INTRODUCTION:

Tolperisone hydrochloride (TOL), chemically (R, S) 2-methyl-1-(4 methyl phenyl)-3- (1-piperidyl) propane -1 one (Fig. 1), is a piperidine derivative¹. It is a centrally acting muscle relaxant for the symptomatic treatment of spasticity and muscle spasm in different pathological conditions like acute and chronic muscle spasm, electroconvulsive therapy, neurological conditions and orthopedic manipulation². TOL is official in Japanese Pharmacopoeia³ which describes potentiometric titration method for its estimation. Various methods like colorimetric^4,5, UV spectrophotometric⁶, extractive spectroscopic⁷, HPTLC⁸ and HPLC^9,10 are reported in literature for estimation of TOL in pharmaceutical dosage forms as well as in biological fluids.

Diclofenac sodium (DIC), chemically sodium (o-{(2,6-dichlorophenyl) amino} phenyl) acetate (Fig. 2)¹, is a synthetic non-steroidal anti-inflammatory drug widely used in clinical medicine for the treatment of inflammatory conditions such as rheumatoid arthritis, osteo arthritis and ankylosing spondylitis². Various analytical methods to estimate DIC have been reported in literature. These include HPLC^11,12 and HPTLC¹³.

The combined dosage form of TOL and DIC is available in the market for the treatment of muscle pain or spasm. Literature survey reveals that, no method is reported for the simultaneous determination of these drugs in combined dosage form. The aim of present work was to develop simple, accurate, precise and rapid UV spectrophotometric methods for simultaneous estimation of TOL and DIC in combined tablet dosage form.

Fig. 1 Structure of Tolperisone hydrochloride

Fig. 2 Structure of Diclofenac sodium

MATERIALS AND METHODS:

Apparatus

UV-visible double beam spectrophotometer (Shimadzu, Japan, UV-1800) with UVProbe 2.33 software was used for spectral measurements. The spectral bandwidth of 0.5 nm, scanning range of 200-400 nm and medium scanning speed was selected.

Materials

TOL gift sample (100.1% w/w) was kindly provided by Themis Medicare Ltd., Haridwar, Uttarakhand, India. DIC was gifted by Torrent Pharma Ltd., Ahmedabad, Gujarat, India. Marketed tablet formulations containing TOL 150 mg and DIC 50 mg (TOLPIDOL-D) manufactured by Themis Medicare Ltd were purchased from local market. Methanol used was of analytical grade. Distilled water was used throughout the experiment. Calibrated instruments and glasswares were employed throughout the work.

Preparation of Stock Solutions

Standard stock solutions of TOL and DIC, each having concentration of 1000 µg/ml, were prepared in methanol. From these solutions, 100 µg/ml of TOL and 10 µg/ml of DIC standard solutions were prepared by dilution with distilled water.

Preparation of Solutions for Calibration Curve

Standard solutions having concentration 6, 9, 12, 15, 18 and 21 µg/ml of TOL and 2, 3, 4, 5, 6 and 7 µg/ml of DIC were prepared by diluting the stock solutions with distilled water for preparing calibration curves.

Determination of Absorbance Maxima (λ_max) and Zero Crossing Point (ZCP)

The solutions of TOL (15 µg/ml) and DIC (5 µg/ml) were scanned separately in the UV range of 200-400 nm. Their zero order spectra were converted to first order derivative spectra and overlain to determine their Zero Crossing Point (ZCP) setting Δλ = 5 and scaling factor 10.

First Order Derivative Method

Calibration curves were plotted at 276 nm for TOL and 261 nm for DIC. The regression equations of the two drugs were determined using calibration curve equations. The concentration of TOL and DIC in test solution was determined from the respective regression equations.

Stability in Standard Solution

The standard solutions of TOL and DIC were analyzed after storing at 0, 1, 2, 4 and 6 hour at room temperature (25 ˚C) after preparation by the developed method.

Preparation of test solution from Tablet Powder

Twenty tablets were accurately weighed and average weight was calculated. The tablets were triturated to a fine powder. An accurately weighed quantity of powder equivalent to 15 mg TOL or 5 mg DIC was transferred to a 100 ml volumetric flask and sonicated with 10 ml methanol for 5 min. The volume was made up to 100 ml using distilled water. The solution was filtered through whatman filter paper no. 41 and 10 ml of the filtrate was diluted upto 100 ml with distilled water to obtain a solution having concentration of 15 µg/ml of TOL or 5 µg/ml of DIC. The absorbance of test solution was measured at selected wavelengths and the concentrations of the two drugs were estimated using first order derivative spectroscopic method.

Validation of the proposed method¹⁴

The proposed methods were validated according to the International Conference on Harmonization (ICH) guidelines.

Linearity

The calibration curves were plotted over a concentration range of 6-21 µg/ml and 2-7 µg/ml for TOL and DIC respectively. The absorbance of the solutions was measured at 261 and 276 nm against distilled water as blank. The calibration curves were constructed by plotting absorbance versus respective concentrations and the regression equations were calculated.

Reproducibility

The intraday and interday precision of the proposed methods was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days for 3 different concentrations of standard mixture solutions of TOL and DIC for the developed method.

Recovery Study

The accuracy of the method was determined by the standard addition method. Known amounts of standard solutions of TOL and DIC were added at 80%, 100% and 120 % level to prequantified test solutions containing 15 µg/ml of TOL and 5 µg/ml of DIC. The amounts of TOL and DIC were estimated by each method. The experiment was repeated 5 times for the developed method.

LOD and LOQ

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ /S

Where, σ = standard deviation of intercept of calibration curve and S = mean slope of 5 calibration curves.

RESULTS AND DISCUSSION

Determination of λ_max and ZCP

First order overlain spectra of TOL (15 µg/ml) and DIC (5 µg/ml) found to have zero crossing point at 261 nm and 276 nm respectively (Fig. 3). The absorbance of solutions was measured at 276 nm (ZCP of DIC) and at 261 nm (ZCP of TOL) for estimation of TOL and DIC, respectively. The calibration curves were constructed by plotting absorbance versus concentration and the regression equations were generated (Fig. 4 and 5). Therefore, it can be used for the simultaneous estimation of these drugs.

Fig. 3 First order overlain spectra of TOL (15 μg/ml) and DIC (5 μg/ml) (ZCP are indicated)

Fig. 4 Calibration curve of TOL at 276 nm

Fig. 5 Calibration curve of DIC at 261 nm

Stability in Standard Solution

Standard solutions of TOL and DIC were found to be stable in distilled water upto 6 hours of preparation when stored at room temperature (25˚ C).

Validation of the method

Results of validation studies are summarized in Table 1. The accuracy of the method was confirmed by recovery studies from tablet at 80%, 100% and 120% levels of standard additions and the results are depicted in Table 2. Recovery in the range of 100 – 101% justifies the accuracy of the method.

Analysis of Marketed Formulation

The method was applied to marketed tablet (TOLPIDOL-D) containing TOL 150 mg and DIC 50 mg. The results are depicted in Table 3. Results shows that this method can be successfully applied to marketed formulations.

Table 1 Summary of Validation Parameters

SR. NO.	PARAMETER	FIRST ORDER DERIVATIVE METHOD
SR. NO.	PARAMETER	TOL	DIC
1	Wavelength (nm)	276	261
2	Linearity (µg/ml)	6-21	2-7
3	Regression Equation
	Slope	-0.0214	0.0080
	Intercept	0.0014	-0.0010
4	Correlation Co-efficient (r²)	0.9997	0.9989
5	Precision (%RSD)
	Intraday (n=9)	0.38	1.18
	Interday (n=9)	0.37	1.04
6	LOD (µg/ml)	0.015	0.041
7	LOQ (µg/ml)	0.047	0.124

Table 2 Results of Recovery Studies

LEVEL	% RECOVERY
LEVEL	TOL	DIC
80% (n=5)	100.56	100.33
100% (n=5)	100.61	100.67
120% (n=5)	100.11	100.44

Table 3 Results of Analysis of Tablet

DRUG

FIRST ORDER DERIVATIVE METHOD

% ± SD (n=3)

TOL

100.18 ± 0.14

DIC

100.67 ± 0.25

CONCLUSION:

The results show that the developed spectrophotometric method is sensitive, precise and specific for simultaneous estimation of TOL and DIC. The proposed method is simple and eco-friendly compared to HPLC and HPTLC. The methods can be applied to determine TOL and DIC content in commercial combined tablet dosage forms.

ACKNOWLEDGEMENTS:

The authors are thankful to Themis Medicare Ltd., Haridwar, Uttarakhand and Torrent Pharma Ltd., Ahmedabad, Gujarat for providing TOL and DIC respectively for research. The authors are also thankful to The Principal, Maliba Pharmacy College for providing all the facilities to carry out the work.

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Received on 01.11.2012 Modified on 25.11.2012

Asian J. Research Chem. 5(12): Dec., 2012; Page 1448-1451