INTRODUCTION:

A novel oral antidiabetic medication called empagliflozin HCl is primarily prescribed to treat type 2 diabetes. It functions by inhibiting sodium glucose cotransporter-2 (SGLT-2) ^1-4 selectively. The kidney is the focus of the novel pharmacological class of SGLT2-inhibitors, which decrease blood glucose levels by decreasing renal glucose reabsorption and raising urine glucose excretion. ^5-8. A search of the literature indicated that different analytical procedures for empagliflozin HCl have been published, utilising liquid chromatography ^11–27 and spectrophotometric techniques ^9–10, for both human plasma and bulk and/or pharmaceutical dose forms.

The International Conference on Harmonisation (ICH) developed recommendations for risk assessment, quality by design (QbD), and quality by review (Q8, Q9, and Q10) in consideration of future requirements. A novel approach to creating and evaluating high-quality pharmaceutical goods is called "quality by design." These days, developing and analysing pharmaceutical products with quality features is an integral component of the modern approach. Quality by Design (QbD) is a major option to improve product quality, but it is difficult to apply on an industrial scale. The creation of a design space and control strategy is key to the quality by design concept. Finding the design points that can provide results in accordance with specifications and predetermined conditions with potential for repeatability, accuracy, and repeatability is one of the design's benefits. Several mathematical models are used to analyze mathematical data to generate a design space^28-29.

As a result, the goal of the current study was to create an analytical technique for the determination of empagliflozin HCl using the QbD methodology that would be easy to use, affordable, and time-efficient. One such technique is High Performance liquid Chromatography. When applied to analytical methods, Quality by Design (QbD) principles may yield more robust methods that yield consistent, dependable, and high-quality data throughout the process. This, in turn, may reduce the number of method incidents in routine environments. In the end, this would save time and money by reducing the amount of time spent on investigations.

Figure: 1 Structure of Empagliflozin Hydrochloride

MATERIALS AND METHOD:

Materials: Empagliflozin HCl received as gift sample from Lupin Pharmaceutical Ltd, Pune, M.S. and other chemicals were used of analytical grade (Merck).

Instrumentation:

An Agilent 1260 Infinity instrument including an implicit degasser and an autosampler (AS-4050). Additionally, a PDA (Shimadzu, SPD-20A) detector with a wavelength of 228 nm was part of the system. Software for programming Open Lab EZ Chrome was used to gather and handle data. With a C18 column, chromatographic separation was carried out.

Chromatographic conditions:

Following various preliminary steps, a C18 column with a portable stage was selected. The mobile phase used in the experiment consisted of a combination of 0.05 M potassium dihydrogen orthophosphate buffer pH 3 and acetonitrile in a 40:60 v/v ratio. The mobile phase was pre-mixed, degassed, and filtered using a 0.4μm nylon filter. A finder was set to 228nm in frequency, and the flow rate was maintained at 1.0 ml/min. Using an autosampler with a variable circle volume of 0-100 µl, 20 µl was infused in this method. Because of the framework's segment stove, section temperatures could be programmed throughout the run. It was decided to keep the segment temperature at 40 °C throughout the procedure following an initial run at various temperatures.

Preparation of stock solution:

A 10 ml volumetric flask containing a little amount of 0.05 M potassium dihydrogen orthophosphate buffer pH 3 and acetonitrile in the ratio of (40:60 v/v) was filled with precisely weighed 10 mg of empagliflozin HCl. To achieve a 1000 ppm concentration, the volume was increased to 10 ml using the same mobile phase composition.

Preparation of working solution:

Take out 1 millilitre (ml) of the stock solution, transfer it to a volumetric flask, and dilute it with the mobile phase to make 10 millilitres (100 ppm). After that, the mixture is sonicated for thirty minutes.

Method development:

Selection and Preparation of Mobile Phase:

Various amounts and flow rates of mobile phases comprising methanol, water, acetonitrile, and cradles at different pH values were tried. With a flexible stage consisting of 40 sections of potassium dihydrogen orthophosphate buffer pH 3 and 80 pieces of acetonitrile, good peaks were obtained at a stream pace of 1.0 ml/min. Before being inserted into the framework, the two components of the portable stage were vacuum-separated through 0.45µm film channels and sonicated for 30 minutes.

Preparation of Standard Stock Solutions:

The drug's standard solutions were made with acetonitrile in a 40:60 v/v ratio and 0.05 M potassium dihydrogen orthophosphate buffer pH 3. To create standard stock solutions with a concentration of 1000 μg/mL, 10 mg of the medication was weighed and then dissolved in diluents in 10-milliliter volumetric flasks. To get the necessary medication concentrations, diluent was added to the normal stock solutions. Every day, all solutions—including the stock solution—were made from scratch.

Preparation of Calibration Curve:

The drug's standard stock arrangements were transferred to a 10 mL volumetric flask and appropriately diluted using a 40:60 v/v ratio of acetonitrile to 0.05 M potassium dihydrogen orthophosphate buffer pH 3. Aliquots were obtained so as to obtain final fixations within the range of 10–30 μg/mL.

Plotting the peak area of the drug chromatograph on the x-axis and the top areas recorded for each concentration on the y-axis allowed for the creation of the drug calibration curve. Calculations were made for the calibration curve's slope, Y-intercept, and correlation coefficient.

Experimental Design:

Factorial Design:

A 2-factor, 3-level design used is suitable for exploring quadratic response surfaces and constructing second order polynomial models with Design Expert®

Table 1: Coded Values for Independent Variables

Name of Factor	Coded Values	Levels
Name of Factor	Coded Values	Small	Medium	High
Detection wavelength (nm)	A	226 nm	228 nm	230 nm
Flow rate (ml/min)	B	0.8	1.0	1.2

Table 2: Different Batches with their Respective Composition

Batch Code	Detection wavelength (nm)	Flow rate (ml/min)
B1	226	0.8
B2	226	1.0
B3	226	1.2
B4	228	0.8
B5	228	1.0
B6	228	1.2
B7	230	0.8
B8	230	1.0
B9	230	1.2

Validation of analytical method:

According to ICH Q2 (R1) recommendations [30], the suggested RP-HPLC method of analysis was verified for criteria such system appropriateness, specificity, linearity, precision, accuracy, and robustness, as well as limit of detection (LOD) and limit of quantitation (LOQ).

System suitability:

Before sample analysis can start, the chromatographic systems that will be employed for the analysis must pass system suitability limits. After setting up the chromatographic system, give the RP-HPLC system forty minutes to stabilise. To assess the system suitability parameters, such as resolution (NLT 2.0), tailing factor (NMT 1.5), theoretical plate count (NLT 3000), and percentage RSD for peak area of six replicate injections of the Empagliflozin HCl standard (% RSD NMT 2.0), inject the blank preparation (single injection) and the standard preparation (six replicates). Then, record the chromatograms. After analysis, the tailing factor, percent RSD, and theoretical plates were found to be satisfactory. Table 3 presents the ideal chromatographic conditions as well as the system suitability data.

Linearity:

Empagliflozin HCL was produced as a stock solution of 1000 μg/mL in 0.05 M Potassium-Di- Hydrogen Phosphate Buffer pH 3: Acetonitrile (60:40) v/v. Working standard solutions for the drug, ranging from 10 to 150 μg/mL, were created from this stock and introduced into the HPLC apparatus. The medication has been shown to exhibit linearity in the 10-150 μg/mL range. Plotting the peak regions of the medication under study against its concentration produced the calibration graph, which was produced by repeat analysis at all concentration levels. The Microsoft Excel® tool was then used to determine the linearity of the relationship.

Precision:

For each of the drugs, the precision of the developed method was confirmed. The peak regions identified by a real study of six simulated infusions of a typical centralisation of every drug. By calculating the RSD, the accuracy of the method was also verified with regard to the intra- and inter-day variation in the peak zones.

Accuracy:

To evaluate the method's accuracy for the substance, a known concentration of the drug was spiked at three distinct concentration levels: 50%, 100%, and 150%. The difference between the theoretical and predicted values was then compared to the concentration determined by the method.

LOD and LOQ:

The lowest quantity in a sample that can be identified under the specified experimental conditions, but not necessarily measured, is known as the limit of detection. The lowest analyte concentration in a sample that can be found with reasonable accuracy and precision is known as the limit of quantitation. The limit of quantitation and the limit of detection were determined using the following formula. LOD is equal to 3.3 σ/S and LOQ is equal to 10 σ/S, where σ is the response standard deviation and S is the calibration curve's slope. Table 4 displays the LOD and LOQ values. The new method's sensitivity was validated by the LOD and LOQ values.

RESULTS AND DISCUSSION:

Method Development:

Chromatographic Separation:

Following several experiments, the ideal chromatographic conditions were determined by taking into account the system suitability factors. The results of this process are listed in Table 3, and the representative HPLC chromatograms of the drug and blank are displayed in Figs. 2 and 3, respectively. According to ICH Q2 (R1) recommendations, all system suitability parameter findings correlate within acceptance criteria.

Table 3. The system suitability data and the optimum chromatographic conditions.

Parameter	Chromatographic conditions
Instrument	Agilent 1260 Infinity instrument with Autosampler (AS-4050)
Column	Discovery C18 (250mm x 4.6ID, Particle size: 5 micron)
Detector	PDA (Shimadzu, SPD-20A) detector
Diluents	0.05 M Potassium - Di- Hydrogen Phosphate Buffer pH 3: Acetonitrile (60:40)
Mobile phase	0.05 M Potassium - Di- Hydrogen Phosphate Buffer pH 3: Acetonitrile (60:40)
Flow rate	1.0 ml/min
Detection wave length	UV at 228 nm.
Run time	5 minutes
Column Temperature	40 ^oC
Volume of injection loop	20 µL
Retention time (tR)	2.31 min
Theoretical plates [th.pl] (Efficiency)	6915
% RSD Tailing factor (asymmetry)	1.32
% RSD of minimum 5 replicate of calibration standard.	1.6

Figure 2: Chromatogram of blank solution

Figure 3: Chromatogram of drug Empagliflozin HCL

Figure 4 Calibration curve of Empagliflozin HCl

Linearity:

Under proposed experimental conditions, the relationship between the area and concentration of Empagliflozin HCL was studied. Linearity was checked by preparing standard solutions at 5 different concentration levels of each of Empagliflozin HCL. Standard solutions (40, 40,80,120,160 µg/mL) of Empagliflozin HCL were injected into the RP-HPLC system to get the chromatograms. The average peak area and retention time were recorded. The calibration curve was constructed between concentrations versus peak area by the prepared concentration of 20-160 µg/mL of stock solution. The linearity range was found to be 20-160 µg/mL and the calibration graph of Empagliflozin HCL shown in Figure 4. Results show that a phenomenal correlation exists between peak area and concentration of drug within the linearity range. The Summary of validation parameters are shown in Table 4.

Table 4. Summary of validation parameters.

Parameter	Result
Linearity range (µg/mL)	20-160 µg/mL
Liner Regression equation	y = 189532x
R²	0.9899
Intraday precision (% RSD)	1.14 %
Interday precision (% RSD)	1.12 %
Recovery	99.80 %
LOD (µg/mL)	0.312
LOQ (µg/mL)	0.662
Robustness	Robust

Accuracy:

The accuracy of the method was found to be good with the overall % RSD for recovery at 50 %, 100 % and 150 % levels were all within the limits which indicate that the proposed method was found to be accurate. The results are tabulated in table 5.

Table 5: Results of Accuracy (%Recovery)

Sr. no.	% Assay level of recovery	Amount of drug added (μg/mL)	*Mean (± SD) amount found (μg/ml)**	% Recovery
1	50%	50	148.32 ± 0.66	99.72
2	100%	100	196.21 ± 0.45	98.06
3	150%	150	297.77 ± 0.11	99.25

Precision:

The value of Empagliflozin HCl were found within limit, which indicates that the developed method is precise.

Table 6: Result of Precision (% Recovery)

Sr. No.	Evaluation Parameter	Results	Acceptance Criteria
1	% Assay values obtained by six test solutions (Average)	99.98	NLT 98% and NMT 102 %
2	% RSD for Assay values obtained by six test solutions	1.4	NMT 2.0 %

Specificity:

The value of Empagliflozin HCl were found within Limit, which indicates that the developed method is specific.

Table 7: Result of Specificity

Sr. No.	Results	Acceptance Criteria
1	Retention time of Empagliflozin HCl peak in test solution is comparable to that in standard solution.	Retention time of Empagliflozin peak in test solution should be comparable to that in standard solution.
2	Peak purity of standard and test solution is within acceptance criteria	NLT 97.0

LOD and LOQ:

Method validation following ICH guidelines indicated that the developed method had high sensitivity with LOD of 0.312 μg/mL and LOQ of 0.662 μg/mL.

CONCLUSION:

The current study focusses on systematic QbD and the creation of an easy-to-use, quick, accurate, and affordable RP-HPLC method for the simultaneous measurement of empagliflozin HCl. The experimental design delineates the process of scouting essential components, such as flow velocity and detecting wavelength. The 0.05 M Potassium-Di- Hydrogen Phosphate Buffer pH 3: Acetonitrile in the ratio of 40:60 in the optimised model confirms the eligibility for drug estimation. The mobile phase's flow rate and column temperature were optimised to 1 ml/min and 40 °C, respectively. The validation study verified that the approach was resilient, linear, exact, accurate, selective, and specific, which helped to justify the choice of the ideal conditions. As a result, applying the response surface technique offers improved insight for developing methods and conducting robustness tests. Under regulatory flexibility, this created technique is suitable for regulatory submission and satisfies the design space concept.

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Received on 08.04.2025 Revised on 17.07.2025

Accepted on 22.09.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5411-5415.

DOI: 10.52711/0974-360X.2025.00780

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.