Simultaneous UV-Spectrophotometric Estimation of Aceclofenac and Cyclobenzaprine HCl by three different methods

 

Mahesh Mukund Deshpande*, Machindra Jayram Chavan

Department of Pharmaceutical Chemistry, Amrutvahini College of Pharmacy, Sangamner, M.S., India.

*Corresponding Author E-mail: mahesh_deshpande11@rediffmail.com

 

ABSTRACT:

Background: An Aceclofenac and Cyclobenzaprine HCl is NSAID and antidipressive agent, a rare combination. Objective: The aim was to develop and validate new UV-spectrophotometric methods. Method: In first method (M-I) simultaneous estimation with forced degradation was performed. In second method (M-II) simultaneous estimation by derivatization technique was performed. In third method (M III) the simple simultaneous estimation was performed. The abbreviations ACE and CYC are used for Aceclofenac and Cyclobenzaprine HCl in the current manuscript. Results: In method I, the linearity range was obtained as 5 - 25µg/ml with correlation coefficient 1 respectively. The results were 99.45% 99.50% 99.97% 99.78% 99.36% 99.67% respectively for both the drugs. The forced degradation for both drugs by applying the method was carried out. In method II, the derivatization with 2% ninhydrin was carried out. The correlation coefficient was obtained as 1 for both the drugs respectively. The recovery study values were obtained as 95.60%, 95.95%, and 95.53% 95.85% 98.82% 98.33% respectively for both the drugs. In the method III, the correlation coefficient of both the drugs was 1. The recovery study values were obtained as 99.56-100.32% 99.05 – 100.2% for both drugs respectively. Conclusion: All values were obtained in the acceptable range of as per ICH guidelines. The forced degradation was useful to obtain stability indicating data for this combination. The derivatization technique is first time used for the analysis of this combination. The one-way analysis of variance (ANNOVA) shows no significant difference among these three methods. As no methods are available for this new and recent combination, these methods can be routinely used for the analysis of these drugs.

 

KEYWORDS: Aceclofenac (ACE), Cyclobenzaprine HCl (CYC), UV-spectrophotometric, Forced degradation, Derivatization, ICH guidelines.

 

 


INTRODUCTION: 

The aceclofenac sodium2-[(2-{2-[(2, 6-dichlorophenyl) amino] phenyl} acetyl) oxy] acetic acid and cyclobenzaprine HCl dimethyl(3{tricyclo[9.4.0.0ł,⁸]pentadeca-1(15),3,5,7,9,11,13-heptaen-2ylidene}propyl)amine are NSAID and muscle relaxant, are given  in combination.1 An Aceclofenac is widely employed NSAID for the treatment of osteoarthritis, ankylosing spondylitis, rheumatoid arthritis.2 The different analytical methods including UV spectrophotometric, RP-HPLC,HPTLC were available for Aceclofenac and Cyclobenzaprine HCl separately or in combination of other drug.3-18

 

There was one HPLC method was developed for estimation of aceclofenac and cyclobenzaprine HCl in combination.19 Therefore, the simple, economical, and robust UV-spectrophotometric methods on two different UV-spectrophotometers were developed and validated as per ICH guidelines.20 The three methods were presented as M- I, M-II and M- III. The abbreviations ACE and CYC are used for Aceclofenac and Cyclobenzaprine HCl in the current manuscript.

 

M- I

UV-Spectrophotometric Simultaneous Estimation and Forced Degradation of Aceclofenac and Cyclobenzaprine HCl in combination M-1.1 Chemicals and Instruments:

The standard API of both the drugs was obtained as a gift sample for research purpose. The tablet formulation was procured from market containing Aceclofenac 200 mg and 15mg Cyclobenzaprine HCl. The Shimadzu-1800 and Lab India 3000 spectrophotometers were employed for the analysis. The D1201LH sonicator was used.

 

M-1.2 Preparation of standard stock solution:

The standard stock solutions100µg/ml of both the drugs was prepared in ethanol. The standard stock solutions were further diluted to 5, 10, 15, 20, 25µg/ml.The maximum wavelengths 275nm and 290nm were used toanalyze aceclofenac and cyclobenzaprine HCl respectively.

 

M-1.3 Estimation of drugs in tablet:

The 20 tablets were weighed and average weight determined.The tablets were crushed and the amount equivalent to 200mg of aceclofenac and 15mg of cyclobenzaprine HCl were taken from tablet formulation and diluted to obtain 200 and 15µg/ml of aceclofenac and cyclobenzaprine HCl. The solution was filtered through Whatman filter paper to remove the excipients from tablet.

 

M-1.4 Validation of Method:

M-1.4.1 Linearity:

The linearity range was set in between 5-25µg/ml by diluting the standard stock solutions of both drugs respectively.The correlation coefficients (R2) for ACE and CYC were 1at 275 and 290nm respectively.

Fig. (M 1.1-M 1.4)

 

 

Fig. M-1.1 UV spectra of ACE

 

 

Fig.M-1.2 UV spectra of CYC

 

Fig.M-1.3 Linearity of ACE

 

 

Fig.  M-1.4 Linearity of CYC

 

M-1.4.2 Precision:

The method precision was determined by taking five replicates at three concentration levels 5, 10, 15 µg/ml respectively. The % RSD values obtained were 0.64, 0.77, 0.83 for aceclofenac and 0.59, 0.43, 1.06 for cyclobenzaprine respectively.

 

M-1.4.3 Accuracy:

The accuracy at three concentration level Low, Middle and High of linearity range was determined. The % RSD values were 0.98, 0.12, 0.25of 5, 15, 25 µg/ml respectively for aceclofenac and 0.91, 0.28, 0.21 of 5, 15, 25 µg/ml respectively for cyclobenzaprine  

 

M-1.4.4 Sensitivity:

The sensitivity in the form of limit of detection and limit of quantitation were determined by using Standard deviation of precision and slope of calibration curve added in formula. The values of LOD were 0.10, 0.089 µg/ml and LOQ were 0.32, 0.27 µg/ml for aceclofenac and Cyclobenzaprine HCl respectively.

 

M-1.4.5 Recovery:

The recovery of the method was determined at 80,100,120% levels by standard addition method. The % mean recoveries were found to be 99.45, 99.50, 99.97 % and 99.78, 99.36, 99.67% respectively for aceclofenac and cyclobenzaprine HCl respectively at three concentration levels.

M-1.5 Degradation study:

M-1.5.1 Degradation by acid hydrolysis:

The acidic degradation was carried out by adding 5ml of 1N HCl to 1ml standard stock solution of both drugs separately.The solutions were kept aside for three hours and volume was made up to 10ml with ethanol. Then the solutions were analyzed at their respective wavelengths.

 

M-1.5.2 Degradation by base hydrolysis:

The base hydrolysis degradation was carried out by adding 5ml of 1N NaOH to 1ml standard stock solution of both drugs separately. The solutions were kept aside for three hours and volume was made up to 10ml with ethanol. Then the solutions were analyzed at their respective wavelengths.

 

M-1.5.3 Degradation by Hydrogen peroxide (Oxidation):

The oxidation degradation was carried out by adding 5 ml of 6% Hydrogen Peroxide to 1ml standard stock solution of both drugs separately. The solutions were kept aside for three hours and volume was made up to 10ml with ethanol. Then the solutions were analyzed at their respective wavelengths.

 

M-1.5.4 Degradation by Heat:

Both the drugs were exposed to 500C for eight hours. After that the standard solutions of both the drugs were prepared in ethanol and analyzed at their respective wavelengths.

 

M-1.5.5 Results of forced degradation study:

The acid degradation was found to be 2.4 and 5.5% for ACE and CYC respectively. The base degradation was found to be 3.8 and 2.3% for ACE and CYC respectively. The Oxidation degradation was found to be 11.57 and 8.57% for ACE and CYC respectively. The thermal degradation was found to be 26.76 and 22.63% for ACE and CYC respectively.

 

M-II

Use of derivatization technique for simultaneous estimation of Aceclofenac and Cyclobenzaprine HCl by UV:

M-2.1 Chemicals and Instruments:

The Ninhydrin was used to carryout derivatization of drugs. The formulation was procured from market. The Shimadzu UV 1800 was used for analysis of the samples.

 

M-2.2 Preparation and derivatization of standard:

The standard stock solution 100µg/ml of both the drugs was prepared in ethanol. The standard stock solutions were further diluted to 5, 10, 15, 20, 25µg/ml. The maximum wavelengths 275nm and 290nm were used to analyze aceclofenac and cyclobenzaprine HCl respectively. The dilutions for formulation was prepared by using same methodology that was employed for M-I. The working standard was derivatized by adding 2ml of Ninhydrin in 1ml of standard mix well and volume was made up to the mark of volumetric flask.The filtrate from Whatman filter paper of formulation was also derivatized by 2ml of Ninhydrin. The solution was warmed at 800C for 20min. on temperature bath and then cooled and volume was made up to the mark with diluent.The wavelength maximum were 349nm and 342 nm respectively used for analysis of ACE and CYC on UV-Spectrophotometer.

 

M-2.4 Validation of Method:

M-2.4.1 Linearity:

The linearity of both the drugs was determined by preparing the series of dilutions of stock solutions in the concentration range of 5-25µg/ml.Five replicates of each concentration were prepared and mean absorbance of each dilution was taken for linearity. The correlation coefficient was found to be 0.997 for both the drugs.

Fig. (M 2.1-M-2.4)

 

Fig.M-2.1 UV spectrum of ACE

 

Fig.M-2.2 Calibration Curve of ACE

 

Fig.M-2.3 UV spectrum of CYC

 

 

Fig.M-2.4 Calibration Curve of CYC

 

 

M-2.4.2 Precision:

The precision was determined at three concentration levels of linearity range that was low, medium and high. Five replicates of each low, medium and high of both drugs were analyzed whose % RSD values were 1.17, 1.2, 0.24 for ACE and 1.7, 1.2, 0.24 for CYC respectively.

 

M-2.4.3 Sensitivity:

The limit of detection for ACE and CYC was found to be 0.20 and 0.22 µg/ml respectively.

The limit of quantitation for ACE and CYC was found to be 0.62 and 0.66 µg/ml respectively.

 

M-2.4.4 Recovery:

The accuracy in the form of recovery was determined at three concentration levels 80,100,120% in the triplicates by standard addition method. The results of recovery were found to be 95.60, 95.95, 95.53% for ACE and 95.85, 98.82, 98.33% for CYC.

 

 

M- III

UV spectrophotometric simultaneous estimation method for Aceclofenac and Cyclobenzaprine HCl

M-3.1 Chemicals, instrument and reagents:

The Merk 99% methanol was used for analysis and all other chemicals were AR grade. The formulation Flexabenz plus tablet containing 200 mg of ACE and 15 mg of CYC were purchased from market. The Lab India-3092 UV-spectrophotometer was used for the analysis.

 

M-3.2 Validation of Method:

M-3.2.1 Linearity:

The linearity of both the drugs was analyzed in the concentration range 5-25 µg/ml with correlation coefficient 0.9994 and 0.9998 for ACE and CYC respectively. Fig. (M-3.1to M-3.2)

 

M-3.2.2 Precision:

The inter-day and intra-day precision of both drugs was determined whose % RSD values were 0.91-1.07% and 0.78-1.13% for ACE and CYC and 0.89-1.19% and 0.90-1.78% ACE and CYC respectively at three concentration levels by taking three replicates.

 

M-3.2.3 Sensitivity:

The limit of detection was found to be 1.05 and 1.45 µg/ml for ACE and CYC respectively. The limit of quantitation was found to be 3.55 and 2.25 µg/ml for ACE and CYC.

 

M-3.2.4 Assay of tablet formulation:

The assay of tablet formulation was found to be 99.76%±1.013 and 99.51%±0.959 for ACE and CYC respectively at five replicates each.

 

M-3.2.5 Accuracy:

The accuracy in the form of recovery was determined by standard addition method. The % recoveries were found to be 99.56-100.32% and 99.05-100.2% respectively for ACE and CYC. The % RSD of three replicates was found to be 1.37, 1.64 and 1.06, 1.18 respectively for ACE and CYC.

 

 

Fig.M-3.1 Calibration curve of CYC

 

Fig.M-3.2 Calibration Curve of ACE

 

Comparison among the methods:

The one-way ANNOVA was performed for comparing these three methods.The GraphPad InStat software was used to calculate one-way ANNOVA. The following values of recovery were taken for one-way ANNOVA.

 

Table No.1 Comparision of recoveries of M-I to M-III:

 

Method

Level of recoveryvalues

80%

100%

120%

ACE

CYC

ACE

CYC

ACE

CYC

M-I

99.45

99.78

99.50

99.63

99.97

99.67

M-II

95.60

95.85

95.95

98.82

95.53

98.33

M-III

99.06

99.04

99.56

99.05

99.62

99.32

 

The P values obtained for ACE and CYC were 0.9796 and 0.6296 respectively, which shows no significant difference among these three methods.

 

CONCLUSION:

The all above mentioned spectrophotometric methods were developed and validated as per ICH guidelines. The values obtained for the parameter’s linearity, accuracy, precision, sensitivity, recoveries were within range of acceptable limit as per ICH guidelines. In M-I the forced degradation profile provides the information of stability of both drugs in formulation and further stability indicating of both the drugs. The derivatization technique was also carried out which shows strong absorption of both the drugs in UV-visible region.In all three methods ethanol (Method I and II) and methanol (Method III) employed as solvent and estimation was performed on double beam spectrophotometer.The forced degradation was useful to obtain stability indicating data for this combination. The derivatization technique is first time used for the analysis of this combination. The one-way analysis of variance (ANNOVA) shows no significant difference among these three methods.All methods prove simple and economicalas well as no methods are available for this new and recent combination, hence can be routinely employed for the estimation of aceclofenac and cyclobenzaprine HCl in bulk and in formulation.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest with this investigation.

 

ACKNOWLEDGEMENT:

The authors are thankful to the management of Amrutvahini College of Pharmacy, Sangamner, M.S., and India

 

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Received on 07.12.2022            Modified on 16.08.2023

Accepted on 09.01.2024           © RJPT All right reserved

Research J. Pharm. and Tech. 2024; 17(3):1095-1099.

DOI: 10.52711/0974-360X.2024.00171