INTRODUCTION:

Tizanidine is a short-acting drug for the management of spasticity. Tizanidine is an agonist at alpha 2-adrenergic receptor sites and presumably reduces spasticity by increasing presynaptic inhibition of motor neurons. In animal models, tizanidine has no direct effect on skeletal muscle fibers or the neuromuscular junction, and no major effect on monosynaptic spinal reflexes. The effects of tizanidine are greatest on polysynaptic pathways. Tizanidine 5-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-2,1,3-benzothiadiazol-4-amine (Figure 1). The overall effect of these actions is thought to reduce facilitation of spinal motor neurons.

Tizanidine reduces spasticity by increasing presynaptic inhibition of motor neurons through agonist action at α2-adrenergic receptor sites.

Ibuprofen, a propionic acid derivative, is a prototypical non steroidal anti-inflammatory agent (NSAIA) with analgesic and antipyretic properties. The exact mechanism of action of ibuprofen is unknown. Ibuprofen is a non-selective inhibitor of cyclooxygenase, an enzyme involved in prostaglandin synthesis via the arachidonic acid pathway. Ibuprofen 2-[4-(2-methylpropyl)phenyl]propanoic acid (Figure 2). Its pharmacological effects¹ are believed to be due to inhibition cylooxygenase-2 (COX-2) which decreases the synthesis of prostaglandins involved in mediating inflammation, pain, fever and swelling. Antipyretic effects may be due to action on the hypothalamus, resulting in an increased peripheral blood flow, vasodilation, and subsequent heat dissipation. Inhibition of COX-1 is thought to cause some of the side effects of ibuprofen including GI ulceration.

Figure 1. Structure of Tizanidine

Figure 2. Structure of Ibuprofen

MATERIALS AND METHODS:

Tizanidine and Ibuprofen bulk drugs was procured as a gift sample from Chandra lab Hyd Pvt. Ltd. Methanol used for analysis was of AR Grade and Distilled Water. Shimadzu UV-Vis (double beam) spectrophotometer² was used for spectrophotometric analysis. It was connected to a personal computer having UV Probe Ver.2.10 software and provided with 1 cm quartz cells. HPLC used Shimadzu (LC 20 AT VP), Soft ware: Spinchrom Detector: Prominence UV Vis detector.

Instrument:

The instrument used was Agilent 1220 HPLC instrument. The instrument is consisting of Agilent 1220 LC pump and variable wavelength programmable UV detector and 20µl inject port.

Chromatographic conditions:

Intersil ODS C18 column³ (250×4.6mm) 5.0µm, flow rate was 1.0ml/min, mobile phase ratio was (40:60 v/v) Potassium dihydrogen Phosphate Buffer: Acetonitrile pH 3.2 (pH was adjusted with Ortho-phosphoric acid), detection wavelength was 269nm. Injection volume was 20µl and analysis was performed at ambient temperature.

Preparation of buffer:

6.75gm of potassium di hydrogen phosphate (KH₂PO₄) was weighed and dissolved in 1000ml of water and volume was made up to 1000ml with water. Adjust the pH to 3.2 using ortho phosphoric acid. The buffer was filtered through 0.45µ filters to remove all fine particles and gases.

Preparation of standard stock solution of Tizanidine:

10mg of Tizanidine was weighed and transferred in to 100ml volumetric flask and dissolved in methanol and then make up to the mark with methanol and prepare 10 µg/ml of solution by diluting 1ml to 10ml with methanol.

Preparation of standard stock solution of Ibuprofen:

10mg of Ibuprofen was weighed in to 100ml volumetric flask and dissolved in Methanol and then dilute up to the mark with methanol and prepare 10µg/ml of solution by diluting 1ml to 10ml with methanol.

Determination of Working Wavelength:

The wavelength of maximum absorption (λ_max) of the drug, 10μg/ml solution of the drugs in methanol were scanned using UV-Visible spectrophotometer within the wavelength region of 200–400nm against methanol as blank. The absorption curve shows characteristic absorption maxima at nm for Tizanidine, nm for Ibuprofen and 269nm for the combination.

Figure 3: UV-Overlap spectrum of Ibuprofen and Tizanidine

Figure 4: Optimized Chromatogram Tizanidine and Ibuprofen

Validation of Proposed Method:

The Proposed method was validated as per ICH guidelines⁴. The solutions of the drugs were prepared as per the earlier adopted procedure given in the experiment.

1. Linearity and range:

Linearity of Tizanidine and Ibuprofen was observed in both methods the range of 0.6-1.4µg/ml and 120-280 µg/ml. Detection wavelength used was 269nm.

Table 1: linearity data

	Tizanidine		Ibuprofen
S. No.	Concn (µg/ml)	Area	Concn (µg/ml)	Area
1	0.6	85.711	120	5220.620
2	0.8	136.654	160	6487.143
3	1.0	194.120	200	7731.788
4	1.2	244.595	240	9173.107
5	1.4	293.548	280	10435.854
Slope	261.8		32.79
Intercept	70.88		1251.5
Correlation coefficient	0.999		0.999

Figure 5: Linearity graphs of Tizanidine and Ibuprofen

2. Precision:

Intraday and Interday Precision studies on RP-HPLC and UV method for Tizanidine and Ibuprofen⁵ which shows the high precision % amount in between 98% to 102% indicates to analytical method that concluded.

Table 2: Method precision of Tizanidine and Ibuprofen

Tizanidine			Ibuprofen
S.No.	Rt	Area	Rt	Area
1	2.503	142.769	4.180	7699.608
2	2.517	146.627	4.223	7795.276
3	2.510	147.866	4.240	7823.707
4	2.500	147.904	4.243	7876.643
5	2.483	147.327	4.297	7810.702
6	2.503	144.349	4.180	7851.824
Avg	2.5027	146.140	4.227	7809.627
Stdev	0.0114	2.112	0.044	61.288
%RSD	0.46	1.45	1.05	0.78

Figure 6: Chromatogram of precision injection

3. Robustness:

The changes were did flow rate (± 1 ml/min), PH of mobile phase composition (± 1 ml/min), and Wavelength (± 1 ml/min),. % RSD for peak area was calculated which should be less than 2%. The result shown in analytical method that concluded.

Figure 7: Chromatogram of Tizanidine and Ibuprofen for Robustness (0.8 ml/min)

Table 3: Results of Robustness study

Parameter

Tizanidine

Ibuprofen

Retention time(min)

Tailing factor

Retention time(min)

Tailing factor

Flow Rate

0.8 ml/min

1.0 ml/min

1.2 ml/min

2.927

2.503

2.120

1.075

1.080

1.021

5.127

4.180

3.717

1.630

1.550

1.556

Wavelength

267nm

269nm

271nm

2.473

2.503

2.473

1.050

1.080

1.050

4.333

4.180

4.333

1.585

1.550

1.500

4. Ruggedness:

It includes change in analysts, chemicals, laboratories, instruments, source of reagents, solvents. A validation parameters ruggedness is the degree of reproducibility produce in a obtain test result under the analysis of same limitation⁶ and same conditions. The ruggedness of the method was studied by changing the experimental condition⁷.

Figure 8: Chromatogram of Analyst 01 standard preparation

Table 4: Ruggedness data of Ibuprofen and Tizanidine

Ruggedness		Tizanidine	Ibuprofen
% RSD		0.21%	0.64%
Assay	Analyst-1	98.06	99.35
Assay	Analyst-2	97.76	100.26

5. Assay Results:

Figure 9: Chromatogram of Assay sample preparation

Table 5: Assay Results

Tizanidine			Ibuprofen
	Standard Area	Sample Area	Standard Area	Sample Area
Injection-1	193.706	142.562	7826.434	7716.294
Injection-2	195.827	174.597	7818.800	7829.617
Injection-3	195.161	232.244	7827.481	8109.044
Injection-4	191.310	211.418	8127.142	8311.593
Injection-5	195.128	214.801	8349.883	8280.792
Average Area	194.226	195.124	7989.948	8049.468
Tablet average weight	525		525
Standard weight	1		200
Sample weight	262.5		262.5
Label amount	2		400
std. purity	99.2		99.3
Amount found in mg	1.99		400.16
Assay(%purity)	99.66		100.04

CONCLUSION:

A method was developed on trial and error basis by changing the variables wherever required. Finally a method was optimized and the conditions were determined. Method was developed by using RP HPLC Method During this optimization at every trial a new combination of mobile phase was tried to overcome the drawbacks of the previous run. Finally the method was optimized at trial 5, the optimized method was using mobile phase water acetonitrile and methanol at 269nm and validated as per ICH guidelines.

The method was validated for system suitability, linearity, precision, accuracy, specificity, robustness, LOD and LOQ. The system suitability parameters were within limit, hence it was concluded that the system was suitable to perform the assay. The method shows linearity between the concentration range of 0.6-1.4µg/ ml for Tizanidine and 120-280µg/ml for Ibuprofen. The % recovery of Tizanidine and Ibuprofen were found to be in the range of 98.0% - 102.0%. As there was no interference due to mobile phase, the method was found to be specific. The method was robust as observed from insignificant variation in the results of analysis by changes in Flow rate and wavelength variation separately and analysis being performed by different analysts.

The present method is validated and the results are better than previous methods which are performed on these drugs.

Hence it can be concluded that the proposed method was a good approach for obtaining reliable results and found to be suitable for the routine analysis of Tizanidine and Ibuprofen in Bulk drug and Pharmaceutical formulation.

Finally we propose the method is accurate, precise, linear, specific, and robust. So we recommend this method for the routine analysis of Tizanidine and Ibuprofen in bulk and its dosage forms.

ACKNOWLEDGMENTS:

The authors express their sincere thanks to KJR College of Pharmacy., Burugupudi, Korukonda, and Andhra Pradesh, India.

CONFLICT OF INTEREST:

None.

REFERENCES:

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5. Maryadele J.O. Neil.Eds, The Merck Index, An Encyclopedia of Chemical, Drugs and Biologicals, 13th edition, Published by Merck Research Lab, Division of Merck and co. Inc., Whitehouse Station, NJ: 2006:148. NJ: 2006:86.

6. Kumar Nallasivan P, Saranya K.C, Siva Kumar R, Jeevanantham S, and Venkatnarayanan R. Simultaneous determination of Ibuprofen and Tizanidine in bulk drug and its combined dosage form by spectrophotometry. Der Pharmacia Lettre, 2010, 2(6), pg.no. 289-295.

7. Riddhi Gondalia, Rajashree Mashru, and Pankaj Savaliya. Development and Validation of Spectrophotometric Methods for Simultaneous Estimation of Ibuprofen and Paracetamol in Soft gelatin capsule by Simultaneous Equation Method. International Journal of ChemTech Research; Oct 2010, Vol.2, Issue 4, pg.no.1881

Received on 28.04.2022 Modified on 03.06.2022

Asian J. Pharm. Ana. 2022; 12(4):248-252.

DOI: 10.52711/2231-5675.2022.00040