Development and Validation of a New Derivative UV Spectrophotometric Method for Simultaneous Quantification of Tizanidine and Aceclofenac in Tablets

 

G. Sowjanya*, D. Parimala Pallavi, Oindrila Mazumder, S. Praveen Kumar

Department of Pharmaceutical Analysis, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam-530045, Andhra Pradesh, India

 

ABSTRACT:

The present study deals with development and validation of a new derivative UV spectrophotometric method for simultaneous quantification of Tizanidine and Aceclofenac in tablets. Tizanidine and Aceclofenac were obtained as gift samples and a simple derivative method was developed in borate buffer pH 9.0 using double beam UV/Visible spectrophotometer. The absorbance of Tizanidine was measured at 250nm (zero crossing point of Aceclofenac) and for Aceclofenac, 308.92nm (zero crossing point of Tizanidine) was selected. The linearity of Tizanidine and Aceclofenac was found to be in the range of 0.1-40 µg/mL and 2-100 µg/mL respectively. The %RSD in precision study was found to be 0.014-1.92% (intraday) and 0.014-1.44% (inter day) indicating that the method was precise. The % recovery for both the drugs in combination was obtained in the range of 98 - 101.36 % and found no interference from excipients. The method was also found to be sensitive as observed from the LOD and LOQ values. The proposed method was validated as per ICH guidelines and can be conveniently used for quantification of Tizanidine and Aceclofenac in tablets.

 

 

INTRODUCTION:

Chemically Tizanidine¹ is 5-chloro-N-(4,5-dihydro-1H-imidazol-2-y1)2,1,3-benzothiadiazol 4-amine which is a short acting drug for the management of spasticity. It is an agonist at 2- adrenergic receptor sites and presumably reduces spasticity by increasing presynaptic inhibition of motor neurons. Aceclofenac¹ chemically defined as 2-[2-(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy acetic acid is used as an effective NSAID having pronounced analgesic, antipyretic anti-inflammatory property. It is used in the treatment of acute low back pain and for rheumatic disorders.

 

Tizanidine and Aceclofenac (Fig. 1a and 1b) are official in Indian Pharmacopoeia² but analytical methods for simultaneous quantification of Tizanidine and Aceclofenac are not reported in Pharmacopoeia. Hence an attempt has been made to develop and validate a simple derivative UV spectroscopic method for simultaneous estimation of these drugs in combination dosage forms.

 

 

Fig.1a: Structure of Tizanidine

 

Fig. 1b: Structure of Aceclofenac.

 

A systematic literature review was executed to determine various methods and techniques previously used for the estimation of Tizanidine and Aceclofenac. Few methods were reported for individual determination of Tizanidine including UV-Spectrophotometric³ and ultra-HPLC method using UV detection^4. Several methods were reported for estimation of Aceclofenac and some of the recent works include UV-visible spectrometry^5-7 and HPLC^8-10. Few reverse phase HPLC methods^11,12 and UV- Spectrometric methods^13,14 were reported for the simultaneous quantification of Tizanidine and Aceclofenac and so a simple derivative UV spectroscopic method was developed in the present work.

 

MATERIAL AND METHODS:

The pure form of Tizanidine and Aceclofenac drugs were collected from Dr. Reddy’s laboratories. The commercial tablets of combined Tizanidine and Aceclofenac (Zerodol-MR) were purchased from the local pharmaceutical store. All the reagents and chemicals used like boric acid, sodium hydroxide, methanol and water (AR grade) were manufactured by Qualigens. Spectral measurements were made on a SHIMADZU double beam UV-Visible spectrophotometer (UV 1800) by using 1cm matched quartz cells. Shimadzu (AUX 220) electronic balance was used for weighing the samples.

 

Preparation of Borate buffer pH 9.0:

Borate buffer pH 9.0 was prepared by dissolving 6.20g of boric acid in 500ml of water. It was adjusted to pH 9.0 with 1M sodium hydroxide (about 41.5ml) and diluted to 1000 ml with water.

 

Preparation of stock solution:

Standard stock solution of Tizanidine and Aceclofenac was prepared by dissolving 25mg of pure Tizanidine and Aceclofenac drugs in 25mL distilled water and methanol respectively in separate 25mL volumetric flasks to get a concentration of 1mg/mL (1000 µg/mL) solutions individually.

 

 

 

Preparation of working standard and series of standard solutions:

From the stock solution prepared above 1mL was transferred into separate 100mL volumetric flasks and diluted with borate buffer pH 9.0 to produce working standard solutions of 100µg/mL concentration. From the above working standard solutions suitable aliquots were taken and diluted with borate buffer.

 

Preparation of sample solution (from tablet):

For the analysis of commercial formulation, 20 tablets of combined Tizanidine and Aceclofenac were taken and the tablets were crushed into powder. The powder equivalent to 10mg of Aceclofenac was taken in a 10 mL volumetric flask, dissolved in about 6ml of a mixture of distilled water and methanol respectively and sonicated for 30 minutes. The volume was made up to 10mL with methanol and it was filtered to get a solution of 1000µg/mL. From this solution 1mL was transferred separately into 10mL volumetric flasks and made up to volume with borate buffer pH 9.0. These solutions were further diluted in borate buffer pH 9.0 and scanned in the UV region.

 

Optimization:

Selection of solvent:

For the selection of the suitable solvent, a 10µg/mL solutions of Tizanidine and Aceclofenac were diluted in acetate buffer pH 5.0, phosphate buffer pH 2.0 and borate buffer pH 9.0 and scanned in the UV region. After analyzing the spectra, it was found that the spectra obtained with borate buffer pH 9.0 were discrete with no overlapping.

 

Selection of Zero Crossing Points (ZCP):

Derivative spectrophotometry¹⁵ involves the conversion of a normal spectrum (fundamental, zero-order spectrum) to its first, second or higher derivative spectra by differentiating absorbance of the sample with respect to wavelength. The differentiation of zero-order spectrum can lead to separation of overlapped signals, elimination of background caused by presence of other compounds in a sample, improvement of resolution of mixtures as it enhances the detectability of minor spectral features, and enhancement of sensitivity and specificity. Derivative spectra yield a more characteristic profile in comparison to the parent one; new maxima and minima appear and measurement at points where derivative spectra cross the X-axis termed as zero crossing points help in eliminating the interferences.

 

From the zero order spectra of Tizanidine and Aceclofenac obtained in the UV range of 200-400nm, the spectra were derivatized using the inbuilt software into first order spectra for which the derivative absorbance (at minima) was calculated at the corresponding zero crossing points and calibration curves were plotted. The standard graph for Aceclofenac was plotted at 308.92 nm (ZCP of Tizanidine) and at 250nm for Tizanidine (ZCP of Aceclofenac).

 

METHOD VALIDATION:

The developed method was validated in terms of linearity, precision, accuracy, limit of detection and limit of quantification as per the ICH Q2 (R1).¹⁶

 

Linearity:

Aliquots of working standard solutions of Tizanidine (0.01mL – 4mL) and Aceclofenac (0.2 – 9mL) were pipetted out into individual 10 mL volumetric flasks and made up with borate buffer pH 9.0. The working standard solution along with these solutions were scanned in the UV range and the absorbance for each solution was measured at their λ_max using the borate buffer as blank. These absorbance values were used to calculate the molar absorptivity value. The obtained spectra were derivatized using the inbuilt software into first order spectra for which the derivative absorbance (dA/dλ) at minima was calculated at the corresponding zero crossing points and calibration curves were plotted. The standard graph for Aceclofenac was plotted at 308.92nm (ZCP of Tizanidine) and the curve for Tizanidine was plotted at 250nm (ZCP of Aceclofenac) by taking concentration on x-axis and dA/dλ on y-axis. The regression analysis was performed and the correlation coefficients were noted down.

 

Precision:

To check the degree of repeatability of the method, suitable statistical evaluation was carried out. Three different concentrations were prepared from the formulation in triplicates and the derivative absorbance of each solution was measured at 250nm and 308.92nm, on the same day at different time intervals and on a different day for intra and inter day study, respectively. The mean, standard deviation (SD) and relative standard deviation (RSD) for the assay obtained in precision studies were calculated and observations are listed.

 

ACCURACY:

Recovery studies were carried out by applying the method to a tablet dosage form to which known amounts of standard Tizanidine and Aceclofenac corresponding to 80, 100 and 120% of label claim has been added (standard addition). At each level the solutions were prepared in triplicate and the dA/dλ was measured at 250 nm and 308.92 nm for Tizanidine and Aceclofenac respectively in each solution. The percentage recovery was calculated and the RSD values were recorded.

Limit of Detection (LOD) & Limit of Quantitation (LOQ):

LOD is the lowest concentration of analyte that an analytical process can reliably differentiate from background levels calculated as 3.3 σ/S. LOQ is the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability calculated as 10 σ/S.

 

Where,

σ is the standard deviation of the absorbance of the sample

S is the slope of the related calibration graphs

 

Assay:

The developed method was also applied for quantification of Tizanidine and Aceclofenac in marketed dosage forms. The above prepared sample solution was suitably diluted with borate buffer pH 9.0 and the solution was scanned against a borate blank. The spectra obtained were derivatized and the dA/dλ of the mixture was measured at 250nm and 308.92nm. The values obtained were incorporated into the regression equation and the concentrations of both the drugs were calculated.

 

RESULTS AND DISCUSSION:

A simple and sensitive derivative UV spectroscopic method was developed and validated for the simultaneous quantification of Tizanidine and Aceclofenac in bulk and tablets. Several buffers were used in the method optimization and finally borate buffer pH 9.0 was chosen as the ideal buffer. The drug solutions prepared in borate buffer were scanned in the range of 200-400nm and the absorption spectra were recorded. These spectra were derivatized to get first order derivative spectra and the zero crossing points (ZCP) were recorded at 308.92nm for Tizanidine where Aceclofenac showed absorbance and 250nm for Aceclofenac where Tizanidine showed absorbance in the selected solvent.

 

The method obeyed linearity in the range of 0.1–40 µg/mL for Tizanidine and 2-100 µg/mL for Aceclofenac as observed from the regression analysis. The regression equation was found to be y = 0.003x + 0.001 (R² = 0.999) at 250 nm for Tizanidine and y = 0.0004x – 0.0006 (R² = 0.998) at 308.92 nm for Aceclofenac. The overlain first order derivative spectra of pure Tizanidine and Aceclofenac are given in Fig. 2a and 2b. The linearity data of both the drugs is given in table 1a and 1b, the corresponding linearity graphs are given in Fig. 3a and 3b.

 

Fig. 2a: Overlain first order derivative spectra of Tizanidine (at 250 nm)	Fig. 2b: Overlain first order derivative spectra of Aceclofenac (at 308.92 nm)

 

Table 1a: Linearity table of Tizanidine (at 250 nm)

Conc. (µg/mL)	dA/dλ
0.1	0.001
1	0.007
2	0.008
4	0.016
8	0.032
16	0.063
24	0.095
32	0.126
40	0.157

 

Table 1b: Linearity table of Aceclofenac (at 308.92 nm)

Conc. (µg/mL)	dA/dλ
2	0.0008
5	0.002
10	0.004
20	0.008
40	0.015
60	0.024
80	0.031
100	0.039
-	-

Fig. 3a: Linearity graph of Tizanidine (at 250 nm)	Fig. 3b: Linearity graph of Aceclofenac (at 308.92 nm)

 

The LOD and LOQ of Tizanidine was found to be 0.572 µg/mL and 1.732µg/mL respectively whereas the LOD and LOQ of Aceclofenac was found to be 0.352µg/mL and 1.056µg/mL respectively.

 

 

Based on the dA/dλ values and the calculated assay values, the % RSD in precision study was found to be in the range of 0.014 – 1.92 (intraday) and 0.014 – 1.44 (interday) and the method is said to be precise as the % RSD is within the limits (less than 2.0). The corresponding data is given in table 2.

 

 

 

 

Table 2: Intraday and interday precision data

Tablet conc. (µg/mL)		*Conc. found (µg/mL)				Assay (% w/w) ± SD, % RSD		Assay (%w/w) ± SD, %  RSD
		Intraday		Interday		Intraday		Interday
Tz	Ac	Tz	Ac	Tz	Ac	Tz	Ac	Tz	Ac
0.5	25	0.52	26	0.520	26	104.5 ± 0.015, 0.014	104 ±1.9, 1.92	104.5 ± 0.015,  0.014	105 ± 1, 0.95
1	50	1.02	51.5	1.02	51.6	104.4 ±1.21, 1.16	103.07 ± 0.93, 0.90	105 ± 1.50, 1.44	102.8 ± 0.76, 0.74
1.5	75	1.59	79.3	1.604	80.4	106.5 ± 0.924,  0.87	105.7 ± 1.03, 0.97	107.1 ± 0.92, 0.86	106.3 ± 0.89, 0.84

Tz: Tizanidine              Ac: Aceclofenac          * Mean of three determinations

 

Recovery studies were carried out by applying the method to powder form of the tablet and the results are tabulated in table 3. % recovery of Tizanidine and Aceclofenac in combination obtained in the range of 98 - 101.36 % indicate that the method is accurate.

 

Table 3: Accuracy data

Level (%)	Tablet conc. (µg/mL)		Std. drug added (µg/mL)		*Conc. found (µg/mL)		Recovery (%) ± SD, % RSD
	Tz	Ac	Tz	Ac	Tz	Ac	Tz	Ac
80	1	50	0.8	40	1.78	89.1	99 ± 0.98, 0.99	99.37 ± 0.981, 0.987
100	1	50	1	50	1.98	99.5	99.1 ± 0.751, 0.76	99.83 ±1.443, 1.445
120	1	50	1.2	60	2.11	111	99.4 ± 0.693, 0.694	100.6 ±1.311, 1.30

Tz: Tizanidine              Ac: Aceclofenac          * Mean of three determinations

 

The developed method was applied for the assay in formulation (Zerodol-MR) and the percentage purity obtained against the label claim is given in table 4 along with the overlain spectra in Fig. 4.

 

Table 4: Assay in tablets

Brand	Drug	Label claim (mg)	Amount obtained (mg)	Assay (% w/w) ± SD
Zerodol- MR	Tizanidine	2.0	1.974	98.7 ± 0.75
	Aceclofenac	100.0	99.1	99.1 ± 1.44

 

Fig. 4: Overlain spectra of Tizanidine and Aceclofenac in pure and tablet form (1 & 50 µg/mL)

 

Table 5: Summary of optical characteristics and validation parameters

Parameters	Tizanidine	Aceclofenac
Linearity (µg/mL)	0.1- 40	2 -100
Method precision (% RSD)	0.014 – 1.92 (Intraday)
	0.014 – 1.44 (Inter day)
Accuracy (% Recovery)	98 - 101.36
R2	0.999	0.998
y = mx+c	0.003x + 0.001	0.0004x - 0.0006
LOD (µg/mL)	0.572	0.352
LOQ (µg/mL)	1.732	1.056
Molar absorptivity (L mol-1 cm-1)	16992.5	7103.81

CONCLUSION:

A simple, precise, accurate first order derivative UV Spectroscopic method was developed and validated for the determination of Tizanidine and Aceclofenac. The method was found to be economical in terms of usage of solvents and yet sensitive compared to the existing methods. The developed method was also applied for the assay of Tizanidine and Aceclofenac in tablets and there was no interference from the excipients as observed from the recovery values. Hence the developed method can be used in the regular quality control and simultaneous quantification of Tizanidine and Aceclofenac in API and pharmaceutical dosage form. 

 

ACKNOWLEDGEMENTS:

The authors are thankful to GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam for supporting the research work.

 

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Accepted on 28.09.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):569-574.