Determination of Diacerein by a Simple Isocratic RP-HPLC Method

 

Arvind B Karadi^1* Kalpana Jyothi M¹, Shiva Kumar CH¹, Appala Raju S.¹, Shobha Manjunath¹ and Ashok L. Ganure²

¹Matoshree Tarabai Rampure Institute of Pharmaceutical Sciences, Gulbarga-585 105 (K.S.) India

²K.J. College of Pharmacy, Vadasma (North Gujarat), India.

ABSTRACT:

A Simple and precise reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the quantitative determination of Diacerein in bulk and its pharmaceutical formulations. Chromatographic separation was performed by using column XDB C₈ (4.6x 150 mm, 5µm) with a mobile phase comprising of a mixture of phosphate buffer and Acetonitrile (50:50,v/v) and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 0.8 ml/min with detection at 254nm. Separation was completed in less than 10 min. As per ICH guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, ruggedness and robustness. Linearity of Diacerein was found to be in the range of 10-100µg/ml and correlation coefficient was found to be 0.999. The retention time of Diacerein was 3.3 min; the separation was performed at an ambient temperature. The limit of Detection and limit of quantitation for Diacerein was found to be 3.6 and 9.95 (S/N ratio). There was no significant difference in the intraday and inter day analysis of Diacerein determined for five different concentrations using this method. The RSD value 0.3% indicates a high precision of the analytical method. The proposed method was simple, sensitive, precise, accurate and useful for routine quality control analysis.

 

 

 

INTRODUCTION:

Diacerein^1,2 is chemically 9,10-dihydro-4,5-dihydroxy-9,10-dioxo-2-anthroic acid diacetate.It is an anti arthritic drug. It is used in the treatment of osteo arthritis, multiple sclerosis, amyotropic lateral sclerosis³.

 

DIACEREIN

 

Literature survey reveals that few analytical techniques utilizing HPLC, spectrophotometry have been reported to quantify Diacerein in bulk drug^4-6.

 

The aim of this study is to develop a new HPLC method for the quantitative estimation of Diacerein in bulk drug and its formulations with high sensitivity, accuracy, precision and economical. The above method is sensitive, accurate and precise, can be used for routine quality control of this drug.

 

MATERIALS AND METHODS:-

Standards and reagents:

Diacerein was obtained from Glenmark Pharmaceuticals Ltd., Bangalore, India. All solvents used were of HPLC grade and reagents used were of AR grade.

 

Chromatographic system and conditions:

Waters 2695 separation module with Displacement pumps DNX 2491, UV-Visible (waters 2487), and Empower 2 software.

 

Column: XDB C₈ (4.6 X 150 mm, 5µm)

Chromatographic separations were achieved using a guard column and analytical column C₈ (4.6x 150mm, 5µm). The mobile phase consisting of phosphate buffer of pH 3.0 and Acetonitrile in the ratio of 50:50 was passed through 0.45µ membrane filter and degassed by ultrasonication under vacuum before use. The flow rate was maintained at 0.8 ml/min and the eluent was monitored at 254nm. The injection volume was 10µl. All separation was performed at an ambient temperature.

 

Preparation of Standard Solution:-

Weigh accurately about 25 mg of Diacerein and transferred into a 100 ml volumetric flask, about 70 ml of mobile phase  (phosphate buffer and Acetonitrile in ratio of 50:50) was added and sonicated for 20min to dissolve it completely and the volume was made  up to the mark with the mobile phase. Further working solutions of 100µg/ml was prepared with mobile phase. A typical chromatogram was shown in Fig 1.

 

Fig 1: A typical chromatogram For Diacerein (Standard)

 

Preparation of Sample solution:

20 capsules each containing 50mg were weighed and powder was collected. An accurately weighed portion of the powder equivalent to 25mg was transferred in to 100ml volumetric flask. About 70ml of mobile phase was added and sonicated  for 20 min to dissolve it completely and the volume was made  up to the mark with the mobile phase. Mix well and filter through 0.45 µ membrane filter paper. Further working solution of 50 µg/ml prepared with mobile phase. The procedure was repeated for 6 times. A typical chromatogram was shown in Fig 2 and results are shown in Table 2.

 

Fig 2: A typical chromatogram for Diacerein (Sample)

Calibration curve:

Aliquots of Diacerein containing 0.1, 0.25, 0.5, 0.75 and 1ml (1ml = 100µg/ml) were transferred into a series of 10ml volumetric flasks and volume was adjusted up to the mark with mobile phase to obtain concentrations in the range of 10-100µg/ml. All measurements were repeated for five times. Each concentration and calibration curve was constructed by plotting the peak area Vs the drug concentration. The area exhibited linear responses with r² = 0.999 for Diacerein. They were shown in Table 1.

 

Table 1: Optical characteristics of Diacerein

Parameters	Method
Beer’s Law Limit	10-100µg/ml
Correlation coefficient (r )	0.999
Standard Error of estimation	4668.38
Standard deviation	10438.5
% RSD	0.3
No. of Theoretical plates	3441
Tailing factor	0.84
LOD	3.60
LOQ	9.95
HETP	0.0435
Range of errors Confidence limits with 0.05 level Confidence limits with 0.01 level	±11040.7 ±16334.66

 

Table 2: Calibration of HPLC method for the estimation of Diacerein

Linearity Level	Concentration	Mean Peak Area(n=5)
I	10 µg/ml	828852
II	25 µg/ml	1807192
III	50 µg/ml	3524156
IV	75 µg/ml	5419123
V	100 µg/ml	7228727

 

Validation of HPLC method:

1)      Specificity and selectivity:  The specificity of the HPLC method was determined by comparing the chromatograms of the standard and sample solutions. The parameters like retention time, resolution and tailing factor were calculated. Good correlation was found between the results of standard and sample solution. The method is selective, showed no interfering peaks around the retention time, base line showed any significant noise.

 

Fig 3: Calibration curve for Diacerein

Table 3: Intra day and Inter day precision for Diacerein assay in Pharmaceutical  dosage forms by the proposed HPLC method

Concentration (µg/ml)	Peak area ( Intra day)	Mean (n=5)	SD	CV	Peak area (Inter day)	Mean (n=5)	SD	CV
50	3493139	3509727.6	10438.5	0.3	3538073	3545137.6	5818.5	0.16
50	3507028				3544186
50	3513450				3542571
50	3514476				3547112
50	3520544				3553746

 

Table 4: Recovery of Diacerein using Proposed HPLC method

Concentration (at Specification Level)	Peak Area	Amount Added (mg)	Amount Found (mg)	Recovery	Mean Recovery
50%	1814801	12.3	12.47	101.4%	99.9%
100%	3535310	24.6	24.29	98.7%
150%	5451403	37.6	37.46	99.6%

 

Table 5:  Robustness Table for peak Areas and Retention time

Parameters		Mobile Phase Ratio		Flow Rate		Temperature
		Peak Area	RT	Peak Area	RT	Peak Area	RT
Initial	Diacerein	3544380 (50:50)	3.359	3544380  (0.8ml/min)	3.359	3544380 (250c)	3.359
Changed	1	3252426 (45:55)	3.417	3544380  (0.8ml/min)	3.359	3544380 (250c)	3.359
		3242838 (55:45)	2.717	3544380 (0.8ml/min)	3.359	3544380 (250c)	3.359
	2	3544380 (50:50)	3.359	4167292 (0.7ml/min)	3.846	3544380 (250c)	3.359
		3544380 (50:50)	3.359	3237722 (0.9ml/min)	3.000	3544380 (250c)	3.359
	3	3544380 (50:50)	3.359	3544380 (0.8ml/min)	3.359	3558112 (230c)	3.369
		3544380 (50:50)	3.359	3544380 (0.8ml/min)	3.359	35558941 (270c)	3.372
% Deviation		8.23	1.72	17.57	14.49	0.38	0.29

 

 

2)      Precision: Precision of the method was studied as intra day and inter day variations. Intra day variation was determined by analyzing three different concentrations for three times within a day. Inter day precision was assessed using same concentration of drug for three different days, over a period of a week. The results were shown in Table 3.

 

3)      Accuracy: The accuracy of an analytical method is the closeness of the test result obtained by that method to true value. Recovery experiments were performed sample solutions; a known amount of standard drug solution of Diacerein was added to the pre analyzed samples and subjected them to the proposed HPLC method. The results are shown in Table-4.

 

4)      LOD and LOQ:

The sensitivity measurement of Diacerein by using the proposed method was estimated in terms of LOD and LOQ. The LOD and LOQ were calculated by using equation LOD = 3.3σ/S and LOQ=10σ /S where σ = Standard deviation of the peak are of the drug, S = Slope of the corresponding calibration curve. The LOD and LOQ were found to be 3.60 and 9.95.

 

5)      Robustness:  The robustness study was done by making small changes in the method like change in mobile phase ratio, flow rate and temperature. There is no significant impact on retention time and tailing factor was found. The results were shown in Table 5.

 

6)      Ruggedness: Appropriate concentration of standard stock solution was prepared and analyzed by two different analysts on different days using different columns (Agilent C₈ 4.6 X 150 mm, 5µm and YMC C₈ 4.6 X 150 mm, 5µm) using same operational and environmental conditions. Peak area was measured for same concentration solutions for five times. The %RSD of two sets of data (0.3and0.2) indicates the ruggedness of the method.

 

RESULTS AND Discussion:

The proposed HPLC method is simple, accurate, reproducible and useful for determination of Diacerein in pharmaceutical dosage formulations (capsules). The results of the assay showed good agreement with labeled claim. The recovery of the drug was determined at 50,100 and 150% level. The %recovery of Diacerein was found to be 99.6 to 101.4% whish shows high accuracy of the proposed method. Inter day and intra day precision was determined by analyzing the drug sample at three different concentration levels. The results are presented in the form of %RSD which is below 1.00 showed high precision of proposed HPLC method. The system suitability parameter for the proposed method was studied and verifies that the resolution and reproducibility of the chromatographic system is adequate for the analysis.

 

Conclusion:

On the basis of the results of assay and validation parameters the proposed method was simple, specific, fast, accurate and precise for estimation of Diacerein in bulk drug and pharmaceutical formulations and can be applied for the routine estimation of Diacerein (capsules).

 

ACKNOWLEDGEMENT:

The Authors are thankful to Glenmark Pharmaceuticals limited, Bangalore, India  for providing Diacerein as a gift sample and Principal, Management of H.K.E .Society’s college of pharmacy, Gulbarga for providing necessary facilities to carry out the present work.

 

REFERENCES:

1.       O’ Neil, M.J., Ed., The Merck Index- An Encyclopedia of Chemicals, Drug and Biologicals, 14^th Edn.,Merck and Co.,Inc., 2006; 503.

2.       Sweetman SC, editor, Martindale the complete Drug reference, Pharmaceutical Press, London, 35^th Edn, 2007; 35.

3.       CIMS, Multi Media Pvt. Ltd, Bangalore, July – Sep, 2008; 200.

4.       Valerio Giannellini, Francesco Salvatore, Gianluca Bartolucci.A validated HPLC stability indicating method for the determination of diacerhein in bulk drug substance. Journal of Pharmaceutical and Biomedical Analysis, 2005; 39, 776-780.

5.       Silvia HM, Lutiane Parcianello, Marcella Z, Arend. Development and validation of a dissolution method with spectrometric analysis for diacerhein capsules. Sci Pharm 2008; 76: 541-554.

6.       Janhavi Rao, Kanchan Chauhan, KR Mahadik. A stability indicating high performance liquid chromatographic method for the determination of diacerein in capsules. Indian Journal of Pharmaceutical Sciences. 2009; 71; 24-29.

 

 

 

Received on 12.01.2010        Modified on 09.02.2010

Accepted on 20.03.2010        © AJRC All right reserved