INTRODUCTION:

Tolperisone Hydrochloride is approved by FDA as a generic drug manufacturing for regulatory market and non-regulated market to Taj Pharmaceuticals, Mumbai on 7^th October 2010. It is piperidine derivative centrally acting muscle relaxant drug. Generic formulations of this drug are available in market now. Tolperisone acts at the level of spinal cord by blocking sodium channels and calcium channels. It exerts spinal reflex inhibitory action predominantly via a pre synaptic inhibition of the transmitter release from the primary afferent endings via a combined action on voltage-gated sodium and calcium channels.

It causes preferential antinociceptive activity against thermal stimulation that is likely to be attributed to its local anesthetic action. It well absorbed in the stomach, duodenum and jejunum. It contain below structure:

Its IUPAC name is 1-(4-methyl-phenyl)-2-methyl-3-(1-piperidino)-1-propanone hydrochloride. Its molecular weight and molecular weight are C₁₆H₂₃NO · HCl and 281.82 g/mole respectively. It is Very soluble in acetic acid, Freely soluble in acetone and water, Soluble in acetic anhydride, Slightly soluble in acetone, Partially insoluble in diethyl ether. IUPAC name of Diclofenac sodium is Sodium{2-[(2,6-dichlorophenyl) amino] phenyl}acetate and it structure is define as below:

Its molecular formula and molecular weight are C₁₄H₁₀Cl₂NNaO₂and 318.13 g/mole. It is Very slightly soluble in cold water, considerably more soluble in hot water soluble in ethanol and ethyl acetate, very slightly soluble in chloroform, slightly soluble in ether, practically insoluble in petroleum ether, pentane and benzene.^[1-2]Up to now some analytical methods developed on Tolperisone, HCL and Diclofenac Sodium. But no stability indicting method developed by HPLC and here developed method was validated by ICH guideline.^[3-25]

MATERIALS AND METHODS:

Establishment of the Optimum Conditions of the Method:

In chromatographic methods, various conditions are used for the development of method. Various chromatographic conditions used during the development of analytical method should be optimizing for developing simple, sensitive, accurate and precise method. The parameters which are optimized are as follows:

Selection of Solvent:

Diclofenac Sodium and Tolperisone Hydrochloride is freely soluble in the methanol. Hence, methanol is selected as solvent for the drug.

Selection of Column for Diclofenac Sodium and Tolperisone Hydrochloride:

Diclofenac Sodium and Tolperisone Hydrochloride is soluble in methanol, hence polar in nature. So, C₁₈ column is suitable for analysis. The column used is Kromacil RP-C18 column (150mm×4.6mm, 5µm).

Selection of Wavelength for Detection:

The optimum wavelength for detection was set at 275nm for Diclofenac Sodium and 255nm for Tolperisone Hydrochloride which gives maximum resolution and minimum tailing.

Selection of Mobile Phase:

The column was allowed to saturate with the mobile phase. Various mobile phases comprising of Methanol, Acetonitrile and Water were tried in different compositions at different-different flow rates and different pH to achieve sharp peak of Diclofenac Sodium and Tolperisone Hydrochloride.

Optimization of pH:

pH is most effective factor in the HPLC as it affects the retention time, resolution and intensity of the peak of drugs. Hence, various pH range are tried to get the best pH for the selected mobile phase. pH range for trial is selected from the pKa of the drugs. After trying the various pH range the best pH for the selected mobile phase was 3.0. At this pH it gives the robust result and fulfills all the criteria.

Optimization of Flow Rate:

Flow rate is also very important during the method development of HPLC. As it affects the retention time and tailing factor. So, flow rate must be optimized. Mobile phase is also checked for the result at various flow rates like 0.5 ml/min, 0.75 ml/min, and 1ml/min. It was found that flow rate 0.5 ml/min gives the suitable result at selected mobile phase. Hence, 0.5 ml/min is selected as flow/rate for this particular RP-HPLC method. Mixture of Methanol: Acetonitrile: water(80: 16: 4 v/v/v) Flow rate: 0.5 ml/min (pH-3 adjust with orthophosphoric acid) was selected as mobile phase because all system suitability parameters were fulfill, that is tailing was less than 2, number of theoretical plates were greater than 2000 and retention time of analyte was 6.19 min.

Preparation of solutions:

Preparation of Mobile Phase:

In separate bottle methanol (80ml), acetonitrile (16ml) and water (4ml) were sonicated for 10 min and filter through 0.2 µm syringe filter and these components are used as mobile phase in binary gradient mode.

Preparation of Standard Stock Solution:

Weigh accurately 15mg of Tolperisone HCl and 5mg of Diclofenac sodium and transferred to 10 ml volumetric flask. Methanol (HPLC GRADE) was added to dissolve the drug and final volume was made with the same solvent to obtain a concentration 1500 µg/ml for Tolperisone HCl and 500µg/ml for Diclofenac sodium, these solutions were used for analysis.

Preparation of Sample Solution:

Weigh tablet powder equivalent to 15 mg of TolperisoneHCland5mg Diclofenac sodium transferred to 10 ml volumetric flask. Methanol (HPLC GRADE) was added to dissolve the drug and final volume was made with the same solvent to obtain a concentration 1500 µg/ml for Tolperisone HCl and 500µg/ml for Diclofenac sodium and this solution was used for analysis.

RP-HPLC method validation parameters:

Linearity:

Linearity of Tolperisone HCl and Diclofenac Sodium was performed over a concentration range of 0.15, 1.5, 15 150 and 1500µg/ml for Tolperisone HCl and 0.05, 0.5, 5.0, 50 and 500µg/ml for Diclofenac sodium. Solutions for analysis were prepared by serial dilution of stock solution (tolperisone HCl 15000µg/ml and Diclofenac sodium 5000µg/ml) by taking 1 ml from stock solution and dilute up to 10 ml with mobile phase. These solutions were injected in column to get the chromatogram.

Accuracy:

Accuracy is done to check that whether the method gives the accurate result or not. Accuracy is also termed as % Recovery. Accuracy is measured by standard addition method. The solutions were made from the tablet in such a manner that it contains 30 µg/ml of TolperisoneHCland10 µg/ml of Diclofenac sodium Then 80%, 100% and 120% of API were added to the solutions and then chromatograms were taken and finally the % recovery was calculated. This experiment was performed three times for continuous evaluation.

Precision:

Repeatability:

The final solution containing30 µg/ml of TolperisoneHCland10 µg/ml of Diclofenac sodium was prepared and injected 6 times into the column to get the chromatogram. The peak area were measured of the chromatograms and % RSD was calculated.

Intraday Precision:

For Intraday Precision, three aliquot of standard stock solutions were prepared containing final concentration 15, 30 and 45µg/ml for Tolperisone HCl and 5, 10 and 15 µg/ml Diclofenac sodiun were injected three times in a same day. % RSD was calculated was calculated for both the drug.

Interday Precision:

For Interday Precision, three aliquot of standard stock solutions were prepared containing final concentration 15, 30 and 45 µg/ml for Tolperisone HCl and 5, 10 and 15 µg/ml Diclofenac sodium were injected three times in on different day. % RSD was calculated was calculated for both the drug.

System Suitability Test Parameters (n= 6):

A system suitability test of the chromatographic system was performed before each validation run to ensure the suitability of the method whenever it will be used for estimation. Six replicate injections of standard preparation were injected and retention time, theoretical plate and tailing factor were determined in terms of % RSD for the same. Acceptance criteria for system suitability test that is of retention time between 2-10 minutes is suitable, theoretical plates not less than 2000 and tailing factor not more than 2.0.

Assay:

Assay of dosage form is the one of critical quality parameter to assure the quality of dosage form. Some interference of the excipients with the peaks of interest appeared; hence the proposed is applicable for the routine estimation of Tolperisone HCl and Diclofenac Sodium in pharmaceutical dosage forms. The method was successfully applied for the estimation of contents in the tablet. 30 µg/ml of TolperisoneHCland10 µg/ml of Diclofenac sodium were used for measurement of peak area and the result is expressed in terms of % purity of drug substances.

Specificity:

The specificity of the method was determined by checking the interference of placebo with analyte. The chromatograms of blank, mobile phase and placebo do not show any interference at the retention time of Tolperisone HCl and Diclofenac Sodium as it can be seen from respective chromatograms. Only one peak of Tolperisone HCl and Diclofenac Sodium were obtained in the spectral evaluation which indicates that there was no interference from excipients in the proposed method. Further, peak purity data clearly shows that there is no any other interference in the developed method.

Robustness:

It means some deliberate changes have been made to the method parameter and check any significant changes obtained in the results or not. If not than the method is robust. Here, pH and wavelength were changed to check the robustness of the developed method.

Limit of Detectionand Limit of Quantification:

LOD: It is the lowest amount of analyte in a sample that can be detected but not necessarily quantities under the stated experimental conditions. Limit of detection can be calculated using following equation as per ICH guidelines.

LOD=3.3×N/S

LOQ: It is the lowest concentration of analyte in a sample that can be determined with the acceptable precision and accuracy under stated experimental conditions. Limit of quantification can be calculated using following equation asper ICH guidelines.

LOQ=10×N/S

Where, N is the standard deviation of the peak areas of the drug and S is the slope of the corresponding calibration curve.

Stability study degradation studies by RP-HPLC method:

All degradation studies were done at a drug concentration of 150 µg/ml for Tolperisone HCl and 50 µg/ml for Diclofeanc sodium.

Forced Degradation under Acidic Condition:

To 1 ml of stock solution, 1 ml of 0.1 N HCl was added in a 10 ml of volumetric flask and volume was brought up to the mark with methanol. The volumetric flask was kept under normal condition for 12 hours for degradation. After 12hours, 1 ml of solution was withdrawn and neutralize with 0.1 N NaOH. Resulting solution was analyzed for acid degradation study.

Forced Degradation under Alkaline Condition:

To 1 ml of stock solution, 1 ml of 0.1 N NaOH was added in a 10 ml of volumetric flask and volume was brought up to the mark with methanol. The volumetric flask was kept under normal condition for 12 hours for degradation. After 12hours, 1 ml of solution was withdrawn and neutralize with 0.1 N HCl. Resulting solution was analyzed for alkaline degradation study.

Forced Degradation under Oxidative Condition:

To 1 ml of stock solution, 1 ml of 3 % hydrogen peroxide was added in a 10 ml of volumetric flask and volume was brought up to the mark with methanol. The volumetric flask was kept under normal condition for 12 hours for degradation. After 12 hours, solution was heated in a boiling water bath to remove excess hydrogen peroxide. Resulting solution was analyzed for oxidative degradation study.

Forced Degradation under Thermal Condition:

For thermal decomposition drug powder was kept at 60°Cfor 24 hours. From that powder solution having concentration of 150µg/ml for Tolperisone HCl and 5µg/ml for Diclofenac Sodium were prepared and analyzed for thermal degradation study.

Forced Degradation under Direct Sunlight:

A sample of drug were exposed to a direct sunlight. Drugs were kept in petridish for time period of 48 hours. From that powder solution having concentration of 150µg/ml for Tolperisone HCl and 5 µg/ml for Diclofenac sodium were prepared and analyzed for sunlight degradation study.

RESULT AND DISCUSSION:

Method development (HPLC):

For method development of Tolperisone HCl and Diclofenac sodium by HPLC various factors are optimized. These Optimized factors.

Table 1: RP-HPLC optimized chromatographic conditions

Parameters	Chromatographic condition
Mode of elution	Gradient
Mobile phase	Methanol : Acetonitrile: Water (80: 16: 4) adjust pH 3.0 with ortho phosphoric acid
Column	KROMACILC₁₈ (250 mm × 4.6 mm, 5μ)
Flow rate	0.5 ml/min
Run time	10 min
Injection volume	20μl
Detection wavelength	275 nm
Retention time	Tolperisone Hydrochloride : 2.93min Diclofenac Sodium : 4.2min
Temperature	25 ±2 °C

Table 2: List of Mobile Phase Trials in RP-HPLC

Sr. No.	Mobile Phase composition	Conclusion
1	Methanol : Acetonitrile (50:50 v/v) Flow rate : 1 ml/min	Tailing was observed in Tolperisone HCl
2	Methanol : Acetonitrile (90:10 v/v) Flow rate: 1 ml/min	Tailing was observed in Tolperisone HCl
3	Methanol : Acetonitrile (80:20 v/v) Flow rate: 1 ml/min	Less Tailing was observed in Tolperisone HCl as compare to previous one
4	Methanol : Acetonitrile (80:20 v/v) Flow rate: 0.5ml/min	Tailing was observedinTolperisoneHCl
5	Acetonitrile : 0.1% Triethylamine (35:65 v/v) Flow rate: 1 ml/min (pH-5 with orthophosphoric acid)	Poor chromatography (blunt peak) was observed for TolperisoneHCl
6	Acetonitrile : Ammonium Acetate Buffer(0.1% Triethyl amine) (30:70 v/v) Flow rate: 1 ml/min (pH-5 with orthophosphoric acid)	Poor chromatography (Broad peak) was observed for TolperisoneHcl
7	Methanol : Acetonitrile : water (50:40:10 v/v/v) Flow rate: 1 ml/min	Tailing was observed in Tolperisone HCl
8	Methanol : Acetonitrile : water (40:40:20 v/v/v) Flow rate: 1 ml/min	Tailing was observed in Tolperisone HCl
9	Methanol : Acetonitrile : water (60:30:10 v/v/v) Flow rate: 1 ml/min	Tailing was observed in Tolperisone HCl
10	Methanol : Acetonitrile : water (70:20:10 v/v/v) Flow rate: 1 ml/min	Tailing observed in Tolperisone HCl
11	Methanol : Acetonitrile : water (70:20:10 v/v/v) Flow rate: 1 ml/min	Tailing observed in Tolperisone HCl
12	Methanol : Acetonitrile : water(50:25:25 v/v/v) Flow rate: 1 ml/min	Tailing observed in Tolperisone HCl
13	Methanol : Acetonitrile: water (70:30:10v/v/v) Flow rate: 0.5 ml/min	Tailing observed in Tolperisone HCl
14	Methanol : Acetonitrile: water (80:10:10v/v/v) Flow rate: 0.7 ml/min	Tailing observed in Tolperisone HCl
15	Methanol : Acetonitrile: water (80:10:10 v/v/v) Flow rate: 0.5 ml/min	Tailing observed in Tolperisone HCl
16	Methanol: Acetonitrile: water (80:10:10 v/v/v) Flow rate: 0.5 ml/min (adjust pH-5 with orthophosphoric acid)	Less Tailing observed in Tolperisone HCl
17	Methanol: Acetonitril: water (80:16:4 v/v/v) Flow rate: 0.5 ml/min (adjust pH-4 with orthophosphoric acid)	Less Tailing observed in Tolperisone HCl but not satisfactory
18	Methanol :Acetonitrile: water(80:16:4 v/v/v) Flow rate: 0.5 ml/min (adjust pH-3 with orthophosphoric acid)	No tailing was observed for any drugs and resolution was good

Table 3: Linearity data of Tolperisone HCl by HPLC

Sr.No	Concentration (μg/ml)	Area
1	0.15	8811
2	1.5	25377
3	15	149358
4	150	832019
5	1500	6680165

Table 4: Linearity data of Diclofenac Sodium by HPLC

Sr.No.	Concentration(μg/ml)	Area
1	0.05	6472
2	0.5	18410
3	5	109496
4	50	606639
5	500	4911852

Precision:

Repeatability:

Repeatability was done to check the difference between results under the same chromatographic condition. Repeatability data of Tolperisone HCl and Diclofenac Sodium are given in table 5.7 and 5.8 respectively. %RSD of Tolperisone HCL and Diclofenac Sodium was found to be 0.021 and 0.034. All parameters are less than 2. This clearly indicates that proposed method is precise.

Table 5 Repeatabilitydata of TolperisoneHCl by HPLC

Sr. No.	Concentration (μg/ml)	Mean± SD	RSD
1	150	839333± 18014.14	0.02

N= 6

Table 6 Repeatability data of Diclofenac Sodium by HPLC

Sr.No.	Concentration (μg/ml)	Mean± SD	RSD
1	50	624113±19643	0.03

Intraday Precision:

% RSD of Tolperisone HCl (15 μg/ml) for intraday precision was found to be 1.27, 1.21, and 1.16 for and 1.39, 1.53 and 1.46 for Diclofenac Sodium which is given in table 5.9 and table 5.10 for Tolperisone HCl and Diclofenac Sodium respectively. All parameters are less than 2. This clearly indicates that proposed method is precise.

Table: 7 Intraday Precision of Tolperisone HCl by HPLC

Sr. no	Time duration
Sr. no	1^sthour	2^nd hour	3^{rd hour}
Mean	147696.8 ±1884.12	148083.2 ±1794.27	147810 ± 1728.93
%RSD	1.27	1.21	1.16

Table: 8 Intraday Precision of Diclofenac Sodium by HPLC

Sr.no	Time duration
Sr.no	1^sthour	2^nd hour	3^{rd hour}
Mean	106463.8 ± 1481.14	104959.3 ± 1609.55	103741 ± 1518.19
%RSD	1.39	1.53	1.46

Interday Precision

% RSD of Tolperisone HCl (15 μg/ml) for interday precision was found to be 1.27, 1.21, and 1.16 for and 1.39, 1.53 and 1.46 for Diclofenac Sodium (5 μg/ml) which is given in table 5.9 and table 5.10 for Tolperisone HCl and Diclofenac Sodium respectively. All parameters of % RSD are less than 2. This clearly indicates that proposed method is precise.

Table: 9 Interday Precision of Tolperisone HCl by HPLC

Sr.no	Time duration
Sr.no	1^sthour	2^nd hour	3^{rd hour}
Mean	147613 ± 4625.84	146159.3 ± 2161.55	145972.8 ± 2087.90
%RSD	1.10	1.47	1.46

Table: 10 Interday Precision of Diclofenac Sodium by HPLC

	Time duration
	1^sthour	2^nd hour	3^{rd hour}
Mean	104061 ± 1303.082	105540 ± 1056.63	105402 ± 1751.80
%RSD	1.25	1.00	1.66

Accuracy:

Accuracy of method is express in terms of recovery study at 80%,100 and 120% level of recovery of three replicate of each of both drug were determine and data of recovery was given in following table. The data shows that proposed method is accurate.

Table 11 Accuracy data of Tolperisone HCl by HPLC

Level of Recovery	Concentration (μg/ml)		Total amount	Area	Amount Recovered	% Recovery	Mean Recovery	Standard Deviation of area	%RSD of area
Level of Recovery	Standard	test	Total amount	Area	Amount Recovered	% Recovery	Mean Recovery	Standard Deviation of area	%RSD of area
80%	12	15	27	186563	26.99	99.99	99.61	710.63	0.38
	12	15	27	186458	26.97	99.91
	12	15	27	185283	26.70	98.92
100%	15	15	30	198721	29.75	99.17	98.70	586.50	0.29
	15	15	30	197562	29.49	98.30
	15	15	30	197985	29.58	98.62
120%	18	15	33	212986	32.98	99.95	99.63	852.26	0.40
	18	15	33	211523	32.65	98.94
	18	15	33	213012	32.98	99.96

Table 12 Accuracy data of Diclofenac Sodium by HPLC

Level of Recovery	concentration (μg/ml)		Total amount	Area	Amount Recovered	% Recovery	Mean Recovery	SD of area	%RSD of area
Level of Recovery	Standard	test	Total amount	Area	Amount Recovered	% Recovery	Mean Recovery	SD of area	%RSD of area
80%	4	5	9	135625	8.97	99.76	99.52	460.70	0.34
	4	5	9	135742	8.99	99.89
	4	5	9	134892	8.90	98.92
100%	5	5	10	145479	9.99	99.90	98.93	959.31	0.66
	5	5	10	143562	9.79	97.93
	5	5	10	144589	9.89	98.98
120%	6	5	11	155269	10.90	99.95	99.09	858.41	0.55
	6	5	11	153568	10.82	98.36
	6	5	11	154217	10.88	98.97

Table 14 Robustness data ofTolperisoneHCl by HPLC

15 (ug/ml)	Mobile phase composition			Flow rate
	Methanol : ACN: water (80:16:4) (pH 3.0)			Flow rate
	80:16:4	82:16:2	78:14:6	0.5ml/min	0.7 ml/min	0.3ml/min
1	149325	149325	149524	149325	140852	149523
2	149256	149825	149148	149256	140189	149425
3	149521	149465	149852	149521	140132	147896
SD	137.47	257.94	352.27	137.47	400.25	912.37
Mean	149367	149538	149508	149367	140391	148948
%RSD	0.09	0.17	0.23	0.09	0.28	0.61

Table 15Robustness data ofDiclofenac Sodium by HPLC

5 ug/ml	Mobile phase composition			Flow rate
	Methanol : ACN: water 80:16:4 (pH 3.0			Flow rate
	80:16:4	82:16:2	78:14:6	0.5 ml/min	0.7 ml/min	0.3 ml/min
1	103452	103256	102351	103452	101230	104523
2	103745	103452	103251	103745	101120	104987
3	103610	103652	101210	103610	100988	105120
SD	146.65	198.00	1022.86	146.65	121.16	313.48
Mean	103602	103453	102270	103602	101112	104876
%RSD	0.14	0.19	1.00	0.14	0.11	0.29

Application of proposed method to the tablet dosage form

No interference of the excipients with the peaks of interest appeared; hence the proposed method is applicable for the routine estimation of Metformin HCl and Linagliptin in tablet dosage forms. Results obtained are shown in following table.

Degradation study :

Acidic decomposition :

Tolperisone HCl is known to be acid liable drug, and undergoes 9.81% degradation by stress acidic condition. While on the other hand Diclofenac Sodium is stable under stress acidic condition because only 1.88 % degradation found within 12 hrs. Three degradants peaks were found with retention time (Rt) 3.4 min, 3.9 min for (Tolperisne HCl), and 6.0 min(for Dilofenac sodium).

Table 16 Assay of Tolperisone HCl and Diclofenac Sodium in tablet dosage formby HPLC

Formulation	Label claimed (mg)	Amount found (mg) per tablet	% Label claim
Tolperisone HCl	15	14.96± 0.40	99.79± 0.08
Diclofenac Sodium	5	4.98± 0.03	99.28± 0.02

Table 17 System suitability data of Tolperisone HCl and Diclofenac Sodium by HPLC

Parameter	Value for Tolperisone HCl	Diclofensc Sodium
Tailing factor	1.30± 0.05	1.20±0.034
No of theoretical plate	4222± 20	5669± 20
Retention time	2.9±0.02	4.2± 0.05
Resolution	8.54±0.05	10.25±0.09

Fig.4. HPLC chromatogram of Tolperisone HCl by acid degradation (12hrs.)

Fig.5 HPLC chromatogram of Diclofenac sodium by acid degradation (12hrs.)

Fig. 6 HPLC chromatograms of Tolperisone HCl and diclofenac sodium by acid degradation(12 hrs).

Alkaline decomposition

Tolperisone HCl shows more liability towards alkaline hydrolysis as compared to Diclofenac sodium. Tolperisone HCl and Diclofenac sodium degraded up to 12.0% and 6.32% with corresponding increase in concentration of degradant peaks. Four degradants peaks were found with retention time of 3.2 min, 3.8 min, 6.0 min and 8.0 min.

Fig.7 HPLC chromatogram of TolperisoneHClby alkali degradation (12hrs.)

Fig.8HPLC chromatogram of Diclofenacsodiumby alkali degradation (12hrs.)

Fig. 9HPLC chromatogram of Tolperisone HCl and Diclofenac sodium by alkali degradation (12hrs.)

Oxidatative degradation:

After exposure to oxidative condition, Diclofenac sodium was found to be stable while Tolperisone HCl shown liability towards stress oxidative condition. Tolperisone HCl degraded up to 13.56% with corresponding increase in concentration of degradant peak. Degradant was found at 3.79 min Rt.

Fig.10HPLC chromatogram of Diclofenac sodium by oxidation degradation (12hrs.)

Fig.11. HPLC chromatogram of Tolperisone HCl by oxidation degradation (12hrs.)

Fig.12. HPLC chromatogram of Tolperisone HCl and Diclofenac sodium by oxidation degradation (12hrs.)

Thermal decomposition

Thermo liable property of Tolperisone HCl was clearly observed as it shows 11.70% degradation in thermal stress condition. In compare to Tolperisone HCl, Diclofenac sodium was more stable. It only shows 1.69% degradation under thermal stress condition. Two new degradant peaks were found at 3.89 min, and 6.8 min. retention time.

Fig.13. HPLC chromatogram of Diclofenac sodium by thermal degradation (12hrs.)

Fig.14HPLC chromatogram of Tolperisone HCl by thermal degradation (12hrs.)

Fig.15 HPLC chromatogram of Tolperisone HCl and Diclofenac sodium by thermal degradation (12hrs.)

Sunlight degradation

Both drugs are very stable under direct sunlight condition. There is no degradant peak in direct sunlight condition even after 24 hrs.

Fig.16. HPLC chromatogram of Diclofenac sodium by sun light degradation (12hrs.)

Fig. 17. HPLC chromatogram of Tolperisone HCl by sun light degradation (12hrs.)

Fig. 18HPLC chromatogram of Tolperisone HCl and Diclofenac sodium by sun light degradation (12hrs.)

Tolperisone HCl was more degrade than Diclofenac sodium in different stress condition. Diclofenac sodium degrade significantly in alkaline condition. Tolperisone HCl degrade significantly in acidic, basic, oxidized and thermal condition.

CONCLUSION:

Review of literature reveals that no stability indicating method reported for simultaneous estimation of Tolperisone HCl and Dcilofenac sodium in combined solid dosage form. So, the RP-HPL method has been developed for simultaneous estimation of Tolperisone HCl and Dcilofenac sodium in combined tablet dosage form. Simple, accurate, precise and reproducible method for simultaneous estimation of Toperisone HCl and Dcilofenac sodium in Tablet dosage form has been developed by HPLC. The proposed method gives good resolution of Tolperisone HCl, Diclofenac Sodium and degradant product in degradation study. So, proposed method can be applied for routine quality control test.

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Received on 01.12.2015 Modified on 20.12.2015

Research J. Pharm. and Tech. 9(1): Jan., 2016; Page 33-43

DOI: 10.5958/0974-360X.2016.00007.X

Drug	LOD(µg/ml)	LOQ(µg/ml)
TolperisoneHCl	0.0062	0.018
Diclofenac Sodium	0.0126	0.037

Sr. No	Stress Type	Retention time of Tolperisone HCl	Retention time of Diclofenac Sodium	No. of extra peaks found	% degradation of Tolperisone HCl	% degradation of Diclofenac Sodium
1	Acid	2.93	4.27	3	9.8	1.88
2	Base	2.92	4.26	4	12.0	6.32
3	Oxidation	2.93	4.27	1	13.56	-
4	Thermal	2.92	4.26	2	11.70	1.69
5	Sunlight	2.93	4.27	-	-	-