American Journal of Multidisciplinary Research & Development (AJMRD)

Volume 05, Issue 04 (April - 2023), PP 09-15

ISSN: 2360-821X
www.ajmrd.com

Research Paper Open Access

HIGH PERFORMANCE CHROMATOGRAPHIC (GPC)

METHOD FOR THE ESTIMATION OF DIACEREIN IN SOLID
DOSAGE FORMS
RAVEENDRAN K.C,
College of Pharmaceutical Sciences, Govt. Medical College, Kozhikode, Kerala, INDIA, 673008.

ABSTRACT: Diacerein is now widely used for the treatment of Osteoarthritis. The mechanism of action differs
from the Non-steroidal anti-inflammatory drugs since it is not related to the inhibition of Prostaglandins. Anti-
osteoarthritis and cartilage stimulating properties have been demonstrated in vivo and in animal models. Diacerein
and Rhein have been shown to inhibit the production of interleukin-I- beta by human monocytes and the effects of
the cytokine onchondrocytes in vivo. Diacerein in therapeutic dose inhibit the stimulation of interleukin-1 beta-
production and production of nitrous oxide. It also significantly reduces severity of pathological changes of
osteoarthritis compared to placebo and increases the expression of transforming growth factor TGF-beta-1 and TGF
beta-2 with potential cartilage repairing properties. From the literature, Diacerein is seemed to be a safe and
promising drug. This research work has been taken up to develop and validate simple, accurate, sensitive and cost-
effective methods for the estimation of Diacerein in dosage forms. A new LC method with UV detection was
developed for the quantitative determination of Diacerein in pharmaceutical dosage forms. The chromatographic
separation was performed on GPC column with a mobile phase of tetra hydro furan. A flow rate of 1.2ml/minute was
used. The resulting chromatogram exhibited a retention time of 5.784 minute. The limit of detection and limit of
quantification was found to be 0.5µg/m land 1µg/ml. A linearity range of 1-10µg/ml was established. The correlation
coefficient was found to be 1. 007. The results of analysis were treated statically, as per ICH guidelines for
validation of analytical procedures and by recovery studies. The results were found to be accurate and not much
interferences from capsule excipients. The active pharmaceutical ingredient was extracted from its finished dosage
forms using tetrahydrofuran. The percentage recovery was found to be 99.9%w/w.

Key words: Diacerein, Rhein, Gel permeation, HPLC, Linearity, Recovery etc

I. INTRODUCTION
Diacerein is an anthraquinone derivative that has been used in Osteoarthritis. Osteoarthritis is a common
disease in the ageing population and results from a complex interplay of genetic, metabolic, biochemical and
biomechanical factors with secondary local inflammation. The process involves the interaction of degradation and
repair of articular cartilage, bone and synovium. The most important cells in the entire process are the
chondrocytes1,2. Primary Osteoarthritis (OA) which can be either localized or generalized , most often idiopathic,
except in rare cases in which a defective gene has been found to cause a familial form of osteoarthritis 3,4.Diacerein
does not alter renal or platelet cyclooxygenase activity and may therefore be tolerated by patients with
prostaglandin dependent renal function5,6.Diacerein and Rhein have been shown to inhibit the production of inter
leukin-1 beta by human monocytes and the effects of the cytokine on chondrocytes in vivo7. They exert
chondroprotective effects in cultured articular cartilage and reduced severity of cartilage, bone and synovial
membrane damage in osteoarthritis8. Studies inhibit that diacerein does not block the synthesis of prostaglandins,
thromboxane of leukotrienes but may actually stimulate prostaglandin synthesis, especially PGF-2 alpha, a
prostaglandin with cytoprotective effect on the gastric mucosa9.10 .Janhavi Rao reported HPLC method of diacerein
in capsule dosage forms by isocratic separation was achieved using a perfect target OD-3,250X4.6 mm id columns
and using UV detection at254nm.The mobile phase was selected phosphate buffer: acetonitrile(40:60)ratio with pH
4.0.Kannappan N, at et al describes validation of Diacerein by High performance liquid chromatography(RP-HPLC)
method using Zorbax CN column and the mobile phases were selected Acetonitrile and buffer pH-3.5,None of the

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 HIGH PERFORMANCE CHROMATOGRAPHIC (GPC) METHOD FOR THE ESTIMATION...

method gives correct values recoveries(approx. 80.30-118.14%w/w) and there it is necessary a method having
simple, good precision, lack of errors, relevant values for estimation of Diacerein in solid dosage form.

II. MATERIALS AND METHODS

Chemicals and Reagents
Tetrahydro furan HPLC grade
Diacerein R.S, Obtained from Orbit Life Sciences Pvt Ltd, Mumbai
Commercially available pharmaceutical dosage form of Diacerein 50 mg Capsules, Dycerein- Marketed by
Glenmark Pharmaceuticals, Mumbai, INDIA.
Chromatographic system Parameters
Column : Waters Styragel HR-0.5 GPC column
Dimension : 7.8 x300 mm
Stationary Phase : Styrene-divinyl benzene copolymer particles
Pump : Waters 600 series
Injector : Waters 717 plus auto sampler
Flow rate : 1.2 ml/ min
PREPARATION OF STANDARD SOLUTION
Weighed accurately 10 mg of Diacerein R.S and transferred to a 10 ml standard flask, dissolved and made
up to the volume with tetrahydrofuran. The solution had concentration of 1 mg/ml (Solution A). From this, 1ml was
pipette out, transferred in to a 10 ml standard flask, and made up to the volume with tetra hydro furan. The resulting
solution had a concentration of 100µg/ml (solution B). From this solution accurately pipette out0.4,0.6,0.8 in to
three 10ml standard flasks and made up to the volume using tetrahydrofuran to get a concentration of 4,6,8µg/ml
respectively (Solution C)
Development chromatogram and determination of retention time
After initial stabilization, standard solutions of Diacerein were taken in the micro-syringe and injected
20µl of each solution separately in to the column. The retention time was found to be 5.784 minutes.
Table.1: Determination of Linearity range of Diacerein R.S
SL.NO. Final concentration
(µg/ml) Peak height Peak Area
1 4 31575 361368
2 6 43018 491748
3 8 65173 746588

Inference: The calibration curve was found to be linear in the concentration range of 2-10µg/ml.
STATISTICAL EVALUTION OF CALIBRATION PLOT
The data above was used to derive a regression equation of the peak area Y on the concentration X and peak height
Y on the concentration X by the principles of least squares and the equation is as follows.
Y=aX +b
Peak area wise
Linear regression Y=100730 +533229
Correlation coefficient was found to be 1.0007

Figure 1. Linearity Plot of Calibration of concentration with Peak Height

Figure.2. Linearity plot of concentration vis Peak Width

Accuracy
The accuracy of an analytical procedure is defined as the closeness of the measured value to the true value.
In a method with high accuracy, a sample whose true value is known is analysed and measured value should ideally
be identical to the true value. Accuracy was determined by carrying out a recovery study and the method was found
to be accurate.
Limit of detection (LOD)
The detection, limit of an individual analytical procedure is the smallest level of analyte that gives a
measurable response. The limit of detection of Diacerein by proposed method was found to be 0.5µg/m/

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 HIGH PERFORMANCE CHROMATOGRAPHIC (GPC) METHOD FOR THE ESTIMATION...

Limit of quantification (LOQ)

The limit of quantification of an individual analytical procedure is the smallest concentration of analyte
which gives a response that can be accurately quantified. The limit of quantitation of Diacerein by the proposed
method was found to be 1µg.ml.
Linearity
The linearity of an individual analytical procedure is a measure of how well a calibration plot of response
v/s concentration approximates a straight line.
The calibration curve of Diacerein by the proposed method was found to be linear in the range of 1-10µg/ml
Range of the proposed method was found to be 9µg/ml

ESTIMATION OF DIACEREIN IN DOSAGE FORMS

Details of analysed capsules
Batch No. DJ1245
Mfg. by: Glenmark Pharmaceuticals, Mumbai, INDIA.

Method
The content of 20 capsules was accurately weighed and the average weight calculated. A weight of the
powder equivalent to 10 mg was accurately weighed out and transferred to a stoppered flask. Extracted three times
successively with 2.5ml of Tetrahydrofuran HPLC grade and filtered through Whatman No. filter paper and the
combined extract was then made up to 10 ml with Tetrahydrofuran. The resulting solution had a concentration of 1
mg/ml (Solution A). From the resulting solution, pipetted 1 ml in to another 10ml standard flask and made up to the
volume with Tetra hydro furan. The resulting solution had a concentration of 100µg/ml (Solution B). From the
solute on B, accurately pipetted out 0.4,0.6,0.8 ml and transferred to three 10 ml standard flask and made up to the
volume with Tetrahydrofuran. The final solutions had concentrations of 4,6,8µg/ml respectively.
The chromatogram was generated after injection of 20µl of each solution to the column, under the same
chromatographic conditions as mentioned above. The peak arears and peak heights obtained from the chromatogram
are tabulated.
Table.2: showing Calibration of Diacerein capsule
SL CONC Peak Peak Concentration Active Average
NO. µg/ml area height from calibration content/capsule Content/capsules
curve

1 4 363571 32361 3.864 49.9

2 6 484702 43061 5.151 49.9 49.9

3
8 747918 65992 7.727 49.8

CALCULATIONS:
Label Claim = 50 mg
Average weight of the capsule content = 0.3925 g
Weigh equivalent to 10 mg = 0.0785 g
Weight taken = 0.0785g
Average content of Diacerein determined by the proposed method = 49.9 mg
Percentage Label Claim = 49.9X 100
50 = 99.9 %W/W
Standard deviation =0.1529
Coefficient of variation= 0.3079
Standard error = 0.0882.

III. RECOVERY STUDIES

The content of twenty capsules was accurately weighed and average weight found out. Finely powdered the
contents of the capsules in a glass mortar. A weight equivalent to 5 mg was accurately weighed and transferred in to
a stoppered flask, add 5 mg of Diacerein R.S. Extracted three times successively with 2.5 ml of tetrahydrofuran
HPLC grade, and filtered through Whatman No.1 filter paper and the combined extract was then made up to 10 ml
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with Tetrahydrofuran. The resulting solution had a concentration of 1mg/ml (Solution A). From the resulting
solution pipetted 1 ml in to another 10 ml standard flask and made up to the volume with tetrahydro furan. The
resulting solution had a concentration of 100µg/ml (Solution B). From the solution B, accurately pipetted out
0.4,0.6,0, 0.8 ml and transferred to three 10 ml standard flask and made up to the volume with tetrahydrofuran. The
final solution had a concentration of 4,6,8µg/ml respectively. Chromatogram generated after injection 20µl each of
4,6, and 8µg/ml solution to column, under the same chromatographic conditions were evaluated and the peak area
obtained from the chromatogram are tabulated.
Table.3: showing Concentration v/s Peak area of Dy cerein
Serial No. Concentration Peak Area Percentage
(µg/ml) Standard Sample recovery
(Dyce rein)
1 4 360172 361368 100.3
2 6 492748 491588 99.8
3 8 749588 745895 99.5

CALCULATIONS
Weight equivalent of Diacerein in Capsule powder taken = 5 mg
Weight of Diacerein R.S taken = 5 mg
Total amount of Diacerein theoretically =10 mg
Average value of percentage Recovery = 99.87%
The following statistical parameters were evaluated
Standard deviation = 0.3512
Coefficient of variation = 0.3518
Standard error = 0.2028

Table.4: Concentration v/s peak area of Neo cerein

Serial No. Concentration Peak area Percentage
Standard Sample Recovery
(Neo cerein)
1 4 360571 361368 100.3
2 6 491748 500511 100.1
3 8 749288 744554 99.4

Weight equivalent of Diacerein in capsule powder taken =5 mg

Weight of Diacerein R.S taken = 5 mg
Total amount Diacerein theoretically = 10 mg
Average value of percentage Recovery = 99.9%
The following statistical parameters were evaluated
Standard deviation = 0.4740
Coefficient of variation = 0.4744
Standard error = 0.2726
COMPARISON OF THE PROPOSED METHODS WITH THE PUBLISHED METHOD: -
Diacerein in dosage forms were analysed by the HPLC method as given in published work, and a comparison was
done between the proposed method and already published method.
Reagent used
1.Acetnitrile: HPLC grade supplied by Qalinge’s Fine Chemicals, Mumbai
2.Phosphate buffer
HPLC variables
Column: Kromasil C-18, 5 µm ,250x 4.6 mm I’d column
Pumps: -Jasco 2080 plus
Injector: Jasco Autosampler 2055
Detector : UV 254 nm
Mobile phase: Phosphate buffer: Acetonitrile (40:60)
Flow rate: 1.5 ml/mn
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Retention time: 4.9 minutes

Table.5: Showing Concentration v/s Peak Area

SL. Conc. Peak area Label Average
NO. Diacerein standard sample claim %
1 4 366726 324419 88.46
2 6 529342 495547 93.6 91.78
3 8 703613 656331 93.28

Table.6: Showing concentration v/s Peak height

SL Conc. Peak Height Label Average
No. Diacerein Standard Sample Claim %
1 4 76483 69900 91.39
2 6 112509 109726 97.52 94.34
3 8 153247 144215 94.10

Table.7:
COMPARISON OF THE PROPOSED METHOD WITH THE PUBLISHED METHOD
Method Content % Label % Recovery
(mg) claim
HPLC
49.9 99.90 99.85
Published method
a) Peak area wise 45.30 91.4 -
b) Peak height wise 45.96 92.2 -

IV. RESULTS AND DISCUSSION

Diacerein is now widely used for the treatment of Osteoarthritis. The mechanism of action defers from the
Nonsteroidal anti-inflammatory drugs since it is not related to the inhibition of Prostaglandins. Anti-osteoarthritis
and cartilage stimulating properties have been demonstrated in vivo and in animal models. Diacerein and Rhein have
been shown to inhibit the production of interleukin-I beta by human monocytes and the effects of the cytokine on
chondrocytes in vivo. Diacerein in therapeutic dose inhibit the stimulation of interleukin-1 beta production and
production of nitrous oxide. It also significantly reduces severity of pathological changes of osteoarthritis compared
to placebo and increases the expression of transforming growth factor TGF-beta-1 and TGF beta-2 with potential
cartilage repairing properties. From the literature, Diacerein is seemed to be a safe and promising drug.
Review of literature suggest that only a few analytical methods have been developed for the estimation of Diacerein
in dosage forms. There are no official methods for its quantification. Only One HPLC method with UV detection
were published for its estimation 0f Diacerein in capsules.
This research work has been taken up to develop and validate simple, sensitive and cost-effective methods
for the estimation of Diacerein in dosage forms. A new LC method with UV detection was developed for the
quantitative determination of Diacerein in pharmaceutical dosage forms. The chromatographic separation was
performed on GPC column with a mobile phase of tetra hydro furan. A flow rate of 1.2ml/minute was used. The
resulting chromatogram exhibited a retention time of 5.784 minute. The limit of detection and limit of quantification
was found to be 0.5µg/m land 1µg/ml. A linearity range of 1-10µg/ml was established. The correlation coefficient
was found to be 1. 007.The results of analysis were treated statically, as per ICH guidelines for validation of
analytical procedures and by recovery studies. The results were found to be accurate and not much interferences
from capsule excipients. The active pharmaceutical ingredient was extracted from its finished dosage forms using
tetrahydrofuran. The percentage recovery was found to be 99.9%w/w.
Comparison of the proposed method with published method
The proposed method was compared with published HPLC method for analysis of Diacerein in dosage
forms using phosphate buffer: Acetonitrile (40:60). The percentage label claim found to be 91.4(Peak area wise).
The proposed method was found to be accurate and reproducible than the published method. Moreover, the
proposed HPLC method with UV detection was found to be more sensitive than the published method. This method
shows better accuracy, Linearity range of 1-10µg/ml indicates reliability of the method. A low LOD value of 0.5µg

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suggest that the method may extended to determine the concentration of Diacerein in body fluids also, after
optimization of system parameters and revalidation.

V. CONCLUSION
The efficient development and validation of Analytical methods are critical elements in the development of
pharmaceuticals he desirable characteristics for an analytical method include high sensitivity and selectivity,
accuracy, precision and robustness.
The HPLC method demonstrated herein are applicable to the estimation of Diacerein in pure as well as in
existing dosage forms. In order to ensure that the data generated with each of the above methods are accurate and
precise, the experiments have been performed on calibrated equipment using suitable reference standards. To prove
and document the reliability of the methods, validation as per ICH guidelines has been carried out to a possible
extent.
The proposed HPLC method provides simple, accurate and reproducible quantitative method for routine in vitro
tests of Diacerein dosage forms. Finally, as mentioned no pharmacopoeial methods for determination of Diacerein in
dosage forms have been reported yet. Hence the proposed method can be considered as simple and specific method
for the estimation of Diacerein in pure and dosage forms.

Table.8: REPORT OF COMPARISON BETWEEN THE PROPOSED METHOD

Method Content/ % Accuracy Repeatabilit Linearit Correlat LOD

Brand used Capsule Label 100% y y range ion
(mg) claim Level N=3 coeffici
±SD % CV ent

HPLC
Diacerein 49.9 99.90 100.20 - 1-10 1.007 0.5
50 ±0.4500 µg/ml µg/ml

REFERENCES
[1]. Miller D.R, Osteoarthritis: Pharmacotherapy self-assessment program.4th Edition, American
College of Clinical Pharmacy 2007:225-227.
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Arthritis Research &Therapy 2008,10, R71 doi:1186/ar2444.
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[5]. www.ncbi.nih.gov/PubMed/11037895, html.
[6]. www.Jkscience.org/archive/volume83/Diacerein.html.
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Prakashan: Mumbai,2002.
[8]. Fidelix T.S, Soares B.G, Trivashani V.F, Diacerein for Osteoarthritis, Cochrane Data base
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[13]. Cartidin monograph-Ranbaxy Laboratories Ltd.

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[14]. N. Kannappan, Madhukar A.R, Srinivasan R, L.A Srinivas, C.H Naveen Kumar, Mannavalan - Analytical
method development and validation of Diacerein tablets by RP-HPLC.
[15]. Dycerin monograph Orbit life sciences, Pvt. Ltd.,
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