INTRODUCTION:

Tofisopam (Figure 1), is a 2,3-Benodiazipene, prescribed as second line treatment for anxiety. Unlike 1,4- and, 1,5- benzodiazepine, tofisopam lack anticonvulsant, sedative, and motor skill impairment properties¹. Tofisopam enhances the effect of muscimol like 1,4-benodiazepines, it is also claimed to be non-dependence causing and can be used in treatment of schizophrenia². As per the literature survey several spectrophotometric, and chromatographic methods were developed and validated for the estimation of Tofisopam in bulk and pharmaceutical formulation using different organic solvents, columns, and at different wavelengths^3-15. The methods for determining tofisopam in biological fluids and profiling impurities in pharmaceutical formulations were also published, although only a small number of chromatographic methods were reported with extended run times using different solvents^16-18.

Hence, a simple, specific, accurate, and reliable approach for determining tofisopam by RP-HPLC technique for estimation of tofisopam in bulk and pharmaceutical formulation is required.

Fig. 1: Structure of Tofisopam.

MATERIALS AND METHOD:

Materials:

Tofisopam was procured from Symed labs, HPLC grade methanol and AR grade orthophosphoric acid was procured from Sigma Aldrich, and Merck Millipore was used to obtain HPLC grade water.

Chromatographic conditions

An Agilent 1220 Infinity-II liquid chromatographic system equipped with a PDA detector, autosampler, and Agilent Control Panel software was used. The separation was achieved on a Phenomenex Luna C18 (250×4.60mm, 5µ) chromatographic column at 1mL/min flow rate using methanol: 0.1% OPA in water (90:10% v/v) at 238nm detection wavelength.

METHOD DEVELOPMENT^19-28:

Preparation of Standard Working solution:

10mg drug was dissolved in 10ml methanol to obtain 1000µg/ml standard stock solution. 1ml of standard stock solution was diluted to 10ml mobile phase followed by further dilution to obtain 10µg/ml as the final working standard.

Selection of detection wavelength

A solution containing 10µg/ml tofisopam was prepared and scanned in the range of 200-400nm. Tofisopam was detected at 238 and 309nm (Figure 2), the results with regard to retention time, and area were compared, 238nm exhibited better results than 309nm. Hence 238nm was selected for further analysis.

Fig. 2: UV spectrum of tofisopam

Selection of Mobile Phase:

Mobile phase was selected on the basis of solubility of tofisopam. Tofisopam was soluble in acetonitrile, methanol and chloroform and was very slightly soluble in water. Preliminary trials were carried out on combination solvents with different ratios such as methanol:water (50:50% v/v), methanol:water (75:25% v/v), methanol:0.1% OPA in water (90:10% v/v), amongst the above the third trial exhibited the satisfactory results and was selected for the analysis. The spectrum was obtained with maximum absorbance at 238 and 309nm.

Preparation of 0.1% OPA in water:

1mL of OPA was transferred to 1000mL volumetric flask and and the volume was made upto to the mark with water

Preparation of sample solution (Pharmaceutical Formulation):

Ten tablets, each containing 50mg of tofisopam were weighed and powdered. Powder equivalent to 10mg of tofisopam was transferred to 10ml volumetric flask, diluted in methanol, sonicated for a minute and made upto the mark with methanol (1000µg/ml). The solution was further diluted in mobile phase to obtain final concentration of 10µg/ml in mobile phase. The drug content in the tablet dosage form was determined.

RESULTS AND DISCUSSION:

Optimization of Chromatographic Conditions:

The separation was achieved on Phenomenix Luna C18 (250×4.6mm, 5µ) chromatographic column using mobile phase methanol:0.1% OPA in water in the ratio of 90:10%v/v. The retention time was 3.16min±0.02min, (Figure 3) tailing factor was less than 2 and the theoretical plates were more than 8000.

Fig. 3: Chromatogram of optimized chromatographic condition.

METHOD VALIDATION:

System Suitability:

To demonstrate the system suitability for tofisopam six replicate injections of 10µg/ml was injected into the chromatographic system, the %RSD of 0.616% was observed.

Linearity

The linear response was confirmed using 6 concentration levels ranging from 10µg/ml to 60µg/ml. The calibration curve was obtained by plotting peak area versus concentration. The regression equation y = 43.027x+32.115 was obtained with the correlation coefficient (r²) 0.9996. (Figure 4)

Fig. 4: Calibration curve of tofisopam in the concentration range of 10-60µg/ml

Limit of Detection and Limit of Quantitation:

LODs and LOQs may also be computed using the slope of the calibration curve (S) and the standard deviation of the response (σ) of the curve. according to the formula: LOD = 3.3(σ /S) and LOQ = 10(σ /S). The LOD of tofisopam was found to be 2.757µg/ml and LOQ was 8.855µg/ml.

Precision:

The newly developed method was found to be precise upon Inter day and Intraday studies. For Interday studies, six replicate injections of 10µg/ml tofisopam were injected on three consecutive days and the %RSD was calculated. The %RSD of all the Interday analysis was less than 2% (Table no. 1). All the replicates were prepared separately on each day.

Table no. 1: Results of Interday precision

Conc. (µg/ml)	Area on Day-I	Area on Day-II	Area on Day-III
10	751.856	762.248	757.523
10	754.925	759.478	758.404
10	751.175	758.774	758.565
10	752.497	759.443	759.443
10	751.893	760.837	760.837
10	753.021	759.396	759.396
Mean	751.895	760.029	759.028
SD	0.786	1.281	1.136
%RSD	0.175 %	0.168 %	0.150 %

For Intraday studies, like interday, 6 replicate injections of 10µg/ml tofisopam were injected at 1^st, 4^th, and 8^th, hour, the %RSD was calculated (Table no. 2)

Table no. 2: Results of Intraday precision

Conc. (µg/ml)	Area on Day-I	Area on Day-II	Area on Day-III
10	762.277	762.953	757.765
10	760.796	759.544	759.249
10	763.143	759.861	761.373
10	761.536	758.881	759.539
10	762.745	760.652	760.198
10	760.364	759.998	759.863
Mean	761.810	760.315	759.665
SD	1.101	1.416	1.186
%RSD	0.145%	0.186%	0.156%

Accuracy:

Accuracy or recovery studies were performed by standard addition method, in this the sample of 10µg/ml was spiked with 50%, 100% and 150% of standard and % recovery was calculated. The results of the same are presented in table no. 3.

Table no. 3: Results of recovery studies.

Level	% Recovery (%)	Mean % Recovery (%)
50% R¹	97.662	98.495
50% R²	99.564
50% R³	98.260
100% R¹	103.592	103.294
100% R²	103.163
100% R³	103.128
150% R¹	101.147	101.621
150% R²	101.050
150% R³	102.667

*R¹: Replicate 1, R²: Replicate 2, R³: replicate 3.

Assay:

10 tablets, each tablet containing 50mg of tofisopam were weighed and powdered. The powder containing tofisopam equivalent to 10mg was weighed and diluted to obtain 10µg/ml concentration. The diluted sample was then introduced into chromatographic system and % purity was determined. The% purity was found to be 101%.

Robustness:

The robustness was assessed by altering the method parameters,and changing the mobile phase ratio and detection wavelength. The method was found to be robust and reproducible upon slight parametric variations. The results are presented in table no. 4

Ruggedness:

The potency of the method was assessed by human variation. Another analyst was assigned to carry out the analysis and the %RSD with respect to peak area was calculated for both the analysts. The results were found to be reliable and reproducible. The results are presented in table no. 5.

Table no. 4: Results of Robustness Studies.

	Mobile phase ratio Methanol:0.1% OPA in water			Detection wavelength
Change in method Parameters	88:12% v/v	90:10% v/v	92: 8% v/v	236nm	238nm	240nm
%RSD of peak area	0.183%	0.158%	0.178%	0.163%	0.179%	0.167%

Table no. 5: Results of Ruggedness studies.

Analyst	Analyst-I				Analyst-II
Conc (µg/ml)	RT	Area	TP	TF	RT	Area	TP	TF
10	3.17	771.54	2872.00	0.81	3.17	774.94	2856.00	0.81
10	3.17	773.05	2840.00	0.82	3.17	775.95	2840.00	0.82
10	3.17	774.50	2841.00	0.82	3.17	777.98	2807.00	0.82
Mean	3.17	773.03	2851.00	0.82	3.17	776.29	2834.33	0.82
SD	0.00	1.48	18.19	0.01	0.00	1.55	24.99	0.01
%RSD	0.03%	0.19%	0.64%	0.74%	0.02%	0.20%	0.88%	0.70%

CONCLUSION:

The newly devised RP-HPLC method for determining tofisopam in bulk and pharmaceutical formulations was found to be simple, specific, economical, and accurate. The ability to determine tofisopam in both pure and pharmaceutical formulations has been demonstrated to be a reliable and useful technique. All validation parameters were determined to be within acceptable limits in accordance with ICH Q2R1 Guidelines. It may be used in the laboratory for tofisopam analysis every day with a high level of precision and accuracy.

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Received on 06.04.2023 Modified on 23.08.2023

Research J. Pharm. and Tech 2024; 17(4):1449-1452.

DOI: 10.52711/0974-360X.2024.00229