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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.
INTRODUCTION
Glucosamine (C6H13NO5) is an amino sugar and a prominent Since glucosamine is a precursor for glycosamino glycans,
precursor in the biochemical synthesis of glycosylated proteins and and it’s a major component of joint cartilage, supplemental glu-
lipids. Glucosamine is part of the structure of the polysaccharides cosamine may help to prevent cartilage degeneration and treat arthri-
chitosan and chitin, which compose the exoskeletons of crustaceans tis. A Cochrane, 2005 meta-analysis of glucosamine for osteoarthritis
and other arthropods, cell walls in fungi and many higher organisms. found that only “Rotta” preparations (including older studies) found
Glucosamine is one of the most abundant monosaccharides 1. It is beneficial effects for pain and functional impairment.3 It also found
produced commercially by the hydrolysis of crustacean exoskeletons that when only the studies using the highest-quality design were
or, less commonly by fermentation of a grain such as corn or wheat. In considered, there was no effect above placebo4. In addition, in vitro
the US it is one of the most commonly used non-vitamin, non-mineral, analysis of glucosamine has revealed that glucosamine inhibits carti-
natural products used by adults as a complementary or alternative lage cell characteristics 5. Studies reporting beneficial effects have
medicine2. 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.
*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
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
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
8.184
4.299
AU
0.10
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
0.16
AU
0.14
System Suitability solution: 0.12
0.10
0.06
dard solution is not more than 2.0 and the %RSD of five replicate 0.04
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
Glucosamine Linearity
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- 4. Dahmer S, Schiller RM, Glucosamine, Am Fam Physician, 2008, 78
(4): 471–6.
ferent concentrations from 84ppm to 504ppm of Glucosamine sul-
5. Terry DE, Rees-Milton K, Smith P, Carran J, Pezeshki P, Woods C,
phate with a Correlation coefficient of 0.9999 and 5.6 to 33.6ppm of Greer P, Anastassiades TP, N-acylation of glucosamine modulates
Diacerein with correlation coefficient of 0.9998, hence this result indi- chondrocyte growth, proteoglycan synthesis, and gene expression,
cates that the method is suitable for analysis. Based on the System J. Rheumatol, 2005, 32 (9): 1775–86.
6. Clegg DO, Reda DJ, Harris CL, Glucosamine, chondroitin sulfate, and
suitability and the Linearity conformations this method can be used
the two in combination for painful knee osteoarthritis, N. Engl. J.
for analysis. Med. 2006, 354 (8): 795–808.
7. Rozendaal RM, Koes BW, van Osch GJ, Effect of glucosamine sulfate
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