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Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 76 

Research Article  

Development and validation of novel stability indicating RP HPLC Method for 

quantitative Estimation of Empagliflozin in Tablets 

Ausuf Ahmad1, Zahra Maryam2 

From, 1Department of Clinical and Pharmaceutical Sciences, School of Life and Medical Sciences, University of Hertfordshire, 

Hatfield, UK, 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir 26470, Turkey. 

Correspondence to: Zahra Maryam, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir 

26470, Turkey. Tel: +90-5411666124, E-mail: zahra.maryam489@gmail.com 

ABSTRACT 

The current study was conducted to develop and validate a novel method for the qualitative and quantitative determination of 

Empagliflozin in bulk granules and tablets. This method was successively applied to the analysis of the approved dosage form of 

Empagliflozin available in the market with the brand name of Jardiance tablets. The developed method was found to be precise, 

economic, and rapid with a run time of 20 minutes. The Empagliflozin peak was eluted at 5.0 minutes. This elution was done on the 

C18 column (150 X 4.6mm) having a 5μm particle size using an isocratic mobile phase, a mixture of phosphate buffer pH 3.0 and 

methanol in the ratio of 70:30 at a flow rate of 1.0ml/min with PDA detector (maximum wavelength of 224nm) and column 

temperature 30°C. The method was found to be linear with LOD and LOQ, 0.068 μg and 0.35 μg, respectively. The correlation 

coefficient R2 calculated was 0.996. Precision and accuracy of the method evaluated by interday, intraday, and recovery study was 

found to be within limits. The forced degradation study proved the stability of the method. Objectives: This research article is aimed 

at developing a new method for developing and validating a novel method for the qualitative and quantitative determination of 

Empagliflozin in bulk granules and tablets.  Methods: To conduct this research, all the validation parameters were employed according 

to International Council for Harmonization (ICH) guidelines and all the validation parameters e.g., Accuracy, specificity, linearity, 

LOD, LOQ, robustness, and degradation studies were performed. Results: Results of the proposed technique were found to be 

trustworthy, accurate, suitable, and robust for everyday application. Conclusion: Conclusively the results of the study indicated that 

the current method is simple, precise, accurate, stable, and economic, and thus can be used for routine quality control testing of 

Empagliflozin.   

Keywords: Empagliflozin, RP-HPLC, Method Validation, Qualitative Determination 

hemically, Empagliflozin is (1-chloro-4[b d-

glucopyranos-1-y1]-2-[4-([1]-tetrahydrofuran-3-yl-

oxy) benzyl]-benzene [1-2]. Empagliflozin controls the 

excess glucose level in the blood. It is an inhibitor of sodium-

glucose cotransporter-2(SGLT-2) and promotes the excretion 

of excess glucose in urine by reabsorbing it in the kidney, thus 

is used in the treatment of type 2 diabetes. [1,3-4]. The 

empirical formula of Empagliflozin is C23H27ClO7 and its 

molecular mass is 450.91 a.m.u [5-6].  Empagliflozin is 

available in 10 mg and 25mg oral tablet dosage forms alone or 

in combination with other drugs. Jardiance, Glyxambi, Synjardy, 

and Synjardy XR are examples of Empagliflozin tablets [7-10].   

 
Figure 1 – Structure of Empagliflozin 

The development of a quantitative method and its validation 

are fundamental and essential phases in pharmaceutical analysis 

to ensure that the results of the proposed technique are 

trustworthy, accurate, suitable, and robust for everyday 

application. The literature review revealed that Empagliflozin 

can be detected both qualitatively and quantitatively by using 

Liquid Chromatography (LC), Ultraviolet spectrophotometer 

(UV), High-Performance liquid chromatography (HPLC), and 

Ultra Performance Liquid Chromatography (UPLC) techniques 

[11-15]. However, few reports are available for the RP-HPLC 

measurement of empagliflozin in pharmaceutical preparations. 

In this article, we discussed the validation of a novel technique 

for precise Empagliflozin quantification in pharmaceutical 

dosage forms and bulk pharmaceuticals.  

The validation is important as Drug regulatory governing 

bodies play a vital role and have placed emphasis on drug 

manufacturing companies to offer comprehensive information 

regarding the process for the validation of analytical methods 

C 

mailto:zahra.maryam489@gmail.com


Ahmad et al                                                                                               RP-HPLC Method Validation of Empagliflozin 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 77 

[15]. We performed the validation in accordance with ICH [16-

18]. The current strategy, which is brand-new to the market, 

uses several mobile phase compositions to estimate 

Empagliflozin in a quality control laboratory. In accordance 

with the recommendations of the International Council for 

Harmonization, the method's performance was validated. 

This study was conducted to develop and validate the novel, 

accurate, precise, and stability-indicating RP-HPLC method for 

the analysis of Empagliflozin in bulk granules and tablets. In 

the development stage, the wavelength detection study was 

conducted by scanning the sample between 200nm and 400nm. 

A major peak was found at 224 nm (λ max). Then the method 

was validated for validation parameters I-e accuracy, precision 

(repeatability, intermediate precision), specificity, the limit of 

detection, the limit of quantification, linearity, range, and 

robustness as per ICH and United States Pharmacopeia (USP) 

guidelines using HPLC [11, 19]. Stress study was performed in 

acid, base, peroxide solutions, thermal, and photolytic stress 

conditions to check the stability of dosage form as well as the 

robustness of the method [20-21].  

MATERIALS AND METHODS 

All the chemicals, Methanol, Acetonitrile, Hydrochloric Acid, 

Hydrogen Peroxide, Sodium Hydroxide, and Monobasic 

hydrogen phosphate were purchased from Science Center Merk 

distributor Islamabad. All the chemicals were used without any 

further purification. 

Instrumentation: Gradient HPLC model LC20 (Shimadzu 

Japan) (LC20AT) equipped with PDA detector (SPD M-20A), 

autosampler (SIL-20AHT), Column oven (CTO-20A) and 

degassing unit (DGU-20ASR) was used for the study. 

Computer-supported software-based Spectrophotometer model 

1800 (Shimadzu Japan) for wavelength detection and FT-IR 

model Alpha (Germany) with ATR diamond ATR module for 

identification of Empagliflozin were employed. Calibrated 

analytical balance Metler Tolendo was used for weighing 

purposes. PH meter was served for the adjustment of the pH of 

the buffer for the mobile phase. A neutronic Photostability 

chamber was used for the photostability study, and thermal 

degradation was checked using a memart oven. An ultrasonic 

bath was used to dissolve the samples. 

Chromatographic Conditions: In the isocratic mobile phase, 

the mixture of phosphate buffer pH 3.0 and methanol in the ratio 

of 70:30 with a flow rate of 1.0 ml/minute and column 

temperature of 30 °C was used. The flow rate was set at 1.0 

ml/minute and the column temperature was kept at 30 °C. We 

kept the Injection volume at 20 µL. The mobile phase was also 

used as diluent and chromatograms were recorded at 224nm. 

The summary of chromatographic conditions is mentioned in 

Table 1.  

Table 1 – Chromatographic conditions 

Preparation of solutions 

Preparation of Mobile Phase: 6.8 grams of monobasic 

potassium phosphate was dissolved in 900 ml deionized water 

in a 1000ml volumetric flask, the pH was adjusted to 3.0 with 

orthophosphoric acid and the solution was filtered through a 

0.5-micron filter and degassed. Then phosphate buffer and 

methanol were mixed in a ratio of 70:30 to make the isocratic 

mobile phase.   

Preparation of standard solution: After weighing carefully, 

the Empagliflozin working standard equivalent to 25mg, was 

dissolved in a diluent in 50 ml volumetric flask. The solution 

was filtered and 2ml of the filtrate was taken to 50 ml 

volumetric flask, mixed well, and the volume was made with 

diluent to 50 ml and sonicated for 5.0 minutes. 

Preparation of sample solution: We took 20 tablets, weighed, 

and powdered them. Then the powder equivalent to 25 mg 

Empagliflozin was dissolved in diluent and the volume was 

made up to 50 ml in the volumetric flask. After filtration, 2.0 

ml of this solution was transferred to another 50 ml volumetric 

flask, mixed well with diluent, and sonicated for 5.0 minutes. 

Method Development: We performed some preliminary tests 

for the development of a stable and effective method for 

qualitative and quantitative estimation of Empagliflozin. First, 

the wavelength was detected using a UV-Visible 

spectrophotometer by scanning the sample and standard 

solution between 200nm to 400nm. A major peak of 

Empagliflozin was observed at 224nm (Figure 2).  

 

Figure 2 – UV spectrum of Empagliflozin 

Mobile phase Phosphate buffer:  Methanol (70:30%V/V) pH 

adjusted to 3.0 

Column ODS C18, 250×4.6 mm, 5μ 

Wavelength 224 nm 

Flow rate 1.0 mL/min 

Injection 

volume 

20 μL 

Run time 10 min 

Diluent Phosphate buffer:  Methanol (70:30%V/V) pH 

adjusted to 3.0 



Ahmad et al                                                                                               RP-HPLC Method Validation of Empagliflozin 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 78 

Then many injections of the sample were applied on HPLC 

using water, methanol and acetonitrile, and phosphate buffer in 

different compositions (to determine the optimal mobile phase 

combination) as mobile phase. The mobile phases tested 

included methanol and Acetonitrile in the ratios of 60:40 and 

70:30, phosphate buffer, and methanol with 65:35, 60:40, and 

70:30 ratios to observe the separation and characteristics of the 

peak. It was observed that the best symmetry of peak with less 

retention time and low pressure of HPLC pump was achieved 

using phosphate buffer and methanol in the ratio of 70:30 as 

mobile phase. The optimum flow rate selected after hit and trial 

basis was 1.0 ml per minute.                                                 

System Suitability: A suitability study of the system was 

conducted to confirm its performance.  

 

Figure 3 – Suitability chromatogram 

All critical parameters including percentage Relative standard 

deviation (RSD), tailing factor, USP plate count, and USP 

resolution were verified by injecting six injections of standard 

solution. The results of system suitability are depicted in Table 2.     

Table 2 – System suitability 

 

 

Figure 4 – Specificity chromatogram 

Specificity: The analytical method should have the ability to 

resolve all its process impurities, known and unknown 

degradation impurities during stress study. A placebo should 

not interfere with the retention times of active drugs and 

Impurities. The specificity of the method was confirmed by 

injecting the blank, placebo, and standard solution.  

Linearity: The linearity of the method was checked by 

injecting 5 standard solutions of concentration ranging from 25 

µg/ml to 125 µg/ml. Then the graph was plotted between 

concentration and peak areas. A straight line was obtained 

which confirmed the linear response of the method (Fig. 5). 

 

Figure 5 – Linearity curve 

The intercept, slope, correlation coefficient of regression, and 

sum of squares were calculated. The linearity results are 

presented in Table 3. 

Precision 

a) Repeatability: For repeatability testing, we applied six 

replicate injections of the same concentration of 

sample solution on the same day. All the parameters 

including percentage RSD, tailing factor and USP 

plate were checked and are mentioned in Table 4.  

Accuracy: The accuracy of a method describes how close the 

results are to a true value. The accuracy study was conducted 

by calculating the percentage recovery of Empagliflozin. It was 

processed by injecting three replicates of three different 

concentrations of the analyte. In the end, the percentage 

recovery of the analyte was determined to confirm the accuracy 

of the system and depicted in Table 6. 

Table 3 – Linearity 

 

y = 845241x
R² = 0.9976

0

1000000

2000000

3000000

4000000

5000000

6000000

0 1 2 3 4 5 6 7



Ahmad et al                                                                                               RP-HPLC Method Validation of Empagliflozin 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 79 

Table 4 - Repeatability 

 

Table 5 – Intermediate precision 

 

 

Figure 6 – Accuracy chromatogram 

Limit of Detection (LOD) & Limit of Quantitation (LOQ): 

Qualitative analysis of an analyte in a tablet at the lowest 

possible concentration is called the limit of detection. It can be 

calculated by the formula LOD = 3.3σ/S, where σ is the standard 

deviation of response and S is the slope of the calibration curve. 

Whereas the lowest possible concentration of an active 

ingredient in a tablet that can be detected quantitatively is called 

the limit of quantitation. In the recent study, the limit of 

quantitation was calculated in the sample with acceptable 

precision and accuracy limits by the formula LOQ=10σ/S. The 

results of the limit of detection of Empagliflozin calculated in 

XR tablets were found to be 0.068μg/ml while the limit of 

quantitation came out to be 0.35 μg/ml. 

 

Figure 7 – Limit of detection 

 

Figure 8 – Limit of Quantitation 

Robustness: The robustness of the analytical method is the 

capacity to remain unchanged by a small change in conditions. 

High system robustness ensures a positive indication in normal 

use. The robustness of the method was checked by making 

minor changes in the composition of the mobile phase, flow 

rate, temperature of the column, and the pH of the buffer. It was 

confirmed that no significant changes occur after deliberately 

changing the system conditions. The robustness results are 

depicted in Table 7.  

Table 6 – Accuracy of the system 

 



Ahmad et al                                                                                               RP-HPLC Method Validation of Empagliflozin 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 80 

Table 7 – Robustness of the method 

 

Table 8 – Stress stability study 

 

Stress stability study: This study was conducted to assess the 

stability of the method under stressed conditions. The forced 

degradation parameters like acid, alkali, oxidative, photolytic, 

thermal, and hydrolytic analyses were carried out under the 

scope of this study. 

Acid Degradation study: In the acid degradation study, 1ml of 

2N HCl was added to 1ml of stock solution of Empagliflozin 

and the mixture was refluxed for 30 minutes at 60°C. The 

resultant solution was diluted with diluent to 5 µg/ml. Then 

three replicate injections of 10 μl solution were injected into the 

chromatograph and chromatograms were observed to check the 

stability of the solution. The percentage degradation was 

checked by comparing the results with the standard. 

Alkali Degradation Study: This study was conducted by 

taking 1ml of stock solution and 1ml of 2N NaOH solution and 

the mixture was refluxed for 30 minutes at 60°C. Then the 

solution was diluted to 5 µg/ml. Three replicate injections of 10 

μl solution were then injected into the chromatograph and the 

behavior of the peak area was observed. This peak area was then 

compared with that of the standard. 

Dry Heat degradation study: Thermal degradation was 

carried out by placing powder of Empagliflozin in a petri dish 

at 105°C for 1 hour. Then 5 µg/ml solution of Empagliflozin 

was made and triplicate injections were applied to the 

chromatograph and the degradation was observed by comparing 

the peak areas with the standard solution. 

Photo stability: For photolytic degradation, a standard stock 

solution of Empagliflozin was placed in a photostability 

chamber for 5 days. Then after 5 days, the powder was diluted 

to make the concentration of about 5 µg/ml and then triplicate 

injections were applied to the chromatograph and the results for 

possible degradation were observed. The standard solution kept 

in normal condition was studied versus the photolytic standard 

solution and then the percentage degradation was calculated. 

The results of degradation studies were depicted in Table 8. 

RESULTS AND DISCUSSION 

With the advancements in technology and instrumentation, 

there comes the requirement to design an efficient, dependable, 

and selective quantification approach for active pharmaceutical 

components. Though numerous analytical techniques have been 

employed to estimate drugs, researchers continue to develop 

alternative and more efficient HPLC methods to quantify a wide 

range of compounds. 

We also employed a novel method for the pharmaceutical 

assay of empagliflozin tablets. We developed an accurate, 

simple, and stable reverse phase HPLC method for both 

qualitative and quantitative estimation of Empagliflozin using 

different ratios of mobile phase, different chromatographic 

conditions, diluents, and flow rates for analysis. In optimal 

conditions of isocratic mobile phase with the combination of 

phosphate buffer pH 3.0 and methanol (70:30), C18 column 

(150X4.6mm) with particle size 5μm and flow rate of 1.0 ml 

per minute, accurate and precise results were obtained. The 

results of system suitability, specificity, linearity, and 

robustness were all within good acceptable limits. There was no 

interaction of solvents like mobile phase, diluents, and 

excipients with the peak of Empagliflozin. Additionally, 

statistical analysis revealed negligible differences between the 

peak areas during precision, suitability, and repeatability 

studies. A fit value of 0.9996 was obtained for the regression 

equation y = 845241x, indicating a strong correlation. It was 

discovered that the minimal detection and quantification limits 

were 0.068 µg/ml and 0.35 µg/ml, respectively. Due to the 

proper selection and composition of the mobile phase, the 

reduced detection values in our investigation were noted. Lower 

standard deviations and coefficients of variation were found in 

the drug assay results, indicating good procedure accuracy. 

The peak purity of a sample depends on the selectivity and 

specificity of an analyte. For confirmation of peak purity, the 

drug solution was also tested against a placebo mixture (with 

excipients used in tablet formulations).A lack of co-elution 

peaks during the drug sample's retention time indicated that the 

current approach was suggestive of both selectivity and 

specificity. Since there was no peak of any additive during the 

analytical run of empagliflozin, the devised method can be 

effectively used to quantify the medicine in the marketed 

tablets. The underlying methodology was confirmed to be 

reliable because no concentration change was seen when the 

flow rate, column temperature, or mobile phase composition 



Ahmad et al                                                                                               RP-HPLC Method Validation of Empagliflozin 

Vol 2 | Issue 2 | Apr – Jun 2023                                                                                     Indian J Pharm Drug Studies | 81 

was changed. Previous studies also confirmed the reliability of 

analytical methods, frequently by altering the solvent 

composition, flow rate, and column temperature to a limited 

extent [22]. Based on the results, it can be concluded that the 

present method can be employed in pharmaceutical labs for 

routine quality control testing and can serve as a basis for 

further modifications in the method in the future. 

CONCLUSION 

Validation parameters mentioned in the recent study were 

checked according to ICH guidelines and satisfactory results 

were obtained. A stress stability study confirmed that the 

method is stability-indicating. Therefore, based on the study 

conducted it can be ensured that the method can be applied to 

the pharmaceutical lab for routine analysis of tablet dosage 

form.  

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How to cite this article: Ahmad A. Zahra M.  

Development and validation of novel stability indicating 

RP HPLC Method for quantitative Estimation of 

Empagliflozin in Tablets. Indian J Pharm Drug Studies. 

2023: 2(2); 76-81.  

Funding: None                    Conflict of Interest: None Stated 

 

https://www.drugs.com/monograph/empagliflozin.html
http://dx.doi.org/10.1155/2021/9983477
http://dx.doi.org/10.1007/s40262-013-0126-x
http://dx.doi.org/10.1007/s40262-013-0126-x
http://dx.doi.org/10.5958/2231-5675.2020.00021.6
http://dx.doi.org/10.5958/2231-5675.2016.00030.2

