Analytical Method Development for Simultaneous Determination of
Glucosamine Sulphate And Diacerein From A Mixture Using Derivative And
Derivative Ratio Spectroscopy
Helen Chattopadhyay^{a}, Sanhita Basu Mallick^{b}
, Sriparna Datta
*.
^{a}
Research Scholar, Dept. of Chemical Technology, 92, A.P.C. Road, University
of Calcutta, Kolkata – 700 009,India.
^{b}
M.Tech final year student, Dept. of Chemical Technology, 92, A.P.C. Road,
University of Calcutta, Kolkata – 700 009,India.
* Associate Professor, Dept. of Chemical Technology, 92,A.P.C. Road,
University of Calcutta, Kolkata – 700 009,India.
Corresponding Author:
sriparnadatta2014@gmail.com
ABSTRACT
The aim of the present work is to develop easy, fast and sensitive
analytical method for the analysis of simultaneous estimation of
Glucosamine sulphate (GS) & Diacerein(DC). The different concentration
of GS & DC in the range of 840µg/mL and 2.520µg/mL respectively were
analyzed. First derivative spectra of the mixtures containing different
ratio of both the drugs were measured at 433.0 nm & 401.50 nm for GS
and DC respectively. The concentration of the drugs was calculated from
their corresponding regression equations measuring the intensity of the
signals of the derivative ratio spectrum at 299.0 nm for GS and at 430.0 nm
for DC. Limit of detection and limit of quantification for
GS and DC were tested in triplicate which was found experimentally
detectable. The average percent recovery was 100.5899.46% for DC and
101.0297.89% for GS. Percent relative standard deviation values of the
methods were found within the acceptable range for both the drugs. It was
observed that the deviation of the mean initial absorbance was less than
2.64% for analytical solution stability study which was within the
acceptable range (±3%) indicating the stability of the analytical
solutions. These methods were validated as per ICH guideline. Hence the
proposed method can be used for the routine analysis of the drugs in a
mixture.
Keywords:
Glucosamine sulphate, Diacerein, Derivative spectrophotometry, Derivative
Ratio Spectrophotometry, Method validation.
INTRODUCTION
Osteoarthritis (OA) is a chronic degenerative disorder of multifactorial
etiology characterized by loss of articular cartilage, hypertrophy of bone
at the margins, subchondral sclerosis and range of biochemical and
morphological alterations of the synovial membrane andjoint capsule.[1]
Present day therapy for OA combines nonpharmacologic and pharmacologic
treatments aimed at symptomatic relief. Although there are no medications
or surgical interventions yet proven to cure OA,considerable research is
currently being directed at disease modification.[2]
Glucosamine is an amino sugar and a prominent precursor in the biochemical
synthesis of glycosylated proteins and lipids and also major component of
joint cartilage. Glucosamine supplements are widely used to prevent
cartilage degeneration in the treatment of arthritis. The molecular
mechanism of glucosamine was also intensely investigated. Glucosamine
decreases the activity of nuclear κB (NFκB) that mediates intracellular
signaling of cytokines and MMPs in stimulated chondrocytes.Glucosamine at a
concentration of 10µg/ml significantly reduced IL1βinduced mRNA
expression of cJunNterminal kinase (JNK), NOS and cyclooxygenase2
(COX2) that synthesizes PGE2 from arachidonic acid, in equine
chondrocytes. It also suppresses the IL1βinduced phosphorylation of p38
mitogenactivated protein kinase (MAPK) in human synoviocytes. Glucosamine
also modulates cell function. Glucosamine hydrochloride inhibited
chondrocytes proliferation at a high concentration (e.g. >5mM) depending
on its culture condition, however, it enhanced expression of matrix
components. Glucosamine was revealed to inhibit ossification of mouse
chondrogenic cells (ATDC5) and induced sulfated glucosaminoglycan by
regulating chondrogenic master genes, Smad2 and Smad4.Glucosamine has a
large amount of in vitro evidence that supports symptommodifying and
structuremodifying effects in clinical use. [3]
Diacerein, also known as diacetylrhein an anthraquinone derivative is a
symptomatic slowacting OA drug. [4] It inhibits interleukin1, which has
demonstrated efficacy on functional manifestation of osteoarthritis and on
the structural component.[5]. In vitro studies have shown that diacerein
not only inhibits IL1β [68], but also stimulates the production of
cartilage growth factors such as transforming growth factor β, even in the
presence of IL1β [9].
A number of studies have shown that the use of diacerein and glucosamine in
combination is beneficial, because both drugs reduce symptoms and change
the articular structure in OA. [10]
Derivative Ratio spectrophotometric method has some reported advantages of
being able to suppress matrix effects, ease of operation and obtaining
results rapidly and offer greater selectivity in the simultaneous
determination of two or more compounds [1113]. Although results from this
method are sometimes not as accurate as those from the HPLC method, it is
still regarded as a good analytical method without prior separation to
determine coexisting similar components in a simple system [14]. It is an
analytical technique of good utility and offers better background
correction than normal spectrophotometry for resolving binary mixtures and
some ternary mixtures [1520].In Derivative Ratio Spectrophotometric method
the spectrum for a mixture is divided by the standard spectra for each of
the analyte and hence the quotient becomes a spectrum that is independent
of the analyte concentration. The use of standardized spectra as divisors
minimizes experimental errors and background noise. Easy measurements on
separate peaks, higher values of the analytical signals and no need to work
only at zerocrossing points (sometimes coexisting compounds have no
maximum or minimum at these wavelengths) are advantages for derivative
ratio spectrophotometry in comparison with the zerocrossing derivative
spectrophotometry. Also, the presence of a lot of maxima and minima in
derivative ratio spectra seems to be an advantage ,since these wavelengths
give an opportunity for the determination of these compounds in the
presence of other active compounds and excipients that may interfere.[21]
In this method, overlap of the spectra in a certain region is desirable,
because upon dividing of one spectrum by another, the error increases when
one of the absorbance approaches zero [2224]. This method permits the use
of the wavelength of greatest sensitivity either at maximum or at minimum.
The review of the literature revealed that there is no Spectrophotometric
method available for the combination of Glucosamine sulphate and Diacerein.
Aim of present work is to develop a simple, economic, reproducible and
rapid method for simultaneous estimation of this binary drug formulation.
MATERIAL AND METHODS
1. Chemicals and reagents:
Glucosamine sulphate and Diacerein both were supplied by Biogen Extracts
Pvt. Ltd. ( Bangalore,India),Ninhydrine (AR) (Spectrochem Pvt.Ltd.) was
purchased from local supplier.Phosphate buffer was prepared by USP method.
Throughout the experiment we used Demineralised water.
2. Preparation of the drug solutions:
Different concentration of glucosamine sulphate (GS) and Diacerein (DC)
solutions in pH=7.4 phosphate buffer were prepared. Definite amount of 0.2%
ninhydrin solution and pH=6 phosphate buffer were added to each solution.
All the reaction mixtures were kept in water bath for 30 minutes, after
which they were transferred to ice bath to stop the reaction and were
allowed to stand at room temperature for another 10 minutes [25]. The
solutions were scanned seperately in the wavelength range of 200nm to
700nm. in spectrum mode. (Shimadzu UV probe 2.42 version software was used)
.
3. First derivative Spectrophotometric method (D1):
The obtained zero order absorption spectra were derivatized from first to
fourth order. First order derivative (n=1) with Δλ = 5nm spectra showed
good sensitivity and linearity hence we calculated the the zero crossing
wavelengths from all the spectra.
4. First derivative of the ratio Spectrophotometric method (RD1):
The ratio spectra of different GS standards at increasing concentrations
was obtained by dividing each with the stored spectrum of the standard
solution of DC (15.0μg/ml) (computer aided) are shown in [Figure 3a] and
the first derivative of these spectra traced with the interval of Δλ = 5nm
were illustrated in [Figure 3b]. When the Δλ values increases, the signal
amplitude decreases slightly; Δλ =5 nm was considered to be the optimum
value. The ratio and derivative ratio spectra of the solutions of DC at
different concentrations traced with the interval of Δλ = 5nm by using the
standard spectrum of GS (20.0 μg/ml) as divisor (computer aided), were
demonstrated in [Figure 4a] and [Figure 4b], respectively. Divisor
concentrations of both GS and DC were optimum in terms of sensitivity,
repeatability and signal to noise ratio.
5. Assay of GS and DC mixture:
The absorption spectrum was recorded for the laboratory prepared mixtures,
against water as blank. The mixtures were analyzed by the developed D1 and
RD1 methods.
For the simultaneous determination of GS and DC by the proposed method, it
was necessary to study the influence of the variables: concentration of the
standard spectrum divisor, number of points for the smoothing function and
the Δλ for measuring the first derivative of the ratio spectra.The
influence of these different parameters was studied to optimize the signal
of the derivative ratio spectra; i.e., to give good selectivity and higher
sensitivity in the determination. Medium scan speed was chosen throughout
the work.
6. Validation of the method:
Linearity
: Spectrophotometric analysis of GS & DC were performed in the range of
840µg/mL and 2.520 µg/mL respectively. The limit of detection (LOD) and
limit of quantification (LOQ) were determined by 3.3 σ/s and 10 σ/s
criteria respectively; where σ is the standard deviation of the analytical
signal and s is the slope of the corresponding calibration curve.
Accuracy
: Accuracy of the method was determined by percent recovery method. The
percent recovery of the added pure drugs was calculated as;
% Recovery = ((DtDs)/Da) X 100;
where Dt is the total drug concentration measured after standard addition;
Ds is the drug concentration in the formulation mixture; Da is the drug
concentration added.
Precision
: Intraday and Interday precision of the proposed methods were determined
and percent relative standard deviation (%RSD) was calculated.
Stability of the analytical solution
: This was evaluated by comparing the values of the freshly prepared drug
solutions and the same solution after 24 hrs.
RESULTS AND DISCUSSION
Derivative Method:
The absorption (zeroorder) UV spectra of GS & DC are shown in the
figure. They exhibited UV absorption with maxima at 431nm and 402 nm for DC
& GS respectively. So, the absorption spectra of GS and DC are strongly
overlapped over the range of 402431 nm (Fig. 1). The zero order spectrum
of the mixture shows that GS can be quantified (with more or less accuracy)
from the mixture but DC cannot be determined from this spectrum. So we have
to opt for these D1 and RD1 method.
Figure 1
: zero order spectrum for GS ,DC & mixture of GS and DC
First derivative Spectrophotometric method (D1)
:
The first derivative spectra of both GS and DC (Figure 2a & 2b) are
shown below. The zerocrossing method is the most common procedure for
conducting analytical calibration in derivative spectrophotometry, so 433.0
nm and 401.50 nm of the spectra were selected as zero crossing wavelengths
for analysis of GS & DC respectively.
Figure 2a.
First derivative UV absorption spectra
Figure 2b.
First derivative UV absorption spectra Of Glucosamine sulphate KCl of
Diacerein
First derivative of the ratio spectra (RD1):
Figure 3a & 3b show the ratio spectra of different concentrations of GS
(spectra divided by the spectrum of a 15.0µg/mL of DC) and DC (spectra
divided by the spectrum of a 20.0µg/mL of GS ) while, Figure 4a & 4b
show their first derivatives. The concentration of the drugs was calculated
from their corresponding regression equations measuring the intensity of
the signals of the derivative ratio spectrum. Wavelength 299.0 nm (maxima)
was selected for the quantification of GS in GS+DC mixture. Again
wavelength 430.0 nm (maxima) was selected for the quantification of DC in
DC+GS mixture. Measured analytical signals at these wavelengths were
proportional to the concentrations of these drugs.
Figure 3a.
Ratio spectrum of GS
Figure 3b
. Ratio spectrum of DC
Figure 3b. Ratio spectrum of DC
Figure 4a.
First Derivative Ratio spectra of KCL
Figure 4b.
First Derivative Ratio spectra of Glucosamine sulphate Diacerein
Statistical Analysis of Results:
Concentration Ranges and Calibration Graphs:
A critical evaluation of the proposed Derivative and Derivative Ratio
Spectrophotometric methods was performed by statistical analysis of the
experimental data. Concentration range(µg/mL), Wavelength (nm), Correlation
coefficient (R^{2}), Slope, Intercept , LOD , LOQ and percentage
relative standard deviation (%RSD) of Intraday & Interday precision are
given in table 1.The linearity of the calibration graphs and the adherence
of the system to Beer’s law were validated by the high values of the
correlation coefficients and the small intercepts. Detection and
quantification limits of the two drugs using the proposed methods were
calculated and were verified and they were found experimentally detectable.
%RSD values of the developed methods do not exceed 2% which demonstrates
the good precision and repeatability of the developed
methods. [26]
Table 1:
validation parameters for first and ratio first derivative spectroscopic
methods
Parameters assessed

Method D1

Method RD1

GS

DC

GS

DC

Concentration range(µg/mL)

840

2.520

840

2.520

Wavelength (nm)

433

401.50

299.0

430.0

Correlation coefficient (R^{2})

0.975

0.999

0.995

0.999

Slope

1.551

4.358

9.21

151.9

Intercept

+0.045

0.016

+1.771

+0.483

LOD

8.7963X10^{3}

5.12X10^{3}

1.2273X10^{2}

6.009X10^{3}

LOQ

0.144

3.692X10^{2}

0.175

0.0449

Intraday precision (%RSD)

0.475

0.46

0.4165

0.469

Interday precision (%RSD)

1.37

0.56

1.88

1.02

Accuracy:
The basic concentration level of sample solution selected for spiking of
the drugs standard solution was 1.2mg of GS and 0.375 mg of DC. Addition of
standard drug solution to preanalysed sample solution at three different
concentration levels (80 %, 100 % and 120 %) were within the range of
linearity for both the drugs.
Table 2:
Recovery studies
Amount of
base drug(mg)

Amount of pure
drug added (mg)

% Recovery

Method D1

Method RD1

GS

DC

GS

DC

GS

DC

GS

DC

1.2

0.375

0.96

0.300

102.06

100.5

99.50

101.01

1.2

0.375

1.2

0.375

97.27

99.6

101.80

96.71

1.2

0.375

1.44

0.45

94.32

101.64

101.77

100.68

Average

97.88

100.58

101.02

99.47

Percent relative standard deviation (%RSD)

3.906

1.01

1.305

2.405

Assay of the mixture:
The proposed methods were successfully applied to the analysis of GS and DC
in synthetic mixtures and results are mentioned in table 3.
Table 3:
Results of Assay of Laboratory mixture by first & ratio first
derivative spectroscopic methods
Weight taken
(mg)

% Amount found

Method D1

Method RD1

GS

DC

GS

DC

GS

DC

2.0

0.25

103.48

97.48

99.79

96.43

1.6

0.375

101.7

104.3

97.07

99.59

0.8

0.75

100.8

102.06

98.2

100.50

Average

101.99

101.28

98.35

98.84

%RSD

1.363

3.476

1.366

2.136

The Derivative and Derivative Ratio Spectrophotometric methods enhance the
detectability of the minor features of the UV absorption spectrum of one of
the component in the presence of other. The proposed methods were validated
as per ICH guideline and LOD & LOQ for method D1 & RD1 for both the
drugs were calculated and tested in triplicate. RSD values found were well
within the acceptable range which indicated that these methods had good
repeatability and reproducibility. Results of recovery studies showed good
accuracy and reproducibility which was evident from the data as results
were close to 100 % and standard deviation was low. For analytical solution
stability study it was observed that the deviation of the mean initial
absorbance was less than 2.64% which was within the acceptable range (±3%)
indicating the stability of the analytical solutions..
CONCLUSION
The present methods were simple, rapid and sensitive for the simultaneous
estimation of Glucosamine sulphate & Diacerein in bulk drugs as well as
in their mixture without prior separation. Thus these methods can be
considered alternative tools for the routine analysis for the mixture of
the combination of the drugs.
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