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.

Figure 1 Structure of Tolperisone hydrochloride

Figure 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 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., Ahmadabad, 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 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 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 to determine λ_max and isoabsorptive point of both the drugs.

Spectrometric Methods :

Method 1: Simultaneous Equation Method:

Calibration curves were plotted at 261 nm and 276 nm. The absorptivity coefficients of TOL and DIC were determined using calibration curve equations. Simultaneous equations were computed using absorptivity coefficients of TOL and DIC. The absorbance and absorptivity values at the particular wavelength were substituted in the following equations to obtain the concentration of test solution.

……………(Equation 1)

……………(Equation 2)

Where,

= concentration of TOL

= concentration of DIC

= Absorbance of sample at 261 nm and 276 nm respectively

= Absorptivity of TOL at 261 nm and 276 nm respectively

, = Absorptivity of DIC at 261 nm and 276 nm respectively

Criteria for obtaining maximum precision are that following ratios should lie outside the range 0.1-2.0 for precise determination of TOL and DIC.

and ……………(Equation 3)

Method 2: Q- Absorbance Ratio Method

Calibration curves were plotted at 261 nm and 293.5 nm. The absorptivity coefficients of TOL and DIC were calculated using calibration curve equations. The absorbance of test solution and absorptivity values at the particular wavelength were substituted in the following equations to obtain the concentration.

……………(Equation 4)

……………(Equation 5)

Where,

= concentration of TOL

= concentration of DIC

= Absorbance of test at 293.5 nm (isoabsorptive point)

= Absorptivity of TOL and DIC at 293.5 nm respectively

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 a mixture of 20 ml methanol and 30 ml water for 5 min. The volume was made up to 100 ml using 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 simultaneous equation method and absorbance ratio method.

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 three methods.

Validation of the proposed method¹⁴ :

The proposed methods were validated according to the International Conference on Harmonization (ICH) guidelines¹⁴.

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 for Simultaneous Equation Method and at 261 and 293.5 nm for Q-Absorbance Ratio Method against 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 all three methods.

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 9 µg/ml of TOL and 3 µg/ml of DIC. The amounts of TOL and DIC were estimated by each method. The experiment was repeated 5 times for both methods.

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 :

The overlain spectra of TOL (15 µg/ml) and DIC (5 µg/ml) were found to have λ_max at 261 nm and 276 nm respectively and isoabsorptive point was observed at 293.5 nm (Fig. 3). All these wavelengths are quite separated from each other. Therefore, it can be used for the simultaneous estimation of these drugs. The criteria for obtaining maximum precision by Simultaneous Equation Method (Equation 3) should be outside the range 0.1 to 2.0. Calculated results found to be outside the range 0.1-2.0 for both TOL and DIC. Therefore, Simultaneous Estimation Method can be applied for simultaneous estimation of TOL and DIC.

Figure 3 Zero order overlain spectra of TOL (15 μg/ml) and DIC (5 μg/ml) (λ_maxand isoabsorptive points are indicated) (Absorbance vs. wavelength graph)

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 methods was confirmed by recovery studies from tablet at 3 different levels of standard additions and the results are depicted in Table 2. Recovery in the range of 99 – 101% justifies the accuracy of both the methods.

Analysis of Marketed Formulation :

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

Table 1 Summary of Validation Parameters

Sr. No.	Parameter	Simultaneous Equation Method				Q-Absorbance Ratio Method
Sr. No.	Parameter	TOL		DIC		TOL		DIC
1	Wavelength (nm)	261	276	261	276	261	293.5	261	293.5
2	Linearity (µg/ml)	6-21	6-21	2-7	2-7	6-21	6-21	2-7	2-7
3	Regression Equation
	Slope	0.0547	0.0286	0.0210	0.0284	0.0547	0.0064	0.0210	0.0177
	Intercept	0.0045	0.0049	0.0094	0.0065	0.0045	0.0033	0.0094	0.0045
4	Correlation Co-efficient (r²)	0.9995	0.9994	0.9999	0.9999	0.9995	0.9994	0.9999	0.9998
5	Precision (%RSD)
	Intraday (n=9)	0.08		0.90		0.13		0.94
	Interday (n=9)	0.07		0.60		0.13		0.60
6	LOD (µg/ml)	0.030	0.035	0.016	0.012	0.030	0.052	0.016	0.019
7	LOQ (µg/ml)	0.091	0.105	0.048	0.035	0.091	0.157	0.048	0.057

Table 2 Results of Recovery Studies

Level	% Recovery
	Simultaneous Equation Method		Q-Absorbance Ratio Method
	TOL	DIC	TOL	DIC
80% (n=5)	100.00	99.33	100.44	99.67
100% (n=5)	100.72	99.17	100.67	99.33
120% (n=5)	100.11	99.89	100.22	99.33

Table 3 Results of Analysis of Tablet

Drug

Simultaneous Equation Method% ± SD (n=3)

Q-Analysis Method

% ± SD (n=3)

TOL

100.47 ± 0.14

100.38 ± 0.14

DIC

100.80 ± 0.43

100.80 ± 0.16

Statistical comparison of the developed methods :

Both the analytical methods were compared using statistical analysis. F-test was applied which did not show significant difference between the experimental values obtained by the three methods. The calculated values were found to be 0.95 and 0.25 for TOL and DIC respectively which are less than value in the F-table i.e. 4.06 at 5% level of significance.

CONCLUSION:

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

ACKNOLEDGEMENTS:

The authors are thankful to Themis Medicare Ltd., Haridwar, Uttarakhand and Torrent Pharma Ltd., Ahmadabad, 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.

REFERENCES:

1. The Merck Index, 14th Edition, Published by Merck research laboratories, Division of Merck and Co., INC., Whitehouse station, NJ, USA, 522, 1636.

2. The Martindale: The Complete Drug Reference, Sweetman SC, 35^th Edition, Pharmaceutical Press, London, UK, 38, 1738.

3. Japanese Pharmacopoeia, The ministry of Health, Labour and Welfare, Prefectural office in Japan, 15th edition, 2006, 1190.

4. Jagathi V, Shaiba M, Raghavi K, “Spectrophotometric methods for the determination of Tolperisone”, IJPI’s Journal of Analytical Chemistry, 2011, 1(2), 37-39.

5. Praveen S, Anupama P, Jagathi V, “Spectrophotometric determination of Tolperisone using 2, 4-dinitrophenylhydrazine reagent”, Indian Journal of Research and Pharmaceutical Science, 2010, 1(3), 317-320.

6. Nimila IC, Balan P, Chiranjeevi N, “Method development and statistical validation of UV spectrophotometric method for tolperisone hydrochloride in bulk and tablet dosage form”, Journal of Pharmacy Research, 2011, 4(5), 1356-1357.

7. Jagathi V, Shaiba M, Raghavi K, “Assay of tolperisone by extractive spectrophotometry”, Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2010, 1(3), 654-657.

8. Liawruangrath S, Liawruangrath B, “High performance thin layer chromatographic determination of tolperisone hydrochloride”, Journal of Pharmaceutical and Biomedical Analysis, 1999, 20(1), 401-404.

9. Jung-Woo B, Young-Seo P, Uy-Dong S, “HPLC Determination of tolperisone in human plasma”, Archives of Pharmaceutical Research, 29(4), 339-342.

10. Murali M, Satyanarayana PVV, “Simple validated isocratic RP –HPLC method for estimation of Tolperisone in bulk and pharmaceutical dosage form”, Scholars Research Library, Der Pharma Chemica, 2011, 3(5), 13-19.

11. Dhaneshwar SR, Bhusari VK, “Validated HPLC Method for Simultaneous Quantitation of Diclofenac Sodium and Misoprostol in Bulk Drug and Formulation”, Pelagia Research Library, 2010, 1, 110-118.

12. Kubala T, Gambhir B, Borst SI, “A specific stability indicating HPLC method to determine Diclofenac Sodium in raw materials and pharmaceutical solid dosage forms”, Informa Healthcare, 1993, 19, 749-757.

13. Shah SA, Savale SS, Shishoo CJ, “Sensitive HPTLC method for monitoring dissolution profiles of Diclofenac from different tablets containing combined Diclofenac and Acetaminophen”, JPC - Journal of Planar Chromatography - Modern TLC, 2003, 16, 36-44.

14. ICH, Q2 (R1) Validation of Analytical Procedure: Text and Methodology, International Conference on Harmonization, Geneva, Switzerland; 2005.

Received on 15.12.2012 Modified on 01.01.2013

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 187-190