RP-HPLC Method Development and Validation for Determination of Safinamide in Bulk and Pharmaceutical Formulation

Safinamide (SAF) is used for the usage of Parkinson's disease. After Alzheimer's disease, Parkinson's disease (PD) is the one further common chronic progressive neurological condition in the ageing ^[5]. SAF works by increasing the amount of dopamine, a chemical messenger specifically needed to control movement in the brain. It is an orally available derivative from chemical class of α-amino amides, with multiple mechanisms of action involving inhibition of MAO-B and Dopamine reuptake used in the treatment of epilepsy and Parkinson's disease ^[2]. Besides MAO-B inhibition, SAF exhibits novel anticonvulsant activities, including sodium channel blockade, calcium channel blockade and glutamate release inhibition ^[3]. SAF is a unique molecule with several mechanisms of action and an excessive therapeutic index ^[4] used with additional medication (levodopa/carbidopa) to treat symptoms of Parkinson's disease. It can help improve symptoms such as shakiness, stiffness, and difficulty moving ^[6]. It was approved in Europe on February 2015, in the United States on March 2017, and in Canada on January 2019^[5]. Safinamide has been associated with a low rate of serum enzyme increases all over treatment, but has not been similar to cases of clinically apparent acute liver injury ^[6].

Literature survey reveals an authorized chiral liquid chromatographic method for the enantiomeric separation of safinamide mesylate^[1] Except this, several analytical methods, encompassing HPTLC^[7], UPLC-MS/MS^[8-10], RP-HPLC^[2-6], and spectrophotometric methods^[11-13], have been employed for the analysis of different formulations of safinamide as indicated by detail literature survey. The present study designates the development and validation of a simple, specific, sensitive, accurate and precise RP – HPLC ecofriendly technique for the estimation of SAF in tablet dosage form. The anticipated method is elevated and validated according to ICH guidelines ^[6]. The IUPAC name of Safinamide is ((2S)-2-[[4-[(3-fluorophenyl) methoxy] phenyl] methylamino] propanamide. The Chemical Structure of SAF is exposed in Fig. No. 01.

Fig. No. 01: Chemical Structure of Safinamide

MATERIALS AND METHODS

Chemicals Used:

In method development and validation of stabilizers succeeding chemicals and reagents were used.

Table No. 1: List of chemicals

Table No. 2: List of instruments

HPLC Method Development

Selection of mobile phase:

Each mobile phase was vacuum degassed and filtered over 0.45µ membrane filter. The mobile phase was allowed to equilibrate while waiting for mobile phase over baseline was found. The standard solution having solution of SAF was run using different individual solvents were tried to become a good and stable peak. From the various mobile phases tried, mobile phase containing ACN and Water (0.1% OPA) was selected since it gave sharp, well resolved peaks with symmetry within the limits and significant reproducible retention time for Safinamide.

Selection of Column:

Selection of column is the essential part in the method development. By applying column chemistry, the most suitable column C18 (100mmX 4.6mm, 2.5µm particle size) was selected.

Selection of detection Wavelength:

Standard solutions were scanned in the range of 200-400nm, against 10 ml ACN and volume make with water solvent system as reference Safinamide were showed absorbance maxima (lambda max) at 225 nm respectively.

Diluent Preparation:

The Mobile phase was utilized as the diluent.  

Preparation of standard stock solution:

Accurately weight and transfer 10 mg of Safinamide  working standard into 10 ml volumetric flask as about diluent  Acetonitrile completely and make volume up to the mark with the same solvent to get 1000 µg/ml standard  (stock solution) and 15 min sonicate to dissolve it and remove the unwanted gas, further an aliquots portion of SAF stock solution were assorted in volumetric flask in 10 ml and volume was make up to the mark with mobile phase from the subsequent solution 0.1-0.5 ml was transferred to 10 ml volumetric flask and the volume was make up to the mark with 95% ACN :05 % 0.1% OPA), prepared in (95% ACN : 05%  buffer pH 3.0 with OPA) solvent. 

Method Validation

The developed method was validated as stated by ICH guidelines.

Specificity:

For specificity study solutions of Safinamide were prepared and injected into HPLC system and chromatogram recorded.

Linearity:

Preparation of standard stock solution for Linearity:

10 mg of Safinamide were weighed and transferred to 10 mL volumetric flask and make up the volume up to the mark with diluent. Sonicate for 10 min with occasional swirling. 0.1 ml of this solution diluted up to 10 ml volumetric flask with diluents was added to make up the volume.

Preparation of linearity solution:

Sample-I (10 µg/ml of Safinamide): Take 0.1 ml from stock solution and make up the volume with diluent upto 10 ml.

Sample-II (20 µg/ml of Safinamide): Take 0.2 ml from stock solution and make up the volume with diluent upto 10 ml.

Sample-III (30 µg/ml of Safinamide): Take 0.3 ml from stock solution and make up the volume with diluent upto 10 ml.

Sample-IV (40 µg/ml of Safinamide): Take 0.4 ml from stock solution and make up the volume with diluent upto 10 ml.

Sample-V (50 µg/ml of Safinamide): Take 0.5 ml from stock solution and make up the volume with diluent upto 10 ml.

Procedure

Inject each sample into the chromatographic system ^[21] and measure the peak area.

Plot a graph of peak area V/S concentration (Concentration on X-axis and Peak area on Y-axis) and calculate the correlation coefficient.

Accuracy (Recovery):

Preparation of standard stock solution:

10 mg of Safinamide working standards were weighed and transferred to 10 mL volumetric flask & diluents was added to make up the volume 0.1 ml of this solution diluted up to 10 ml with diluents.

Procedure:

The accuracy was determined by Safinamide (equivalent to 10 mg of SAF (80 %, 100 % and 120 % of the label claimed, correspondingly) to quantity equivalent to average weight of marketed preparation. This sample mixture containing 10 mg of SAF were diluted or triturated and then subjected to chromatographic analysis using the described method. The resultant mixtures were analyzed in triplicates done three days. The % recovery of added drug was taken as a measure of accuracy ^[15].

Table No. 3: Table of Accuracy for RP-HPLC Method

Repeatability of system was determined with the sample solution containing 30 μg/ml of Safinamide. Sample was injected, peak areas measured and %RSD was calculated. It was repeated for two times ^[14].

Procedure:

10 mg weight of sample (1000 μg/ml of Safinamide) were weighed and transferred to 10 mL volumetric flask & diluent was added to make up the volume. Sonicated for 10 min with occasional swirling. Above solution was filtered over and done with 0.45 μ membrane filter. Transfer 0.3 ml from this solution in volumetric flask and diluted up to 10 ml with diluent.  

Precision:

Precision of an analytical technique is the degree of agreement between distinct test results when the procedure is useful repetitively to several test group of a homogenous sample. To calculate the midway precision of the method, Precision was accomplished on altered days by upholding same conditions.

Intra-day precision (Day I):

Sample solutions containing Safinamide in three different concentration (10 µg/ml, 30 µg/ml, 50 µg/ml) concentration and were analyzed three times on the same day and %R.S.D was calculated.

Inter-day precision (Day II):

Sample solutions containing Safinamide in three different concentration (10 µg/ml, 30 µg/ml, 50 µg/ml) concentration and were analyzed three times on the different day and %R.S.D was calculated ^[24].

Analysis of marketed formulation:

For the determine of content of Safinamide  in marketed tablet pharmaceutical formulation (label claim 10 mg of Safinamide), 10 mg weighed and average weight of tablet was calculated 74.32 mg and sample equivalent to 14.86 gm of the drug was extracted from the tablet powder with 10 ml ACN. To ensure complete extraction it was sonicate for 15 min. 0.2 ml of supernatant was then diluted up to 10 ml with mobile phase. The resultant solution was injected in HPLC and peak area of drug was noted.

(Take 14.864 mg crushed powder of tablet in 10 ml volumetric flaks and make up to 10 ml with Acetonitrile  Means 14.864 mg in 10 ml Acetonitrile= 1000 µg/ml Safinamide  stock2)

Regression equation was generated by peak areas of standard solutions. Using the regression equation and peak area of the sample the amount of Safinamide in the sample was calculated.

Robustness:

The analysis was executed in different conditions to find the inconsistency of test results. The subsequent conditions are tested for discrepancy of results.

Preparation of standard stock solution:

1000 µg/ml of Safinamide working standards were transferred to 0.3 mL in volumetric flask & this solution diluted up to 10 ml with diluent. The mobile phase composition was changed in (±1 %) proportion of ACN:0.1% OPA  in the mobile phase composition 94:06, 96:04 instead of 95:05 and the flow rate was changed in (±0.1 ml/min) 0.9 ml/min, 1.1 ml/min instead of 1.0 ml/min also the change in detection wavelength (±1 nm) 224 nm, 226 nm instead of 225 nm and the effect of the results were examined it was performed using 30 µg/ml solution of Safinamide  in duplicate.

Ruggedness:

The degree of reproducibility of test outcome acquires by the analysis of equivalent sample under variability of Condition. For example, different analyst, laboratory as well as different instrument.

Detection Limit and Quantitation Limit:  

Based on the S.D. of the response and the slope of calibration curve, the detection limit (DL) and quantitation limit (QL) was calculated as,   

Detection Limit   = 

Quantitation Limit =

Where, σ = the S.D. of regression lines, S = slope. 

The slope (S) may be predictable from the calibration curve and S.D. was used should be intended from the y-intercepts of regression line in calibration curve.  

RESULTS AND DISCUSSION:

Spectroscopy

UV absorption of 20 mcg solution of Safinamide in Acetonitrile was generated and absorbance was taken in the range of 200-400 nm. λ max of SAF was found to be 225 nm respectively.

Fig. No. 02: UV Spectrum of Safinamide

Analytical Method Development

Optimization of the HPLC method

To estimate SAF six trials were performed from that 6^th trial which provides the optimized result. Therefore, all parameters are anticipated for the development of the RP-HPLC technique. Table No. 4

Principle: Reverse phase liquid chromatography with DAD Detector Fortis (S.K) Gradient System.

Table No. 4: Optimized chromatographic conditions

Optimized chromatogram of Safinamide:

Fig. No. 03: Optimized chromatogram of Safinamide

Table No. 5: Details of chromatogram of Safinamide

Method Validation

All the method validation parameters such as accuracy, linearity, precision, detection limit, quantification limit and robustness were validated as per the ICH guidelines ^[22-23].

Specificity and Selectivity:

The studies should have no interfering from the inessential components and be well determined from them. Specificity is the procedure to identify quantitatively to evaluate in presence of component that may be assessed to be present in the sample medium, while selectivity is the procedure to distinguish qualitatively the analyst in existence of components that may be assessed to be existing in the sample medium.

Fig. No. 04: Chromatogram of Specificity & Selectivity (20 mcg)

Table No. 6: Details for Chromatogram of Specificity & Selectivity

Linearity:

From Safinamide  standard stock solution, different working standard solution (10-50 μg/ml) were prepared in mobile phase 20 μl of sample solution [16] was injected into the chromatographic system using fixed volume loop injector. Chromatograms were recorded. The area of each concentration was recorded shown in Table No. 7

Fig. No. 05: Overlay of Linearity

Table No. 7: Linearity data for Safinamide

Regression equation data for Safinamide 

The plot of Concentration (on X axis) V/S Average Peak Area (on Y axis) data of Safinamide is a straight line.

Y = mx + c

Slope (m) = 46.16

Intercept (c) = 4.91

Correlation Coefficient (r) = 0.999

Fig. No. 06: Calibration curve of Safinamide

The plot was linear passing through the origin; Correlation Coefficient was obtained less than 1 that concluded.

Accuracy:-

To validate the accuracy of developed method Recovery studies were accomplished. To pre evaluated tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was estimated Table No 8.  

Table No 8: Recovery Studies of Safinamide for RP-HPLC method

*Mean of each 3 reading

RP-HPLC method is established by recovery studies executed at different stages of concentrations (80%, 100% and 120%). The % recovery was found to be contained by 98-102%.

System suitability parameters: (Repeatability)

To determine the resolution and reproducibility of the proposed chromatographic method for determination of Safinamide system suitability parameters were studied. The result shown in below. Table No. 9.

Table No. 9: Repeatability studies on RP-HPLC for Safinamide

Precision:-

The method was established by analyzing various replicates standards of Safinamide. All the solution was analyzed thrice in order to record any intra-day & inter-day disparity in the consequence that determined. The result acquired for intraday and inter day precision is revealed in Table No. 10 respectively.

Table No. 10: Result for Intra-day and Inter day Precision

Analysis of tablet formulation: (Brand name: Safion 50 mg)

Total weight of 20 tablet Powder wt. = 14.86 gm

Avg. Powder Weight 74.32 mg

Eq.wt for 10 mg =   10 x 74.32 / 50 = 14.864 mg

Tablet stock solution: Take 14.864 mg in 10 ml mobile phase i.e. = 1000 µg/ml tab solution.

Weigh 10 mg Safinamide Tablet and calculated the average weight, accurately weigh and transfer the sample equivalent to 14.864 mg SAF into 10 ml volumetric flask. Make volume up to the mark with diluent and sonicate to dissolve it completely. Mix well and filter over 0.45 µm filter. Further pipette out 0.2 ml from the above stock solution transfer to 10 ml volumetric flask and dilute up to the mark with diluents. (20 µg/ml). The simple chromatogram of test SAF Shown in Fig. No. 07 the amounts of SAF per Tablet were calculated by extrapolating the value of area from the calibration curve. Practice of the analysis was recurrent two times with Tablet formulation. Tablet Assay for %Label claim for %RSD Intended, Result was exposed in Table No. 12.

Fig. No. 07: Chromatogram of marketed formulation (20 mcg/ml)

Table No. 11: Details of chromatogram of marketed formulation

Table No. 12: Analysis of marketed formulation

Analysis of marketed formulation were accomplished and % Label Claim was found to be 99.00 % Acceptable are concluded.

Robustness:

The Robustness of a method is its proficiency to continue usual by insignificant measured differences in parameters. To calculate the robustness of the proposed method, insignificant but measured variations in the optimized method parameters were done. The consequence of changes in mobile phase configuration and flow rate, wavelength on retention time and tailing factor of drug peak was deliberate. The results of robustness studies are shown in Table No. 13.

Table No. 13: Result of Robustness Study of Safinamide

Ruggedness:

From stock solution, sample solution of SAF 30 µg/mL was prepared and analyzed by two different analysts using similar operative and conservational circumstances. Peak area was measured for same concentration solutions, two times. The results are indicated in Table No. 14.

Table No. 14: Analysis of Analyst-1 & 2