Development and
Validation of Stability indicating RP-HPLC Method for the simultaneous
Estimation of Sitagliptin and Ertugliflozin in bulk and Tablet Dosage Forms
A. Suneetha*, V.
Mounika, Shaik Mahammad Sajid
Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi Road, Guntur, A.P India.
*Corresponding Author E-mail: drasuneetha@gmail.com
ABSTRACT:
A
new simple, sensitive, accurate, precise and reproducible RP-HPLC method has
been developed for the simultaneous estimation of sitagliptin and ertugliflozin
in bulk and pharmaceutical dosage form using Inertsil ODS (4.6×150mm, 5µ)
column in isocratic mode. The mobile phase consisted 0.1% TFA: Methanol:
Acetonitrile (30: 60: 10). The
detection was carried out at 250nm. The method was linear over the
concentration range for sitagliptin 40-200μg/ml and for ertugliflozin
6-30μg/ml. The recoveries of sitagliptin and ertugliflozin were found to
be 100.26 and 100.18% respectively. The validation of method was carried out
utilizing ICH guidelines. The described HPLC method was successfully employed
for the analysis of pharmaceutical formulation containing combined dosage form.
KEYWORDS: Ertugliflozin, RP
HPLC, Sitagliptin, ICH guidelines.
INTRODUCTION:
A novel class of
anti-diabetic drugs, which are inhibitors of dipeptidyl-peptidase IV (DPP4),
which included sitagliptin, vildagliptin and saxagliptin1-5. Type 2
diabetes mellitus (T2DM) is a progressive disease, for the treatment of many
patients they require combination therapy to maintain over time glycemic levels6-7.
Efficacy and safety of the addition of ertugliflozin in patients with type 2
diabetes mellitus inadequately controlled with metformin and sitagliptin8-10.
Ertugliflozin is an oral sodium glucose transporter 2 inhibitor.
The study assessed the
efficacy and safety of co‑initiation of ertugliflozin and sitagliptin
compared with placebo in patients with T2DM inadequately controlled on diet and
exercise11-12. Ertugliflozin (1S,2S,3S,4R,5S) ‑5[4 chloro
3[4ethoxyphenyl] methyl] phenyl]1 (hydroxyl methyl) 6,7 di oxa bicyclo [3.2.1]
2,3,4 triol13. Sitagliptin chemically7 [(3R) 3
amino 1o xo 4(2,4,5 trifluorophenyl) butyl] 5,6,7,8 tetrahydro3
(trifluoromethyl) 1,2,4 triazolo [4,3a] pyrazine phosphate (1:1)
monohydrate. The structures of ertugliflozin and sitagliptin were shown in
figures 1 and 2. The placebo-adjusted differences in changes from baseline in
systolic blood pressure were not statistically significant. Ertugliflozin is
used for the treatment a higher prevalence of genital mycotic infections
occurred in men and women with ertugliflozin compared with placebo.14-17
Most of the methods were reported for the separation and estimation of
sitagliptin and ertugliflozin individually and combination with other, hence
the authors made an attempt to develop stability indicating RP HPLC method for
estimation of both drugs in bulk and formulations18-28.
Figure1: Structure
of Ertugliflozin
Figure2: Structure
of Sitagliptin
Experimental:
Apparatus:
The HPLC WATERS, software:
Empower 2, 2695 separation module, UV detector, Electronic weighing balance
(Afcoset ER-1000A), pH Meter (Adwa – AD 10100).
Reagents and Chemicals:
All the chemicals and
reagents in this experiment were of analytical grade. Water was double
distilled and filtered with a membrane filter. Methanol – HPLC grade (Merck,
India), Acetonitrile, Tri fluoro acetic acid and potassium dihydrogen ortho
phosphate (SD fine chem, India) were used to prepare mobile phase.
Pharmaceutical grade standard drugs viz., ertugliflozin and sitagliptin were
kindly gifted by Biocon.
Methodology:
Preparation of 0.1% Tri fluoro
acetic acid:
Pipette out 1ml of Trifluro
acetic acid and dissolved in 1000ml of HPLC water, pH was adjusted
up to 3.0 with formic acid. Final solution was filtered through 0.45
m membrane filter
and sonicated for 10 mins.
Preparation of mobile phase:
Accurately measured 300ml
(30%) of buffer solution, 600ml (60%) of HPLC grade methanol and 100ml (10%) of
HPLC grade acetonitrile were mixed and degassed in an ultrasonic water bath for
10 minutes and then filtered through 0.45µm filter under vacuum filtration.
Diluent Preparation:
The Mobile phase is used as a
diluent.
Standard Solution Preparation:
Accurately weighed and transferred
200mg of sitagliptin and 30mg of ertugliflozin into a 100ml clean dry
volumetric flask and made volume up to the mark with the diluent and sonicated
to dissolve it completely (Stock solution A).
From the stock solution A,
serial dilutions were made to obtain the concentrations of 6-30μg/mL for
ertugliflozin and 40-200μg/mL for sitagliptin.
Method
Development:
The
method was developed with different buffers and organic solvents but the
composition of potassium dihydrogen ortho phosphate and methanol showed
symmetrical peaks, with good resolution- high theoretical plates, and low
retention times of both ertugliflozin and sitagliptin. The optimized parameters
were shown in Table 1.
Table 1:
Optimized Chromatographic conditions
|
S. No |
Parameter |
Description/Value |
|
1. |
Stationary Phase |
Inertsil ODS (4.6*150mm, 5µ) |
|
2 |
Mobile Phase |
0.1% TFA: Methanol: Acetonitrile (30: 60: 10) |
|
3 |
Flow rate |
1 ml/min |
|
4 |
Detection Wavelength |
250nm |
|
5 |
Detector |
Photo diode array |
|
6 |
Injection |
Autosampler |
|
7 |
Injection volume |
20 μl |
|
8 |
Column Temperature |
35°C |
|
9 |
Run time |
6 min. |
Method
Validation:
The
proposed RP-HPLC method was validated as per ICH guidelines26.
System Suitability
Constraints:
The system suitability
parameters were showed good theoretical plates 3647 and 4622 for ertugliflozin and
sitagliptin. The tailing factor was found to be less than 2 for both drugs and
good resolution exists between the peaks. The chromatograms were shown in
Figure 3 and results were tabulated in Table 2.
Table 2: Results of system
suitability parameters
|
S. No. |
Name |
RT (min) |
Area (µV sec) |
Height (µV) |
USP resolution |
USP tailing |
USP plate count |
|
1 |
Sitagliptin |
2.798 |
248346 |
45708 |
- |
1.14 |
4622.39 |
|
2 |
Ertugliflozin |
3.587 |
86376 |
247671 |
4.37 |
1.11 |
3647.58 |
Figure
3: Chromatogram for system suitability
Specificity:
The stress degradation studies implies the specificity of the method.
Different parameters were evaluated depending upon the separation between
degradants and active moiety, as well as method ability to analyze analyte in
the presence of other products as shown in Fig. 4, 5, 6. No interfering peaks were found
in blank at retention times of the drugs. Hence the method was specific.
Figure 4: Standard Chromatogram
of Sitagliptin
Figure 5: Standard Chromatogram
of Ertugliflozin
Figure
6: Mixed Standard Chromatogram of Sitagliptin and Ertugliflozin
Linearity:
The calibration curve
was linear over concentration range of 6-30μg/mL for ertugliflozin and
40-200μg/mL for sitagliptin and R2 values were found to be
0.999 for both drugs. The data of linearity were showed in Figures 7and8.
Figure 7: Calibration graph for
Sitagliptin
Figure 8: Calibration graph for
Ertugliflozin
System Precision:
The precision was assessed
through system precision and method precision. The method precision was
estimated through assay. The optimized concentrations of standard and sample
solutions were injected into chromatographic system six times and the % RSD of
peak areas were found to be less than 0.5. There was no significant change in
assay content and system suitability parameters at different conditions of
ruggedness like day to day and system to system variation.
Accuracy:
The accuracy of the
method was performed by standard addition process at three different levels in
triplicate. The concentration of 50% solution showed % mean recovery 100.30 and
100.32 for ertugliflozin and sitagliptin, respectively. The concentration of
100% solution showed % mean recovery 100.66 and 100.28 for ertugliflozin
andsitagliptin, respectively. The concentration of 150% solution showed % mean
recovery 99.88 and 100.02 for ertugliflozin and sitagliptin, respectively. The
results were tabulated in Table 3 and 4.
Table 3: Accuracy data for
Ertugliflozin
|
Spiked Level |
Amount Added (µg/ml) |
Amount recovered (µg/ml) |
% Recovery |
Mean Recovery |
|
50% |
06 |
06.02 |
100.30 |
100.28 |
|
100% |
12 |
12.08 |
100.66 |
|
|
150% |
18 |
17.98 |
99.88 |
Table 4: Accuracy data for
Sitagliptin
|
Spiked Level |
Amount Added (µg/ml) |
Amount recovered (µg/ml) |
% Recovery |
Mean Recovery |
|
50% |
25 |
25.08 |
100.32 |
100.20 |
|
100% |
50 |
50.14 |
100.28 |
|
|
150% |
75 |
75.02 |
100.02 |
Limit of detection and
Limit of quantification:
The limit of detection
and quantification limits performed based on the slope and standard deviation.
The method showed ability to detect ertugliflozin and sitagliptin at low level
of concentrations. The LOD and LOQ were found to be 2.1µg/ml–6.9µg/ml for
ertugliflozin and 3.0µg/ml-10.89µg/ml for sitagliptin.
Robustness:
The robustness of the
method was performed with deliberate change in flow rate, temperature and
mobile phase composition. There was no change in the results which indicates
that the method was more robust.
Force Degradation
Studies:
The stability studies
were implemented on the Ertugliflozin and Sitagliptin. The method showed, there
was no interference of degradants and blank. The developed RP-HPLC method
verifies the proficiency of stability indicating method for the analysis of Ertugliflozin
and Sitagliptin. Different stress indicating studies were conducted with 0.1 N
HCl, refluxed for 3 H at 70ºC, Base (0.1 N NaOH refluxed for 4H at 70ºC), H2O2(3%
H2O2 Stored at room temperature for 2 H), hydrolytic for
6H at 70oC and UV light (near UV 250 nm for 5 days). The results were tabulated in Table 5.
Table 5: Results for Stability
studies of Sitagliptin andErtugliflozin
|
Sample Name |
Sitagliptin |
Ertugliflozin |
||
|
Area |
% Degraded |
Area |
% Degraded |
|
|
Standard |
248748.3 |
|
86512.3 |
|
|
Acid |
229734 |
7.64 |
84527 |
2.29 |
|
Base |
236644 |
4.87 |
84867 |
1.90 |
|
Peroxide |
239842 |
3.58 |
84253 |
2.61 |
|
Thermal |
227462 |
8.56 |
83527 |
3.45 |
|
Photo |
238373 |
4.17 |
83987 |
2.92 |
CONCLUSION:
The
developed and validated RP-HPLC method for the simultaneous estimation of
ertugliflozin and sitagliptin showed low tailing factor and high theoretical
plates, good precision, accuracy and robustness, met the all values within the
limits according to ICH guidelines. Hence it can be used for quality control
analysis.
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Received on 24.02.2020 Modified on11.03.2020
Accepted on
28.03.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020;
10(2): 81-85.
DOI: 10.5958/2231-5675.2020.00014.9