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DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING SIMULTANEOUS ESTIMATION
OF METFORMIN AND ALOGLIPTIN IN TABLETS BY HIGH-PERFORMANCE THIN LAYER
CHROMATOGRAPHY
Original Article
MALATHI SELLAPPAN1*, ARUNADEVI NATARAJAN2
1,2*
Received: 30 Apr 2019, Revised and Accepted: 07 Oct 2020
Department of Pharmaceutical Analysis, PSG College of Pharmacy, Peelamedu, Coimbatore, Tamilnadu, 641004, India
Email: malathisanju@gmail.com
ABSTRACT
Objective: A simple and stability-indicating high-performance thin-layer chromatographic method was developed and validated for the
simultaneous estimation of metformin and alogliptin in tablets.
Methods: The method was developed in TLC aluminum plates pre-coated with silica gel 60F 254
Results: The developed method was found to be suitable for the excellent separation of the drug samples. Calibration curves were linear in the
range of 40-200 ng/spot with a correlation coefficient of 0.996 for metformin and calibration curves were linear in the range of 1-5 ng/spot with a
correlation co-efficient of 0.997 for alogliptin, respectively. Stability study shows that the chromatograms of samples degraded with acid, base,
hydrogen peroxide, dry heat, and photolytic showed well-separated spots of pure metformin and alogliptin as well as some additional peaks at
different Rf values. The method was successively applied to pharmaceutical formulation. No chromatographic interference from the tablet
excipients was found.
as the stationary phase and the solvent system
consists of methanol: chloroform: 0.5% ammonium sulphate [4:4:2, v/v/v]. The system was found to conferred a compact spot for metformin [Rf
value of 0.44±0.02] and alogliptin [Rf value of 0.66 ± 0.22]. Densitometric analysis of metformin and alogliptin was carried out at the wavelength of
254 nm. Forced degradation studies were conducted to know the stability of the drug samples under various stress conditions like acid, base,
peroxide, photolytic degradation according to the ICH guidelines.
Conclusion: The newly developed method can be applied for the identification and quantitative determination of metformin and alogliptin in the
combined dosage form.
Keywords: Type 2 diabetes mellitus, Metformin, Alogliptin, High-performance thin-layer chromatography, Stability indicating, Method validation
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i12.33871. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps.
INTRODUCTION
Metformin hydrochloride is chemically N; N-Dimethylimido
dicarbon imidic diamide. The chemical classification of metformin is
a biguanide. The biguanide classes of anti-diabetic with an anti-
hyperglycemic drugs are used in the treatment of type-2 diabetic
patients. The classes of medications, particularly those which work
via the incretin pathway, achieve glucose-lowering and minimizing
risks ideally. The combination should be well-tolerated, convenient
to take, should have few contraindications, a low risk of
hypoglycemia, weight gain and be reasonably effective over both the
short and long term such as the combination of metformin and
alogliptin is a dipeptidyl peptidase-4 [DPP-4] inhibitor which is used
in combination in the therapy of type 2 diabetes [1].
Fig. 1: Chemical structure of metformin
Fig. 2: Chemical structure of Alogliptin
Alogliptin is an orally administered antidiabetic drug in the
dipeptidyl peptidase-4[DPP-4]. DPP-4 inhibitors represents a new
therapeutic approach to the treatment of type 2 diabetes that
functions to stimulate glucose-dependent insulin release and reduce
glucagon’s levels. This is done through inhibition of the inactivation
of incretins, particularly glucagon-like peptide-1 [GLP-1] and gastric
inhibitory polypeptide [GIP]. Alogliptin inhibits dipeptidyl peptidase
4[DPP-4], which normally degrades the incretin glucose-dependent
insulin tropic polypeptide [GIP] and glucagon-like peptide1 [GLP-1],
thereby improving glycemic control [2]. A combination of both drugs
is recently launched in the market. The chemical structures of the
drugs are shown in fig. 1, fig. 2, respectively. Literature survey reveals
that spectrophotometric [3-5], High-Performance Liquid
chromatography [HPLC] [6-8] and high-performance thin-layer
chromatography [HPTLC] [9-11] methods for the estimation of
metformin alone or in combination with other drugs from
pharmaceutical formulation have been developed whereas
spectrophotometric [12-14], RP-HPLC [15-17], HPTLC [18] and LC-
MS/MS [19] methods for the estimation of alogliptin alone or in
combination with other drugs from pharmaceutical formulation have
been developed. However, no stability-indicating method has been
reported so far simultaneous estimation of both drugs in combined
pharmaceutical dosage form by HPTLC. The current method is the first
method used so far for the simultaneous estimation of the alogliptin
and metformin by HPTLC method. The advantages of HPTLC are, a
large number of samples can be simultaneously analyzed in a shorter
period. Unlike HPLC, this method utilizes fewer quantities of solvents,
thus lowering the cost of analysis.
The ideal stability-indicating chromatographic method should
estimate the drug and also be able to resolve the drug from its
International Journal of Pharmacy and Pharmaceutical Sciences
Print ISSN: 2656-0097 | Online ISSN: 0975-1491 Vol 12, Issue 12, 2020
Sellappan et al.
Int J Pharm Pharm Sci, Vol 12, Issue 12, 68-73
69
degradation products. Hence an attempt has been made to develop
an accurate, rapid and reproducible method for the determination of
metformin and alogliptin in presence of their degradation products
for their content analysis in pharmaceutical dosage forms,
containing this combination as per ICH guidelines [20].
MATERIALS AND METHODS
Metformin Hcl was received from Cadila pharmaceutical,
Ahmedabad; Gujarat. Alogliptin was kindly supplied by Vivan Life
sciences, Thane, Maharashtra. All other chemicals like methanol,
ammonium sulphate and chloroform were used for AR grade.
HPTLC method
Instrumentation
Camag HPTLC system equipped with Linomat V sample applicator
operated under a gentle stream of nitrogen, coupled with 100 µl
HAMILTON syringe and CAMAG TLC scanner 3 controlled by
WINCATS software was used for the application and detection of
spots respectively. The Chromatographic separations of drugs were
performed using pre-coated silica gel TLC aluminum plate 60 F 254
Selection of mobile phase and optimization of condition
[10 cm x10 cm] with 250 µm thickness and a CAMAG twin trough
chamber was used for the chromatographic development.
Initially, chloroform: methanol in ratio 4:6 [v/v] was tried for both drugs
simultaneously. The spots were not developed properly and dragging
was observed. Then chloroform: methanol in the ratio 6: 4 [v/v] was
tried. The developed spots were diffused to the above mobile phase; 2 ml
of 0.5 % ammonium sulphate was added. Both the peaks were
symmetrical and no tailing was observed. Finally, the mobile phase
consisting of chloroform: methanol: 0.5 % ammonium sulphate [4:4:2,
v/v] gave good resolution. By optimizing chromatographic conditions
such as having chamber saturation for 20 min, using 10 ml of the mobile
phase and activating the plates before spotting, we obtain a good
resolution as well as a sharp and symmetrical peak with Rf value of 0.44
±0.02 for metformin and 0.66 ±0.02 for alogliptin.
Chromatographic condition
For chamber saturation, 20 ml of the mobile phase was transferred
into the development tank with the lid closed. The assembly was
aside for 20 min under room temperature. The Plates were
prewashed with methanol and activated at 110 °C for 5 min before
chromatography. The mobile phase consists of methanol:
chloroform: 0.5% ammonium sulphate [4:4:2, v/v/v]. The plate was
air-dried using an air dryer after the application of spots and the
development but before densitometric scanning. The source of
radiation utilized was a deuterium lamp. The evaluation was
performed using peak areas with concentration.
Preparation of standard solutions
A combined standard stock solution containing 4000 µg/ml of
metformin and 100 µg/ml of alogliptin was prepared in methanol.
The application of standards was done by Hamilton syringe with the
help of automatic sample applicator Linomat V on the TLC plate that
gave a concentration, 40-200 ng/spot of metformin and 1-5 ng/spot
of alogliptin, respectively. Each concentration was spotted five times
on the TLC plates. The plates were developed using the previously
described mobile phase. The calibration graph was plotted as peak
areas versus corresponding concentrations.
Analysis of marketed formulation
To determine the content of metformin and alogliptin simultaneously
in conventional tablets, [label claim containing 500 mg metformin and
12.5 mg alogliptin] twenty tablets were accurately weighed. The
average weight was calculated and ground to a fine powder. A quantity
of powder equivalent to 400 mg metformin and 10 mg alogliptin was
transferred into a 100 ml volumetric flask containing 50 ml methanol
was sonicated for 30 min and diluted to mark with the same solvent.
The resulting solution was filtered using 0.45 µm Millifilter. The
aliquots solutions are applied on the TLC plate. The analysis was
repeated for six times. Metformin and alogliptin gave sharp and well-
defined peaks at Rf 0.44 ± 0.02 and 0.66 ± 0.02 respectively when
scanned at 254 nm [fig. 3].
Fig. 3: Densitogram of standard metformin and alogliptin
Validation of HPTLC method
Accuracy
Accuracy of the method was determined by replicates [n=3] analysis
and was carried out using three solutions prepared by standard
addition of pure active pharmaceutical ingredient at three different
concentration levels 80%,100%, and 120%. Accuracy was calculated
by comparing the difference between the spiked value [theoretical
value] and the found value [9, 10].
Precision
Intra-day precision
Intra-day precision was found out by carrying out the analysis of the
standard drug at three different concentrations of 80,120 and 160
ng/spot for metformin; 2, 3 and 4 ng/spot for alogliptin were
selected from linearity range. The intraday analysis was carried on
the same day in three replicates. Each concentration was applied in
duplicate and % RSD was calculated [11, 12].
Inter-day precision
Inter-day precision was found out by carrying out the analysis of the
standard drug at three different concentrations of 80,120 and 160
ng/spot for metformin; 2, 3, and 4 ng/spot for alogliptin were
selected from linearity range. The interday analysis was carried on
three different days in three replicates. Each concentration was
applied in duplicate and % RSD was calculated [11, 12].
Repeatability
Repeatability was determined by applying the corresponding µl of a
standard solution containing 120 ng/spot of metformin and 3
Sellappan et al.
Int J Pharm Pharm Sci, Vol 12, Issue 12, 68-73
70
ng/spot of alogliptin in six replicates and the respective areas were
calculated. The % RSD was calculated [11, 12].
Forced degradation studies
Forced degradation of each drug substance was carried out under acid,
base, hydrolytic, oxidation, photolytic and thermal stress conditions. A
thermal and photodegradation study was carried out in a solid-state.
Solutions were prepared by dissolving drug substances in a small
volume of methanol and later diluted with hydrochloric acid, 0.1N
sodium hydroxide or hydrogen peroxide to achieve a concentration of
100 µg/ml of each metformin and alogliptin [16].
Preparation of acid-induced degradation product
The 40 mg of metformin and 1 mg alogliptin in 10 ml volumetric
flask to add 1 ml of 0.1M hydrochloric acid and make up the volume
with mobile phase then refluxed at 40 °C for 30 min. After
completion of 30 min, about 1 ml of the above solution was taken
and diluted up to 10 ml with methanol. The resultant solution was
applied to TLC plates in triplicates. The chromatograms run as
described in section [16].
Preparation of base induced degradation product
The 40 mg of metformin and 1 mg of alogliptin in 10 ml volumetric
flask to add 1 ml of 0.1M sodium hydroxide and make up the volume
with mobile phase then refluxed at 40 °C for 30 min. After
completion of 30 min, about 1 ml of the above solution was taken
and diluted up to 10 ml with methanol. The resultant solution was
applied to TLC plates in triplicates [17].
Preparation of hydrogen peroxide-induced degradation
product
The 40 mg of metformin and 1 mg of alogliptin in 10 ml volumetric
flask to add 1 ml of 0.1M 30% hydrogen peroxide and make up the
volume with mobile phase then refluxed at 40 °C for 30 min. After
completion of 30 min, about 1 ml of the above solution was taken,
neutralized and diluted up to 10 ml with methanol. The resultant
solutions were applied to TLC plates in triplicates [17].
Preparation of light heat degradation products
The 40 mg of metformin and 1 mg of alogliptin was transferred into
a clean and dry Petridis. The Petridis was placed in direct sunlight
for 5 d. In this study, the drug substance was exposed to direct
sunlight for 5 d to determine the effect of irradiation on the stability
of the drugs in solid-state. Afterward, the drug was transferred into
a 10 ml volumetric flask and make up the volume with the mobile
phase.1 ml of the above solution was transferred into a 10 ml
volumetric flask and diluted with 10 ml using the mobile phase. The
resultant solution was applied to the TLC plate in triplicate [17].
Preparation of dry heat degradation products
Accurately weighed and transferred 40 mg of metformin and 1 mg of
alogliptin was transferred into a clean and dry Petridis. The Petridis
was placed in an oven at 50 °C for 3h. The drug was transferred into
a 10 ml volumetric flask and make up the volume with mobile
phase.1 ml of the above solution was transferred into a 10 ml
volumetric flask and diluted with 10 ml using the mobile phase. The
resultant solution was applied to the TLC plate in triplicate [17].
RESULTS AND DISCUSSION
A novel, simple and precise HPTLC method coupled with
densitometer was developed for the estimation of alogliptin and
metformin present in the marketed formulation. Several analytical
methods have been reported for the determination of metformin
with alogliptin and combined with other drugs in pure and
pharmaceutical dosage forms using spectrophotometry [3-5, 14],
HPLC [6-8, 12, 15-17] Colorimetry [13]. Few methods like LC-
MS/MS for determination of alogliptin and voglibose inhuman
plasma [19] are found in the literature but these methods are not
preferred for routine analysis of alogliptin and metformin in bulk
and formulation studies because of the high cost of analytical
technique and the skilled requirement for sample treatment. There
are some HPTLC methods [9-11, 18] reported in the literature but
they have certain restrictions like requiring a large quantity of
samples and organic solvents or sensitive to microgram
concentration. In this work, new stability-indicating HPTLC method
has been developed for the simultaneous estimation of the alogliptin
and metformin. Initially, chloroform: methanol in ratio 4:6[v/v] was
tried for both drugs simultaneously; the spots were not developed
properly and dragging was observed. Then, chloroform: methanol in
the ratio 6: 4 [v/v] was tried. The developed spots were diffused to
the above mobile phase; 2 ml of 0.5 % ammonium sulphate was
added. Both the peaks were symmetrical and no tailing was
observed. Finally, the mobile phase consisting of chloroform:
methanol: 0.5 % ammonium sulphate [4:4:2, v/v] gave good
resolution. By optimizing chromatographic conditions such as
having chamber saturation for 20 min, using 10 ml of the mobile
phase and activating the plates before spotting and obtain a good
resolution as well as a sharp and symmetrical peak with Rf value of
0.44 ±0.02 for metformin and 0.66 ±0.02 for alogliptin. The method
ensures minimal use of the mobile phase with minimal run time
compared to other reported analytical methods. The ideal stability-
indicating chromatographic method should estimate the drug and
also be able to resolve the drug from its degradation products.
Accuracy results displayed good reproducibility with % RSD values
2. This was found to be accurate as the percent recovery observed
was high i.e. within the range of 98.84-100.4. The LOD of the
developed HPTLC method was found to be 40 ng/spot for metformin
and 1 ng/spot for alogliptin, respectively. The LOQ of the developed
HPTLC method was found to be 130 ng/spot for metformin and 60
ng/spot for alogliptin, respectively. It confirms the method is sensitive.
Besides, the estimation of the marketed preparation of alogliptin and
metfomin with the validated methods showed that the drug contents
separated with no interfering peaks were generated by the excipients
in the marketed formulation as shown in the fig. 5. The method is
versatile and simple for the analysis of alogliptin and metformin in
pure and pharmaceutical formulations. The method confirms minimal
use of the mobile phase with a short run time compared to other
reported analytical methods. The method was found to obey all the
validation parameters as per ICH guidelines [20].
Validation of the developed method
Linearity
Metformin and alogliptin showed linearity in the range of 40-200
ng/spot and 1-5 ng/spot respectively. The slope, intercept and
correlation coefficient values for metformin were found to be
4.3725, 501.5 and 0.997 respectively. The slope, intercept and
correlation coefficient values for alogliptin were found to be 31.7,
205.02 and 0.999 respectively.
Precision
Intra-day and Inter-day was found out by carrying out the analysis
of the standard drug at three different concentrations of 80,120 and
160 ng/spot for metformin 2,3 and 4 ng/spot for alogliptin were
selected from linearity range. Repeatability was determined by
applying the corresponding µl of a standard solution containing 120
ng/spot of metformin and 3 ng/spot of alogliptin in six replicates.
The % RSD was found to be less than 2%.
Accuracy
Accuracy of the method was determined by replicates [n=3]
analysis, carried out using three solutions prepared by standard
addition of pure active pharmaceutical ingredient at three different
concentration levels 80 %, 100 % and 120%. Accuracy was
calculated by comparing the difference between the spiked value
[theoretical value] and the found value. Results are presented in the
term of % recovery of the active pharmaceutical ingredient and data
for both metformin and alogliptin. The summary of validation
parameters was listed in table 1.
Analysis of marketed formulation
The chromatograms of the drugs are extracted from commercial
formulation, exhibited two peaks at Rf value of 0.44 and 0.66 for
metformin and alogliptin, respectively. The results of the analysis of
the marketed formulation are given in table 2.
Sellappan et al.
Int J Pharm Pharm Sci, Vol 12, Issue 12, 68-73
71
Table 1: Summary of validation parameters
Parameters
Metformin
Alogliptin
Linearity range [ng/spot]
40-200
1-5
Correlation coefficient [R2]
0.996
0.997
Slope
4.3725
31.7
Intercept
501.58
205.02
Limit of detection[ng/spot]
40
1
Limit of quantification [ng/spot]
130
6
% Recovery [n=3]
100.4
98.87
Precision[%RSD], [ mean
±
S. D]
Repeatability of application [n=6]
1.18,1197
±
14.2
2.210,801.3
±
17.6
Intra-day [n=3]
0.58,1008.7
±
5.9
2.02,875.8
±
17.7
Inter-day [n=3]
1.45,1221.9
±
1.64,779.6
±
12.8
% RSD =Relative standard deviation, SD=Standard deviation, n= number of injections
Table 2: Analysis of marketed formulation
Component
Amount [ mg]
% Label claim
% RSD*
Labeled
Found
Metformin
500
498
99.6
0.32
Alogliptin
12.5
12
96.0
0.85
*Relative standard deviation; [n=5]
Forced degradation results
Peaks obtained from samples degraded by treatment with acid, base,
hydrogen peroxide, dry heat treatment and photolytic contained well-
separated spots of the pure drugs and same additional peaks at
different R f
values. It is apparent from [fig. 4-8] the spots of
degradation products were well resolved from those of the drugs. The
peaks of the metformin and alogliptin were not significantly shifted in
the presence of the degradation peaks, which indicated the stability-
indicating the nature of the method. The results are shown in table 3.
Fig. 4: Densitogram of acid-treated metformin and alogliptin
Fig. 5: Densitogram of base treated metformin and alogliptin
Sellappan et al.
Int J Pharm Pharm Sci, Vol 12, Issue 12, 68-73
72
Fig. 6: Densitogram of hydrogen peroxide-treated metformin and alogliptin
Fig. 7: Densitogram of dry heat-treated metformin and alogliptin
Fig. 8: Densitogram of light heat-treated metformin and alogliptin
Sellappan et al.
Int J Pharm Pharm Sci, Vol 12, Issue 12, 68-73
73
CONCLUSION
The present study represents an accurate, simple, specific, precise
and stability-indicating HPTLC method has been developed for the
quantitative determination of metformin and alogliptin in bulk drug
and tablet formulation. The developed method was validated based
on ICH guidelines. Non-requirement of skilled personnel to operate
the instruments involved is an added advantage of this method.
Therefore, the method can be applied for routine quality control
analysis of metformin and alogliptin in active pharmaceutical
ingredients and the combined dosage form.
ACKNOWLEDGEMENT
This research was supported by Cadila pharmaceutical, Gujarat, and
Pharmasave pharmacy, Canada.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
Both authors have contributed equally.
CONFLICT OF INTERESTS
Declared none
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