Development and Validation of Stability-indicating RP-HPLC Method for the Simultaneous Estimation of Metformin and Ertugliflozin

Swathi Naraparaju¹*, Sowmya Deeti², Soujanya Chaganti¹, Karuna Devi Barla¹,

Durga Pani Kumar Anumolu²

¹Department of Pharmaceutical Chemistry, Gokaraju Rangaraju College of Pharmacy,

Hyderabad, 500090, Telangana, India.

²Department of Pharmaceutical Analysis, Gokaraju Rangaraju College of Pharmacy,

Hyderabad, 500090, Telangana, India.

*Corresponding Author E-mail: swathi8006@grcp.ac.in, swa.pharma@gmail.com

ABSTRACT:

A rapid, precise and sensitive RP-HPLC strategy was established for the simultaneous analysis of the Metformin and Ertugliflozin in bulk and tablet dosage form. The evolved methodology used Ascentis 150 (150 mm x 4.6 mm, 5 μ) as column and 0.1% OPA:  acetonitrile (60:40) as mobile phase. Temperature of the column at 30 °C and the detection wavelength at 220.0 nm were maintained. The chromatographic responses of Metformin and Ertugliflozin were eluted at 2.267 and 2.803 min, respectively. Validation of the optimized method was done as per the protocols of ICH. Upon utilization of optimum chromatographic conditions, the method showed good linearity for Metformin and Ertugliflozin across the concentration range of 25-150 μg/mL and 0.375-2.25 μg/mL, respectively. The %RSD values in accuracy, precision and robustness studies were observed less than 2.0. The %assay values for both Metformin and Ertugliflozin were found to be within acceptance criteria, when the analytes were estimated in the marketed formulation. The method was also observed to be sensitive. The LOD and LOQ values of Metformin (0.098 and 0.297 μg/mL) and Ertugliflozin (0.001 and 0.004 μg/mL) indicated the same. The method showed less retention times, good linearity, accuracy and robustness. Hence, the developed RP-HPLC methodology can be employed in quality control testing of both metformin and ertugliflozin. 

KEYWORDS: Metformin, Ertugliflozin, RP-HPLC, Precision, Linearity.

INTRODUCTION: 

Metformin (MET) is a biguanide antihyperglycemic agent used as first-line of drug in the control and management of type 2 diabetes mellitus. It is chemically known as 3-(diaminomethylidene)-1,1-dimethylguanidine with C₄H₁₁N₅ as molecular formula and 129.16 g/mol as molecular weight.  It occurs as colouress crystalline solid and soluble freely in water¹.

Ertugliflozin (ERT) is diarylmethane derivative and chemically known as (1S,2S,3S,4R,5S)-5-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol. It has 436.9 g/mol molecular weight with C₂₂H₂₅ClO₇ as molecular formula². It is indicated as monotherapy or in combination with metformin hydrochloride or sitagliptin as an adjunct to control type 2 diabetes³. The chemical structures of MET and ERT are provided in Figure 1.

Figure 1: Chemical structures of (a) Metformin and (b) Ertugliflozin

From the extensive literature review, several analytical methods for the quantification of MET and ERT        alone^4-21 and MET in combination with other antidiabetic drugs were identified^22-42. Though several RP-HPLC methods were reported earlier for the simultaneous quantification of MET and ERT^31-41, high sensitivity and early retention times (RT) were given more attention in the present work. Further, the chromatographic conditions meeting with the above features were determined through optimization. ICH guidelines⁴² were utilized to validate the optimized method and the details were provided in subsequent sections.

MATERIALS AND METHODS:

Instrumentation:

The LC system was from Waters HPLC 2695 and was comprised of automatic sampler to inject the sample, Ascentis 150 column (150mm x 4.6mm, 5µ) for separation, Photodiode Array (PDA) detector for the measurement and connected to Empower 2 Software for controlling the instrumentation and processing the data generated.

Materials:

Active pharmaceutical ingredients, such as MET and ERT were procured from Hetero Labs Pvt Ltd., Hyderabad. Marketed formulation (Segluromet tablets) containing MET (500mg) and ERT (7.5mg) was procured from nearby drug store. HPLC grade water, methanol and acetonitrile were procured from Merck (Mumbai, India). Chemicals, such as potassium dihydrogen phosphate and ortho-phosphoric acid (OPA) used were of analytical grade. Based on the solubility of the both the analytes, mixture containing acetonitrile and water in 50:50 was utilized as diluent throughout the investigation.

Potassium dihydrogen phosphate (0.01N):

Potassium dihydrogen phosphate (1.36g) was precisely measured and shifted to a 1000mL of volumetric flask. The solution was subjected to degas and sonication after adding Milli-Q water. Dilute OPA solution was used to make up the pH to 5.4.

Ortho-phosphoric acid (OPA, 0.1%, v/v):

Ortho-phosphoric acid (1mL) was diluted to produce 1000mL with HPLC grade water in a volumetric flask.

Preparation of standard stock solutions

Precisely measured MET (50mg) was shifted to volumetric flask (50mL), dissolved in a small volume of diluent. After sonication for 10 min, flask was made up with diluent to give 1000µg/mL MET. From this, 1 mL was shifted to 10mL volumetric flask and the volume is made with diluent to give 100µg/mL MET. Ertugliflozin stock solution containing initial and final concentrations 15 and 1.5µg/mL were prepared by dissolving 0.75mg in diluent by adopting above mentioned procedure.

Methods:

The methodology was optimized by using a number of columns and mobile phases in various trials. The λ_max of MET and ERT was 234 and 218nm, respectively. In view of chromatographic parameters, selectivity and sensitivity of the method 220nm was chosen for the investigation and PDA detector was used for measurement. In all the trials, the chromatographic conditions, such as mobile phase flow rate (1mL/min), sample injection volume (10µL), column temperature (30℃) and run time (10min) were maintained constant.

System suitability test:

MET and ERT standard stock solutions were serially diluted with diluent to get 25 and 0.375µg/mL solutions, respectively. These solutions were used to explore variables of system suitability, such as RT, USP plate count, and peak tailing.

Linearity:

The linearity of the procedure was estimated by shifting aliquots of standard solutions in to a group of 10mL volumetric flasks to give span of 25-150µg/mL for MET and 0.375-2.25µg/mL for ERT. Statistical variables, such as slope, intercept and correlation coefficients were computed from respective calibration curves.

Accuracy:

Exactness in the chromatographic responses was ruled by measuring analyte recoveries. Triplicate solutions of the standard analyte solutions at 50, 100 and 150% levels were spiked to fixed samples. The resultants were analyzed by the optimized methodology. The mean percentage recoveries and percentage relative standard deviation (%RSD) were determined statistically.

Precision:

System precision

From a single volumetric flask of working stock solution (MET:100µg/mL; ERT:1.5µg/mL), six injections were given and the peak areas of both the analytes were measured from their chromatograms to determine the system precision.

Method precision

The preciseness in the procedure was arbitrated by analyzing specific concentration (as mentioned above) of MET and ERT repeatedly on the same day to determine intra-day precision (repeatability) and the same concentrations were measured on three alternative days across a week time to ascertain inter-day precision in the method. Peak areas were calculated for six working sample solutions in each case and discrepancies in the corresponding measurements were expressed as %RSD.

Sensitivity:

Responsiveness of the chromatographic methodology was verified by limit of detection (LOD) and limit of quantification (LOQ). The LOD is calculated by multiplying the ratio of standard deviation and slope by 3.3, while LOQ is calculated by multiplying the same with 10.

Specificity:

The particularity in the optimized methodology was decided by looking for the interference between the peaks in placebo and blank at the recorded RT of MET and ERT.

Robustness:

The toughness in the HPLC strategy was ensured with the adoption of the methodology with a few alterations. The mobile phase flow rate (±0.1mL), mobile phase solvent ratio (±5mL), and column temperature (±5℃) were maintained, samples were injected and the %RSD values were computed.

Degradation studies:

Stock solutions of the formulation containing MET (100 µg/mL) and ERT (1.5µg/mL) were utilized for degradation studies.

Acidic degradation:

Stock solution of MET and ERT (1 mL) was added to 2 N Hydrochloric acid (1mL). The resultant is refluxed at 60℃ for 30 min.

Alkali degradation:

A mixture containing MET and ERT (1mL) and 2 N sodium hydroxide (1mL) was refluxed at 60℃ for 30 min.

Oxidative degradation:

The sample solution prepared by adding 20% hydrogen peroxide (1mL) to the stock solution of MET and ERT (1mL) was maintained undisturbed for about half an hour at 60℃ to perform HPLC study.

Dry heat degradation:

Dry heat degradation of the analyte solutions were studied by keeping them in an oven at 105°C for 6 h.

Photo stability studies:

A beaker containing analyte solutions were placed in a UV chamber for week period.

The resultant solutions in all the above tests were diluted to get solutions of the required concentrations and they were injected into HPLC system. The stability of the formulation was judged by analysing the corresponding chromatograms.

Assay:

Twenty tablets of Segluromet bearing MET (500mg) and ERT (7.5mg) were precisely measured and made in to fine powder. The powder analogous with 1 tablet was moved to a 100mL volumetric flask containing 50mL of diluent. After sonication for 25min, volume was made using diluent and screened via HPLC filters. The same were diluted again so as to get solutions with final concentrations of 100µg/mL of MET and 1.5µg/mL of ERT. Replications of sample solutions were subjected to optimized chromatographic separation procedure. The outcome formulation chromatograms were recorded and further details were elaborated in the results and discussion section.

RESULTS AND DISCUSSION:

Method development and optimization:

The HPLC methodology was optimized to determine suitable column and mobile phase for the chromatographic separation of MET and ERT. The details of the chromatographic runs were provided in Table 1. In trial 5, both MET and ERT peaks were resolved properly, their peak shapes were good and high theoretical plate count was noticed without any peak tailing. The RT of MET and ERT were obtained as 2.267 and 2.803 min, respectively. Thus, Ascentis 150 column (150mm x 4.6mm, 5µ) and 0.1% OPA:acetonitrile (60:40, v/v) mobile phase combination was considered to be optimum for the chromatographic separation and same was utilized though out the investigation. The optimized chromatogram obtained in trail 5 was depicted in Figure 2.

Table 1: Details of chromatographic conditions in various trials

Trial	Column	Mobile phase	Observation
1	Waters 150 (150 mm x 4.6 mm, 5 µ)	Methanol: Water (50:50 v/v)	Single peak (metformin) is eluted. Peak shape is not good.
2	Altima 150 column (150 mm x 4.6 mm, 3 µ)	Acetonitrile: Water  (50:50 v/v)	Both the peaks eluted, but peak shape is not good. USP plate count was less than 2000.
3	Altima C18 column (150 mm x 4.6 mm, 5 µ)	Acetonitrile: 0.1% OPA  (50 :50 v/v)	Peaks shape was broad.
4	Altima C18 column (150 mm x 4.6 mm, 5 µ)	Acetonitrile: 0.01 N potassium dihydrogen phosphate  (60:40 v/v)	Peaks are eluted, but metformin peak was eluted near void volume.
5	Ascentis 150 column (150 mm x 4.6 mm, 5 µ)	Acetonitrile: 0.1 %OPA  (40:60 v/v)	Good resolution, no tailing and high theoretical plate count was observed.

Table 2: System suitability parameters data for MET and ERT

Injection	Metformin^a			Ertugliflozin^b
	RT (min)	USP Plate  Count	USP Tailing	RT (min)	USP Plate Count	USP Tailing
Mean^c	2.267	2582.17	1.067	2.804	5608.83	1.36
SD	0.007	23.903	0.019	0.001	84.184	0.023
% RSD	0.302	0.926	1.843	0.037	1.5009	1.677

^aMET = 25 µg/mL, ^bERT = 0.375 µg/mL; ^cMean of six replications; RT: Retention time; SD: Standard deviation; %RSD: %Relative standard deviation; **Acceptance limit: %RSD less than or equals to 2.

Figure 2: Optimized chromatogram of the method

Method validation:

The variables of system suitability, such as retention time of the analytes, USP plate count and tailing factor were evaluated by injecting six replications of analyte solutions MET (25µg/mL) and ERT (0.375µg/mL) separately in to the system. The separation of analytes was achieved by maintaining optimum chromatographic conditions as described earlier. The outcomes of the study were provided in Table 2. Theoretical plate count should not be less than 2000, and %RSD of tailing factor should be less than 2 according to ICH guidelines. All the system suitable parameters were found to be satisfactory as they were within the specification limits.

Six serially diluted concentrations of MET (25-150 µg/mL) and ERT (0.375-2.25µg/mL) each in triplicate were injected and the responses recorded and the illustrations of calibration curves of both the analytes were provided in Figure 3. The linear regression equations were noted as y = 56567x + 7448.1 with R² 0.9995 for MET and y = 194332x-359.27 with R² = 0.9999 for ERT. Thus, both the analytes have shown good linear chromatographic response against measured concentration range.

Sensitiveness in the method was further ensured by detection and quantification limits. The LOD and LOQ of MET were computed 0.098 and 0.297μg/mL, respectively. The LOD and LOQ of ERT were noted as 0.001 and 0.004μg/mL, respectively.

(a)

(b)

Figure 3: Calibration curve of (a) MET (b) ERT

Recovery studies were performed to justify correctness in the optimized methodology. Results obtained upon measuring the responses of the analytes at three distinct levels of standard solutions after adding to pre-quantified samples were presented in Table 3. The mean % recoveries were observed as 99.26-100.72 and 99.87-100.46 for MET and ERT, respectively. The %RSD values for both the analytes at tested levels were noted less than 2, indicating agreeable accuracy of the HPLC method.

MET (100µg/mL) and ERT (1.5µg/mL) were loaded in to chromatographic system multiple times (n=6) to determine the system and method precision. The outcomes of the studies were displayed in Table 4. The preciseness of the method was identified through the results of the study, as the %RSD values were observed to be less than 2 for both the analytes.

Table 3: Accuracy data

Analyte	Level (%)	Amount added (μg/mL)	Amount recovered^a (μg/mL)	% Recovery	%RSD
MET	50	50	50.36±0.52	100.72	1.03
	100	100	99.27±1.24	99.26	1.24
	150	150	150.11±0.23	100.07	0.15
ERT	50	0.75	0.75±0.006	100.4	0.77
	100	1.5	1.507±0.007	100.46	0.38
	150	2.25	2.247±0.011	99.87	0.51

^aMean of three determinations calculated with respect to amount of analyte added; %RSD: Percentage relative standard deviation.

Table 4: Precision data of MET and ERT

Validation Parameter	Mean Peak area±SD		% R.S.D
	MET	ERT	MET	ERT
System Precision	5685080±57376.9	299478±4904.5	1.0	1.6
Intra-day precision (Repeatability)	5678544±32295.4	298581±2108.0	0.6	0.7
Inter-day precision (Intermediate precision)	5628279±34642	296914±2513.9	0.6	0.8

SD: Standard deviation; %RSD: Percentage relative standard deviation.

The chromatograms of blank, placebo and the analytes were recorded to assess the specificity. No interfering peaks were observed in blank and placebo at RT of MET and ERT. Hence, the method was considered to be specific. The outcomes of robustness studies were provided in Table 5. It was observed that slight modifications in the mobile phase flow rate (±0.1mL), composition of mobile phase (±5mL) and temperature of column (±5℃) didn’t affect the chromatographic separation of the analytes. The %RSD of each variable was found to be with in the acceptance criteria (<2) as per ICH protocols. This evidenced the strength and toughness of the methodology.

Table 5: Robustness data

Condition	%RSD
	MET	ERT
Flow rate (0.8 mL/min)	0.8	1.5
Flow rate (1.2 mL/min)	1.30	1.10
Mobile phase (35A:65B)	0.90	0.70
Mobile phase (45A: 55B)	1.10	0.80
Temperature (25 °C)	0.40	0.60
Temperature (35 °C)	0.80	0.60

A: Ortho-phosphoric acid (0.1% ); B: Acetonitrile.

Degradation Studies:

The sturdiness in the methodology was evaluated by subjecting its formulation to various stress conditions. The degraded samples of each test were loaded in to HPLC and the amount of drug degraded was displayed in Table 6. The %drug degraded was observed to be relatively more in alkali degradation for both MET  (8.38%) and ERT (7.78%), while in all other degradation tests it was minimal (< 5%).

Table 6: Degradation studies data

Degradation condition	%Drug degraded
	MET	ERT
Acid	3.67	4.73
Alkali	8.38	7.78
Oxidation	3.39	5.65
Thermal	3.39	3.65
UV	2.90	2.12

Assay

The optimized and validated HPLC methodology was implemented to measure MET and ERT in marketed formulation. The study outcomes were shown in Table 7. The mean %assay of three determinations each for MET and ERT were obtained as 99.75±0.3 and 99.60±0.70, respectively. The %RSD values of the same (0.57 and 0.71) were found to be in line with the standards of ICH protocols. This further approves the utilization of current methodology in the regular analysis of formulations containing MET and ERT.

The contemplated method conditions and the results of the investigation were compared with the literature HPLC methods^31-42. The investigated method was observed to be rapid, as the RT of both the analytes was relatively low. The sensitivity of the methodology was evidenced through its lower LOD and LOQ values and implementation of the method across lower linearity ranges for both the analytes, when compared to the earlier reports.

Table 7: Assay data of MET and ERT

Drug Name (Brand name: Segluromet)	Label claim (mg)	Std. Area (mean±SD) ^a	Sample Area (mean±SD)^a	% Assay	%RSD^b
Metformin	500	5685080±57376.9	5678544±32295.4	99.75±0.3	0.57
Ertugliflozin	7.5	299478±4904.5	2369784±126815	99.60±0.70	0.71

^aMean value of three determinations; ^bPercentage relative standard deviation

CONCLUSIONS:

The current RP-HPLC methodology established to quantify MET and ERT was proved as rapid, sensitive, and precise. Optimum chromatographic conditions needed for separation of analytes were identified as Ascentis 150 column (150mm x 4.6mm, 5µ) and OPA (0.1%):acetonitrile mobile phase (60:40, v/v) by optimization studies. The RT was noted as 2.267min for MET and 2.803min for ERT, when the measurement was done at 220 nm using PDA detector. The optimized method showed good linearity for MET and ERT over the concentration range of 25-100µg/mL and 0.375-1.50 µg/mL, respectively. The sensitivity of the methodology was further evidences in terms of lower LOD values 0.098 and 0.001µg/mL and LOQ values 0.297 and 0.004 µg/mL for MET and ERT, respectively. The deliverables of the validation parameters met with ICH acceptance criteria. The assay results were in accordance with that mentioned under ICH rules, when the methodology was adopted for marketed formulation. In connection with above results, we conclude that this method can be employed for the routine quality control analysis of MET and ERT.

ACKNOWLEDGEMENTS:

The authors are grateful to the Principal, Gokaraju Rangaraju College of Pharmacy and Gokaraju Rangaraju Educational Society for providing necessary infrastructure facilities.

CONFLICT OF INTEREST:

The author (s) confirms that this article content has no conflict of interest.

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Received on 15.09.2023      Revised on 09.10.2024

Accepted on 24.03.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3599-3605.

DOI: 10.52711/0974-360X.2025.00518

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