Development and Validation of a UV Spectrophotometric Method for Stability Evaluation of Metformin

Stability-indicating methods are quantitative analytical techniques that utilize the unique structural, chemical, or biological properties of each active ingredient in a drug product. These methods enable the differentiation between the active ingredient and its degradation products, allowing for accurate measurement of the active content [1]. 

The International Council for Harmonisation (ICH) guideline on “Stability Testing of New Drug Substances and Products” recommends conducting stress testing to reveal the inherent stability characteristics of the active substance [2]. This stress testing should encompass various conditions, including acidic, alkaline, oxidative, thermal, and photolytic environments. An ideal stability-indicating method quantifies the intact drug while effectively separating it from its degradation products [3]. 

To establish the stability of the active pharmaceutical ingredient (API), it is subjected to forced degradation under several conditions: acidic, basic, oxidative, thermal, and photolytic stress. This forced degradation should occur early in the development process to ensure that the method can reliably differentiate between the API and its degradation products. While not every stress condition will necessarily lead to degradation, it is crucial to evaluate each one to assess its potential effects [5,6]. 

Metformin is a biguanide oral hypoglycemic agent primarily used for treating type 2 diabetes mellitus (T2DM). Evidence indicates that, in addition to improving glycemic control, metformin may be associated with reduced cardiovascular mortality and a lower risk of certain cancers. Notably, metformin has not been linked to significant elevations in serum enzymes during therapy and is rarely a cause of idiosyncratic liver injury [7].

Fig. 1 Structure of Metformin

MATERIALS AND METHODS

Chemicals and Reagents

All reagents and solvents employed in this study were of analytical grade. Metformin hydrochloride (bulk drug) and a commercial tablet formulation containing 500 mg of metformin were obtained from a local pharmacy. Methanol and distilled water were used throughout the UV analysis.

Instruments

Analytical work was carried out using a UV–visible spectrophotometer (Shimadzu Model 1700) equipped with 1 cm quartz cells. An analytical balance (Sigma 200/A Super) was used for weighing all materials with high precision.

Preparation of Standard Solution

A standard stock solution of metformin was prepared by dissolving 10 mg of the bulk drug in 100 ml of a methanol:water mixture (9:1 v/v), yielding a concentration of 100 µg/ml. The solution was sonicated until complete dissolution and stored in amber glass containers to prevent photodegradation [8].

Determination of Wavelength of Maximum Absorbance (λmax)

A 10 µg/ml working standard solution was scanned from 200 to 400 nm against the solvent blank. Metformin showed a well-defined absorption maximum (λmax) at 232 nm, which was used for all subsequent measurements [9].

Preparation of Calibration Curve

Aliquots of the standard stock solution were diluted with methanol:water (9:1 v/v) to obtain working standards in the concentration range of 8–18 µg/ml. Each solution was analyzed at 232 nm against the blank.

The calibration curve was constructed by plotting absorbance (A) versus concentration (C, µg/ml) and showed good linearity with the regression equation

A = 0.036C + 0.001 (R² = 0.999).

where A is absorbance and C is concentration (µg/ml). The correlation coefficient (R²) was found to be 0.999, indicating excellent linearity within the validated Beer–Lambert’s law range [10].

Analysis of Commercial Tablet Formulation

Twenty tablets were weighed, finely powdered, and a quantity equivalent to 10 mg of metformin was transferred to a 100 ml volumetric flask. The powder was dissolved in methanol: water (9:1 v/v), sonicated for 15 minutes, and filtered through Whatman filter paper No. 41. An aliquot of the filtrate was diluted to yield a final concentration within 8–18 µg/ml. Absorbance was measured at 232 nm, and the drug content was determined using the calibration curve [11].

Forced Degradation Studies

Forced degradation studies were performed to evaluate the stability of metformin under different stress conditions, following ICH Q1A(R2) recommendations [12]. For all conditions, 10 mg of metformin was weighed accurately and exposed to the following environments:

• Acidic degradation: 10 mg metformin in 10 ml of 0.1 N HCl, refluxed at 60°C for 5 hours. After completion, the sample was neutralized with 0.1 N NaOH.
• Alkaline degradation: 10 mg metformin in 10 ml of 0.1 N NaOH, refluxed at 60°C for 5 hours, followed by neutralization with 0.1 N HCl.
• Neutral degradation: 10 mg metformin dissolved in distilled water, refluxed at 60°C for 5 hours.
• Oxidative degradation: 10 mg metformin treated with 3% hydrogen peroxide solution and kept under ambient light for 12 hours.
• Thermal degradation: Bulk drug placed in a hot air oven at 60°C for 12 hours.
• Photolytic degradation: Bulk drug exposed to direct sunlight for three consecutive days.

After each stress treatment, samples were appropriately diluted with methanol:water (9:1) to yield a concentration within 8–18 µg/ml. The absorbance of each sample was measured at 232 nm, and the percentage of degradation was calculated using the initial absorbance of the unstressed sample as the reference.

RESULTS AND DISCUSSION

Linearity and Calibration Curve

The developed method exhibited excellent linearity within the validated concentration range of 8–18 µg/ml. The absorbance values for each concentration were measured at 232 nm and are presented in Table 1. The calibration plot of absorbance versus concentration showed a straight line with a regression equation:

A = 0.036C + 0.001A = 0.036C + 0.001

where AA is absorbance and CC is concentration (µg/ml). The correlation coefficient (R2 = 0.999R2 = 0.999) indicates strong linearity, confirming adherence to Beer–Lambert’s law.

Table 1. Linearity of Metformin

The absorbance values were below 1.0, ensuring reliable quantitative analysis.

Figure. 2 Linearity of Metformin

Optical characteristics (Table 2) demonstrate the precision and sensitivity of the developed method.

Table 2. Optical Characteristics of Metformin

Low LOD and LOQ values confirm that the method is sufficiently sensitive for quantification of metformin in pharmaceutical dosage forms.

Analysis of Commercial Formulation

The developed method was successfully applied for the assay of a marketed metformin tablet (500 mg). The sample solution (diluted to 10 µg/ml) showed an absorbance corresponding to 99.2% of the labeled claim, demonstrating the method’s applicability and accuracy. No interference from excipients was observed, confirming its selectivity.

Forced Degradation Studies

Forced degradation testing was carried out to evaluate the stability behavior of metformin under a variety of stress conditions, including acid, base, oxidation, thermal, photolytic, and neutral hydrolysis. The percentage of degraded and remaining active drug were calculated by comparing stressed samples with the unstressed reference solution.

Table 3. Summary of Stress Degradation Results

Figure. 3 Stress Degradation Results