Stability Indicating HPLC Meth od for Simultaneous Determination of Diacerein and Aceclofenac


A Suganthi* and TK Ravi

Department of Pharmaceutical Analysis, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore-641 044

*Corresponding Author E-mail:



A simple, specific, accurate and stability-indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of diacerein and aceclofenac, using a RP-18 column and a mobile phase composed of water: acetonitrile (45: 55, v/v) pH 2.4 adjusted with o-phosphoric acid. The retention time of diacerein and aceclofenac were found to be 5.9 min and 14.2 min, respectively. Linearity was established for diacerein and aceclofenac in the range of 1-10 µg/ml and 2-20 µg/ml, respectively. The percentage recoveries of diacerein and aceclofenac were found to be in the range of 98.90-100.05% and 99.60-100.68%, respectively. Both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat, and photolytic degradation. The degradation studies indicated diacerein to be susceptible to H2O2, dry heat, acid, alkaline and neutral hydrolysis, while aceclofenac showed degradation in acid and alkali.The degradation products of diacerein and aceclofenac were well resolved from the pure drugs with significant differences in the retention time values. This method can be successfully employed for simultaneous quantitative analysis of diacerein and aceclofenac in bulk drugs and formulations.


KEYWORDS: Diacerein, aceclofenac, degradation products, HPLC-PDA.



Diacerein (DIA), 1, 8- diacetoxy 3-carboxy anthraquinone  (fig 1) is a novel osteoarthritis drug, yellow powder, almost odorless, partially soluble in alcohol but freely soluble in acetonitrile and dimethyl formamide1. It is not official in pharmacopoeia. Aceclofenac (ACE), 2-[2, 6-dichloro–phenyl amino] phenyl acetoxy acetic acid (fig 2), white crystalline powder, soluble in methanol, acetonitrile and dimethyl formamide, has analgesic activity and a good tolerability profile in a variety of painful conditions 2.It is official in British Pharmacopoeia3. There are many reported methods for analysis of diacerein (DIA) and aceclofenac (ACE) alone or in combination with other drugs in pharmaceutical dosage forms or individually in biological fluids.


DIA and ACE combination tablet is a recently introduced analgesic and anti- inflammatory tablet formulation in Indian market. Literature survey reveals that only two HPLC4,5 methods were found to be reported for determination of diacerein (DIA) and many methods for ACE6-8 individually and in combination with other drugs.


However, no method is reported for simultaneous estimation of these two drugs by reverse phase HPLC.


The International Conference on Harmonization (ICH) guideline entitled ‘Stability testing of new drug substances and products’ requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substances9. An ideal stability-indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine DIA and ACE simultaneously, in presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it was thought necessary to study the stability of DIA and ACE under acidic, alkaline, neutral hydrolysis, oxidative, dry heat, and photolytic conditions. This paper reports validated stability-indicating HPLC method for simultaneous estimation of DIA and ACE in presence of their degradation products. The proposed method is simple, accurate, reproducible, stability-indicating and suitable for routine estimation of DIA and ACE in combined dosage form. The method was validated in compliance with ICH guideline.



DIA and ACE of pharmaceutical grade were kindly supplied as gift samples by Glen mark Pharmaceuticals, Kurkumbh, India and were certified to contain ≥99%(w/w) and 99-101%(w/w) respectively, on dried basis. Acetonitrile and water used were of HPLC grade and were purchased from Merck, India. The tablet formulation (Dycerin A) containing 5 mg of DIA and 10 mg of ACE was procured from local market and used for analysis. The liquid chromatograph mass spectrometer Shimadzu LCMS-2010EV, which consisted of following components: a binary gradient pump, variable wavelength programmable PDA detector with auto sampler system. The chromatographic analysis was performed using Compaq Intel Core-2 DUO HP W/907 software on a pre-packed RP-18 column (250×4.6 mm, 5 µm particle size). In addition, an electronic balance (Shimadzu. Elec.balance BL-220H), a pH meter (Eli co L127), a sonicator (Leclasonic ultrasonic cleaner), a hot air oven (Inlab equipments Ltd) were used in the study. Separation was achieved using a mobile phase consisting of water: acetonitrile (45:55v/v). Solution at a flow rate of 1ml/min and the eluent was monitored using PDA detector at 254 and 275 nm. The column was maintained at ambient temperature and injection volume of 20 µl was used.


Fig1: structure of diacerein (DIA)


Fig 2: structure of aceclofenac (ACE)


Preparation of Stock and Standard solutions:

Stock solutions were prepared by weighing 5 mg of DIA and 10 mg of ACE and transferred to two separate 50 ml volumetric flasks, made up to the volume with HPLC grade acetonitile to obtain a solution containing 100 µg/ml of DIA and 200 µg/ml ACE. Aliquots of standard stock solutions of DIA and ACE were transferred using graduated pipettes into 10 ml volumetric flasks and solutions were made up to  the volume with the mobile phase to give the final concentration of 1-10 µg/ml for DIA and 2-20 µg/ml for ACE.


Calibration curves for DIA and ACE

Apart from individual solution, stock solution containing DIA & ACE in the ratio of 1:2 was also prepared using acetonitrile and the final concentration of 1-10 µg/ml and 2-20 µg/ml of DIA and ACE in different 10 ml volumetric flasks. All the solutions were injected and chromatograms were recorded (fig3). Calibration curves were constructed by plotting peak area versus. concentrations and regression equations were computed for both the drugs. (Table 1)


Analysis of Formulation

To determine the content of DIA and ACE in tablet (label claim 50 mg of DIA and 100 mg of ACE), 20 tablets were weighed. The tablets were crushed together in a mortar to a fine powder. An amount equivalent to 5 mg of DIA and 10 mg of ACE was transferred into a 50 ml dried volumetric flask. HPLC grade acetonitrile was added to dissolve and the volume made with the same. This solution was sonicated for 30 minutes to affect complete dissolution. The solution was filtered using Whatmann filter paper no.1. Appropriate volume of the aliquot was transferred to a 10ml volumetric flask and volume was made up to the mark with mobile phase to obtain the concentration of 5, 10 µg/ml of DIA and 10, 20 µg/ml of ACE. A 20 µl volume of each sample solution was injected into HPLC six times and peak areas were measured under optimized chromatographic conditions.


Forced Degradation Studies:

Forced degradation studies of both the drugs were carried out under conditions of hydrolysis, oxidation, dry heat and photolysis. For each study four samples were prepared, the blank solution stored under normal condition, the blank subjected to stress in the same manner as the drug solution, zero time sample containing the drug which was stored under normal conditions and the drug solution subjected to stress treatment: First the study was conducted separately for DIA and ACE of concentration 5 mg and 10 mg. Secondly it was conducted for bulk drugs in combination. At last the study was applied to the formulation containing 5 mg of DIA and 10 mg of ACE.  For formulation study the tablet powder equivalent to 5 mg of DIA and 10 mg of ACE was transferred to 100 ml round bottom flasks, and treated under acidic, alkaline, neutral, oxidizing, thermal and photolytic stress conditions. When degradation was complete the solution were left to equilibrate to room temperature and an aliquot of sample was withdrawn and diluted with mobile phase to get the concentration equivalent to 10 µg/ml of DIA and 20 µg/ml of  ACE.


Forced degradation with acidic media was performed by heating the drug under reflux with 1M hydrochloric acid for 2 hour. Forced degradation in basic media was performed by heating under reflux with 0.01M NaOH solution for half hour. To study neutral degradation, the drug was dissolved in acetonitrile and heated under reflux with water for 4 hours. Degradation with hydrogen peroxide was performed by treating the drug with 10% H2O2 (w/v) for 24 hour at room temperature. For thermal degradation, solid drugs were kept in Petri dish in oven at 80°C for 5hours.  The photolytic degradation study was also performed by exposing the drug to sunlight for 4 hours.


For HPLC analysis all the degraded sample solutions were diluted with mobile phase to obtain final concentration of 10 µg/ml of DIA and 20 µg/ml of ACE .Then 20 µl solution was injected into the HPLC system and analyzed under the chromatographic condition described earlier.



The mobile phase consisting of water: acetonitrile (45: 55, v/v) having pH 2.4 adjusted with o-phosphoric acid, at 1ml/min flow rate was optimized which gave two sharp, well resolved peak with minimum tailing factor for DIA and ACE (fig 3). The retention times for DIA and ACE were 5.9 and 14.2 min, respectively. UV overlain spectra of both DIA and ACE showed the maximum absorbance at 254 and 275 nm, so these wavelength were selected as the detection wavelength. The calibration curve for DIA and ACE was found to be linear over the range of 1-10 µg/ml and 2-20 µg/ml, respectively. The data of regression analysis of the calibration curves is shown in Table 1. The proposed method was successfully applied to the determination of DIA and ACE in their combined tablet dosage form. The results for the combination were comparable with the corresponding labeled amounts. The developed method was also found to be specific, since it was able to separate other degradants present in the tablet from the two drugs (fig 4). The LOD for DIA and ACE were found to be 25 and 50 ng/ml, respectively. The results for validation and system suitability test parameters are summarized in Table 1.


Fig 3:Chromatogram of mixture of DIA and ACE


Diacerein (DIA) with tR of 5.9 min and aceclofenac  (ACE) with tR of 14.2 min.


Fig 4: Chromatogram of marketed formulation of DIA and ACE under stress


Diacerein (DIA) with tR of 5.9 min and aceclofenac  (ACE) with tR of 14.2 min resolved from other degradants with tR  6.7,7.8,8.7 and 13.1 min, respectively.



Linearity range (μg/ml)





Correlation coefficient



Average of SE of estimation



LOD (ng/ml)



LOQ (μg/ml)



Recovery (%)



Interday precision (%RSD)



Intraday Precision (%RSD)






Retention Time (min)



Theoretical plates



Tailing Factor



DIA is diacerein, ACE is aceclofenac, SE standard error of the mean, SD is standard deviation for n = 6 observations


For robustness evaluation for both the drugs few parameters like flow rate, percentage of acetonitrile in mobile phase and pH of mobile phase were deliberately changed. One factor was changed at one time to estimate the effect. Each factor selected was changed at three levels (-1, 0, +1) with respect to optimized parameters. Robustness of the method was done at the concentration levels 10 and 20 µg/ml for DIA and ACE, respectively and the method was found to be robust.


Forced degradation studies of both the drugs were carried out under conditions of hydrolysis, dry heat oxidation and photolysis. The degradation study indicated that DIA was susceptible to acidic and alkaline hydrolysis more than neutral hydrolysis while it was less stable to H2O2 and direct heat but stable to direct sunlight where as ACE was susceptible to alkaline hydrolysis more than acidic hydrolysis and stable to H2O2, thermal and direct sunlight(fig5-10) .DIA showed 3 degradation peaks immediately after addition of 0.01M sodium hydroxide in addition to drug peak with the retention time of 6.7, 7.8 and 8.7 min. whereas ACE required more time to degrade and gave only one peak at 13.2 min after 1/2 hour under alkaline and acidic hydrolysis. In oxidation and thermal degradation, the drugs degrades as shown by the decreased peak areas when compared to peak areas of the same concentrate of the non degraded peaks. Percent degradation was calculated by comparing the areas of the degraded peaks in each degradation condition with the corresponding areas of the peaks of both the drugs under non degradation condition. Summary of degradation studies of both the drugs is given in Table 2.


Fig 5: Chromatogram of mixture of DIA and ACE degradated with 1M hydrochloric acid




Degradation condition

Time (h/day)

% degradation

tR (min) degradation products





Acid, 1 M HCl (reflux at 70°c)

2 hrs





Base , 0.01 M NaOH(reflux at 70°c)

˝ hr





Neutral , water (reflux at 70°c)

4 hrs





Oxidative,10%v/v H2O2(ambient, in dark)

24 hrs





Dry heat (80°c)

4 hrs





Direct sun light(photolysis)

5 hrs





DIA is diacerein, ACE is aceclofenac,  tstands for retention time, ND represents no degradation       observed.


Fig 6: Chromatogram of mixture of DIA and ACE degradated with 0.01M sodium hydroxide


Fig 7: Chromatogram of mixture of DIA and ACE degradated under neutral hydrolysis


In this reported study, a stability-indicating HPLC method was developed for the simultaneous estimation of DIA and ACE and validated as per ICH guidelines, statistical analysis proved that the method developed was accurate, precise and repeatable. The developed method was found to be simple, sensitive and selective for analysis of DIA and ACE in combination without any interference from the excipients. The method was successfully used for the estimation of drugs in pharmaceutical formulation. Assay results for combined dosage form using proposed method showed 98.90-100.05% of DIA and 99.60-100.68% of ACE. The results indicated the suitability of the method to study stability of DIA and ACE under various forced degradation condition viz. acid, base, neutral, oxidative, dry heat and photolytic degradation products.



It can be concluded that as the method could separate the drugs from their degradation products, it may be employed for analysis of stability samples of DIA and ACE. However characterization of degradation products was not carried out.

Fig 8 Chromatogram of mixture of DIA and ACE degradated  with 10%v/v hydrogen peroxide


Fig 9: Chromatogram of mixture of DIA and ACE degradated  under  dry heat



The authors thank Glenmark Pharmaceuticals, Kurkumbh, India for providing free gift samples for this work.



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Received on 05.01.2010                             Modified on 16.01.2010

Accepted on 28.02.2010                            © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 600-603