Identification and Characterization of Photoproduct Obtained By Photodegradation of Aceclofenac

 

Nilesh Patel1,*, Shailesh Shah2, Ishwarsinh Rathod3, Dhaval Patel4 and Biraju Patel4

1Department of Pharmaceutical Sciences, Saurashtra University, Rajkot-360005, Gujarat, India.               2Department of Quality Assurance, Maliba pharmacy College, Bardoli, Gujarat, India.               3Department of Quality Assurance, L.M. College of Pharmacy, Ahmedabad-380009, Gujarat, India.     4Veerayatan Institute of Pharmacy, Mandvi-370460, Gujarat, India

*Corresponding Author E-mail: nkpatel99@gmail.com

 

ABSTRACT:

Aceclofenac is [[[2-[(2, 6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetic acid. Photodegradation product of aceclofenac in methanol was synthesized in laboratory. Photodegradation product characterized and identified by Mass spectrum, Infrared spectrum and HPTLC study.  Photodegradation of Aceclofenac in methanol gives Methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate (Methyl ester of Aceclofenac) which is also reported in British pharmacopoeia as one of the  impurities.

 

KEYWORDS: Aceclofenac, Photodegradation, Photoproduct.

 


INTRODUCTION:

Aceclofenac, a phenyl acetic acid derivative is used as an anti-inflammatory agent1. Aceclofenac is official in British pharmacopoeia2 and European pharmacopoeia3. Various methods like HPLC4-10, HPTLC11-13, spectrophotometric14-28 and others29-33 are reported in literature for analysis of aceclofenac. The literature survey reveals that no systematic study has been reported for the photodegradation of aceclofenac. Photoproduct of Diclofenac is responsible for dermatitis which is also a phenyl acetic acid derivative14. Therefore an attempt has been made to synthesized, identified and characterized photodegradation product of aceclofenac. The Photodegradation and in vitro phototoxicity of aceclofenac are reported to evaluate phararmacological response of photodegradation product34.

 

EXPERIMENTAL:

Aceclofenac was obtained as a gift sample from Astron Research Ltd, Ahmedabad.  Methanol pre-washed TLC aluminum sheets of silica gel 60 F254 (layer thickness 0.2 mm. 20 x 20 cm, E. Merck, Germany) were used as stationary phase. Toluene, ethyl acetate, glacial acetic acid and methanol (All AR grade, Rankem Labs Ltd) were used for mobile phase preparation.

 

A camag HPTLC system comprising of CAMAG Linomat IV (Semi automatic spotting device), CAMAG twin trough chamber, CAMAG TLC Scanner 3, CAMAG CATS 4 Software and Hamilton 100 ml HPTLC syringe were used for study. UV spectra were taken between 200 to 400 nm wavelengths on UV-VIS 160A SHIMADZU spectrophotometer. Infrared spectrum of photodegradation product was carried out on FTIR 8400S SHIMADZU. Mass spectrum was obtained on LCQ classic, Thermo finnigan mass spectrometer.

 

Identification and Characterization of photodegradation product:

Aceclofenac (500 mg) was dissolved in 10 ml methanol in a 25 ml volumetric flask. The solution was exposed in sunlight for 72 hours during the month of April. The crystal of photodegradation product of aceclofenac was formed and filtered off with Whatman filter paper (No.-41). The collected crystal was dried and recrysatalized thrice from methanol.

 

Characterization of photodegradation product was performed by various methods. All the melting points are taken in open capillaries and are uncorrected. Photodegradation product was dissolved in chloroform (10 µg/ml) and UV spectrum was taken between 200 to 400 nm wavelength. Photodegradation product was mixed with KBr and scanned between 4 000 to 400 cm-1 for FTIR-spectrum. Mass spectrum was obtained on LCQ classic, Thermo finnigan mass spectrophotometer at 4.5 kv, using direct infusion ESI probe with 50-800 m/z range. Crystal of photodegraded product were dissolved in methanol and spotted on silica gel F254 plate and analyzed according to validated HPTLC method15. The purity of peak was determined by comparing spectrum at three different level i.e. peak start, peak apex, peak end.

 

Synthesis of photodegradation product:

Aceclofenac (500 mg) was dissolved in 10 ml methanol in a 25 ml volumetric flask. Add 2 drops of sulphuric acid, mixed well and set aside for 90 min at room temperature (38±2oC). The crystal of methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate was formed in the solution (Figure-1). The crystal was filtered off with Whatman filter paper (No 41). The collected crystal was recrysatalized thrice from methanol were analyzed. 10 µl (100 ng) of synthetic methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate, photodegradation product and   standard solution of aceclofenac were spotted on HPTLC plate and analyzed.

 

Figure-1: Synthesis of photodegradation product

 

RESULTS AND DISCUSSION:

Aceclofenac is an anti-inflammatory drug act as preferential cox-2 enzyme inhibitors. Various HPLC, HPTLC and spectrophotometric method are reported in literature for analysis of aceclofenac. Aceclofenac is not soluble in an aqueous solvent and freely soluble in methanol. Therefore methanol was used as solvent. Developed and validated HPTLC method was used for identification and characterization. Toluene: ethyl acetate: glacial acetic acid:  methanol (6.00:1:00.20.00:0.15 v/v/v/v) is used as mobile phase and methanol pre-washed TLC aluminum sheets of silica gel 60 F254 (layer thickness 0.2 mm,  20 x 20 cm, E. Merck, Germany) were used as stationary phase . The optimized mobile phase gives well-separated peak of aceclofenac at Rf value 0.35±0.03.

 

Photodegradation of aceclofenac was carried out in methanol by keeping the solution in sunlight for 3 days (72 hr). The solid crystal obtained was recrysatalized and characterized with the help of melting point, UV spectrum, IR spectrum, Mass Spectrum and HPTLC and compare with the synthesized methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate.

 

Melting point of photodegradation product is 106-108oC (Aceclofenac: 149-153oC). Overlain UV spectra of both compounds (synthesized and photodegradation product) are identical and exhibits λmax at 278.8nm which indicate that both compound are same (Figure-2). FTIR spectra of photodegradation product of aceclofenac show peaks at 3352 cm-1 due to N-H bond, 1733 cm-1 indicate the presence of ester bond and 1716 indicate presence of ketone group. Overlain FTIR spectra of Photodegradation product and synthesized product was identical, indicates that the photodegradation product of aceclofenac is identical with synthesized methyl-[[[2-[(2,6-dichlorophenyl)amino] phenyl]acetyl]oxy]acetate (Figure-3). Mass spectrum of photodegradation product is taken in positive mode. In positive mode peaks found at 391.9, 390.1, 368, 279.9, 277.9, and 250,215.1. Molecular ion peak (M+) at 368.0 and parent ion peak at 215.1 (Figure-4). The synthesized compound, photodegradation product and pure aceclofenac spotted and analyzed by HPTLC. Spot corresponding to Rf 0.34 is of aceclofenac. Spots at Rf 0.78 are of photodegradation product and synthesized Methyl-[[[2-[(2,6 dichlorophenyl) amino]phenyl] acetyl] oxy]acetate. These indicate that photodegradation product and synthesized product are identical but different than that of aceclofenac. Peak purity spectra of spots reveal that peaks are pure. Correlation coefficient for individual peak purity was 0.9996, 0.9996 and 0.9998 for aceclofenac, photodegradation product and synthesized product respectively. Overlain spectra of photodegradation product of aceclofenac and synthesized methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate are identical indicate that both are same compound (Figure-5). Thus it is confirmed that the product formed on photodegradation of aceclofenac in methyl alcohol is methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate. Normally for esterification acid-alcohol mixture is refluxed. In present work we are getting methyl ester at room temperature by keeping reaction mixture for 90 min without refluxing. This product is one of the compounds in the reported 2 impurity of the aceclofenac.

 

Figure-2: Overlain UV spectra of synthesized and photodegradation product

 


 

Figure-3: Overlain FTIR spectra of synthesized and photodegradation product

 

Figure-4: Mass spectrum of photodegradation product

 


 

Figure-5: Overlain HPTLC spectra of synthesized and photodegradation product

CONCLUSION:

All above facts indicate that the photodegraded product obtained from photodegradation of aceclofenac in methanol is methyl ester of aceclofenac i.e. methyl- [[[2-[(2,6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetate.

 

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Received on 08.12.2010       Modified on 23.01.2010

Accepted on 17.02.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 758-761