Ijpcr 109

INTERNATIONAL JOURNAL OF PHARMACEUTICAL CHEMISTRY RESEARCH
Volume 1 Issue 1 2012 www.earthjournals.org

39
RESEARCH ARTICLE
APPLICATION OF UV SPECTROPHOTOMETRIC AND HPLC
METHODS FOR SIMULTANEOUS DETERMINATION OF
ROSIGLITAZONE MALEATE AND GLICLAZIDE IN COMBINED
TABLET DOSAGE FORM

Dhole S. M.
1
, Khedekar P. B.
2
and Amnerkar N. D
1

1
Department of Pharmaceutical Chemistry, Sharad Pawar College of Pharmacy, Rashtrasant
Tukadoji Maharaj Nagpur University, Nagpur-441110, Maharashtra, India.
2
University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur
University, Nagpur-440033, Maharashtra, India.

Abstract:

Two methods are described for the simultaneous determination of rosiglitazone maleate (ROS) and gliclazide (GLZ)
in binary mixture. The first method was based on spectrophotometric method by using absorption correction
method, correction of absorbance for estimation of GLZ. Two wavelengths, 229 nm where both the drugs having
absorbance and other 320nm (max of ROS) where ROS has absorbance but GLZ shows no interference were
selected for estimation of both drugs using methanol as solvent. The linearity obtained in the range of 2-12 g/mL
and 4-24 g/mL for ROS and GLZ, respectively. The second method was based on HPLC, separation of two drugs
on the reversed phased column C
18
at ambient temperature using a mobile phase consisting of
acetonitrile:methanol:water (40:30:30 v/v, pH adjusted to 3.0 with orthophosphoric acid) at the flow rate of 1
mL/min. Quantitation was achieved with UV detection at 240 nm based on peak area with linear calibration curves
at concentration ranges 1-10 g/mL and 20-100 g/mL for ROS and GLZ, respectively. No chromatographic
interference from the tablet excipients was found. Both methods were validated in terms of accuracy, precision,
ruggedness, limit of detection and limit of quantitation. The high recovery and low coefficients of variation
conforms the suitability of the methods for simultaneous analysis of two drugs in combined tablets. Statistical
analysis proves that the methods were found to be suitable for the routine quality control analysis in pure and
pharmaceutical dosage forms.
Keywords: Absorption correction method; Gliclazide; HPLC; Quantitative Analysis; Rosiglitazone maleate;
Validation.



40
Introduction
Rosiglitazone maleate (ROS), chemically
[()-5-[4-[2-[N-methyl-N(2-pyridyl)amino]-
ethoxy]benzyl]-2,4-dione
thiazolidine]maleate (Fig.1), is a potent oral
antihyperglycemic agent belong to
thiazolidinedione group of antidiabetic drugs
which exert the glucose lowering effect by
binding to peroxisome proliferator activated
receptors gamma (PPAR)
[1-3]
.
Gliclazide (GLZ), chemically (1-(3-
azabicyclo[3.3.0]oct-3-yl)-3-p-
tolylsulfonylurea or 1-
(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-
tosylurea (Fig.2) is hypoglycemic agent of
sulfonyl urea groups of antidiabetic drugs
used in the treatment of type-2 diabetes
mellitus. It reduces blood glucose level by
correcting both defective insulin secretion
and peripheral insulin resistance, increasing
the sensitivity of -cells to glucose,
decreasing hepatic glucose production and
increasing glucose clearance
[3,4]
.
O
S
NH
O
O
N
N
CH
3
.
COOH
COOH

Fig.1. Structure of ROS
NH
S
NH
N
C H
3
O O
O

Fig.2. Structure of GLZ
Literature survey reveals several methods
like UV spectrophotometry, HPLC, HPTLC
are available for the analytical determination
of ROS and GLZ as an individual drug and
in combination with other drugs in
pharmaceutical formulations and in
biological fluids
[5-19]
.The present manuscript
describes two new methods for the
simultaneous determination of two drugs.
The methods were validated as per ICH
norms
[20]
.

Materials and Methods
ROS and GLZ, reference standard were
obtained as a generous gift sample from
Panacea Biotec Ltd., Malpur, Solan (H.P.),
India. Tablets labeled to contain ROS 2mg
and GLZ 80mg, purchased from local
market. All the chemicals used were of AR
and HPLC grade, obtained from Merck
Limited, Mumbai, India.



41
Instrumentation
UV spectrophotometric analyses were
carried out on a Shimadzu 1700 Double
beam UV-Vis spectrophotometer, with 1.0
cm quartz cells.
The HPLC analysis were carried out on
Agilent 1120 Compact LC system composed
of binary pump, manual injector, UV
detector and Ezchrome EliteCompact
software. The column used was Agilent TC-
C18 (250mm4.6mm i.d., 5m partical size)
and the mobile phase consisted of
acetonitrile:methanol:water (40:30:30 v/v,
pH adjusted to 3.0 with orthophosphoric
acid) at a flow rate of 1.0ml/min. Detection
was performed on at 240 nm.
Preparation of standard stock solutions
An accurately weighed quantity 10 mg each
of ROS and GLZ were transferred to two
100 mL volumetric flask dissolved in
methanol and diluted to the mark with same
solvent to obtained standard stock solution
100 g/mL of each drug.
For spectrophotometric analysis, the
working standard solutions of ROS and GLZ
were prepared by taking suitable aliquots of
drug solution from the standard stock
solutions and the volume was made up to 10
mL with methanol to get concentrations of
2-12 g/mL for ROS and 4-24 g/mL GLZ.
For HPLC analysis, the working standard
solutions of ROS and GLZ were prepared by
taking suitable aliquots of drug solution
from the standard stock solutions and the
volume was made up to 10 mL with mobile
phase to get concentrations of 1-10 g/mL
and 20-100 g/mL for ROS and GLZ,
respectively.

Method I: Application of Absorption
correction method

An aliquot portion of standard stock
solutions were diluted with methanol in 10
ml volumetric flask separately to obtain 15
g/mL of each drug. These solutions were
scanned in spectrum mode from 400-200
nm. The overlain spectra of both drugs were
obtained (Fig.3).

Fig.3. Overlain spectra of ROS and GLZ
Two wavelengths from spectra, 229 nm and
320 nm were selected. The absorptivity
values of ROS and GLZ were determined at
selected wavelengths. At 320 nm, estimation
of ROS was done where GLZ has no
interference. The absorbance of working
standard solution of ROS at 229 nm was
calculated using equation (1) and then from
the total absorbance of sample mixture at
wavelength 229 nm, the contribution due to
ROS was subtracted. The calculated
absorbance was called as corrected
absorbance for GLZ. The concentrations of
GLZ (C
y
) at 229 nm using the corrected
absorbance was determine using absorptivity
value.



42
A
x

229 nm
= C
x
x A (1 %, 1 cm)
229 nm of ROS
--------(1)
CA
y

229 nm
= A
229 nm
- A
x

229 nm

--------(2)

Where, C
x
and C
y
: Concentration of ROS
and GLZ, respectively. A
x

229 nm
and A
229 nm

: Absorbance of ROS and mixture of
standard solution, respectively. CA
y

229 nm
:
Corrected absorbance of GLZ at 229 nm.

Preparation of sample solution
For estimation of drugs in the commercial
tablet formulation, twenty tablets were
weighed, and their mean weight was
determined. The tablets were crushed to a
fine powder. For the analysis of ROS, a
standard addition method was used. An
accurately weighed amount 19 mg of pure
ROS was added to finely powdered sample
to bring the concentration ratio of 1:2. A
quantity of powder equivalent to 40 mg of
GLZ was accurately weighed and
transferred to 100mL volumetric flask.
Methanol (25 mL) was added to it and
sonicated for 15 min. The volume was
adjusted up to the mark with methanol and
then filtered through Whatman filter paper
42. An aliquot portion of obtained filtrate
was diluted to 10 mL with methanol to get
final concentration within linearity range.
Absorbance of the resulting solution was
measured at selected wavelengths and the
values were substituted in above equations
to obtain their respective concentrations.
Method I: Application of RP-HPLC
method
An aliquot portion of standard stock
solutions were diluted with mobile phase in
10 mL volumetric flask to obtain 2 g/mL
and 80 g/mL of ROS and GLZ,
respectively. Calibration standard at five
levels were prepared by appropriately
diluting standard stock solutions in the
concentration range of 1-10 g/mL and 20-
100 g/mL for ROS and GLZ, respectively
Preparation of sample solution
Twenty tablets were weighed and their mean
weight was determined. The tablets were
crushed to a fine powder. A quantity of
powder equivalent to 40 mg of GLZ was
accurately weighed and transferred to 100
mL volumetric flask and dissolved in 25 mL
of methanol. Sonication was done for 15
min with swirling and the volume was
adjusted up to the mark with methanol and
then filtered through 0.22 filter paper. An
aliquot portion of obtained filtrate was
diluted to 10ml with mobile phase to get
final concentration within linearity range. A
20 L of sample solution was injected into
the chromatographic system and analyzed
quantitatively. The analysis was repeated for
six times.
Method Validation
The validation procedure was followed the
ICH guidelines and United State
Pharmacopoeia for the analysis of ROS and
GLZ by spectrophotometric and HPLC
methods. The performance parameters
evalutated for these methods were linearity,
precision, accuracy, limits of detection and
quantitation, specificity and ruggedness.
Linearity:
Linearity was studied by analyzing six
standard solutions (n=3) covering the range
of 2-12 g/mL and 4-24 g/mL for ROS and
GLZ, respectively for UV
spectrophotometric and 1-10 g/mL and 20-
100 g/mL for ROS and GLZ, respectively
for HPLC. Standard solutions containing
100 g/mL of each drug in solvent were
prepared in triplicate. Aliquots of these


43
solutions were diluted to six different
concentrations covering the above mention
range. Calibration curves with concentration
verses absorbance or peak area was plotted
for each method and the obtained data were
subjected to regression analysis using the
least squares method.
Precision:
The precision of the reported methods for
the determination of ROS and GLZ were
studied using the repeatability parameters. It
was expressed as relative standard deviation
(R.S.D.) at series of measurements. The
precision was evaluated by analyzing six
independent assays of test sample (n=6), at
final test concentration of analysis. ROS and
GLZ contents and the R.S.D. values were
calculated.
Limit of detection and limit of quantitaton
The limit of detection (LOD) and limit of
quantitaton (LOQ) were separately
determined based on standard deviation of
the y-intercept and the slope of the
calibration curve by using the equations (1)
and (2), respectively.
LOD = 3.3 LOQ =10
S S
where, : standard of y-intercept and S:
slope of calibration curve.
Specificity:
The specificity of the spectrophotometric
and HPLC methods was evaluated through
the analysis of mixture of all excipients
contained in tablet and the results was
evaluated by comparing with standard and
sample solutions for possible interfering
peaks and presence of possible interfering
bands.
Accuracy
The accuracy of the methods was
determined by calculating recoveries of
ROS and GLZ by the standard addition
method at different levels to the pre-
analyzed sample. For that known amounts of
standard solutions of ROSI and GLIM (80,
100, and 120 % of test concentration) were
added to prequantified sample of tablet
dosage form. At each level, samples were
prepared in triplicate and the mean
percentage recovery and R.S.D. value were
determined for both methods.
% Recovery = [(Cs +Cstd) Cs]/Cstd
Where, (Cs +Cstd) is concentration of
recovery solution (drug tablet + reference
standard); Cs is concentration of drug tablet
solution; Cstd is concentration solution of
drug reference standard.
Ruggedness:
Ruggedness of the proposed method was
determined by analysis of sample solution
prepared by proposed methods between
different time intervals, days and analysts.
The % R.S.D. was determined.
Results and Discussion
Method development and optimization

UV spectrophotometric method using
absorption correction method, two
wavelengths were selected from the overlain
spectrum scanned in the range of 400-200
nm using methanol as solvent. Wavelengths
229 nm and 320 nm as absorption maxima
were selected for estimation of GLZ and
ROS, respectively. The absorptivity values
at selected wavelengths are listed in Table 1.



44
Table 1. Absorptivity values
Absorptivity values at
wavelength*
Drug
229 nm 320 nm
ROS 392.50 175.9
GLZ 454.30 -
*Average of five determination.

For HPLC method, the best
chromatographic conditions were adequately
selected to develop a reversed phase liquid
chromatographic method that working in
isocratic mode, allowed the determination of
ROS and GLZ in tablets, without
interference of its common excipients and in
short time as demonstrated in the
chromatogram Fig. 4. Different mobile
phases were investigated, however the
mixture containing
acetonitrile:methanol:water (40:30:30 v/v,
pH adjusted to 3.0 with orthophosphoric
acid)and C18 column was selected that
allowed good separation and formed
symmetrical peak for ROS and GLZ, at a
flow rate of 1ml/min. UV detection was
performed at 240 nm

Fig. 4. Chromatogram of sample solution
of ROS and GLZ
Method validation
Linearity: A linear correlation was found
between absorbance/peak area and the
concentrations of ROS and GLZ .The
regression analysis data are represented in
Table 2. The regression coefficients (r
2
)
obtained were higher than 0.99 for both
drugs, which attest the linearity of the
method.

LOD and LOQ
The LOD and LOQ were found to be 0.56
g/ml and 1.69 g/ml for ROS, 0.74g/ml
and 2.2 g/ml for GLZ, respectively for UV
spectrophotometric method. For HPLC,
LOD and LOQ were found to be 0.34 g/ml
and 0.98g/ml for ROS, 0.67g/ml and 1.8
g/ml for GLZ, respectively.
Precision
Mean contents of ROS and GLZ in precision
analysis (n=6) were very close to labeled
claim of respective drugs The %R.S.D.
value was lower than 2%, assure the
precision of the methods and the results are
shown in Table 3
System suitability: System suitability
parameters such as the number of theoretical
plates and peak tailing are determined. The
results obtained are shown in Table 4.
Accuracy was investigated by means of
recovery studies using the proposed
methods. The percent recoveries after
spiking with additional standard drug afford
recovery in the range of 98-102% and the
results are listed in Table 5. Specificity: Peak
purities higher than 99.0%were obtained for
ROS and GLZ in the chromatogram of
sample solutions and show no interference
due to excipients in the same retention time
of drugs.
The summary of validation parameters is
listed in Table 6.


45

Table 2. Regression analysis data
UV method HPLC Regression
parameters ROS GLZ ROS GLZ
Concentration
range (g/ml)
2-12 4-24 1-10 20-100
Correlation
coefficient (r
2
)
0.9993 0.9992 0.9979 0.9989
Slope 0.0179 0.0456 520.82 278.83
Intercept 0.0005 0.0067 997.12 4673

Table 3. Analysis of formulation using the UV and HPLC methods
UV method HPLC Drug Label
claim
mg/tablet
Parameter
(n=6)
ROS GLZ ROS GLZ
ROS 2 Mean (%) 99.70 99.79 99.25 99.64
Amount
found (mg)
1.994 79.832 1.98 79.71
S.D.
*
0.702 0.739 0.693 0.608
GLZ 80
% R.S.D.
*
0.705 0.741 0.698 0.611

Table 4. Result from System suitability study
Parameters*
ROS GLZ
Retention time (min) 2.51 6.33
Tailing factor 1.22 1.76
Theoretical plates (N) 79834 13557
Resolution - 3.81
Capacity factor 1.35 1.98
*Average of six determinations.
Accuracy

Table 5. Result of Recovery studies


46

Methods Drugs % Amount added % Recovery S.D.(n=3)
UV method ROSI 80 98.72 0.511
100 99.20 0.393
120 99.88 0.732
GL 80 99.35 0.287
100 100.22 0.496
120 98.94 0.514
HPLC
method
ROSI 80 99.12 0.251
100 99.03 0.337
120 99.16 0.350
GLZ 80 99.71 0.589
100 99.92 0.883
120 99.76 0.706
* S.D.: standard deviation.

Analysis of marketed formulation
The proposed validated methods were
successfully applied for determination of
ROS and GLZ in their combined dosage
form. Experimental results of the amount of
ROS and GLZ in tablets, expressed as
percentage of label claim were in good
agreement with the label claims, thereby
suggesting that there is no interference from
the excipients which are normally present in
tablets.
Conclusion
Absorption correction and HPLC methods
were developed for the determination of
ROS and GLZ in combined tablets. Once the
optimized conditions selected, both the
methods were validated and shows good
performances with respect to linearity,
precision and accuracy. Comparing to
HPLC, the developed UV method was less
expensive and short analysis of time. The
developed methods were found to be simple,
specific, rapid, precise and accurate. The
results of this study demonstrated that both
methods could be used for the routine
quality control analysis of ROS and GLZ in
bulk and pharmaceutical dosage form.


47
Table 6. Validation parameters of evaluated methods
UV HPLC
Parameters
ROS GLZ ROS GLZ
Concentration range(g/ml) 2-12 4-24 1-10 20-100
Precision, n=6 (%R.S.D.) 0.693 0.611 0.9995
Recovery, n=9 (%S.D.) 99.270.528 99.50 0.653 99.10 0.067 99.80 0.109
LOD(g/ml) 0.56 0.74 0.34 0.67
LOQ(g/ml) 1.69 2.2 0.98 1.8
Ruggedness(n=3) (%R.S.D.)
Intraday 0.274 0.287 0.235 0.462
Interday 0.269 0.296 0.298 0.541
Analysts 0.259 0.98 0.547 0.594
Specificity Specific Specific
S.D.: Standard deviation; R.S.D.: Relative standard deviation.

Acknowledgements
Authors are thankful to Panacea Biotec Ltd.,
Malpur, Solan (H.P.), India for providing the
gift samples of drugs ROS and GLZ,
respectively and also thankful to Dr. K. P.
Bhusari, Principal, Sharad Pawar College of
Pharmacy, Nagpur for providing
experimental facilities for this
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Volume 1 Issue 1 2012 www.earthjournals.org

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