Varaprasad Bobbarala et al.

/ Journal of Pharmacy Research 2010, 3(2),361-363

Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
HPLC Method Development for Glucosamine Sulphate and Diacerein Formulation
Useni Reddy Mallu1 K. Hussain Reddy 1, Varaprasad Bobbarala2* and Somasekhar Penumajji 3
1Department of Chemistry, Sri Krishnadevaraya University, Anathapur-515003, A.P. India.

2Department of Botany, Andhra University, Visakhapatnam-530003, A.P. India.

3Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Habsiguda, Hyderabad, A.P. India.

Received on: 20-09-2009; Revised on: 16-10-2009; Accepted on:15-12-2009

ABSTRACT
A simple, specific, sensitive, and rapid high performance liquid chromatography (HPLC) method for the determination of Glucosamine
sulphate and Diacerein for assay was developed. Glucosamine and Diacerein were baseline separated and quantitated on C8 reversed phase
column (4.6×250mm, 5.0µm), using a mobile phase composed of a phosphate buffer-acetonitrile (55:45v/v, pH 3.0) delivered at a flow rate of
0.6mL/min, and with UV detection ( λ excitation = 195nm). The method was proven to be linear over a Glucosamine concentration range of 84 to
504µg/mL with a mean correlation coefficient of 0.9999 and a Diacerein concentration range of 5.6 to 33.6µg/mL with a mean correlation
coefficient of 0.9998.

Keywords: High performance liquid chromatography (HPLC), Glucosamine, Diacerein.

INTRODUCTION

Glucosamine (C6H13NO5) is an amino sugar and a prominent
precursor in the biochemical synthesis of glycosylated proteins and
lipids. Glucosamine is part of the structure of the polysaccharides
chitosan and chitin, which compose the exoskeletons of crustaceans
and other arthropods, cell walls in fungi and many higher organisms.
Glucosamine is one of the most abundant monosaccharides 1. It is
produced commercially by the hydrolysis of crustacean exoskeletons
or, less commonly by fermentation of a grain such as corn or wheat. In
the US it is one of the most commonly used non-vitamin, non-mineral,
natural products used by adults as a complementary or alternative
medicine2.
Since glucosamine is a precursor for glycosamino glycans,
and it’s a major component of joint cartilage, supplemental glu-
cosamine may help to prevent cartilage degeneration and treat arthri-
tis. A Cochrane, 2005 meta-analysis of glucosamine for osteoarthritis
found that only “Rotta” preparations (including older studies) found
beneficial effects for pain and functional impairment.3 It also found
that when only the studies using the highest-quality design were
considered, there was no effect above placebo4. In addition, in vitro
analysis of glucosamine has revealed that glucosamine inhibits carti-
lage cell characteristics 5. Studies reporting beneficial effects have
generally used glucosamine sulfate4. Chondroitin sulfate is some-
times used in conjunction, and animal studies suggest that chon-
droitin may increase its efficacy.4 Two recent randomized, double-
blind controlled trials 6,7 have found no effect beyond placebo in re-
ducing pain, while one found an effect beyond placebo8.

Diacerein, also known as diacetylrhein, is a drug used in the

treatment of osteoarthritis. It works by inhibiting interleukin-1. A 2005
Cochrane review found diacerein to be slightly, but significantly, more
effective than placebo in diacerein has a small effect in improving
pain and slowing the progress of osteoarthritis (in the hip)9.
Figure 1: Structure of Glucosamine sulphate. Potassium chloride

*Corresponding author.
Dr. Varaprasad Bobbarala
# 36-92-248/23, Sreenivasa Nagar,
Kancharapalem, Visakhapatnam-530008,
Andhrapradesh, India.
Mobile No: 91-9949129539
E-mail: varaprasadphd@rediffmail.com
Figure2: Structure of Diacerein

Journal of Pharmacy Research Vol.3.Issue 2.February 2010 361-363

 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2010, 3(2),361-363
As Glucosamine is sensitive to UV detector and the Diacerein
is generally estimated using titrimetry, for a formulation of Glucosamine Where in as is the Area of Glucosamine sulphate potassium
and Diacerein a quantitative HPLC method is required to estimate the chloride standard solution, At is the Area of Glucosamine sulphate
amount of the Glucosamine and Diacerein present in the tablet. potassium chloride test sample solution, WT is the Weight taken for
test solution preparation in mg, WS is the Glucosamine working stan-
MATERIALS AND METHODS dard weight taken for standard solution preparation in mg and P is the
Chromatographic Conditions: Potency of Glucosamine sulphate Potassium chloride on as such
basis.
Chromatographic separations were performed using isocratic Percentage (%) Diacerein =
At X WS X 5 X 100 X 25 X P X Avg.wt of tablet X 100
elution at ambient temperature. The Waters HPLC system (Waters
As X 250 X 25 X WT X 5 X 100 X Label Claim
Corporation, Milford, MA, USA) consisted of a model 2965 with UV
detector, and a computer running Waters Empower 3.2 software. The
Where in As is the As is the Area of Diacerein standard
mobile phase was composed of a mixture of Buffer (Add 1.0ml H 3PO4
solution, At is the Area of Diacerein test sample solution, WS is the
in to 2000mL of HPLC water and adjust pH of solution to 3.0± 0.1 with
Diacerein working standard weight taken for standard solution prepa-
dilute Potassium hydroxide solution, degas it with 0.45µ filter) and
ration in mg, WT is the Weight taken for test solution preparation in
acetonitrile (55:45, v/v) and degassed. Glucosamine and Diacerein
mg and P is the Potency of Diacerein working standard.
were separated on a reversed phase C8 column (4.6mm x 250mm, and
5.0µ). The flow rate was set at 0.6mL/min and the injection volume
RESULTS AND DISCUSSION
was 20µL. Glucosamine and Diacerein UV measurements were made
at a wavelength of 195nm.
According to the data obtained the retention time 4.3 corre-
sponds to Glucosamine sulphate and 8.2 corresponds to Diacerein.
Preparation of Standards:
Chromatogram.1 confirms the system suitability of the method. Five
replicated injections of the standard shows that the area of both
Diacerein Stock solutions was prepared by adding 28mg of
Glucosamine and Diacerin is consistent in all the injections and passes
Diacerein working standard in to 250ml volumetric flask add 150ml of
the system suitability (% RSD) of not more than 2%.
diluent (Mix HPLC water and Acetonitrile in the ratio of 25:75 (v/v) mix
and degas.) and sonicate to dissolve the content, then make up to the 0.20

8.178
volume with mobile phase, mix. Blank is prepared by mixing diluent

4.250
AU

0.10

and mobile phase in the ratio of 50:50 (v/v). Glucosamine stock solu- 0.00

tion was prepared by adding 84mg of Glucosamine sulphate potas- 0.20

8.290
4.364
sium chloride working standard in to 50ml volumetric flask add 15ml
AU

0.10

of diluent and 20ml of mobile phase, and sonicate to dissolve the 0.00
0.20
content, and make up to the volume with mobile phase. Pipette out

8.182
4.264
AU

0.10
each 5.0ml of Diacerein standard stock solution and Glucosamine
standard stock solution in to a 25ml volumetric flask make up to 0.00
0.20

8.153
volume with mobile phase, mix.
4.289
AU

0.10

0.00

Test solution preparation: 0.20

8.184
4.299
AU

0.10

Take 20tablets weigh and crush. Weigh accurately equiva- 0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
lent to 168mg of Glucosamine sulphate potassium chloride in to 100ml Minutes

of volumetric flask and add 40ml of diluent and 30ml of mobile phase Chromatogram.1: System suitability
sonicate for 10min and dilute to volume with mobile phase and further
dilute 5 ml of the centrifuged supernatant solution in to 25ml with
mobile phase. 0.30
Diaceren - 8.171

0.28

Procedure: 0.26

0.24
Separately inject equal volume of about 20µL of blank, stan- 0.22

dard preparation in replicate and sample preparation once in to the 0.20

HPLC system and record the chromatograms and calculate. 0.18

Glucosamine - 4.294

0.16
AU

0.14
System Suitability solution: 0.12

0.10

The tailing factor of both active substance peaks in stan- 0.08

0.06
dard solution is not more than 2.0 and the %RSD of five replicate 0.04

injections is not more than 2.0%. 0.02

Calculation: 0.00

Percentage (%) of Glucosamine = 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40
Minutes
6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80

At X WS X 5 X 100 X 25 X P X Avg. Wt of tablet X 100

As X 50 X 25 X WT X 5 X 100 X Label Claim Chromatogram.2: Linearity

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 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2010, 3(2),361-363
Table.1: Linearity of Glucosamine Graph.1: Linearity graph of Glucosamine

Glucosamine Linearity

Linearity Level Concentration(ppm) Area

1 84 163314
2 168 325890
3 252 483224
4 336 632558
5 420 801700
6 504 965128
Correlation co-efficient 0.9999

Table.2: Linearity of Diacerein Graph.2: Linearity graph of Diacerein

Diacerein Linearity

Linearity Level Concentration(ppm) Area

1 5.6 809123
2 11.2 1463760
3 16.8 2139499
4 22.4 2890100
5 28.0 3581101
6 33.6 4283911
Correlation co-efficient 0.9998

Chromatogram.2 represents that this method is linear in dif-
ferent concentrations from 84ppm to 504ppm of Glucosamine sul-
phate with a Correlation coefficient of 0.9999 and 5.6 to 33.6ppm of
Diacerein with correlation coefficient of 0.9998, hence this result indi-
cates that the method is suitable for analysis. Based on the System
suitability and the Linearity conformations this method can be used
for analysis.
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.3.Issue 2.February 2010 361-363