INTRODUCTION:

The development and validation of bioanalytical methods are critical for the accurate and reliable quantification of drugs and their metabolites in biological matrices. Among these, the estimation of metformin (MET) and empagliflozin (EPG) in blood plasma is of particular importance due to their widespread use in the management of type 2 diabetes mellitus.

Metformin is a first-line anti hyperglycemic agent, whereas empagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor that enhances glycemic control by promoting renal glucose excretion¹.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used analytical technique due to its high resolution, reproducibility, and sensitivity². RP-HPLC has been successfully employed for the simultaneous estimation of MET and EPG in various matrices, including pharmaceutical formulations and human plasma. The method development involves optimizing chromatographic conditions such as mobile phase composition, flow rate, and detection wavelength to achieve well-resolved peaks and accurate quantification of both analytes³.

Validation of the RP-HPLC method is crucial to ensure its reliability and accuracy in routine analytical applications. The validation process includes assessing various parameters such as precision, accuracy, specificity, sensitivity, linearity, and stability according to regulatory guidelines. Precision ensures consistent results upon repeated analysis, while accuracy confirms the method’s ability to measure the true concentration of analytes⁴. Specificity ensures no interference from endogenous substances, and sensitivity is determined by the method’s ability to detect low concentrations of the drugs. Linearity indicates a proportional response over a range of concentrations, and stability studies ensure that the analytes remain unchanged under different conditions⁵.

This study aims to develop and validate an RP-HPLC method for the simultaneous estimation of metformin and empagliflozin in blood plasma, adhering to regulatory guidelines and ensuring suitability for pharmacokinetic, bioavailability, and therapeutic drug monitoring applications.

Experiment:

Selection of Precipitating Agent:

Selection of precipitation agent is based on the solubility of drug and good protein precipitation property. MET and EPG were found to be soluble in Acetonitrile and methanol and Acetonitrile having good precipitating property of so the Acetonitrile was selected as the protein precipitation and extraction agent.

Selection of mobile phase:

Initially to estimate MET and EPG in fix dosage form the number of mobile phase in different ratio were tried. A result was shown in Table 1. Taking into consideration the system suitability parameters like Retention Time, Tailing Factor, Number of Theoretical Plates and HETP, the mobile phase found to be most suitable for analysis was 20mM KH₂PO₄: ACN (pH 4.0) in the ratio of 20:80 v/v. The mobile phase was filtered through a 0.45m filter paper to remove particulate matter and then degassed by sonication. Flow rate employed for analysis was 1.0ml/min.

Table 1: Selection of mobile phase

Mobile Phase	Ratio	Retention Time
ACN: Water	50 : 50 v/v	Poor Resolution
ACN: Methanol	50 : 50 v/v	Poor Resolution
20mM KH₂PO₄: acetonitrile (pH 4.0)	20:80 v/v	Most Suitable

Procedure for preparation of mobile phase:

20mM KH₂PO₄: ACN (pH 4.0) in the ratio of 20:80 v/v, pH 4.0 with Ortho Phosphoric Acid filtered through a 0.45m filter paper.

Table 2: Mobile phase selection

Mobile Phase	Ratio	Retention Time
Mobile Phase	Remark
ACN: Water	50 : 50 v/v	Poor Resolution
ACN: Methanol	50 : 50 v/v	Poor Resolution
20mM KH₂PO₄: ACN (pH 4.0)	20:80 v/v	Most Suitable

Selection of diluent:

Diluent used for preparation of sample were compatible with mobile phase and no any significant affect retention and resolution of analyte. After various trials Acetonitrile was used as diluents.

Selection of separation variable:

Table 3: Separation variable

Variable	Condition
Column
Dimension.	250mm x 4.60mm
Particle Size	5m
Bonded Phase	Octadecylsilane (C₁₈)
Mobile Phase
20mM KH₂PO₄	20
Acetonitrile	80
Diluent	Acetonitrile
Flow rate	1.0ml/min
Temperature	Ambient
Sample Size	20ml
Detection wavelength	254mm
Retention time
MET	5.150±0.024min.
EPG	7.162±0.014min.

Extraction of drug sample:

Accurately weighed 10mg of MET was transferred into 50ml volumetric flasks separately and dissolved in 10 ml of plasma, then volume was made up to 50ml with acetonitrile and vortex it to get complete precipitation of plasma protein. Stand it aside for few minute, precipitate of protein settled down then collect the supernatant layer and add 10ml of acetonitrile in precipitate to complete removal of drug. Centrifuge the collected supernatant layer at 6000rpm for 7min at 4^oC and then filtered by Whatmann filter paper (no.41). Concentration of EPG in acetonitrile was 200µg/ml (stock- A).

Preparation of Sub Stock Solution:

5ml of solution was taken from stock-A of MET and transferred into 10ml volumetric flask separately and diluted up to 10ml with diluent (Acetonitrile) to give concentration of 100µg/ml (Stock-B).

Preparation of Different Solution:

1ml, 2ml, 3ml, 4ml and 5ml of stock-B was taken separately in 10ml volumetric flask and volume was made up to 10ml with (Acetonitrile). This gives the solutions of 10µg/ml, 20µg/ml, 30µg/ml, 40µg/ml, 50µg/ml for MET. In same manner 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml, 10µg/ml of EPG also prepared.

System Suitability Parameters:⁶

Separation variables were set and mobile phase was allowed to saturate the column at 1.00ml/min. After complete saturation of column, three replicates of working standard of MET 20mg/ml and 4mg/ml EPG was injected separately. Peak report and column performance report were recorded for all chromatogram.

Table 4: System Suitability Parameters

Parameters	% MEAN±SD*
Parameters	Metformin	Empagliflozin
No. of Theoretical Plates	3537.667±47.647	3274.333±23.989
Tailing Factor	1.140±0.021	1.043±0.038
Retention time	5.150±0.024	7.162±0.014

Validation of developed method:^7-22

Linearity:¹¹

Linearity of analytical procedure is its ability (within a given range) to obtain test, which are directly proportional to area of analyte in the sample. The calibration plot was contracted after analysis of five different (from 10 to 50µg/ml) and (2 to 10µg/ml) concentrations and areas for each concentration were recorded three times, and mean area was calculated. The regression equation and correlation coefficient of curve are given and the standard calibration curve of the drug is shown in figure 5.36. From the mean of AUC observed and respective concentration value, the response ratio (response factor) was found by dividing the AUC with respective concentration (Table given below).

Table 5: Linearity

Parameter	Metformin	Empagliflozin
Concentration (μg/ml)	10-50	2-10
Correlation Coefficient (r²)*	0.999	0.999
Slope (m)*	15.33	51.64
Intercept (c)*	1.346	-1.404

Specificity:

Specificity of the method was carried out to assess unequivocally the analyte presence of the components that might be expected to be present, such as impurities, degradation products and matrix components.

Accuracy:¹²

Recovery studies were performed to validate the accuracy of developed method to reanalyzed sample solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed.

Table 6: Results of recovery study

% Level	% MEAN±SD*
	Metformin	Empagliflozin
80%	98.66±0.666	98.84±1.012
100%	99.27±0.528	95.32±0.995
120%	98.55±0.779	99.01±0.482

* Value of three replicate and three concentrations

Precision:¹³

Repeatability:

The repeatability was performed for five replicate at five concentrations in linearity range 10, 20, 30, 40 and 50mg/ml for MET and 2, 4, 6, 8 and 10mg/ml for EPG indicates the precision under the same operating condition over short interval time.

Intermediate Precision (Day To Day Precision):

Intermediate precision was also performed within laboratory variation on different days in five replicate at five concentrations. Results of day to day intermediate precision for MET and EPG reported in table 7.

Table 7: Results of precision

Parameter	% MEAN±SD*
Parameter	Metformin	Empagliflozin
Repeatability	99.13±0.019	96.84±0.074
Intermediate precision
Day to day precision	98.86±0.120	96.06±0.118

Robustness¹⁴

As per ICH norms, small, but deliberate variations in concentration of the mobile phase were made to check the method’s capacity to remain unaffected. The ratio of mobile phase was change from, 20mM KH₂PO₄: acetonitrile (20:80% v/v), to (15:85 % v/v).

Table 8: Results of Robustness

Parameter	% MEAN±SD*
Parameter	Metformin	Empagliflozin
Robustness	99.17±0.102	96.31±0.115

Detection Limit and Quantitation Limit¹⁵

The LOD and LOQ of developed method were calculated based on the standard deviation of response and slope of the linearity curve.

Table 9: Results of LOD and LOQ

Name	LOD (mg/ml)	LOQ (mg/ml)
Metformin	0.25	0.75
Empagliflozin	0.15	0.45

Analysis of both the drugs in tablets formulation:¹⁶

Amount equal to 250mg of MET (6.25mg of EPG) from the tablets of Jardiance Met was taken in 100ml volumetric flask. This was than dissolve in 10ml of plasma by sonication for about 10 minutes. The volume is made up to the mark by mobile phase and filtered by whatmann filter paper (no.41) and the filtrate was used to prepare samples of different concentration.

Table 10: Analysis of tablet sample

Tablet Jardiance Met	MET*	EPG*
Label Claim (mg)	500 mg	12.5 mg
% Found (mg)	495.65	12.45
% Assay	99.13	99.6
% RSD	0.123	0.225

CONCLUSION:

Overall, the developed RP-HPLC method stands out as a comprehensive tool for the simultaneous estimation of metformin and empagliflozin in blood plasma. Its robustness, precision, accuracy, sensitivity, linearity, specificity, and stability collectively affirm its suitability for routine bioanalytical applications. Consequently, this method can be confidently employed in clinical settings for therapeutic drug monitoring, as well as in research environments for pharmacokinetic and bioavailability studies, ensuring high-quality and reliable analytical results.

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Received on 13.07.2024 Revised on 05.11.2024

Accepted on 18.01.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3699-3702.

DOI: 10.52711/0974-360X.2025.00532

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