Reversed-Phase Liquid Chromatographic
Method for Simultaneous Determination of Metformin
and Repaglinide in Pharmaceutical Preparations
T.M.
Kalyankar1, and R.B. Kakde2*
1School of Pharmacy, S.R.T. Marathawada University, Nanded,
Maharashtra, India.
2Departments of Pharmaceutical
Sciences, Rastrasant Tukadoji
Maharaj Nagpur University, Nagpur, Maharashtra,
India.
ABSTRACT:
A simple, rapid, precise and
accurate reversed phase high performance liquid chromatographic method has been
developed for the simultaneous determination of metformin
in combination with repaglinide. This method uses a Hypersil ODS C18
(250mm×4.6mm×5µ
particle Size) analytical column, a mobile phase of acetonitrile
and buffer containing 0.05 M ammonium acetate in the ratio 60:40 (v/v), and pH
adjusted to 5.0 with orthophosphoric acid. The
instrument was set with a flow rate of 1 ml/min and PDA detector wavelength at
271 nm. The retention times for metformin and repaglinide are 3.13 min and 10.01min respectively. The
method was validated as per the ICH guidelines. The linearity range for metformin and repaglinide were
found to be 200-1200 and 0.5-3.0 µg/ml respectively. The percentage recovery
for metformin and repaglinide
are in the range between 99.25–99.48 and 98.58–100.26 respectively. The correlation coefficients of metformin and repaglinide were
0.999, and 0.999, respectively. The relative standard deviation for six
replicates was always less than 2%. The statistical analysis proves that the
method was suitable for analysis of metformin and repaglinide as a bulk drug and in pharmaceutical
formulation without any interference from the excipients.
KEYWORDS: Antidiabetic drugs, Metformin,
Repaglinide, Validation and RP-HPLC.
INTRODUCTION:
Diabetes is one of the
costliest health problems in the world. Globally, diabetes is likely to be the
Fourth leading cause of death.1 Approximately
90% of people with diabetes have type 2 diabetes. It usually begins as insulin
resistance, a disorder in which the cells do not use insulin properly. As the
need for insulin rises; the pancreas gradually loses its ability to produce
insulin.
Type II diabetes is
associated with older age, obesity, family history of gestational diabetes,
impaired glucose metabolism, physical inactivity and race/ ethnicity.2
If the glycemic target level is not achieved with one
oral agent alone, combination oral and/or insulin therapy is recommended.3,
4 Combination oral therapy becomes an obvious choice when glycemic control is not achieved with conventional
monotherapy.5 The advantages of oral dose combinations as compared
to their components which are taken alone are lower cost and better patient
compliance.6, 7
Combination therapy has been
shown to achieve greater blood glucose lowering than monotherapy
because different classes have different and complimentary mechanisms of
action. Therefore, it is more logical to add another drug than replace the
existing drug. The rapid introduction of combination therapy with two or three
complementary oral anti diabetics help in targeting the dual effect and also
reduced adverse effects.8
Chemically, Metformin (fig. -1A) is 1, 1-Dimethylbiguanide and Repaglinide
(fig. -1B) is S (+)
2-ethoxy-4(2((3-methyl-1-(2-(1-piperidinyl) phenyl)-butyl) amino)-2-oxoethyl)
benzoic acid. Metformin is an anti-diabetic drug from the biguanide
class of oral hypoglycemic agents, given orally in the treatment of non–insulin
dependent diabetes mellitus.9 Metformin reduces free fatty acid oxidation because
enhanced free fatty acid oxidation in diabetes contributes to increased hepatic
glucose production and development of insulin resistance while Repaglinide lowers blood glucose
levels by stimulating the release of insulin from the pancreas. This action is
depends upon functioning beta (ß) cells in the pancreatic islets.10
Fig. 1. Structures of Antidiabetic Drugs: Metformin (A)
and Repaglinide (B)
Literature survey reveals
that several analytical methods are available for determination of metformine and repaglinide
individually or in combinations with other drugs in pharmaceutical dosage forms
and also in biological samples11-15. No method has been reported for
determination of metformine and repaglinide
in combination. The attempt was made to develop a simple chromatographic method
for simultaneous estimation of these two drugs in a tablet formulation. The
method described is rapid, precise and accurate and can be used for routine
analysis of tablets. The developed method was then validated as per ICH
guidelines.16-17
EXPERIMENTAL:
Instrumentation:
The
LC system was from Perkin Elmer Quaternary pump Series 200 and was comprised of
auto sampler injector; and an Intelligence PDA detector connected to the Total
Chrome Navigator version 6.3. For controlling the instrumentation as well as
processing the data generated was used.
Material and reagents:
Metformin (MET) was obtained from Macleoides
Research Laboratory (Mumbai, Maharashtra, India) and Repaglinide
(RGN) was obtained from Wallace Pharmaceuticals, Pvt. Ltd. (Goa, India). Acetonitrile (HPLC grade), Ammonium acetate (AR grade),
Methanol (HPLC grade), Orthophosphoric acid (AR
grade) were obtained from Rankem Pvt. Ltd. Delhi,
India. The 0.45 µm membrane filter was
used throughout the experiment. The tablets of MET in combination with RGN (Eurepa-MF) were purchased from Local market. Double
distilled water was used through out the experiment.
Other chemicals used in the experiment were of analytical or HPLC grade.
Chromatographic
conditions:
An isocratic mobile phase
consists of acetonitrile and ammonium acetate buffer
(pH 5.0) in the ratio of 60:40 v/v, flowing through the column at a constant
flow rate of 1.0 ml/min. A Hypersil ODS C18
column (250mm × 4.6mm, 5 μ) was used as the stationary phase. MET and RGN
have different λmax but considering the
chromatographic parameter, sensitivity, and selectivity of the method for these
drugs, 271 nm was selected as the detection wavelength for PDA detector. The
injection volume was 10 µl.
Mobile phase:
The mobile phase consisted
of Acetonitrile and Ammonium acetate buffer in the
ratio 60:40 (v/v). The pH of the buffer was adjusted to 5.0 with orthophosphoric acid. The buffer used in the mobile phase
consisted of 0.05 M Ammonium Phosphate in double distilled water. The mobile
phase was premixed and filtered through a 0.45-µm membrane filter and degassed.
Standard stock solutions:
Metformin:
An
accurately weighed 50 mg of MET was transferred in a 50 mL
volumetric flask, and dissolved with methanol. The final stock solutions was sonicated for 20 min and filtered through 0.45 µm membrane
filter.
Repaglinide:
An
accurately weighed 50 mg of RGN was transferred in a 50 mL
volumetric flask, and dissolved with methanol. The final stock solutions was sonicated for 20 min and filtered through 0.45 µm membrane
filter.
Preparation of standard mixed solutions:
An
accurately weighed 500 mg of MET was transferred in a 50 mL
volumetric flask, and 25ml of methanol was added. To this 1ml of standard stock
solution of RGN was added. The volume was made upto
the mark with methanol. The contents were sonicated
for 20 min, and then filtered through 0.45μm membrane filter.
Calibration curve solutions:
From
the mentioned stock solutions of MET and RGN calibration curve solutions
containing 200 - 1200 µg/ml of MET and 0.5-3.0 µg/ml of RGN in each calibration
level were prepared.
Preparation of sample solutions
Twenty
tablets were weighed and finely powdered. A quantity equivalent to one tablet
containing 500 mg of MET and 1 mg of RGN was transferred in a 50 mL volumetric flask and volume was made with methanol. The
contents were sonicated with methanol for 20 min, and
filtered through 0.45μm membrane filter.
RESULTS
AND DISCUSSION:
Optimization of
chromatographic conditions:
The chromatographic method
was optimized by Different experiments were performed to achieve the adequate
retentions and resolution for the peaks of RGN and MET. To set the adequate
retentions and resolution, the effects of the mobile phase components, changes
in ionic strength were studied, initially methanol and water in different
ratios were tried. But MET gave broad peak shape While RGN gave no peak, so
water was replaced by potassium dihydrogen buffer
(0.2 M), and mixture of methanol and potassium dihydrogen
phosphate buffer in different ratios were tried. It was found that both peak
shows broad peaks finally Acetonitrile: 0.05M Ammonium
acetate buffer pH 5.0 adjusted with OPA in ratio of 60: 40 v/v gave acceptable
retention time (3.13 min for MET and 10.01 min for RGN) and good resolution for
MET and RGN was found to be 12.43 at the flow rate of 1.0 ml/min. gave adequate
retentions and resolution, and the chromatographic run was 15 min.
Validation of the method:
Specificity:
The
specificity of the method was checked by a peak purity test of the sample
preparation done by PDA detector. The peak purity for RGN and MET was found to be 999. The result
of the peak purity analysis shows that the peaks of the analytes
were pure and also the formation excipients were not
interfering with the analyte peaks.
Calibration and linearity:
The
standard solutions containing 200 - 1200 µg/ml of MET and 0.5 - 3.0 µg/ml of
RGN in each linearity level were prepared. Linearity solutions were injected in
triplicate. In the simultaneous determination, the calibration graphs were
found to be linear for both the analytes in the
mentioned concentrations. The coefficient of correlation was found to be 0.999
and 0.999 for MET and RGN, respectively.
Precision:
The precision of the method
was studied by determining the concentrations of each ingredient in the tablets
six times. In the precision study, % relative standard deviation of the MET and
RGN were found to be 1.024 and 0.768 respectively. The results of precision study indicate that the
method is reproducible.
Intermediate precision:
Intermediate
precision of the method was done by analyzing the sample six times on different
days, by different chemists, using different analytical column of the make, and
different HPLC systems. The percentage assay was calculated using the
calibration curve. The assay results are shown in Table I.
Table I. Assay Results of Active Ingredients in Tablets
|
Set |
Ingre dients |
Label claim (mg) |
Found (mg)
† |
% Label claim ± %RSD |
|
Precision |
MET |
500 |
496.35 |
99.27 ± 1.024 |
|
RGN |
1 |
0.994 |
99.41 ± 0.768 |
|
|
Intermediate
precision |
MET |
500 |
499.55 |
99.91 ± 1.022 |
|
RGN |
1 |
0.995 |
99.53 ± 0.711 |
†
Average of six analyses
Accuracy:
The accuracy of the method
was studied by recovery experiments. The recovery experiments were performed by
adding known amounts of the pure drug. The recovery was done at three levels:
80%, 100%, and 120% of the label claim. Three samples were prepared for each
recovery level. The recovery values for RGN and MET ranged from 99.25–99.48 and
98.58–100.26, respectively (Table II). The average recovery of three levels for
RGN and MET were 99.35 and 99.33 respectively.
Table II. Results of the Recovery Tests for the Drugs
|
Level of addition (%) |
Amount added (mg) |
Recovery (%)* |
Average Recovery† |
|||
|
MET |
RGN |
MET |
RGN |
MET |
RGN |
|
|
80 |
400 |
0.8 |
99.25±1.74 |
98.58±1.66 |
99.33 |
99.35 |
|
100 |
500 |
1 |
99.48±0.94 |
99.23±0.52 |
||
|
120 |
600 |
1.2 |
99.28±0.36 |
100.26±1.8 |
||
*
RSD shown in parenthesis.
†
Average recovery = average of three levels, nine determinations
Determination of the limits of detection
and Quantitation:
For
determining the limits of detection (LOD) and quantitation
(LOQ), the method based on the residual standard deviation (SD) of a regression
line and slope was adopted. To determine the LOD and LOQ, a specific
calibration curve was studied using samples containing the analytes
in the range of the detection and quantitation
limits. The LOD for RGN and MET were 0.00016 and 0.00037 µg/mL,
and the LOQ were 0.00048 and 0.0012 µg/ml respectively.
System suitability:
For
system suitability studies, five replicate injections of mixed standard
solutions were injected, and the parameters like RSD of peak area ratio, column
efficiency, resolution, and tailing factor of the peaks were calculated.
Results are shown in Table III.
Table III. System Suitability Parameters
|
Parameters |
MET |
RGN |
|
Retention
time (min) |
3.13 |
10.01 |
|
Tailing
Factor |
1.47 |
1.06 |
|
Theoretical
Plates |
6669.01 |
13757.05 |
|
Resolution |
12.43 |
|
Table IV. Summary of Robustness Study
|
Chromatographic
changes |
||||||||
|
Flow Rate (ml/min) |
Level |
RT |
Tailing factor |
% drug Found |
||||
|
MET |
RGN |
MET |
RGN |
MET |
RGN |
|||
|
0.8 |
-1 |
3.60 |
10.64 |
1.32 |
1.13 |
99.63 |
100.20 |
|
|
1.0 |
0 |
3.50 |
10.63 |
1.47 |
1.06 |
100.16 |
99.50 |
|
|
1.2 |
+1 |
3.36 |
9.94 |
1.34 |
1.08 |
99.95 |
99.01 |
|
|
MEAN± S.D. 99.74 ± 0.54 99.5 ± 0.59 |
||||||||
|
% of ACN in the mobile phase (v/v) |
Level |
RT |
Tailing factor |
% drug Found |
||||
|
MET |
RGN |
MET |
RGN |
MET |
RGN |
|||
|
58 |
-1 |
3.38 |
9.60 |
1.37 |
1.15 |
99.74 |
98.77 |
|
|
60 |
0 |
3.50 |
10.63 |
1.47 |
1.06 |
100.16 |
99.50 |
|
|
62 |
+1 |
3.48 |
9.83 |
1.32 |
1.09 |
99.77 |
98.40 |
|
|
MEAN ± S.D. 99.89 ±
0.23 98.92 ± 0.60 |
||||||||
|
Temperature |
Level |
RT |
Tailing factor |
% drug Found |
||||
|
MET |
RGN |
MET |
RGN |
MET |
RGN |
|||
|
28 |
-1 |
3.42 |
10.58 |
1.29 |
1.08 |
100.02 |
100.21 |
|
|
29 |
0 |
3.50 |
10.63 |
1.47 |
1.06 |
100.16 |
99.50 |
|
|
30 |
+1 |
3.39 |
10.52 |
1.36 |
1.11 |
100.01 |
100.03 |
|
|
MEAN± S.D. 100.06 ± 0.08 99.81 ± 0.54 |
||||||||
|
pH |
Level |
RT |
Tailing factor |
% drug Found |
||||
|
MET |
RGN |
MET |
RGN |
MET |
RGN |
|||
|
4.8 |
-1 |
3.38 |
11.37 |
1.32 |
1.11 |
100.00 |
100.10 |
|
|
5.0 |
0 |
3.50 |
10.63 |
1.47 |
1.06 |
100.16 |
99.50 |
|
|
5.2 |
+1 |
3.36 |
9.74 |
1.35 |
1.13 |
99.26 |
100.01 |
|
|
MEAN± S.D. 99.80 ± 0.48 99.90 ± 0.27 |
||||||||
* Mean of three levels (n = 3)
Fig. 2. A typical
chromatogram of Metformin and Repaglinide
Robustness:
To evaluate robustness of
the developed method, few parameters were deliberately varied. These parameters
included variation of flow rate, percentage of Acetonitrile
in the mobile phase, pH of buffer and temperature. Each factor selected was
changed at three levels (-1, 0, +1). One factor was changed at one time to
estimate the effect. The results are shown in Table IV
Determination of active ingredients in
tablets:
The
contents of two drugs in tablets were determined by the proposed method using a
calibration curve. The determinations were done in two sets, one for precision
and the second for intermediate precision, and six samples were prepared for
each set. The results are shown in table III. The chromatogram of the tablet
sample is shown in Figure 2.
CONCLUSION:
The developed method is
suitable for simultaneous determination of repaglinide
and metformin in the pharmaceutical preparations as
well as in bulk drugs. The method is validated as per the ICH guidelines and
shown to be specific, accurate and precise. The method can be used in the
quality control departments for the analysis of repaglinide
and metformin in combination.
ACKNOWLEDGMENTS:
The authors are thankful to Macleoides Research Laboratory, Mumbai, Maharashtra, India
and Wallace Pharmaceuticals, Pvt. Ltd, Goa, India, for providing gift samples
of pure drugs. The authors are also grateful to the School of Pharmacy S.R.T.M.
University Nanded, Maharashtra, India for providing
the facilities for this research work.
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Received on 18.08.2011
Accepted
on 12.09.2011
©
A&V Publication all right reserved
Research Journal of
Pharmaceutical Dosage Forms and Technology. 3(5): Sept.-Oct. 2011, 233-237