INTRODUCTION:

Nimesulide a relatively COX-2 selective inhibitor, a nonsteroidal anti-inflammatory drug (NSAID), chemically it is N-(4-Nitro-2-phenoxyphenyl) methane-sulfonamide, which has analgesic and antipyretic properties. It is used in a variety of inflammatory ,pain and fever states in the treatment of acute pain, in the symptomatic treatment of osteoarthritis and primary dysmenorrhea in adolescents and adults above 12 years old [1]. Structure of Nimesulide is shown in Figure 1.

Figure No. 1 Structure of Nimesulide

Figure No. 2 Structure of Tizanidine hydrochloride

It is official in the British Pharmacopoeia [2]. Nimesulide is yellow needle-like crystals, with a melting point of 143°C to 144°C. The molecular weight of Nimesulide is 308.31 g/mol and the molecular formula is C₁₃-H₁₂-N₂-O₅-S. After oral administration the drug is rapidly and extensively absorbed. It is approved for used in treatment of musculoskeletal disorder, dysmenorrhea, thrombophlebitis and dental pain, inflammation. Some HPLC[3,4] and spectrophotometric[5,6] methods have been reported in the literature for its estimation.

Tizanidine hydrochloride is an agonist at α2-adrenergic receptor sites and presumably reduces spasticity by increasing presynaptic inhibition of motor neurons. Tizanidine is a skeletal muscle relaxant, used to treat tightness of muscle, cramping and spasm. It is official in The United States Pharmacopoeia [7] and Indian Pharmacopoeia [8]. Chemically it is 5-chloro-N-(4,5 dihydro-1H-imidazol-2yl)-2,1,3 benzothiadiazol-4-amine hydrochloride. It is a white to off-white, fine crystalline powder, slightly soluble in water and methanol, solubility in water decreases as the pH increases. It is odorless or with a faint characteristic odour. It has molecular formula C₉H₈CN₅S- HCl and molecular weight is 290.2g/mol. Tizanidine is a short-acting drug for the management of spasticity . It is used for the treatment of spasticity due to multiple sclerosis and spinal cord injury . Structure of Tizanidine is shown in figure no.2.

Literature survey reveals that there are some spectrophotometric methods with various other combinations of Nimesulide and Tizanidine along with paracetamol[9]and Tizanidine with different combinations [10]. There are also some methods available for the estimation of Nimesulide and Tizanidine by a spectrophotometer using multiple wavelengths [11]. Very few methods are reported for the simultaneous estimation of nimesulide and tizanidine in their combined dosage form [12,13]. There are some HPLC methods available for estimation of single component of drug of Tizanidine in The United States Pharmacopoeia [14] and also in Indian Pharmacopoeia [8]. Analytical methods for simultaneous estimation of Nimesulide and Tizanidine by reverse phase chromatography have been reported [15]. In this method the mobile phase was a mixture of water and methanol with a pH adjustment of 4.15 with orthophosphoric acid.

The present study describes the determination of Nimesulide and Tizanidine by using reverse phase chromatography, simpler mobile phase, C18 column, and a UV detector. The HPLC methods using the most commonly used mobile phase, shorter run time, easily available columns and simple detectors like the UV detectors are preferred. The use of HPLC is now a day is very much preferred in routine quality control analysis. It is important that well validated HPLC methods are to be developed for simultaneously estimating Nimesulide and Tizanidine.

The aim of this study is development of a simple, precise, rapid and accurate reverse phase HPLC method for the simultaneous estimation of Nimesulide and Tizanidine in pharmaceutical tablet dosage form. The method was validated as per ICH guidelines [16- 19]

MATERIALS AND METHODS:

Materials and Reagents:

Nimesulide and Tizanidine Hydrochloride were obtained from Balaji Drugs Surat. Methanol HPLC grade, ACN HPLC grade, Water HPLC grade was obtained from Merck India Ltd, Mumbai. All solvents and solutions were filtered through membrane filter 0.45μm pore size and degassed before use. Pharmaceutical formulations were purchased from local market.

Instrumentation:

UV-Visible Spectrophotometer Shimadzu, HPLC instrument use for the work was an Agilent 1100, Ultra sonicator, pH meter, Electronic balance, Syringe, HPLC Column C 18, 250X4.6 mm, 5µwere used for the work.

Chromatographic condition:

The HPLC system Agilent 1100 was used where separation has been done on C 18, 250X4.6 mm, 5µ column. 40°C temperature was maintained. The mobile phase contain acetonitrile and water (ACN: WATER) (60:40).pH 4.5 with ortho phosphoric acid (OPA), the flow rate was set at 1.0ml /min, and UV detection at 305 (Isobestic point), mobile phase was sonicated for 25 min. before use.

Preparation of standard solution:

Standard stock solution was prepared by transferring 10mg Tizanidine and 500mg Nimesulide in 100ml volumetric flask and dissolved in methanol. The volume was made up to the mark.

Preparation of working standard solution:

0.02 ml aliquot of standard solution was diluted to 10ml with mobile phase. To get 10μg/ml of Nimesulide and 0.2μg/ml of Tizanidine working standard solution.

Preparation of Tablet solution:

Twenty tablets were weighed and powdered. Accurately weighted quantity of powder equivalent to 100mg of Nimesulide and 2mg of Tizanidine was transferred in 100ml volumetric flask and dissolved in methanol and volume was adjusted to 100ml with methanol. The solution was shaken for 10 min. this solution was filtered through a 0.45 µm nylon filter. A 0.02ml of the filtrate was transferred to a 10 ml volumetric flask and diluted up to the mark with mobile phase. pH was adjusted to 4.5 with orthophosphoric acid. The flow rate was kept at 1ml/min and 305nm wavelength was selected for the determination.

Selection of wavelength:

Spectrophotometric study of Nimesulide and Tizanidine was carried out in methanol. The λ-max was determined on Shimadzu UV-Visible spectrophotometer in the range200-400nm. 20ugm/ml solutions of both Nimesulide and Tizanidine were prepared for the study.

Method development and optimization:

Several mobile phases with different ratios were initially trialed in order to have both eluents on the same chromatogram.The suitability of the column and mobile phase used in the optimized method have been decided based upon the basis of the selectivity, sensitivity as well as acceptable chromatographic parameters of the produced peaks in terms of peak sharpness, peak symmetry, tailing factor and resolution between the two peaks. We used the mobile phase as solvent for all samples to ensure minimum noise and to eliminate any unwanted solvent peaks.

Validation Methods:

The optimized method for simultaneous determination of Nimesulide and Tizanidine has been validated as per ICH guidelines Q2 (R1) for evaluating Accuracy, Precision, Linearity, Limit of Quantitation, Limit of detection, Robustness.

Accuracy:

Accuracy of the method was calculated by performing recovery studies. The recovery experiments were performed by adding known amounts of the drug in the placebo. It is carried out by preparing the samples at concentration levels of 80 %, 100 %, and 120% of test concentration. Recoveries (%), RSD (%) were calculated for each concentration.

Pricision:

Precision were assessed by applying 6 different samples of nimesulide and Tizanidine on the same day with same chromatographic condition as intraday precision. Interday precision was studied by applying 6 independent determinations on 3 different days.

Linearity:

Standard Nimesulide and Tizanidine stock solution were diluted in the range of 10-50μg/ml and 0.2-1.0μg/ml in different volumetric flask respectively. Chromatograms for each were prepared and calibration curve were plotted for peak area of each component against respective concentration of each corresponding drug. Its slope and intercept were determined to confirm linearity.

Limit of detection:

Based on the S.D. of the response and the slope of calibration curve, the detection limit (Tizanidine and Nimesulide) was calculated as,

LOD = 3.3 σ/s

Where,

σ = the S.D. of the y-intercepts of regression lines.

S = the slope of the calibration curve.

The slope S may be estimated from the calibration curve and S.D. was used should be calculated from the y-intercepts of regression line in calibration curve.

Limit of Quantitation:

Based on the S.D. of the response and the slope of calibration curve, the quantitation limit (QL) was calculated as,

LOQ = 10Q/S

Where,

σ = the S.D. of the y-intercepts of regression lines.

S = the slope of the calibration curve.

The slope S may be estimated from the calibration curve and S.D. was used should be calculated from the y-intercepts of regression line in calibration curve.

System suitability:

System suitability was performed as per USP to confirm suitability and reproducibility of the system. The parameters with repeatability, tailing factors, numbers of theoretical plates and resolution between Nimesulide and Tizanidine were assessed by injecting Nimesulide and Tizanidine working solutions.

Robustness:

Small changes in the flow rate (±1 ml/min), ratio of mobile phase (±1 ml/min) and detection wave length (±1 nm) were carried out and effects on the results were examined. The mean and % RSD of peak areas were calculated.

RESULT AND DISCUSSION-

In the present study Nimesulide and Tizanidine both anti-inflammatory drugs are studied. Both have solubility in water, methanol and acetonitrile. To separate the Nimesulide and Tizanidine different composition of mobile were tried. Different composition like water: acetonitrile (50:50), buffer: methanol (60:40), methanol: water (70: 30), methanol: water (80: 20), acetonitrile: water (60:40) etc. were tried with a stainless steel column Agilent C18 (250 mm x 4.6 mm) with particle size 5 μm_.

Wavelength selection:

Nimesulide has λ max at 307 nm and Tizanidine has λ max at 230nm and 320nm in methanol. Wavelength selected for the determination was 305nm at this wavelength both Nimesulide and Tizanidine shows maximum absorbance. As shown in figure no.3 UV result.

Figure No 3 .UV result for Tizanidine and Nimesulide

Figure No 4. Chromatogram of Tizanidine and Nimesulide working mixture

Figure No 5. Chromatogram of marketed formulation

Method Development:

By observing the typical chromatogram of Nimesulide and Tizanidine the retention times were obtain at 6.309 min. and 2.492 min. for the drugs respectively. As shown in figure no.4Developed method was applied on marketed formulation as shown in figure no.5

Validation Methods:

Accuracy:

The prepared developed method signified a very good accuracy as indicated by acceptance limit of less than 2 %. The recovery data for the accuracy study is provided in table no.1

Table No1. Accuracy result for Tizanidine and Nimesulide

	Tizanidine			Nimesulide
Sample no.	80% Recovery	100%	120%	80% Recovery	100%	120%
Sample no.	80% Recovery	Recovery	Recovery	80% Recovery	Recovery	Recovery
1	97.83	101.55	99.77	99.37	101.53	100.41
2	98.26	101.52	100.2	99.34	100.7	100.4
3	98.26	101.39	100	100	100.07	100.15
Mean	98.12	101.49	99.99	99.57	100.7	100.32
SD	0.2	0.06	0.17	0.3	0.6	0.12
%RSD	0.2	0.06	0.17	0.3	0.6	0.11

Precision-

Table No 2. Intraday and Interday Precision Result for Tizanidine and Nimesulide

	Tizanidine		Nimesulide
Set	Intraday (n=6)	Interday (n=6)	Intraday (n=6)	Interday (n=6)
1	100.3	101.1	100.8	99.8
2	100.3	99.9	101.3	100.5
3	100.4	100.9	100.7	101.0
4	100.7	100.5	101.3	100.6
5	100.8	99.5	101.1	99.1
6	100.3	99.5	99.3	100.6
Mean	100.46	100.23	100.75	100.26
SD	0.21	0.64	0.69	0.63
%RSD	0.2	0.64	0.68	0.63

Linearity-

Standard Nimesulide and Tizanidine stock solution were diluted in the range of 10-50μg/ml and 0.2-10μg/ml in different volumetric flask respectively. Chromatograms for each were prepared and calibration curve were plotted for peak area of each component against respective concentration of each corresponding drug. Its slope and intercept were determined to confirm linearity. As shown in figure no.6 and figure no.7.

Figure No 6. Linearity graph of Nimesulide

Figure No 7. Linearity graph of Tizanidine

Limit of Quantitation and Limit of Detection:

LOQ for nimesulide and Tizanidine were found as 29.75μg/ml and o.9649μg/ml respectively similarly LOD was 9.79μg/ml and 0.3184μg/ml

System Suitability:

The result obtain by 6 replicate injections conclude that all the parameters are within acceptable range. As shown in table no. 3.

Table No.3 Result of system suitability parameter

Sr. No.	Retention time		Area		Numbers of Plates
Sr. No.	Tizanidine	Nimesulide	Tizanidine	Nimesulide	Tizanidine	Nimesulide
1	2.487	6.235	1457.09	1.42	6764	15025
2	2.534	6.495	1477.88	1.43	5206	13827
3	2.482	6.192	1464.88	1.43	7050	15230
4	2.530	6.465	1448.58	1.42	5360	14061
5	2.533	6.493	1475.80	1.42	5202	13825
6	2.476	6.189	1456.82	1.43	6852	14863
Mean	2.507	6.344	1463.51	1.43	6072.3	14471.8
SD	0.028	0.1406	10.553	0.005	82.23	58.42
%RSD	1.11	2.21	0.72	0.34	1.35	0.4

CONCLUSION:

The research renders a very simple, exceptionally cost-effective, extremely accurate, quite precise and highly reproducible method for the routine quality analysis of Tizanidine and Nimesulide in tablets. The proposed method is capable of giving faster elution of the analyte with good resolution. Both drugs peaks have purity angles lesser than their purity threshold indicating peaks purity. The percentage recovery and precision studies showed that the method was accurate and precise.

ACKNOWLEDGEMENT:

Authors are thankful to Management and Principal of Mandsaur University, Mandsaur for providing timely support for the research work.

REFERENCE:

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Received on 02.12.2019 Modified on 10.02.2020

Research J. Pharm. and Tech 2020; 13(9):4207-4212.

DOI: 10.5958/0974-360X.2020.00743.X

Changing Parameter	Nimesulide(%RSD)	Tizanidine (%RSD)
Mobile Phase (69:31)	0.23	0.10
Mobile Phase (71:29)	0.47	0.59
Flow Rate (-0.1)	0.16	0.08
Flow Rate (+0.1)	0.59	0.05
Wavelength(-1)	0.53	0.54
Wavelength (+1)	0.50	0.50

Sr. No.	Parameters	Observations for Tizanidine	Observations for Nimesulide
1	Accuracy	% Mean recovery is between 99.12 to 101.49	% mean recovery is between99.57 to100.7
2	Precision	% RSD for Interday and Intraday precision 0.64 and 0.2	% RSD for Interday and Intraday precision 0.63 and 0.68
3	Linearity	Regression equation is y=2397.3x+39.499 R²=0.9993	Regression equation is y=429.15x+1343.4 R²=0.9996
4	LOD and LOQ	LOD=0.3184 and LOQ=0.9649	LOD= 9.79 and LOQ=29.75
5	Robustness	The % variation change in mobile phase, flow rate and wavelength is within limits	The % variation change in mobile phase, flow rate and wavelength is within limits