INTRODUCTION:

Tolperisone Hydrochloride, chemically known as 2-methyl-1-(4-methylphenyl)-3-piperidin-1-ylpropan-1-one hydrochloride, is an centrally acting muscle relaxant. Etoricoxib, chemically known as 5-chloro-2-(6-methylpyridin-3-yl)-3-(4-(methylsulfonyl)-phenyl] pyridine, is a non-steroidal anti-inflammatory drug used to relieve pain and inflammation^1,2.

Literature survey reveals that LC methods are available for estimation of Tolperisone³ and Etoricoxib⁴ as well as UV methods for determination of Tolperisone with other drugs⁵. Large number of spectrophotometric methods available for Etoricoxib determination alongwith other drugs^6-11. To best of our knowledge, we could not found multicomponent estimation of Tolperisone and Etoricoxib together by spectrophotometry. The purpose of present work describes the development of a simple, precise, accurate and economic spectrophotometric methods for the simultaneous estimation of Tolperisone and Etoricoxib in fixed dose combination¹¹. The developed method was validated in accordance with ICH guidelines^12-22 and successfully employed for the assay of Tolperisone and Etoricoxib in fixed dose combination.

a) b)

Fig. 1: Structure of a) Tolperisone and b) Etoricoxib

Theory²¹

Simultaneous equation method is used where a sample contain two absorbing drugs; each of which absorbs at the λ_max of each other i.e. λ₁ and λ₂, it may be possible to determine both the drugs by the technique of simultaneous equation method provided that certain criteria apply.

Where,

Cx and Cy = Concentration of Tolperisone and Etoricoxib in sample solution.

A₁ and A₂ = absorbance of sample solution at wavelength 1 and 2.

ay_l and ay₂= absorptivity of Tolperisone at wavelength 1 and 2 of standard solution.

ax₁and ax₂= absorptivity of Etoricoxib at wavelength 1 and 2 of standard solution.

Absorption ratio method is modification of simultaneous equation method. It is based upon fact that the ratio of absorbance at any two wavelengths is constant value independent of concentration or pathlengths. For absorption ratio method, isobestic point (where absorptivity of both drugs remains constant) is considered as (λ1) and the maximum absorbance (λmax) of Etoricoxib is considered as (λ2).

Where,

Cx and C_Y - Concentrations of Tolperisone and Etoricoxib, respectively.

Qx - Ratio of absorptivity of Etoricoxib at λ1 and λ2nm.

QY - Ratio of absorptivity of TPS at λ1 and λ2nm.

Qm - Ratio of absorbance of sample solution at λ1 and λ2nm.

A - Absorbance of sample solution at Isobestic point.

ax₁or ay₁ – absorbtivity of Tolperisone and Etoricoxib at isobestic wavelength First Derivative spectrum (D¹) is a plot of the rate of change of absorbance with wavelength against wavelength, i.e plot of ΔA/Δλ vs λ. The first order derivatives for Tolperisone and Etoricoxib, were obtained from zero order spectra and measurements are done as zero crossing point of other drug.

MATERIALS AND METHODS:

Instrumentation:

A double beam UV/Visible spectrophotometer (Shimadzu 1780) with matching pair of 1cm quartz cells was used to record all spectra. Band width was kept 1 nm and scanning was carried at speed of 200nm/min. UV Probe software was used to do all data collection. MS-Excel was used to do all statistical calculations.

Reagents and Materials:

Tolperisone and Etoricoxib active pharmaceutical ingredients were kindly gifted by United Pharmatech Pvt Ltd, Roorkee, Uttarakhand, India. Methanol AR Grade was procured from S. D. Fine Chemicals Ltd., Mumbai, India.

Preparation of Standard Stock Solution:

Stock solution of Tolperisone and Etoricoxib were prepared by dissolving accurately weighed 10mg of standard drugs in 10ml of methanol, separately. The concentration of Tolperisone and Etoricoxib were 1000 μg/ml from which further 1 ml was pipetted and diluted to 10ml to achieve concentration of 100μg/ml of Tolperisone and Etoricoxib, respectively.

Methodology:

The working standard solutions of Tolperisone and Etoricoxib were prepared separately in methanol having concentration of 10μg/ml by dilution of stock solution. They were scanned in the wavelength range of 200-400 nm against solvent methanol as blank. The absorption spectra thus obtained were derivatised to first order. From the overlain spectra of both the drugs (Fig. 2) drugs show maximum absorbance at 255nm (λ1) for Tolperisone and 275nm (λ2) for Etoricoxib in methanol so these wavelengths were selected for further analysis by simultaneous equation method. For absorbance ratio method 245nm was selected as λ1 (isobestic point) and 275nm as λ2. For first order derivative spectroscopy wavelengths selected for quantitation were 275nm for Tolperisone (zero cross point of Etoricoxib) and 315nm for Etoricoxib (zero cross point of Tolperisone). The overlain first derivative spectra of Tolperisone and Etoricoxib is given in Fig. 3.

Fig. 2: Overlain zero order spectra of Tolperisone and Etoricoxib (10 μg/ml each)

Fig. 3: Overlain first order derivative spectra of Tolperisone and Etoricoxib (10 μg/ml each)

Validation of Proposed Method:²²

The proposed method was validated according to the ICH Q2 (R1) guidelines.

Linearity:

Accurately measured volumes of standard stock solutions (Concentration 100μg/ml) of both drugs (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0ml for Tolperisone and 0.2, 0.4, 0.6, 0.8, 1 and 1.2ml for Etoricoxib) were transferred to a series of 10ml of volumetric flasks and diluted to the mark with methanol. The calibration curves were plotted over a concentration range of 5-30μg/ml for Tolperisone and 2-12μg/ml for Etoricoxib. The calibration curves were constructed by plotting absorbance versus concentrations and the regression equations were calculated.

Precision:

The precision of the method was demonstrated by intra-day (repeatability) and inter-day (intermediate precision) variation studies. For the intra-day studies, 6 replicates at assay concentrations (10μg/ml for Tolperisone and 4 μg/ml for Etoricoxib) were analyzed in a day and percentage relative standard deviation (%RSD) was calculated. For the inter day variation studies, 3 different concentrations (10, 20 and 30μg/ml for Tolperisone and 4, 8 and 12μg/ml for Etoricoxib) were analyzed on 3 consecutive days and %RSD was calculated. The results obtained for and inter day variations were found to be within limits (less than 2% RSD).

Analysis of Marketed Formulation (Assay):

Twenty tablets (Tolpevac ET, Prevego Healthcare and Research; labelled to contain Tolperisone 150mg and Etoricoxib 60mg) were and powdered. The quantity of tablet powder equivalent to 10mg of Tolperisone was weighed, dissolved in 10ml of methanol and filtered. The 1ml of filtrate (concentration 1000μg/ml of Tolperisone) was further diluted to 10ml (concentration 100μg/ml of Tolperisone). This Solution was further diluted to get 10 μg/ml of Tolperisone (4μg/ml of Etoricoxib). Zero order absorbance spectra were recorded and further converted to first derivative absorbance spectra and quantification was done by respective methods. The analysis procedure was repeated six times.

Accuracy Study:

The accuracy of the method was determined by calculating recovery of Tolperisone and Etoricoxib by the standard addition method. Known amounts of standard solutions of Tolperisone and Etoricoxib were added at 50, 100 and 150% level to prequantified sample solutions of both drugs (10μg/ml for Tolperisone and 4 μg/ml for Etoricoxib). The experiment was repeated for three times.

Limit of Detection and Limit of Quantification:

LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S, respectively; where σ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot.

RESULTS AND DISCUSSION:

Three spectrophotometric methods viz Simultaneous Equation Method, Absorbance Ratio Method and First Derivative Method were developed. The developed methods were validated as per ICH guidelines. Drugs obey the Beers-Lambert’s law at these selected wavelengths of maximum and found to be linear in the range of 5-30µg/ml for Tolperisone while 2-12µg/ml for Etoricoxib. The precision was carried out at two level viz intra-day and inter-day. Recovery study was carried out on the developed method and the recovery was found to be in the range of 99.0–101.0%. Robustness of the methods was checked by change in wavelength of analysis. The results are summarized in Table 1 and Table 2.

Table 1: Validation Results for Tolperisone HCl

Sr. No.	Validation Parameter	Results of Tolperisone HCl
Sr. No.	Validation Parameter	SEM	ARM	FD
1.	Linearity	y = 0.0081x + 0.0419 R² = 0.9968 @ 255 nm y = 0.0874x - 0.1218 R² = 0.9857 @ 275 nm	y = 0.127x + 0.045 R² = 0.999 @ 245 nm (Isobestic λ)	y = -0.0357x + 0.0713 R² = 0.9985 @ 275 nm (ZCP of ETO)
2.	Range (µg/ml)	5 - 30	5 - 30	5 - 30
3.	Assay (Mean ± % RSD)	99.941 ± 0.472	100.147± 0.253	99.906 ± 0.195
4.	Precision	%RSD	%RSD	%RSD
	Intraday precision	0.190 – 0.843 %	0.318 – 0.747 %	0.544 – 0.787
	Interday precision	0.300 – 0.816 %	0.235 – 0.496 %	0.302 – 0.529
5.	Accuracy	Mean % Recovery ± RSD
	50%	99.952± 0.773	99.587± 0.717	100.072± 0.333
	100%	100.275± 0.286	100.043± 0.385	100.089± 0.176
	150%	100.310± 0.422	99.696± 0.456	100.353± 0.466
6.	LOD	0.387 µg/ml	0.682 µg/ml	0.182 µg/ml
7.	LOQ	1.171 µg/ml	2.067 µg/ml	0.551 µg/ml
8.	Robustness	Robust	Robust	Robust

Table 2: Validation Results for Etoricoxib

Sr. No.	Validation Parameter	Results of Etoricoxib HCl
Sr. No.	Validation Parameter	SEM	ARM	FD
1.	Linearity	y = 0.0369x + 0.0172 R² = 0.9956 @ 255 nm y = 0.0434x + 0.0157 R² = 0.9955 @ 275 nm	y = 0.127x + 0.045 R² = 0.999 @ 245 nm (Isobestic λ)	y = -0.0084x + 0.0046 R² = 0.9975 @ 315 nm (ZCP of TOL)
2.	Range (µg/ml)	2 - 12	2 - 12	2 - 12
3.	Assay (Mean ± %RSD)	100.117±0.362	99.719±0.582	100.238 ± 0.280
4.	Precision	%RSD	%RSD	% RSD
	Intraday precision	0.275 – 0.408%	0.213 – 1.012%	0.341 – 0.670%
	Interday precision	0.284 – 1.195%	0.228 – 0.603%	0.236 – 0.527%
5.	Accuracy	Mean % Recovery ± RSD
	50%	99.927 ± 0.099	99.608 ± 0.206	100.698 ± 0.318
	100%	100.016 ± 0.629	100.187 ± 0.513	99.865 ± 0.651
	150%	99.708 ± 0.739	99.944 ± 0.161	99.610 ± 0.250
6.	LOD (µg/ml)	0.350	0.373	0.223
7.	LOQ (µg/ml)	1.059	1.129	0.676
8.	Robustness	Robust	Robust	Robust

CONCLUSION:

Based on the results, obtained from the analysis of described methods, it can be concluded that the methods are simple, economic and robust. The results of the analysis of fixed dose combination by the proposed method are reproducible and reliable. The additives usually present in the pharmaceutical formulation of the assayed sample did not interfere with determination of Tolperisone and Etoricoxib. The method can be used for the routine analysis of these drugs in pharmaceutical formulation.

CONFLICT OF INTEREST:

All authors declare that there is no Conflict of Interest.

REFERENCES:

1. Indian Pharmacopoeia, 8^th Edition, 2018, Volume II, Pg no. 2021, 2022.

2. Japanese Pharmacopoeia, 18^th edition, 2021.

3. Chhalotiya U K, Bhatt K.K, Shah D A, Baldania S L, Patel S B. Development of stability indicating method LC for the estimation of Tolperisone in bulk and pharmaceutical dosage form. Journal of Applied Chemistry. 2013; 1:1-7.

4. Hartman R, Abrahim A, Clausen A, Mao B, Crocker LS, Ge Z. Development and validation of an HPLC method for the impurity and quantitative analysis of etoricoxib. J. Liquid Chrom. and Related Tech. 2003; 26(15): 2551-66.

5. Patel MG, Parmar RR, Nayak PP, Shah DA. The simultaneous estimation of Paracetamol and Tolperisone Hydrochloride in tablet by UV Spectrophotometric methods. Journal of pharmaceutical science and Bioscientific Research. 2012; 2(2): 63-67.

6. Singh S, et al. A simple Ultraviolet spectrophotometric method for the determination of etoricoxib in dosage formulations. Journal of Advanced Pharmaceutical Technology and Research. 2012; 3(4): 237-240.

7. Kutty S V, Haribabu Y, Akhil M, Nihila K. Method development and validation of simultaneous estimation of pregabalin and etoricoxib in bulk and pharmaceutical dosage form by UV-Visible spectroscopy. Indian Drugs. 2023; 60(7): 93-95.

8. Pratima K Syal, Madhusmita Sahoo, Dipali M Mehendre, Snehal S Ingale, Vishnu P Choudhari, Bhanudas S Kuchekar. Simultaneous Spectrophotometric Determination of Drotaverine and Etoricoxib in Combined Tablet Dosage Form by Ratio Derivative and Absorption Corrected Method and its Application to Dissolution Study. Research Journal of Pharmacy and Technology. 2011; 4(2): 298-301.

9. Kumari N, et al. Development and validation of an analytical method for the simultaneous estimation of Etoricoxib and Thiocolchicoside in tablet dosage form by UV Spectrophotometric method. Research Journal of Pharmacy and Technology. 2022; 15(7): 3051-3056.

10. Kamal M F, et al. Simultaneous MEKC-DAD and smart spectrophotometric assays of thiocolchicoside and etoricoxib in challenging concentration ratio mixtures. Reviews in Analytical Chemistry. 2022; 41(1): 137- 145.

11. Poonam H. Chaube, Santosh V. Gandhi, Padmanabh B. Deshpande, Veena G. Kulkarni. High Performance Thin Layer Chromatographic Analysis of Paracetamol and Etoricoxib in Spiked Human Plasma. Research J. Pharm. and Tech. 2011; 4(8): 1303-1306.

12. Santosh V. Gandhi, Parag L. Khairnar1, Atul P. Chaudhari. Application of Multivariate Calibration Methods for Simultaneous Determination of Drugs in Fixed Dose Combination.Asian J. Pharm. Ana. 2018; 8(1):01-06.

13. Santosh V. Gandhi, Pratidnya S. Sonawane. Chemometric–Assisted UV Spectrophotometric Method for Determination of Cefixime Trihydrate and Cloxacillin Sodium in Pharmaceutical Dosage Form. Asian J. Research Chem. 2018; 11(4): 705-709.

14. Mahima R Sengar, Santosh V Gandhi, Vivek Rajmane, Upasana P Patil, Bhargav B Gandhi. Simultaneous Determination of Cefuroxime Axetil and Potassium Clavulanate in Tablet Dosage Form by Spectrophotometry. Research J. Pharm. and Tech. 2010; 3(1): 251-253.

15. Nilesh V. Gaikwad, Padmanabh B. Deshpande, Santosh V. Gandhi, Kapil K. Khandagale. High Performance Thin Layer Chromatographic Determination of Spironolactone and Torsemide in Combined Tablet Dosage Form. Research J. Pharm. and Tech. 2010; 3 (4): 1106-1108.

16. Vandana S. Bhavnani, Padmanabh B. Deshpande, Santosh V. Gandhi, Prajakta Pawar, Ashish K. Gaikwad. High Performance Thin Layer Chromatographic Determination of Metoprolol Succinate and Olmesartan in Combined Capsule Dosage Form. Research Journal of Pharmacy and Technology, 2012; 5(11): 1461-1464.

17. Vandana S. Bhavnani, Padmanabh B. Deshpande, Santosh V. Gandhi, Prajakta Pawar, Ashish K. Gaikwad. High Performance Thin Layer Chromatographic Determination of Metoprolol Succinate and Olmesartan in Combined Capsule Dosage Form. Research J. Pharm. and Tech. 2012; 5(11): 1461-1464.

18. Abhijit D. Dhiware, Padmanabh B. Deshpande, Santosh V. Gandhi, Sujith Nair, Suvarna Vanjari. A Simple and Sensitive RP-HPLC Method for Simultaneous Estimation of Atorvastatin Calcium, Ezetimibe and Fenofibrate in Combined Tablet Dosage Form. Research J. Pharm. and Tech. 2013; 6(10): 1085-1088.

19. Anamika Singh, Jaya Dwivedi, Santosh V. Gandhi. Development and Validation of Stability-Indicating RP-HPLC Method for Determination of Metoprolol Succinate and Olmesartan Medoxomil in Bulk and in Formulation. Research J. Pharm. and Tech. 2014; 7(12): 1368-1373.

20. Santosh V. Gandhi, Deepak Patil, Atul A. Baravkar. Comparison of Chemometric assisted UV Spectrophotometric and RP-HPLC Method for the simultaneous determination of Ofloxacin and Tinidazole in their Combined dosage form. Research Journal of Pharmacy and Technology. 2021; 14(11):5713-8.

21. Practical Pharmaceutical Chemistry, 4^th edition, Part Two, A.H. Beckett and J.B. Stenlake, 275-300

22. ICH Guideline for Validation of Analytical Procedures: Text and Methodology Q2 (R1), 2005.

Received on 29.07.2025 Revised on 30.08.2025

Accepted on 25.09.2025 Published on 18.10.2025

Available online from November 03, 2025

Res. J. Pharma. Dosage Forms and Tech.2025; 17(4):257-261.

DOI: 10.52711/0975-4377.2025.00035

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