INTRODUCTION:

Metformin is a biguanide antihyperglycemic agent and first-line pharmacotherapy used in the management of type II diabetes. Metformin was first approved in Canada in 1972, and received subsequent FDA approval in the US in 1995.^1-4

Metformin hydrochloride is chemically N, N-Dimethylimido dicarbonimidic diamide (fig. 1), with a mode of action and uses similar to other biguanides.⁵

Evogliptin tartrate is a selective DPP-IV (dipeptidyl peptidase-4) inhibitor and it is prescribed in diabetes mellitus. It is also used as anti-atherosclerosis drug when it targets arterial inflammation. It is a β-amino amide derivative.⁶It is chemically (3R)-4-[(3R)-3-amino-4-(2, 4, 5-trifluorophenyl) butanoyl]-3-[(2-methylpropan-2-yl) oxymethyl] piperazin-2-one; (2R, 3R)-2, 3-dihydroxybutanedioic acid, (fig. 1).⁷

(A) (B)

Figure 1: Chemical structure of Metformin hydrochloride (A) and Evogliptin tartrate (B)

The literature review shows that the official methods for estimation by liquid chromatography method for Metformin hydrochloride in Indian Pharmacopoeia 2018.⁸It also reveals many reported UV, HPLC, and HPTLC methods have been reported for Metformin hydrochloride alone and in combination with other drugs.^9-21 For Evogliptin tartrate, UV and RP-HPLC method is reported for its estimation.^22-25

However, no method has been reported for the simultaneous determination of Metformin hydrochloride and Evogliptin tartrate in combination. UV spectrophotometer methods are favored over other analytical techniques due to their wide range of applications, ease of use, robustness, and simplicity. Furthermore, analysis can be carried out in areas outside of the main laboratory when needed using portable UV spectrophotometers. As a result, the development of such a method might be utilized to estimate the combined dosage form of both medications. The proposed method is fast, simple, environment-friendly, precise, and reproductive and may be used in tablet dosage for routine analysis of these two drugs simultaneously. The method was developed in distilled water, making the method cheap and environment-friendly. Following International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, the proposed approach is optimized and validated. There has been a successful attempt in simultaneously estimating both of these drugs utilizing the UV spectrophotometer simultaneous equation approach in this work.^{27, 28}

MATERIALS AND METHODS:

Instruments and apparatus:

UV- visible spectrophotometer, double beam, 1800 (Shimadzu); Analytical balance, model AUX 220 (Shimadzu); Ultra sonicator (Fast clean ultrasonic cleaner) Volumetric flasks– 10mL, 50mL and 100mL; Pipettes – Micropipette of 1mL and 10mL; Beakers – 250mL, 500mL.

All instruments and glass wares were calibrated.

Reagents and materials:

Metformin hydrochloride (Gifted by, Shiva Health care, Mehsana, India); Evogliptin tartrate (Gifted by, Alkem Laboratories Ltd., Mumbai, India); Marketed formulation (Valera M 500, Alkem Laboratories Ltd., Mumbai, India); Whatman filter paper no. 41.

Spectrophotometric conditions:

Mode: Spectrum; Scan speed: Medium; Wavelength range: 400- 200nm; Absorbance scale: 0.00°A – 2.00°A; Initial base line correction: Distilled water (H₂O)

Preparation of stock solution:

Stock solution of Metformin hydrochloride:

Accurately weighed quantity of 10mg of Metformin hydrochloride was transferred into volumetric flask of 100ml, then dissolved and diluted up to the mark with distilled water to give a stock solution having strength 100μg/ml.

Stock solution of Evogliptin tartrate:

Accurately weighed quantity of 50mg of Evogliptin tartrate was transferred into volumetric flask of 50ml, then dissolved and diluted up to the mark with distilled water to give a stock solution having strength 1000 μg/ml.

Working solution of Metformin hydrochloride:

Accurately weighed 1mL stock solution of Metformin hydrochloride were transferred to 10mL volumetric flasks, dissolved in and diluted to the mark with distilled water to obtain standard solution of 10mg/mL.

Working solution of Evogliptin tartrate:

Accurately weighed 1mL stock solution of Evogliptin tartrate were transferred to 10mL volumetric flasks, dissolved in and diluted to the mark with distilled water to obtain standard solution of 100mg/mL.

Preparation of sample solutions:

Twenty tablets were weighed and powdered. Powder equivalent to 50mg of Metformin HCl and 0.5mg of Evogliptin tartrate (standard addition of EGT) was weighed and transferred into a 100mL of volumetric flask. Transferred the 30mL of distilled water into the volumetric flask and sonicated for 10minutes, then dissolved and diluted up to mark with distilled water. The solution was filtered using Whatman filter paper no. 41 and first few drops of filtrate were discarded. Transfer 0.2mL of this solution into a 10ml volumetric flask and diluted to mark with distilled water.

Determination of wavelength:

Determination of wavelength of Metformin hydrochloride:

By appropriate dilution of distilled water with 1.0mL of working standard solution of Metformin hydrochloride (100μg/mL) was pipette out in 10mL volumetric flask to have a concentration of 10μg/mL. Prepared solutions were scanned in the range of 200- 400nm on shimadzu double beam UV visible spectrophotometer using distilled water as a blank to determine the wavelength of maximum absorption for Metformin hydrochloride was at 233nm.

Determination of wavelength of Evogliptin tartrate:

By appropriate dilution of distilled water with 1.0mL of working standard solution of Evogliptin tartrate (1000 μg/mL) was pipette out in 10mL volumetric flask to have a concentration of 100μg/mL. Prepared solutions were scanned in the range of 200- 400nm on shimadzu double beam UV visible spectrophotometer using distilled water as a blank to determine the wavelength of maximum absorption for Evogliptin tartrate was at 267nm.

Method validation:

Linearity:

Linearity is expressed in terms of correlation co-efficient of linear regression analysis.

Linearity of Metformin hydrochloride:

Stock standard solutions of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of Metformin HCl were transferred in a separate series of 10mL volumetric flasks and diluted to the mark with distilled water. The absorbance of Metformin HCl was recorded at λ1. Calibration curves were constructed by plotting absorbance versus concentrations.

Linearity of Evogliptin tartrate:

Stock standard solutions of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL Evogliptin tartrate were transferred in a separate series of 10mL volumetric flasks and diluted to the mark with distilled water. The absorbance of Evogliptin tartrate was recorded at λ2. Calibration curves were constructed by plotting absorbance versus concentrations.

Accuracy:

Accuracy of the method was carried out by applying the standard addition method in which drug sample (Metformin HCl and Evogliptin tartrate combination tablet) spiked with known amount of Metformin HCl and Evogliptin tartrate standard corresponding to 80, 100 and 120% of drug sample, mixed and the powder was extracted and analyzed. The amount of Metformin HCl and Evogliptin tartrate were determined by applying obtained values to the regression equation of the calibration curve.

Precision:

Precision of Metformin hydrochloride:

The repeatability was checked by repeatedly injecting 4 μg/mL of Metformin hydrochloride solutions and recording the responses.

a. Intra- day precision:

The intra- day precisions of the proposed method were determined by measuring the corresponding responses 3 times on the same day for 3 different concentration of Metformin hydrochloride (i.e., 4, 6 and 8μg/mL).

b. Inter- day precision:

The inter- day precisions of the proposed method were determined by measuring the responses on 3 different days for 3 different concentration of Metformin hydrochloride (i.e., 4, 6 and 8μg/mL).

Precision of Evogliptin tartrate:

The repeatability was checked by repeatedly injecting 40 μg/mL of Evogliptin tartrate solutions and recording the responses.

a. Intra- day precision:

The intra- day precisions of the proposed method were determined by measuring the corresponding responses 3 times on the same day for 3 different concentrations of Evogliptin tartrate (i.e., 40, 60 and 80μg/mL).

b. Inter- day precision:

The inter- day precisions of the proposed method were determined by measuring the responses on 3 different days for 3 different concentrations of Evogliptin tartrate (i.e., 40, 60 and 80μg/mL).

Robustness:

Robustness of Metformin hydrochloride:

The robustness was studied by analyzing the samples of Metformin hydrochloride by deliberate but slight variation in the method parameters. The change in the response of Metformin hydrochloride was noted. Robustness of the method was studied by changing the wavelength by ±2nm of concentration 4 μg/mL. The changes in the response of Metformin hydrochloride were noted and compared with the original one.

Robustness of Evogliptin tartrate:

The robustness was studied by analyzing the samples of Evogliptin tartrate by deliberate but slight variation in the method parameters. The change in the response of Evogliptin tartrate was noted. Robustness of the method was studied by changing the wavelength by ±2 of concentration 40μg/mL. The changes in the response of Evogliptin tartrate were noted and compared with the original one.

Limit of Detection and Limit of Quantification:

LOD and LOQ of the drug were calculated using the following equations designated by International Conference on Harmonization (ICH) guideline: LOD= 3.3 × σ/ S and LOQ= 10 × σ/ S Where, σ = Standard deviation of the response, S = Slope of calibration curve.

Assay of the pharmaceutical formulation:

Probability of the proposed method was tested by analyzing the commercially available tablet dosage form Valera M tablet (500mg). Appropriate three different eliquots from sample solution were dilute with distilled water. The amount of Evogliptin tartrate and Metformin hydrochloride present in the sample was determined by the simultaneous equation.

The total amount of Metformin HCl and Evogliptin tartrate can be determined as:

C_x= A₂ ay₁– A₁ ay₂/ax₂ ay₁ – ax₁ ay₂

C_y = A₁ ax₂ – A₂ ax₁/ax₂ ay₁ – ax₁ ay₂

Where, C_x = concentration of x sample

C_y = concentration of y sample

ax₁ and ax₂ = absorptivities of the x compound

ay₁and ay₂ = absorptivities of the y compound

A₁and A₂ = absorbance of dilute sample

RESULTS AND DISCUSSION:

Selection of wavelength:

Standard working solutions were prepared 02 – 12 μg/mL Metformin HCl and 20 – 120μg/mL Evogliptin tartrate in distilled water by appropriate dilution. The spectra have been recorded in the 200 – 400 range and find iso- absorptive point at 250nm (fig. 2).

Figure 2: Overlay spectra of Metformin HCl and Evogliptin tartrate

Selection of solvents:

The selection for a common solvent was made after assessing the solubility of both the drugs. The selected solvent is distilled water as it shows special characteristics of the drug.

Validation of proposed method:

The proposed method was validated for the simultaneous determination of Metformin hydrochloride and Evogliptin tartrate using following parameters.

Figure 3: Calibration curve of Metformin hydrochloride at 233 nm

Figure 4: Calibration curve of Evogliptin tartrate at 267 nm

Linearity:

Linear correlation was obtained between absorbance vs. concentrations of Metformin hydrochloride in concentration range 02-12µg/mL and Evogliptin tartrate in concentration range 20-120µg/mL respectively. The percentage RSD for 233nm and 267nm was found to be in the range of 0.12–0.99 and 1.09–1.88 for MFH and 0.26–1.86 and 0.61-1.53 for EGT respectively for proposed method as shown in table 3 and 4. Regression parameters are mentioned in table 1 of both drugs. The calibration curve of the MFH at 233nm and 267nm are shown in fig.3. The calibration curve of the EGT at 233 nm and 267nm are shown in fig. 4.

Accuracy:

The recovery experiment was performed by the standard addition method.

Accuracy of Metformin hydrochloride:

The mean recoveries were in the range of 98.02%- 100.74% for Metformin HCl. The low value of standard deviation indicates that the proposed method is accurate. Results of recovery studies are shown in table 1.

Accuracy of Evogliptin tartrate:

The mean recoveries were in the range of 99.09%- 101.5% for Evogliptin tartrate. The low value of standard deviation indicates that the proposed method is accurate. Results of recovery studies are shown in table 1.

Table 1: Validation parameters of developed method

Parameters	Metformin hydrochloride	Evogliptin tartrate
Parameters	At 233 nm	At 267 nm
Concentration Range (µg/ mL)	2 – 12	20 – 120
Slope	0.104	0.004
Intercept	-0.005	0.017
Correlation co-efficient (R²)	0.999	0.999
Linearity (% RSD, n=6)	0.12 – 0.99	0.61 – 1.53
Precision (% RSD)
Intraday Precision (% RSD)	0.73-0.75	0.57-0.83
Interday Precision (% RSD)	0.35-1.18	0.52-0.83
Accuracy (% Recovery)	98.02-100.74	99.09-101.5
Robustness	Robust	Robust
Limit of Detection	0.127 μg/mL	0.386 μg/mL
Limit of Quantification	2.393 μg/mL	7.253 μg/mL

Method precision:

Repeatability:

The % RSD values for Metformin HCl and Evogliptin tartrate were found to be 1.37 and 1.53 respectively, which indicates the method is precise.

Intra- day and inter- day of Metformin hydrochloride:

The %RSD for intra-day precision was found to be in the range of 0.73 – 0.75 while inter-day precision was found to be in the range of 0.57 – 0.83 (table 1), which indicates the method, is precise.

Intra- day and inter- day of Evogliptin tartrate:

The % RSD for intra-day precision was found to be in the range of 0.35 – 1.18 while inter-day precision was found to be in the range of 0.52 – 1.81 (table 1), which indicates the method, is precise.

Limit of detection and limit of quantification:

LOD and LOQ of Metformin hydrochloride:

The limit of detection (LOD) was found to be 0.127 µg/mL; while the limit of quantification (LOQ) was found to be 0.386µg/mL for Metformin HCl.

LOD and LOQ of Evogliptin tartrate:

The limit of detection (LOD) was found to be 2.393 µg/mL; while the limit of quantification (LOQ) was found to be 7.253µg/mL for Evogliptin tartrate.

Robustness:

Robustness of Metformin hydrochloride:

The robustness % RSD was found to be in the range of 0.52 – 0.99 for Metformin HCl, which indicates the method is robust.

Robustness of Evogliptin tartrate:

The robustness was found to be in the range of 1.06 – 1.18 for Evogliptin tartrate, which indicates the method is robust.

Assay of the pharmaceutical formulation:

The proposed validated method was successfully applied to determine Metformin HCl and Evogliptin tartrate in their combined dosage form. The results obtained for Metformin HCl and Evogliptin tartrate are comparable with the corresponding labeled amounts as shown in table 2.

Table 2: Analysis of marketed formulation by the proposed method

Sr. No.

Drug

Amount taken (mg)

Amount found (mg)

% Assay± SD (n= 3)

% RSD

Metformin hydrochloride

500

499.9

99.98

± 1.160

1.16

Evogliptin tartrate

4.7

94 ± 1.527

1.63

CONCLUSION:

A simultaneous UV Spectrophotometric method has been developed and validated for the estimation of Metformin hydrochloride and Evogliptin tartrate in pharmaceutical dosage form. The proposed method has been found to be simple, accurate, and precise. Hence the developed method can be used successfully for the routine analysis of Metformin hydrochloride and Evogliptin tartrate in pharmaceutical dosage form.

ABBREVIATIONS:

UV- Ultraviolet, ICH- International conference on Harmonization, LOD- Limit of Detection, LOQ- Limit of Quantitation, M- Meter, mm- Millimeter, μm- Micron, μL- Micro liter, mL- Milliliter, μg- Microgram, MFH- Metformin hydrochloride, EGT- Evogliptin tartrate

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Received on 16.08.2024 Revised on 17.11.2024

Accepted on 18.01.2025 Published on 28.02.2025

Available online from March 04, 2025

Asian Journal of Pharmaceutical Analysis. 2025;15(1):45-50.

DOI: 10.52711/2231-5675.2025.00008

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