INTRODUCTION:

Aceclofenac (ACF), [[[2-[(2, 6-Dichlorophenyl) amino] phenyl] acetyl] oxy] acetic acid is a non steroidal anti-inflammatory drug with good analgesic and anti-rheumatic properties. It is used in various pain conditions like rheumatoid arthritis, osteoarthritis and ankylosing spondylatis.Tizanidine hydrochloride (TZH), 5-chloro-4-(2-imidazolin-2-ylamino)-2, 1, benzothiadiazole hydrochloride is a centrally acting muscle relaxant and an alpha-2-adrenergic agonist that acts mainly at the level of spinal cord.¹ACF and TZH are available in combined dosage form. The combination is more effective than aceclofenac alone and has a favorable safety profile in the treatment of acute low back pain. Combination is used for pain relief and management of rheumatoid arthritis.

Literature survey reveals that assay of ACF in bulk and dosage form is official in BP²and IP³. Several analytical techniques like Spectrophotometry⁴, HPLC^4,5,Densitometric⁵ methods have been reported for determination of ACF alone and spectrophotometric⁶, HPLC^6,7 methods are available with its combination with other drugs.

While TZH is official in IP⁸ and USP⁹, HPLC¹⁰ methods are available for TZH alone and spectrophotometric¹¹, RP-HPLC¹²methods are available for combination of TZH with other drugs. The present work describes three simple, precise and accurate spectrophotometric methods for estimation of ACF and TZH in combined dosage form.

MATERIAL AND METHOD:

An UV-Visible double beam spectrophotometer of Jasco, model V-530 with a pair of 1 cm matched quartz cell was used. Pure drug samples of ACF and TZH were supplied by Lupin Pharmaceuticals as gift samples. Tablets of combined dosage form i.e. Starnac- TZ (Invision Intra Sciences, Bangalore) were procured from the local market. Methanol AR grade and Distilled water were used.

PROCEDURE:

Preparation of Standard Stock solutions:

The stock solutions (100µg/ml) of ACF and TZH were prepared by dissolving accurately about 10 mg of drug in 25 ml methanol and then the volume was adjusted to 100 ml with distilled water separately. Further dilutions were made in distilled water.

Preparation of Mixed Standard solution:

For all the methods, mixed standards in the Beer- Lambert’s range for each drug in the ratio of 1:1 were prepared by diluting appropriate volumes from the standard stock solutions.

Method І: Simultaneous equation method:

From the overlain spectra (shown in Fig. 1) of ACF (10 µg/ml) and TZH (10 µ/ml), two wavelengths i.e. 273.5 nm as λmax of ACF and 227.5 nm as λmax of TZH were selected, at which both drugs showed absorbance. The linearity was found in the range 5-40 µg/ml and 5-30 µg/ml for ACF and TZH respectively.

Fig. No. 1: Overlain Spectra of ACF and TZH (10 µg /ml each)

The absorptivity of these two drugs was determined at 273.5 nm and 227.5 nm. A set of two simultaneous equations were formed using absorptivity values as given below; at selected wavelengths. The concentrations of two drugs in the mixture were calculated using set of two simultaneous equations.

A₁ at 273.5 nm = 274.1Cx + 20.66Cy………..1

A₂ at 227.5 nm = 415.3Cx + 658.5Cy………..2

Where, Cx and Cy are concentrations of ACF and TZH, in g/100 ml, respectively in sample solution. A₁and A₂ are absorbances of sample solutions at 273.5 nm and 227.5 nm, respectively. The absorptivities of ACF and TZH at 273.5 nm are 274.1 and 20.66 respectively. The absorptivities of ACF and TZH at 227.5 nm are 415.3 and 658.5 respectively. The absorptivities reported are the mean of six independent determinations.

By applying the Cramer’s rule and matrices to equations (1) and (2), concentrations Cx and Cy can be obtained as;

Cx = 658.5A₁-20.66A₂/ 171914.75………….3

Cy = 274.1A₂-415.3A₁/ 171914.75………….4

Method II: Graphical absorbance ratio / Q-Analysis method:

Absorbance ratio method uses the ratio of absorbances at two selected wavelengths, one at iso-absorptive point and the other being λ _max of one of two components. From the overlain spectra of two drugs, it is evident that ACF and TZH have iso-absorptive point at 218.5 nm and λ _max of TZH is at 227.5 nm. Standard solutions of each drug having concentration 10 µg/ml each and mixture in ratio of 1:1 were prepared separately in methanol: water (25:75) and the absorbances at 218.5 nm (iso-absorptive point) and at 227.5 nm (λ_max of TZH) were measured and absorptivity coefficients were calculated using calibration curve.

The concentration of two drugs in the mixture can be calculated using equations,

C_X = Q_M-Q_Y /Q_X-Q_Y. A₁ /ax₁,

C_Y = Q_M-Q_X /Q_Y-Q_X.A₂ / ax₁,

Where,

A₁ and A₂ are absorbance of mixture at 218.5 nm and 227.5 nm, ax₁ and ay₁, are absorptivity of ACF and TZH respectively at 218.5 nm, ax₂ and ay₂ are absorptivity values of ACF and TZH, respectively, at 227.5 nm and

Q_M = A₂ /A₁,

Q_Y = ay₂ /ay₁,

Q_X = ax₂ /ax₁.

Method III: Simultaneous equation using AUC method:

In the simultaneous equation using AUC method, the 'X' values of 10 µg/ml of ACF and 10 µg/ml of TZH were determined at the selected wavelength ranges, 270-275 nm and 225-230 nm (Fig. 2). The 'X' values were determined as,

X= Area under curve of component between the selected wavelength range. …………………...…… 5

Concentration of the component in gm/l

A set of two simultaneous equations were framed using these 'X' values are given below,

A₁ = 1397.5 C₁ + 2134.8 C₂……........6

A₂ = 63.0 C₁ + 3307.2 C₂………….....7

Where, C₁ and C₂ are the concentrations of ACF and TZH, respectively in g/100ml in the sample solution. A₁ and A₂ are the areas for mixed standard solutions at the wavelength range, 270-275 nm and 225-230 nm, respectively.

Fig. No. 2: AUC Spectra for Mixture (ACF and TZH 10 µg /ml each)

The 'X' values at 270-275 nm for ACF and TZH were found to be 1397.5 and 2134.8, respectively, while at 225-230 nm for ACF and TZH were found to be 63.0 and 3307.2, respectively. The 'X' values reported are the mean of six independent determinations.

By applying Cramer's rule and Matrices in Eqns. 6 and 7, concentrations C₁ and C₂ can be obtained as,

C₁ = (A₁× 3307.2) - (A2 × 2134.8) / 44.6….8

C₂ = (A2 ×1397.5) – (A1 × 63.0) / 44.6……9

Area under curve (A₁ and A₂) of sample solution containing 10 µg /ml of ACF and 10 µg/ml of TZH were recorded between 270-275 nm and 225-230 nm, respectively. The concentrations of two drugs in the sample were determined by substituting A₁ and A₂ values of sample solution in Eqns. 8 and 9, respectively.

Analysis of Tablet formulation:

Marketed tablets Starnac- TZ containing 100 mg of ACF and 2 mg of TZH were used. As the absorbance of TZH was very low in the concentration available tablet, standard addition ^13,14 was used by adding 9.8 mg of TZH and the calculations were made by subtracting standard (9.8 mg) readings from tablet readings after standard addition.

Twenty tablets were weighed and average weight was calculated. The tablets were triturated to a fine powder. An accurately weighed quantity of powder equivalent to 10.0 mg of ACF and 2 mg of TZH was transferred to 100 ml volumetric flask. To this 9.8 mg of pure TZH powder was added and dissolved in 25 ml of methanol by sonicating for 10 mins and volume was adjusted up to 100 ml with Distilled Water. The solution was filtered through Whatmann filter paper No. 41 and aliquot portion of filtrate was diluted to obtain a solution of 10μg/ml and 10μg/ml of ACF and TZH respectively. The absorbance of sample solution was measured at selected wavelengths. The content of ACF and TZH in sample solution of tablet was calculated. The results of analysis are mentioned in table no. 1.

VALIDATION OF METHODS:

The methods were validated with respect to linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy.

Table 1. Analysis of Tablet formulation

Method	Label Claim (mg/ tablet)		Amount Found (mg/ tablet)		% of Label Claim Estimated*		Standard Deviation		% R.S.D.
	ACF	TZH	ACF	TZH	ACF	TZH	ACF	TZH	ACF	TZH
I	100	2	99.93	2.010	99.93	100.50	1.3030	0.0322	1.3039	0.0320
II	100	2	99.69	2.006	99.69	100.45	0.9205	0.0250	0.9233	0.9163
III	100	2	99.76	2.006	99.76	100.35	0.7633	0.0273	0.7651	0.2720

*Average of six determinations

Table 2. Spectral and linearity characteristics data

Parameters	Method І		Method ІI		Method ІII
Parameters	ACF	TZH	ACF	TZH	ACF	TZH
Slope	0.0264	0.0604	0.0252	0.0604	0.1583	0.1903
Intercept	0.0065	0.0671	0.0051	0.0671	0.0715	0.0375
Correlation coefficient	0.9998	0.9994	0.9997	0.9994	0.9996	0.9999
Linearity range (µg/ml)	5-40	5-30	5-30	5-30	5-40	5-40

Methods I, II and III are simultaneous equation, absorbance ratio method and area under curve methods respectively. ACF and TZH indicate Aceclofenac and Tizanidine HCl respectively.

Table 3. Validation Parameters

Parameters	Method І		Method ІI		Method ІII
	ACF	TZH	ACF	TZH	ACF	TZH
Wavelength selected (nm)	273.5	227.5	218.5	227.5	270-275	225-230
LOD (µg / ml)	0.1704	0.3415	0.1732	0.2401	0.3596	0.3415
LOQ (µg / ml)	0.5165	1.0350	0.5213	0.7210	1.0897	1.0295
Intra day %± S.D.	100.1±0.94	98.8±0.664	100.4±0.155	102.1±0.100	98.7±0.144	99.1±0.065
Inter day %± S.D.	99.9±1.145	100.3±1.40	99.9±0.168	101.6±0.170	98.5±0.165	101.1±0.07

*Average of six determinations.

Table 4. Results of recovery studies

Method

Level of % recovery

% Mean recovery*

Standard Deviation

% R.S.D

ACF

TZH

ACF

TZH

ACF

TZH

Method І

100

120

97.40

100.01

100.99

98.45

99.03

100.38

0.8940

1.0002

0.9904

0.8429

0.6301

1.2402

0.9178

0.9999

0.9807

0.8562

0.6363

1.2300

Method ІI

100

120

99.80

99.67

100.65

99.69

99.81

100.2

0.807

0.361

0.285

0.391

0.319

0.186

0.810

0.358

0.280

0.395

0.319

0.189

Method ІII

100

120

99.15

99.58

99.42

99.62

100.58

100.36

0.1451

0.1508

0.0793

0.7325

0.9587

1.5373

0.1463

0.1514

0.0798

0.7353

0.9531

1.5317

*Average of three determinations.

Linearity:

The linearity range was estimated after analysis of different concentrations of both the drugs. The calibration curves were taken in the range of 5-40 mg/ml for ACF and TZH. The correlation coefficient of the linearity was determined. The results are shown in Table 2.

LOD and LOQ:

LOD and LOQ of the methods were established according to ICH definitions. The results are shown in Table 3.

Precision:

The precision of the method was established by carrying out the analysis of the analytes using the proposed developed methods. Analysis of the binary mixture of ACF and TZH on same day (intra-day precision) and on consecutive days (inter-day precision) was carried out. The low value of standard deviation showed that the methods were precise. The results are shown in Table 4.

Accuracy:

To check the accuracy of the developed methods and to study the interference of formulation excipients, analytical recovery experiments were carried out by addition of standard drug solution to preanalyzed sample at three different levels, i.e. 80, 100, 120% levels. From the total amount of drug found, the percentage recovery was calculated. The results revealed no interference of excipients. The results of recovery studies were summarized in Table 3.

RESULT AND DISCUSSION:

With respect to the validation results obtained from the above developed methods, it can be said that the two methods described here are simple, accurate and could be used for rapid and reliable determination of Aceclofenac and Tizanidine HCl in routine laboratory analysis. Both the drug show good regression values at their respective wavelengths. Recovery was in the range of 98-102 %; the value of standard deviation and % R. S. D are found to be < 2%; shows the high precession of the method.

ACKNOWLEDGEMENT:

The authors are thankful to Lupin Pharmaceuticals for providing drug samples of ACF and TZH. The authors are also thankful to the management of PES’s Modern College of Pharmacy for providing necessary facilities.

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