Development and
Validation of an Analytical Method for the Estimation of Metformin and
Teneligliptin in its Bulk and Tablet Dosage Form by using RP-HPLC
Prem Kumar Bichala1*,
K. Jeevan Kumar2, R. Suthakaran3, Ch. Shankar3
1Associate
Professor, Department of Pharmaceutical Analysis, Vijaya College of Pharmacy,
Munaganoor – 501511,
Hyderabad, Telangana, India
2Department of
Pharmaceutical Analysis, Vijaya College of Pharmacy, Munaganoor – 501511,
Hyderabad, Telangana, India
3Department of
Pharmaceutical Analysis, Vijaya College of Pharmacy, Munaganoor – 501511,
Hyderabad, Telangana, India
*Corresponding Author E-mail: prembichala@gmail.com
ABSTRACT:
Teneligliptin in tablet dosage
form, using Column Inertsil ODS column C8 (4.6 x 250mm), 5µm particle size,
mobile phase Phosphate buffer (PH-3): Acetonitrile (50:50) v/v, detection
wavelength at 240nm, at flow rate of 1.0ml/min and the retention time 3.608 and
5.148 min. The correlation coefficient was found to be 0.999 for both the
drugs. The Recovery studies were performed for Metformin and Teneligliptin in
the range of 50% - 150 %. The % recovery of Metformin and Teneligliptin
found to be 99.97 and 100.64 respectively. % RSD for Precision studies of
Metformin and Teneligliptin was found to be 0.4 and 0.8. Forced Degradation
studies were conducted according to the ICH guidelines and the Drug Product was
found to be stable in all conditions. Hence we recommend that this method can
be a good approach for the quantification of Metformin and Teneligliptin in
combination dosage form and can be adopted for the routine quality control
analysis of these drugs.
KEYWORDS: Metformin and
Teneligliptin, RP-HPLC, ICH.
INTRODUCTION:
Metformin:2
Metformin was discovered in
1922.French physician Jean Sterne began study in humans in the 1950s.It was
introduced as a medication in France in 1957 and the United States in
1995.Metformin is on the World Health Organization's List of Essential
Medicines, which lists the most effective and safe medicines needed in a health
system Metformin
is the most widely used medication for diabetes taken by mouth
|
Molecular Formula |
C4H11N5 |
|
|
Molecular Weight |
: |
129.1636 g/mol |
|
Brand name |
: |
Apo-Metformin (Apotex) |
|
Category |
: |
Anti- hyperglycemic agents / Anti-diabetic drug |
Fig 1: Chemical structure of Metformin
Teneligliptin:3
Teneligliptin is a pharmaceutical drug for the treatment
of type 2 diabetes mellitus. It
belongs to the class of anti-diabetic drugs known as dipeptidyl peptidase-4inhibitors or "gliptins"
IUPAC Name:
1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-4-[(3S,5S)-5-(1,3-thiazolidine-3-carbonyl)
pyrrolidin-3-yl]piperazine
Fig 2: chemical structure of Teneligliptin
MATERIALS AND METHODS:
The various materials and equipments used for the present study are
summarized as follows.
Table 1: Instruments used
|
SL. No |
Instrument |
Model |
|
1 |
HPLC |
WATERS, software: Empower, 2695 separation module.2487 UV detector. |
|
2 |
UV/VIS spectrophotometer |
LABINDIA UV 3000+ |
|
3 |
pH meter |
Adwa – AD 1020 |
|
4 |
Weighing machine |
Afcoset ER-200A |
|
5 |
Pipettes and Burettes |
Borosil |
|
6 |
Beakers |
Borosil |
Table 2: Chemicals used
|
SL. No |
Chemical |
Brand |
|
1 |
Metformin |
Supplied by Pharmatrain |
|
2 |
Teneligliptin |
Supplied by Pharmatrain |
|
3 |
KH2PO4 |
FINAR chemical LTD |
|
4 |
Water and Methanol for HPLC |
MERCK |
|
5 |
Acetonitrile for HPLC |
MERCK |
Optimized Chromatographic
Conditions:
|
Column |
Symmetry column C8 (4.6 x 250mm, 5mm) |
|
Mobile phase |
Water: Methanol (50:50) |
|
Flow rate |
1 ml per min |
|
Wavelength |
240 nm |
|
Injection volume |
20 ml |
|
Run time |
8 min. |
Preparation of mobile phase:8,
9
Mix a mixture of above buffer
500ml (50%) and 500ml Acetonitrile (50%) and degas in ultrasonic water bath for
5 minutes. Filter through 0.45µ filter under vacuum filtration.
Preparation Sample solutions:
8, 9
Accurately weighed and
transferred 62.5mg of Metformin and 2.5mg of Teneligliptin working standard
into a 10ml clean dry volumetric flask add about 7ml of Diluent and sonicate to
dissolve it completely and make volume up to the mark with the same solvent.
(Stock solution)
Further pipette 0.6ml of the
above stock solutions into a 10ml volumetric flask and dilute up to the mark
with diluent.
Standard Solution Preparation:8,9
Accurately weighed and
transferred 125mg of Metformin and 5mg of Teneligliptin working standard into a
10ml clean dry volumetric flask add about 7ml of Diluent and sonicate to
dissolve it completely and make volume up to the mark with the same solvent.
(Stock solution)
Further pipette 0.6ml of the above
stock solutions into a 10mlvolumetric flask and dilute up to the mark with
diluent.
RESULTS AND DISCUSSION:
LINEARITY:5, 6
The linearity range was found
to lie from 250µg/ml to 1250µg/ml of Metformin,10µg/ml to 50µg/ml 0f
Teneligliptin and chromatograms are shown below.
Chromatogram for linearity:
Fig 1: Calibration graph for
Metformin & Teneligliptin
Table 3: Linearity data of
concentration of Metformin and Teneligliptin
|
S. No |
Metformin |
Teneligliptin |
||
|
Concentration (µg/ml) |
Area |
Concentration (µg/ml) |
Area |
|
|
1 |
250 |
244841 |
10 |
29672 |
|
2 |
500 |
525756 |
20 |
68336 |
|
3 |
750 |
856654 |
30 |
113345 |
|
4 |
1000 |
1150925 |
40 |
159680 |
|
5 |
1250 |
1435608 |
50 |
204473 |
PRECISION: 5, 6
Precision of the method was
carried out for both sample solutions as described under experimental work. The
corresponding chromatograms and results are shown below.
Fig 2 : Chromatogram of
Metformin, Teneligliptin showing precision
Fig 3 : Chromatogram of
Metformin, Teneligliptin showing LOD
Fig 4: Chromatogram of
Metformin, Teneligliptin showing LOQ
Table 4 : Results of Precision
for Metformin & Teneligliptin
|
Injec. No. |
Metformin |
Teneligliptin |
||
|
Retention time(min) |
Peak area |
Retention time(min) |
Peak area |
|
|
1 |
3.609 |
852828 |
5.148 |
111368 |
|
2 |
3.603 |
852337 |
5.141 |
112717 |
|
3 |
3.605 |
858355 |
5.137 |
112655 |
|
4 |
3.606 |
852839 |
5.138 |
113939 |
|
5 |
3.603 |
858513 |
5.143 |
112513 |
|
6 |
3.606 |
857582 |
5.145 |
112282 |
|
Mean |
|
855409.0 |
|
112662.3 |
|
SD |
|
12.524.5 |
|
845.7 |
|
%RSD |
|
0.9 |
|
0.8 |
Limit of
Detection for Metformin and Teneligliptin:5, 6
The lowest concentration of
the sample was prepared with respect to the baseline noise and measured the
signal to noise ratio.
Table 5: Results of LOD
|
Drug name |
Baseline noise(µV) |
Signal obtained (µV) |
S/N ratio |
|
Metformin |
66 |
198 |
3.00 |
|
Teneligliptin |
66 |
199 |
3.02 |
Signal to noise ratio shall be
3 for LOD solution
The result obtained is within
the limit.
Limit of Quantification for
Metformin and Teneligliptin:5&6
The lowest concentration of
the sample was prepared with respect to the base line noise and measured the
signal to noise ratio.
Table 6: Results of LOQ
|
Drug name |
Baseline noise(µV) |
Signal obtained (µV) |
S/N ratio |
|
Metformin |
66 |
659 |
9.98 |
|
Teneligliptin |
66 |
660 |
10.00 |
Signal to noise ratio shall be
10 for LOQ solution
The result obtained is within
the limit.
CONCLUSION:
From above review of
literature it was evident that no HPLC method has been reported for the
analysis of Metformin and Teneligliptin. The optimum wavelength for the
determination of Metformin and Teneligliptin was selected at 240nm on the basis
of Isobestic point. Various trails were performed with different mobile phases
in different ratios, but the method was optimized with Phosphate buffer (PH-3):
Acetonitrile (50:50) v/v. The Retention time of Metformin and Teneligliptin
were found to be 3.608 and 5.148 min respectively. The retention time for the
both drugs were considerably less compared to the retention time reported for
the drugs. Therefore above methods has been proven to be linear, precise,
accurate, specific, robust and stable. Hence we recommend that this method can
be a good approach for the quantification of Metformin and Teneligliptin in
combination dosage form and can be adopted for the routine quality control
analysis of these drugs.
REFERENCES:
1.
https://www.drugbank.ca/drugs/DB00331
2.
https://pubchem.ncbi.nlm.nih.gov/compound/teneligliptin
3.
ICH, Text on Validation of Analytical Procedures, ICH – Q2A,
International Conference on Harmonisation, IFPMA, Geneva, 1995; 2-3: A–1 to
A–3.
4.
ICH, Validation of Analytical Procedures Methodology, ICH – Q2B,
International Conference on Harmonisation, 1996; p1-3.
5.
ICH Guidelines, Q2 (R1) Validation of Analytical Procedures Text and
Methodology, 2005; p1-6.
6.
T. N. V. Ganesh Kumar, S. Vidyadhara, Niteen Ashok Narkhede, Y. Sai
Silpa and M. Rajya Lakshmi, Method development, validation, and stability
studies of teneligliptin by RP-HPLC and identification of degradation products
by UPLC tandem mass spectroscopy, Journal of Analytical Science and Technology
volume 7, Article number: 18 (2016)
7. Gadapa Nirupa and Upendra M. Tripathi, RP-HPLC Analytical
Method Development and Validation for Simultaneous Estimation of Three Drugs:
Glimepiride, Pioglitazone, and Metformin and Its Pharmaceutical Dosage Forms, Journal of Chemistry, Volume 2013,
Received on 07.01.2020
Modified on 24.01.2020
Accepted on 18.02.2019 ©Asian Pharma Press
All Right Reserved
Asian J. Pharm. Ana. 2020; 10(1):11-14.
DOI: 10.5958/2231-5675.2020.00003.4