INTRODUCTION:

Aceclofenac is an orally administered non-steroidal anti-inflammatory drug, which possess good analgesic properties and chemically is 2-[[2[-[(2, 6-dichlorophenyl) amino] phenyl] acetyl] oxy] acetic acid¹. Diacerein is chemically 4, 5-diacetyloxy-9, 10-dioxo-anthracene-2-carboxylic acid and is a disease modifying anti-rheumatoid drug used in the treatment of Osteoarthritis and chronic inflammatory arthritis². Many methods have been described in the literature for the determination of aceclofenac with other drugs individually and in combination.^3-7 There are very few reports on analytical methods for estimation of diacerein^8-10 and so far one method has been reported for this combination in plasma¹¹.

No HPTLC method for the simultaneous estimation of Aceclofenac and Diacerein in combined dosage forms has so far been reported. This prompted us to develop simple, accurate, precise and sensitive simultaneous estimation of Aceclofenac and Diacerein. The method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Dycerine a Tablet containing 100 mg of Aceclofenac and 50 mg of Diacerein which is manufactured and marketed by Glenmark pharmaceuticals Ltd. All chemicals and reagents used are of analytical grade.

A Camag HPTLC system comprising of Camag Linomat semi applicator, Hamilton syringe, Camag twin trough chamber, Camag TLC scanner, stationary phase pre coated with Silica gel G 60 F 254 and CATS software for interpretation of the data were used.

HPTLC Instrumentation:

The TLC plates were pre washed with methanol and activated by keeping at 115^° Cfor about 30 min. The samples were spotted in the form of bands of width 5 mm with Hamilton syringe on the pre-coated silica gel G 60 F 254 plate,(10×10cm) slit dimension was kept at 15 min respectively. The mobile phase used was chloroform: methanol (8:2 v/v) in chamber and plate saturation time of 20 min, migration distance was allowed up to 85 mm, linear ascending development was carried out in (10×10cm) twin trough glass chamber. Subsequent to the development, TLC plates were dried in current of air and kept in photo documentation chamber. The images of developed plate were captured at white light, UV 254 nm and UV 366 nm using Camag – Reprostar -3 instrument and the source of radiation was a deuterium lamp.

Preparation of Standard solutions:

A standard solution of aceclofenac and diacerein mixture was prepared with accurately weighed quantities of aceclofenac (50 mg) and Diacerein (25 mg) in to a 10 ml volumetric flask. The content was dissolved in 5 ml of dimethylsulfoxide (DMSO) and then final volume was made up to 10 ml with methanol.

Analysis of pharmaceutical formulation:

The given dycerine ten tablets were powdered using pestle and mortar to fine powder. The quantity of powder equivalent to 100 mg of aceclofenac was weighed, transferred to a 100 ml volumetric flask, and extracted 25 ml of DMSO and methanol mixture. The extracts were filtered through Whatman filter paper, and residue washed with same mixture. The filtered extract and washings were transferred in to 100 ml volumetric flask, and volume was made up to 100 ml with methanol.

Calibration Plots:

On to a pre-washed and activated TLC plate, 2-10 μl of standard stock solution of aceclofenac and diacerein was spotted with Linomat V semi applicator. The plates were developed and scanned. The peak areas of each standard were obtained from the system, and a calibration graph was plotted with concentration vs. peak area. A good linear relationship was obtained over a concentration range of 10-50 μg/spot for aceclofenac, and 5-25 μg/spot for Diacerein.

RESULT AND DISCUSSION:

The method was validated for linearity, accuracy, limit of detection, limit of quantification, inter-day and intra-day assay precision, repeatability of measurement, and repeatability of sample application. From the sample aliquot prepared, 4 and 8 μl solution was applied, and the plate was developed with the mobile phase. A triplicate of those was carried out, and the peak areas were noted. The amount of aceclofenac and diacerein present in the formulation was calculated using the respective calibration graph. The amount obtained and results of analysis shown Table 1.

The mobile phase consisted chloroform: methanol (8:2 v/v) gave R_fvalue of 0.15 ± 0.03 for Aceclofenac and 0.2 ± 0.03 for Diacerein (Fig.1). A good linear relationship was obtained over the concentration range 10- 50 μg/spot of Aceclofenac and 5-25 μg /spot of diacerein respectively. The linear regression data showed a regression coefficient of 0.9982 for aceclofenac and 0.9966 for Diacerein. The repeatability of sample application and measurement of peak area were expressed in terms of % RSD for n = 3 observation. The studies revealed that intra and inter day variation of Aceclofenac and Diacerein is expressed in percentage RSD as shown in Table 2.

TABLE 1: RESULT OF ANALYSIS OF FORMULATION

Drug	Amount (mg /tablet)		% label claim^*	S.D^*
Drug	Labeled	Found^*	% label claim^*	S.D^*
Aceclofenac	100	99.22	99.22	1.46
Diacerein	50	50.77	101.54	1.55

^*An average value ± relative standard deviation of 5 observations.

The LOD with signal/ noise ratio were found to be and 20 ng and 10 ng/spot for Aceclofenac and Diacerein respectively. The LOQ with signal/ noise ratio was found to be and100 ng and 50 ng/spot for Aceclofenac and Diacerein respectively. Intraday assay precision was found to by analysis of standard drug at three times on the same day. Inter day assay precision was carried out using at three different days, and percentage relative standard deviation (%RSD) was calculated. The RSD was found to be less than 2 for both intraday and inter day precision. Repeatability of sample application was assessed by spotting 1 μl of drug solution, six times. From the peak areas, the percentage RSD was determined. The complete validation parameters are shown in Table 2.

Fig 1: Chromatogram of aceclofenac and Diacerein.

Chromatogram showing resolution of aceclofenac (R_f
= 0.38) and Diacerein (R_{f =} 0.66).

TABLE 2: VALIDATION PARAMETERS

PARAMETERS	VALUE
PARAMETERS	ACECLOFENAC	DIACEREIN
R_f	0.38±0.03	0.66±0.03
Linearity (μg/spot)	10-50	5-25
Correlation co efficient	0.9982	0.9966
Accuracy (% RSD)	1.08	1.34
LOD (ng/spot)	20	10
LOQ (ng/spot)	100	50
Precision (% RSD)
Inter day	0.42	0.81
Intra day	0.66	1.21
Repeatability (% RSD)	1.22	1.42

R_f- resolution factor, RSD- related standard deviation, LOD – limit of detection; LOQ – limit of quantification.

The recovery study was carried out at two levels, 50 %, 100 %. To the powdered formulation, the standard drugs of aceclofenac and diacerein were added at 50 % and 100 % levels, dilutions were made and analyzed by the method. The % recovery and % RSD were calculated and found to be within the limit, as listed in Table 3.

CONCLUSION:

Hence the developed HPTLC technique is simple, precise, specific and accurate, statistical analysis proved that the method is repeatable and selective for the simultaneous analysis of Aceclofenac and Diacerein as bulk drugs and in pharmaceutical dosage forms without any interference from the excipients.

ACKNOWLEDGEMENTS:

The authors are grateful to the Management, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, and Dalmiah Research Centre, Coimbatore, for providing the required facilities.

REFERENCES:

1. Shanmugham S, Cendil Kumar A, Vetrichelvan T, Manavalan R, Venkappayya D, and Pande VP. Spectrophotometric method for the estimation of aceclofenac in tablets. Indian drugs 2005; 42: 106- 108.

2. Oneil MJ, Heckelman PE and Koch CB, In: The Merck Index. An Encyclopedia of Chemicals: Drugs and Biologicals. 14^th ed., Whitehouse station, NJ: Merck and Co Inc.; 2006, 503.

3. Garg G, Saraf and Saraf S. Simultaneous estimation of aceclofenac, paracetamol and chlorzoxazone in tablets. Indian J Pharm Sci 2007; 69: 692-694.

4. Srinivasan KK, Alex J, Shirwaikar AA, Jacob S, Sunil Kumar MR and Prabu SL.Simultaneous derivative spectrophotometric estimation of aceclofenac and tramadol with paracetamol in combination solid dosage forms. Indian J Pharm Sci 2007; 69: 540-545.

5. Momin MY, Yeole PG, Puranik MP and Wadher SJ. Reverse phase HPLC method for determination of aceclofenac and paracetamol in tablet dosage form. Indian J Pharm Sci 2006; 68: 387-389.

6. Gopinath R, Rajan S, Meyyanathan SN, Krishnaveni N and Suresh B. A RP-HPLC Method for simultaneous estimation of paracetamol and aceclofenac in tablets. Indian J Pharm Sci 2007; 69:137-140.

7. Sheikh KA, Devkhile AB. Simultaneous estimation of aceclofenac, paracetamol and chlorzoxazone by RP-HPLC in pharmaceutical dosage form. Indian J Pharm Sci. 2008; 46:649.

8. Borgmann SH, Parcianello LM, Arend MZ and Cardoso SG. Direct spectrophotometric determination of diacerhein in capsules. Pharmazie 2007; 62: 483-485.

9. Layek B, Santosh Kumar T, Trivedi R, Mullangi R and Srinivas NR, Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of rhein in human plasma: application to a pharmacokinetic study. Biomedical Chromatograp 2008; 22: 616 – 624.

10. Giannellini V, Salvatore F, Bartolucci G, Coran SA and Alberti MB. A validated HPLC stability-indicating method for the determination of diacerhein in bulk drug substance. J Pharm Biomed Anal 2005; 39: 776–780.

11. Ojha A., Rathod R. and Padh H., Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma. J Chromatograp. B 2009; 877: 1145-1148.

Level	Amount added (mg)		Amount found (mg)^*		% Recovery^*		% RSD^*
Level	Aceclofenac	Diacerein	Aceclofenac	Diacerein	Aceclofenac	Diacerein	Aceclofenac	Diacerein
50 %	50	25	50.89	25.87	101.78	103.48	0.67	1.77
100%	100	50	99.33	49.11	99.33	98.22	1.38	1.43