A simple, specific, and precise high-performance thin-layer chromatographic method has been developed and validated for estimation of Safinamide Mesylate as bulk and in tablet dosage form. The chromatographic development was carried out on aluminum plates precoated with silica gel 60 F254 using a mixture of Toluene: Methanol: Triethylamine (4 : 1 : 0.5 v/v) as mobile phase. Detection was carried out densitometrically at 226 nm. The
Safinamide (SAF) is an orally available derivative from chemical class of
Chemical structure of Safinamide Mesylate.
Literature survey reveals a validated chiral liquid chromatographic method for the enantiomeric separation of safinamide mesylate [
Except this, so far no analytical method was available for estimation of SAF as indicated by detail literature survey. The therapeutic effectiveness and less methods available for its estimation encourage us to undertake this work, so that quantitative estimation of SAF can be done and hence can be used for routine analysis of bulk and formulation as well.
The present study describes the development and validation of a simple, specific, sensitive, accurate precise, and economic HPTLC method for determination of SAF in tablet dosage form. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines [
Safinamide mesylate was kindly gifted from Alkem Pharmaceuticals, Mumbai (Maharashtra), India. Safinamide tablets were obtained from commercial sources within their shelf life period. All the reagents and solvents used were of analytical reagent (AR) grade. Solvents used like toluene, methanol, and triethylamine were of AR grade and obtained from Merck Chemicals.
The samples were spotted in the form of bands of width of 6 mm with a Camag 100
An accurately weighed quantity of 10 mg SAF was transferred to 10 mL volumetric flasks, dissolved in methanol, and volume was made up to mark with the same solvent to obtain a working standard having concentration 1000 ng
Initially, different ratios of methanol, chloroform, n-propanol, and toluene were tried, but tailing of spots was observed. Finally, the mobile phase
Chromatogram of standard safinamide mesylate (
To determine the concentration of SAF in tablets (Label claim: 50 mg per tablet), the contents of 20 tablets were weighed, their mean weight determined, and were finely powdered. The powder equivalent to 10 mg of SAF was weighed. The drug from the powder was extracted with methanol. To ensure complete extraction of the drug, it was sonicated for 20 min and the volume was made up to 10 mL. The resulting solution was filtered using 0.41
Analysis of Tablet formulation (Label claim: 50 mg per tablet).
Concentration | Amount found (ng) | Amount found (%) | % R.S.D. |
---|---|---|---|
1000 | 1008.8 | 100.8 | |
999.76 | 99.97 | ||
995.63 | 99.56 | ||
1006.7 | 100.6 | ||
1011.3 | 101.1 | ||
994.14 | 99.41 | ||
Mean ± S.D. | 0.72 |
The proposed method was validated as per the ICH guidelines in terms of its linearity, accuracy, specificity, intraday and interday precision, robustness, ruggedness, limit of detection (LOD), and limit of quantification (LOQ).
For linearity study, aliquots of 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4
Calibration curve for Safinamide mesylate in concentration 400–2400 ng/band.
The preanalyzed samples of concentration 1000 ng
The specificity of the method was ascertained by analyzing standard drug and formulation. The spot for SAF in formulation was confirmed by comparing the
A typical overlain spectrum of standard drug and drug extracted from tablet scanned at the peak-start, peak-apex, and peak-end positions of the band (Correlation > 0.99).
Repeatability was determined by spotting 1200 ng per spot of SAF. Precision of the method was assessed by spotting 800, 1200, and 1600 ng per spot of SAF on three different times within the same day (intraday) and on three different days (interday).
Robustness of the method was performed by spotting 1200 ng of drug making small deliberate changes in various chromatographic conditions. Mobile phases having different composition of toluene : methanol : TEA (3.8 : 1.2 : 0.5 and 4.3 : 0.7 : 0.2 v/v) were tried, and chromatograms were run. The volume of mobile phase, temperature, and relative humidity was varied in the range of
Ruggedness of the method was performed by spotting 1200 ng of drug by two different analysts maintaining same experimental and environmental conditions.
In order to determine detection and quantification limit, concentrations in the lower part of the linear range of the calibration curve were used. SAF solutions of 400, 480, 560, 640, 720, and 800 ng
The TLC procedure was optimized with a view to develop a method for determination of SAF. Optimization of mobile phase with toluene : methanol : triethylamine (4 : 1 : 0.5) v/v gives sharp and symmetrical peak having
The linear regression data for the calibration curves showed good linear relationship over the concentration range 400–1200 ng
The proposed method when used for extraction and subsequent estimation of drug from tablet dosage form after over spotting with 80, 100, and 120% of additional drug; mean recovery is within acceptable limits, indicating the method is accurate and afforded recovery of 99.44–99.88% (Table
Results of recovery studies.
Initial amount of drug (ng | % of standard drug added | % Recovery* | % R.S.D. |
---|---|---|---|
1000 | 80 | 99.44 | 0.31 |
1000 | 100 | 99.88 | 0.33 |
1000 | 120 | 99.86 | 0.41 |
*Mean of three estimations at each level.
The peak purity of SAF was assessed by comparing the spectra at three different levels, that is, peak start (
The precision of the developed HPTLC method was expressed in terms of % RSD. The results depicted revealed high precision of the method (Table
Intraday and Interday Precision.
Conc. (ng | Intraday | Interday | ||
% Amount found* | % R.S.D. | % Amount found* | % R.S.D. | |
800 | 99.99 | 0.30 | 99.76 | 0.16 |
1200 | 100.04 | 0.24 | 99.97 | 0.13 |
1600 | 100.04 | 0.05 | 100.17 | 0.09 |
*Mean of three estimations at each level.
The standard deviation of peak areas was calculated for each parameter, and % RSD was found to be less than 2% (Table
Robustness of the method.
Parameters | % R.S.D.* |
---|---|
Mobile phase composition | 0.90 |
Mobile phase volume | 0.81 |
Development distance | 0.65 |
Activation of TLC plate | 0.81 |
Duration of saturation | 0.70 |
Time from spotting to chromatography | 0.50 |
Time from chromatography to scanning | 0.43 |
*Mean of three estimations at each level.
The % RSD was found to be less than 2% indicating the method was rugged when estimation was done by two different analysts, Table
Results of ruggedness study.
Analyst | % Amount found of SAF (Mean ± S.D.) | % R.S.D.* |
---|---|---|
I | 0.67 | |
II | 0.66 |
*Mean of three estimations at each level.
Detection limit and quantification limit for SAF were found to be 13.09 ng and 39.67 ng, respectively. This indicates adequate sensitivity of the method as it can be validated in less quantity of drug, and hence the method proves to be economic.
The literature survey promoted us to develop HPTLC method on SAF as no analytical method was reported for it. The HPTLC method was developed and validated as per ICH guidelines, and the method was found to simple, precise, accurate reproducible, and economic, thus can be used for determination of SAF in tablets. Moreover, proposed method also indicates no interference of excipients when applied to tablet dosage form. Future plan includes development of Safinamide mesylate in available combinations along with stability-indicating and forced degradation-studies.
The authors are thankful to Alkem Pharmaceuticals Ltd. (Mumbai, Maharashtra) for providing a gift sample of Safinamide Mesylate.