Online - 2455-3891

Vol 12, Issue 1, 2019 Print - 0974-2441

Research Article

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF NEW STABILITY-INDICATING

REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR
SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND EMPAGLIFLOZIN IN
TABLET DOSAGE FORM

GEETHA SUSMITA A*, RAJITHA G, RAMYA YADAV Y, UMA P

Department of Pharmaceutical Analysis, Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Visvavidyalayam,
Tirupati - 517 502, Chittoor, Andhra Pradesh, India. Email: susmithaadepu@gmail.com
Received: 09 April 2018, Revised and Accepted: 25 September 2018

ABSTRACT

Objective: The objective of this study was to develop and validate a stability-indicating reverse-phase high-performance liquid chromatography (RP-
HPLC) method for the simultaneous estimation of the metformin and empagliflozin in tablet dosage forms.

Methods: The chromatographic conditions were optimized and it was run through Std. BDS (250 mm × 4.6 mm, 5 m) column with mobile phase
consisting of 0.1% orthophosphoric acid buffer: acetonitrile in the ratio of 50:50. The flow rate was 1 ml/min and optimized wavelength was 210 nm.
Temperature was maintained at 30°C.

Results: The retention times of metformin and empagliflozin were found to be 2.588 min and 3.679 min and percentage relative standard deviation
(RSD) of the metformin and empagliflozin was found to be 0.59 and 1.2, respectively. Percentage recovery was in the range of 100.01–100.65% for
metformin and empagliflozin, respectively.

Conclusion: A sensitive, rapid, and specific method has been developed for the simultaneous estimation of metformin and empagliflozin using RP-
HPLC in tablet dosage form.

Keywords: Metformin, Empagliflozin, Reverse-phase high-performance liquid chromatography, Validation, Stability indicating.

© 2019 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/ajpcr.2019.v12i1.26537

INTRODUCTION Combination of metformin and empagliflozin tablets were purchased

from local market.
Metformin is chemically named as N,N-dimethyl imidodicarbonimidic
diamide [1], is a biguanide antihyperglycemic agent used for treating Instrumentation
non-insulin-dependent diabetes mellitus. It improves glycemic Waters HPLC 2965 system equipped with autoinjector and photodiode
control by decreasing hepatic glucose production, decreasing glucose array detector with Empower 2 Software was used for method
absorption, and increasing insulin-mediated glucose uptake. development. UV-visible spectrophotometer PG Instruments T60 with
a special bandwidth of 2 mm and 10 mm and matched quartz was used
Empagliflozin is chemically named as (2S, 3R, 4R, 5S, 6R)-2-[4-chloro-
for spectral measurements of metformin and empagliflozin solutions.
3-({4-[(3S)-oxolan3-yloxy]phenyl}methyl)phenyl]-6-(hydroxymethyl)
oxane-3,4,5-triol [2]. Empagliflozin is a sodium glucose cotransporter-2
Chromatographic conditions
inhibitor, which lowers blood glucose in people with type 2 diabetes by
The chromatographic separation and analysis were performed using a
blocking the reabsorption of glucose in the kidneys and promoting the
Std. BDS column (250×4.6 mm, 5 m particle size) with the mobile phase
excretion of excess glucose in the urine (Fig. 1).
consisting of buffer and acetonitrile in the ratio of 50:50 at a flow rate
of 1 ml/min and detection wavelength was 210 nm. Injection volume
According to the literature survey, there are only few reverse-phase
was 10 ml and column temperature was maintained at 30°C. Optimized
high-performance liquid chromatography (RP-HPLC) methods and
chromatogram was shown in Fig. 2.
ultraviolet (UV) methods available for the estimation of metformin
and empagliflozin individually and in combination with other
Standard preparation
drugs [3-8]. Hence, an attempt was made to develop RP-HPLC method
Accurately weighed and transferred 34 mg of metformin and 2 mg
for simultaneous estimation of metformin and empagliflozin in tablet
of empagliflozin into a 10 ml and 100 ml clean, dry volumetric flasks
formulation to decrease the retention times and run time. It can be
and 3/4 ml of diluent was added and sonicated for 30 minutes. Final
adopted in regular quality control test in industries and laboratories.
volume was made up with diluent. 1 ml solution was pipetted out from
the above two stock solutions and transferred into a 10 ml volumetric
METHODS
flask and made up to final volume with diluent. Typical chromatogram
Chemicals and reagents was shown in Fig. 3.
Acetonitrile, phosphate buffer, ammonium acetate buffer, glacial
acetic acid, potassium dihydrogen phosphate, triethylamine, and Sample preparation
orthophosphoric acid procured from Rankem Chemicals. HPLC A total of 20 tablets were weighed and average weight was calculated
grade methanol and water were purchased from SD Fine chemicals. for each tablet. Then, the tablets were powdered and equivalent weight
 Susmita et al.
Asian J Pharm Clin Res, Vol 12, Issue 1, 2019, 241-244

of one tablet was calculated. It was transferred into a 250 ml volumetric Linearity
flask 200 mL of diluent was added, sonicated for 25 min. Using diluent The linearity test was performed to ensure correlation response of
final volume was made and filtered. 0.2 ml was pipetted out from the concentration range of the analyte and the peak area. The linearity
filtered solution into a 10 ml volumetric flask and final volume was studies were performed using stock solution containing 850 µg/ml and
made with diluent. 5 µg/ml metformin and empagliflozin. These solutions were further
diluted with diluents to yield the concentration range of 2.215–
Assay preparation 1275 ppm and 1.25–7.5 ppm of metformin and empagliflozin. The
Stock solutions of standard preparations were made from the active correlation coefficients were calculated. The calibration plots are
pharmaceutical ingredient (API) and sample preparations were shown in Figs.4 and 5.
prepared from formulation using appropriate diluent. Working solutions
were prepared from stock solutions and both sample and standards were
injected into chromatographic system for the determination of assay.
Precision
The precision of the method was performed by intraday and interday
Validation of HPLC method[9,10] variation studies. The intraday and interday studies were performed by
injecting six injections of test preparations into the chromatographic
System suitability
system. The percentage RSD and SD were calculated. The results are
System suitability was carried out to check the system performance. All presented in Table 2.
the essential characteristics including the percentage RSD, the United
States Pharmacopeia (USP) tailing, the USP plate count, and the USP
resolution were verified. The results are shown in Table 1.
Accuracy
The accuracy was determined by calculating the percentage recoveries
of known amounts of each analyte of metformin and empagliflozin
Specificity
claim to the excipients, and the accuracy results were expressed as
The specificity of the method was carried out by injecting blank, placebo, percentage of analyte recovered. The results are shown in Table 3.
standard, and sample solutions into the chromatographic system. The
interference of the analyte peaks with the excipients was analyzed. Limit of detection (LOD) and Limit of quantification (LOQ)
The limit of detection (LOD) can be defined as the lowest amount of
analyte in a sample can be detected and limit of quantification (LOQ)
was determined as the lowest amount of analyte that was quantified.
These parameters were calculated using the formula,

a b
Fig. 1: (a) Chemical structure of metformin, (b) chemical
structure of empagliflozin

Fig. 4: Calibration plot of metformin

Fig. 2: Optimized chromatogram of metformin and empagliflozin

Fig. 5: Calibration plot of empagliflozin

Table 2: Intraday precision results for metformin and

empagliflozin
Fig. 3: Typical chromatogram of metformin and empagliflozin
S. No. Metformin Empagliflozin
Table 1: System suitability studies of metformin and 1 7921030 250856
2 8015260 245837
empagliflozin
3 8015344 247309
4 7949328 246258
Property Metformin Empagliflozin 5 8044578 241978
RT 2.588 min 3.679 min 6 8012992 249282
Theoretical plates (N) 2886±63.48 4616±63.48 Mean±SD 7993089±47205 246920±3074.3
Tailing factor (T) 1.38±0.117 1.11±0.117 %RSD* 0.59 1.2
RT: Retention time %RSD: Relative standard deviation

242
 Susmita et al.
Asian J Pharm Clin Res, Vol 12, Issue 1, 2019, 241-244

LOD=3.3×s/S and LOQ=10×s/S, Alkali degradation studies

To 1 ml of stock solution metformin and empagliflozin, 1 ml of 2 N
Where, s=Standard deviation and S=Slope of the calibration curve. sodium hydroxide was added and refluxed for 30 min at 60°C. The
resultant solution was diluted to obtain 850 µg/ml and 5 µg/ml
Robustness metformin and empagliflozin and 10 µl were injected into the system,
Robustness of the method was performed under a variety of conditions, and the chromatograms were recorded to assess the stability of the
by deliberate changes such as the composition of the mobile phase, flow sample.
rate, and temperature. The results are shown in Table 4.
Dry heat degradation studies
Forced degradation studies The standard drug solution was placed in oven at 105°C for 6 h to study
Forced degradation studies were performed to assess the stability and dry heat degradation. For HPLC study, the resultant solution was diluted
specificity of the proposed method. Standard and degraded samples to 850 µg/ml and 5 µg/ml metformin and empagliflozin and 10 µl were
were injected into the chromatographic system and the percentage injected into the system, and the chromatograms were recorded to
of drug degraded in solution by applying different conditions such as assess the stability of the sample.
acid, alkali, oxidative, photolytic, thermal, and hydrolytic analysis was
calculated. The results are shown in Table 5. Photolytic studies
The photolytic stability of the drug was studied by exposing the
Oxidative degradation studies 8500 µg/ml and 50 µg/ml solution for UV light by keeping the beaker
To 1 ml of stock solution of metformin and empagliflozin, 1 ml of 20% in UV chamber for 7 days. For HPLC study, the resultant solutions were
hydrogen peroxide was added separately. The solutions were kept for diluted to obtain 850 µg/ml and 5 µg/ml solutions. 10 µl from above
30 min at 60°C. For HPLC study, the resultant solution was diluted to solutions were injected into the system and the chromatograms were
obtain 850 µg/ml and 5 µg/ml metformin and empagliflozin and 10 µl recorded.
were injected into the system and the chromatograms were recorded to
assess the stability of the sample. Hydrolytic degradation studies
Stress testing under neutral conditions was studied by refluxing the drug
Acid degradation studies in water for 6 h at a temperature of 60°C. For HPLC study, the resultant
To 1 ml of stock solution metformin and empagliflozin, 1 ml of 2 N solution was diluted to obtain a 850 µg/ml and 5 µg/ml solution and
hydrochloric acid was added and refluxed for 30 min at 60°C. The 10 µl were injected, and the chromatograms were recorded to assess
resultant solution was diluted to obtain 850 µg/ml and 5 µg/ml the stability of the sample.
metformin and empagliflozin, and 10 µl solutions were injected into the
system and the chromatograms were recorded to assess the stability RESULTS AND DISCUSSION
of sample.
A rapid reverse-phase liquid chromatography method has been
developed for metformin and empagliflozin employing various ratios
Table 3: Accuracy results for metformin and empagliflozin of mobile phases, different chromatographic conditions, and flow
rates for the analysis. Under the optimal conditions of mobile phase
Sample Amount added (ug/ml) Recovery (%) %RSD*
(buffer:acetonitrile) with a ratio of 50:50 and BDS 250 mm×4.6 mm,
Metformin 425 100.64 1.41 5 m particle size column and flow rate of 1 ml/min satisfactory results
850 101.18 1.29 were obtained. The retention time was found to be 2.588 min for
1275 101.89 0.15 metformin and 3.679 min for empagliflozin. The system suitability
Empagliflozin 2.5 101.51 0.97 parameters, plate count, and tailing factor were within the limits
5 99.89 0.47
with good resolution between the analyte peaks. There was no
7.5 100.34 0.96
interference of excipients and mobile phase with the analyte peaks
%RSD: Relative standard deviation that imply the method is specific. The method showed linearity
between the concentration range of 0–1275 (ug/ml) for metformin
Table 4: Robustness data of metformin and empagliflozin and 0–7.5 (ug/ml) empagliflozin. Regression equations were y=9334.0
x+593.4 for metformin and y=48830 x+914.1 for empagliflozin. The
S. No Robustness condition Metformin Empagliflozin calibration plot exhibits a linear relationship between peak area and
%RSD* %RSD* concentration over a wide range. The percentage recoveries were
1 Flow minus (0.9 ml) 0.1 0.3 100.01% and 100.65% for metformin and empagliflozin, respectively.
2 Flow plus (1.1 ml) 0.5 0.7 The method was robust as observed from insignificant variations
3 Mobile phase minus 0.3 0.5 in the results of analysis by changing flow rate, mobile phase, and
4 Mobile phase plus 0.3 0.4 temperature. Limit of detection values was 0.10 ug/ml and 0.31 ug/ml
5 Temperature minus (25°C) 0.1 0.1 for metformin and empagliflozin, respectively. Limit of quantification
6 Temperature plus (35°C) 0.3 0.8 values was 0.01 ug/ml and 0.03 ug/ml, respectively, which shows the
%RSD: Relative standard deviation method developed is sensitive method.

Table 5: Degradation data of metformin and empagliflozin

Type of degradation Metformin Empagliflozin

Area % recovery % degraded Area % recovered % degraded
Acid 7685632 97.45 2.55 239367 98.09 1.91
Base 7757303 96.98 3.02 236010 97.03 2.97
Peroxide 7899186 98.42 1.58 239152 98.02 1.98
Thermal 7924395 99.60 0.4 243284 99.10 0.9
UV 7937101 99.08 0.92 243360 99.23 0.77
Water 7964453 99.08 0.92 244395 99.41 0.59
UV: Ultraviolet

243
 Susmita et al.
Asian J Pharm Clin Res, Vol 12, Issue 1, 2019, 241-244

CONCLUSION 2. Shyamala KN, Mounika J, Nandini B. Validated stability-indicating

RP-HPLC method for determination of Empagliflozin. Pharm Lett
A simple and efficient method was developed for the method 2016;8:457-64.
development and validation for simultaneous estimation of the 3. Venkata SM, Ram BJ, Rajan DS, Adinarayana G, Ramana MK.
metformin and empagliflozin by RP-HPLC in tablet dosage form. Development of a validated HPLC method for the estimation of
Validation was carried out as per the International Conference on metformin HCl and propranolol HCl. Br J Pharm Res 2014;4:1909-22.
Harmonisation guidelines and method validation data showing 4. Lakshmi KS, Rajesh T, Sharma S. Simultaneous determination of
metformin and pioglitazone by reversed phase HPLC in pharmaceutical
satisfactory results. Degradation studies revealed that the method is
dosage forms. Int J Pharm Pharm Sci 2009;1:162-6.
stability indicating. Hence, the proposed method can be applicable for 5. Pandya RH, Rathod R, Maheswari DG. Bioanalytical method
routine quality control analysis of tablet dosage forms in laboratories. development and validation for simultaneous determination of
linagliptin and metformin drugs in human plasma by RP-HPLC
CONFLICTS OF INTEREST method. Pharmacophore 2014;5:202-18.
6. Murthy TG, Geethanjali J. Development of a validated RP-HPLC
All authors have none to declare. method for simultaneous estimation of metformin hydrochloride and
rosuvastatin calcium in bulk and in-house formulation. J Chromatogr
AUTHORS’ CONTRIBUTIONS Sep Tech 2014;5:1-7.
7. Kavitha KY, Geetha G, Hariprasad R, Kaviarasu M, Venkatnarayanan R.
The research was proposed and designed by G.Rajitha. The experimental Development and validation of stability indicating RP-HPLC method
work of the validated method development was carried out by Y.Ramya for the simultaneous estimation of linagliptin and metformin in pure
Yadav and A.Geetha Susmita. The manuscript was drafted by A. Geetha and pharmaceutical dosage form. J Chem Pharm Res 2013;5:230-5.
Susmita which was further edited by G. Rajitha. Authors read and 8. Ramesh J, Kumar NS. Stability indicating RP-HPLC method
approved the final manuscript. development and validation for he simultaneous determination of
vildagliptin and metformin in pharmaceutical dosage form. Int J Pharm
REFERENCES Pharm Sci 2017;9:150-7.
9. Validation of Analytical Procedures. Text and Methodology. Geneva:
1. Kar M, Choudhury PK. HPLC method for estimation of metformin Q2 (R1); 1996.
hydrochloride in formulated microspheres and tablet dosage form. 10. Validation of Analytical Procedures: Text and Methodology. ICH Q2
Indian J Pharm Sci 2009;71:318-20. (R1) Harmonised Tripartite Guideline; 2006;15-64.

244