chromatogram of Accuracy 150%.

Acceptance criteria:

% Recovery for each level and overall recovery: 98.0 to 102.0%

% RSD for each level and overall recovery: NMT 2.0

6) PRECISION:

Precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method is usually expressed as standard deviation or relative standard deviation. Precision was performed on Test sample.

Table 12: Result of Intraday and Inter-Day Precision for Safinamide Mesylate test sample assay

Sr. No	Parameters	Intraday Precision	Interday Precision
1.	Mean	99.16	98.888
2.	STD	1.4813	1.3406
3.	% RSD	1.494	1.356

7) ROBUSTNESS:

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Following changes made under Robustness:

· Change in Wavelength

· Change in flow rate

· Change in column oven temperature

Result of Robustness study of Safinamide:

Table No 13: Robustness study of Safinamide

Change in Parameter	R.T.	Standard area	Asymmetry	Theoretical plates
Wavelength by +3 NM (229 NM)	2.62	5823945	1.44	6960
Wavelength by -3 NM (223 NM)	2.63	5640603	1.41	6814
Flow rate by +10% (1.1 mL/min)	2.41	5492547	1.39	6621
Flow rate by -10% (0.9 mL/min)	2.93	6294053	1.46	7206
Column oven temp by +2ºC (37 ºC)	2.63	5916958	1.42	6527
Column oven temp by -2ºC (33 ºC)	2.62	5926085	1.41	6519

CONCLUSION:

The newly developed analytical method for the estimation of Safinamide Mesylate has proven to be simple, rapid, precise, and highly accurate, with a significantly reduced retention time, making it both cost-effective and highly suitable for routine quality control applications. This method demonstrates excellent stability and reproducibility, ensuring consistent performance across different analytical conditions. It is applicable for the analysis of both bulk drug substances and pharmaceutical dosage forms. The results confirm that the method is not only precise and accurate but also time-efficient, which enhances its cost-effectiveness. It holds significant potential for use in routine quality control and research settings across pharmaceutical industries, academic laboratories, and certified testing facilities.

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Received on 21.06.2025 Revised on 14.07.2025

Accepted on 04.08.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):300-305.

DOI: 10.52711/2231-5675.2025.00047

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Level (%)	Area	Recovered conc (µg/mL)	Added conc (µg/mL)	% Recovery	Mean Recovery	% RSD
50	2976250	15.03	15.12	99.40	99.14	0.572
	2965823	14.98	15.21	98.49
	2961758	14.96	15.03	99.53
100	5946004	30.03	30.15	99.60	99.57	0.714
	6001631	30.32	30.24	100.26
	5917904	29.89	30.24	98.84
150	8874300	44.83	45.18	99.23	100.07	0.912
	9016782	45.55	45.08	101.04
	8917856	45.05	45.08	99.93