Simultaneous determination and validation of Aceclofenac and cyclobenzaprine hydrochloride by area under curve and q-absorbance ratio methods in bulk and pharmaceutical formulations

A simple, accurate, rapid and UV spectrophotometric methods has been developed for the simultaneous estimation of Aceclofenac (ACF) and Cyclobenzaprine hydrochloride (CBP) in bulk and combined tablet dosage form. The methods employed were(A) Area under curve and (B) Q-absorbance ratio method. Method-A involves measurement of peak area in the wavelength range 271-281 nm and 220-230 nm for ACF and CBP respectively. In Method B two wavelengths 276 nm, λ max of Aceclofenac and 257 nm, the isobestic point were selected. In both the methods linearity was found in the concentration range of 6-30 µg/ml for Aceclofenac and 0.45-2.25 µg/ml Cyclobenzaprine hydrochloride. Both the methods were found to be rapid, specific, precise and accurate. Hence these methods can be applied for routine analysis of Aceclofenac and Cyclobenzaprine Hydrochloride in combined dosage form without any interference by the excipients. The above methods are validated according to ICH guidelines.


Introduction
Aceclofenac chemically, a phenylacetic acid derivative, has anti-inflammatory and analgesic properties.It is Nonsteroidal anti-inflammatory drug (NSAIDs) used in various commercial pharmaceutical formulations for the treatment of fever, relief of pain and inflammation in rheumatoid arthritis, osteoarthritis and ankylosing spondylitis and reported to have good anti-rheumatic activity.ACECLO is the glycolic ester of Diclofenac.It is inhibitor of cytokine and works by blocking the action of a substance in the body called cyclooxygenase which involved in the production of prostaglandins and responsible for the generation of pain, swelling and their inflammatory conditions [1][2][3][4].
Cyclobenzaprine exhibits anticholinergic activity, potentiation of norepinephrine, and antagonism of reserpine.Cyclobenzaprine does not directly act on the neuromuscular junction or the muscle but relieves muscle spasms through a central action, possibly at the brain stem level.Cyclobenzaprine binds to the serotonin receptor and is considered a 5-HT2 receptor antagonist that reduces muscle tone by decreasing the activity of descending serotonergic neurons.It is an official drug in USP.Several analytical techniques like RP -HPLC, HPTLC, UPLC and Spectrophotometric method for the estimation of Cyclobenzaprine HCL individually and in other combinations have been reported.[5][6][7].
The combination of Aceclofenac and Cyclobenzaprine hydrochloride is used to relieve pain and relax the muscle [8].
On literature survey, it has been found that several methods have been reported for the estimation of Aceclofenac and Cyclobenzaprine individually and in combination with other drugs.In the view of the need for a suitable method for routine analysis in combined formulations, attempts are being made to develop simple, precise and accurate analytical methods for simultaneous estimation of titled drugs and extend it for their determination in formulation.

Instrument
For UV-Visible Spectroscopy methods, Shimadzu model 1800 double beam UV-Visible Spectrophotometer with spectral band width of 1 ± 0.2nm, wavelength accuracy of ± 0.3nm and a pair of quartz cuvettes having 1cm path length was used. .[9]

Chemicals
Standard Aceclofenac and Cyclobenzaprine hydrochloride was obtained as gift sample from micro labs, Bangalore.Methanol of AR grade, procured from S D FINE CHEM ltd.It was used throughout the experimental work.

Preparation of standard solutions
Preparation of standard solution of Aceclofenac (ACF) And Cyclobenzaprine Hydrochloride (CBP) 100mg each of Aceclofenac and Cyclobenzaprine Hydrochloride were weighed separately and transferred into two different 100ml volumetric flask.Both the drugs were dissolved in 50ml of methanol by ultrasonication and then volume was made up to the mark with methanol to obtain a concentration of 1000µg/mL of each component (stock -A).
From the above stock A solutions, 10 mL of aliquot was pipetted out in a 100 mL volumetric flask for both the drugs and the volume was made upto the mark with the methanol to obtain a concentration of 100µg/ml of each component( stock-B).
From the above stock -B solution further dilutions were made to get the concentration range 6-30µg/mL and 0.45-2.25µg/mLfor Aceclofenac and Cyclobenzaprine hydrochloride respectively.

Preparation of sample solution
20 tablets which contains both ACF and CBP were weighed and powdered.The tablet powder equivalent to 100mg ACF was weighed accurately and dissolves in 70 ml methanol and sonicated for 15mins.The solution was filtered through Whatmann filter paper No. 41, finally the volume was made up to the mark with methanol.Further dilutions were made to bring the concentration of the drugs within the range.

Method A (Area Under Curve method )
In this method the overlain spectra of both drugs wavelength range of 271 nm to 281 nm for Aceclofenac and 220 nm to 230 nm for Cyclobenzaprine Hydrochloride was selected for the analysis.The calibration curve of area v/s concentration was plotted.By applying Cramer's rule and matrix method, concentration of Aceclofenac and Cyclobenzaprine Hydrochloride can be calculated as,

Method B (Q-Absorbance Ratio method)
In this method absorbances are measured at two wavelengths.One being the λmax of Aceclofenac and other being a wavelength of absorptivity of the Cyclobenzaprine Hydrochloride.Then absorbance of both drugs was recorded on selected wavelengths.Concentrations of Aceclofenac & Cyclobenzaprine hydrochloride were calculated by using following equations.CACF = Qm-Qy / Qx-Qy x A1/ax1 CCBP = Qm-Qy / Qy-Qx x A1 /ay1 Where, Qm is ratio of absorbances A1 and A2 of mixture at λ1 and λ2 (isobestic point wavelength) Qx is ratio of absorptivities ax1 and ax2 at λ1 and λ2.Qy is ratio of absorptivities ay1 and ay2 at λ1 and λ2.CACF and CCBP are concentrations of Aceclofenac & Cyclobenzaprine Hydrochloride.

Linearity
In Method A (Fig. 3 to 5) overlay spectra of mixtures were shown.Fig. 3 shows the linearity of both the drugs in their respective wavelengths.The responses for both drugs shows linear concentration range of 6-20µg/ml and 0.45-2.25µg/mlfor ACE and CBP respectively.The regression equation calculated by least square method was y =0.0027x+0.001and y = 0.0667x-0.004with correlation coefficient of both drugs was r 2 = 0.999 and r 2 = 0.999.
In Method B (Fig. 6 to 8) overlay spectra of both drugs and their mixtures were shown.Fig. 6 shows the linearity of both the drugs in their respective wavelengths.The responses of first derivatives both drug shows linear concentration range of 6-30µg/ml and 0.45-2.25µg/mlfor ACF and CBP respectively.The regression equation calculated by least square method was y = -0.008x+0.002and y = -0.341x-0.009with correlation coefficient of both drugs was r 2 = 0.999 and r 2 = 0.999.Summary of validation parameters by developed methods as shown in Table no 1.

Accuracy
Recovery studies was carried out by applying the method to drug sample to which known amount of Aceclofenac and Cyclobenzaprine hydrochloride corresponding to 80, 100, 120% of label claim has been added (standard addition method).The results are tabulated in Table no 2.

Precision:
Precision was studied to find out intra-day and inter-day variations in the test method of ACF and CBP.Intra-day assay precision was found by analysis of standard drug thrice on the same day in different intervals of time.Inter-day assay precision was carried out on three different days and percentage relative standard deviation (%RSD) was calculated.The % RSD were found to be less than 2.0%.Statistical validation data for Intra-day and Inter-day precision methods as shown in Table no 3 and Table no 4.

LOD and LOQ
LOD is the lowest amount of the analyte can be detected but not quantified.
LOQ is the lowest amount of analyte that can be detected and quantified with an acceptable accuracy, precision.In this study, LOD and LOQ were determined based on the standard deviation of the response and the slope of the corresponding curve using the following equations.
The LOD & LOQ were calculated from the followings formulas LOD = 3.3 SD/Slope and LOQ = 10 SD/Slope.

Results and discussion
The selected drugs Aceclofenac and Cyclobenzaprine hydrochloride in Bulk and Formulations were estimated by using both UV spectrophotometric methods and validated as per ICH guidelines.In both the methods linearity response for ACF and CBP was 6-30µg/ml and 0.45-2.25µg/mlrespectively.The % RSD for intraday and inter-day precision was found to be less than 2%.The accuracy of the methods were validated by recovery studies and was found to be significant and under specification limits, with % recovery 99-100%.The assay results were found to be within the acceptable limits.Hence developed methods was found to be precise and sensitive.[13] Figure 1 Chemical structure Aceclofenac

Conclusion
The proposed methods were validated as per ICH guidelines.The standard deviation and % RSD calculated for the proposed methods were low, indicating high degree of precision of the method.The results of the recovery study performed show the high degree of accuracy of proposed methods.Hence, these methods can be employed successfully for the simultaneous estimation of Aceclofenac and Cyclobenzaprine Hydrochloride in routine analysis.

Figure 2
Figure 2 Chemical structure of Cyclobenzaprine

Figure 3
Figure 3 Spectra of ACF and CBP for Area Under Curve method at 271-281nm and 220-230nm

Figure 4 Figure 5 Figure 6 Figure 7 Figure 8
Figure 4 Calibration Curve for ACF between 271-281nm by Area under curve method

Table 1
Summary of Validation Parameters by Developed Methods

Table 2
Statistical Validation Data for Accuracy Determination

Table 3
Statistical Validation Data for Intra-day Precision *n=6

Table 4
Statistical Validation Data for Inter-day Precision *n=3