Introduction

Metformin, a biguanide hypoglycemic agent widely used for the treatment of non-insulin dependent type 2 diabetes mellitus for lowering the blood glucose levels. Several studies reported that metformin also has a potential role in anti-aging, antitumour, cardioprotective and neuroprotective effect (Wang et al., 2017). Metformin has various other benefits in patients with diabetes mellitus such as decrease in hyperinsulinemia, reduction of weight, improvement in lipid profiles, augmented fibrinolysis, and increased endothelial function (Nasri & Rafieian-Kopaei, 2014). The therapeutic effect of metformin will be achieved by, without an increase in insulin concentration and also reduce insulin resistance (Mahadik, Dahia, Mani, Gaikwad, & Gavali, 2012). Metformin targets multiple pathways, including antioxidation, angiogenesis, inflammation, autophagy and cellular survival (Wang et al., 2017). The major mechanism of action of metformin includes reduction of appetite, diminished carbohydrate absorption in the intestine, inhibition of gluconeogenesis in hepatic system and enhanced uptake of glucose by peripheral tissues. The efficacy of the drug is well documented in most of the studies in the form of monotherapy and combination therapy as highly safety, cost effective and an excellent choice in both specialized treatments and primary health care (Papanas & Maltezos, 2009).

Method validation is an analytical process essential for the confirmation of valid measurements used to assure the reliability, quality and consistency of results which is an important factor of good analytical practice. Several international organizations refer method validation guidelines such as European cooperation of Accreditation (EA), European Committee for Normalization (CEN), Codex Committee on Methods of Analysis and Sampling (CCMAS), Cooperation on International Traceability in Analytical Chemistry (CITAC), American Society for Testing and Material (ASTM), Unites States Food and Drug Administration (FDA), Food and Agricultural Organization (FAO) and International Conference on Harmonization (ICH). The four major types of method validation related analytical procedures were categorized as identification of suitable tests, control of impurity tests, qualitative tests and quantitative tests for the samples, drug substances, components of drugs and drug products. The validation also includes and necessary to assess the modifications in synthesis of drug substance and composition of drug products. The authors suggest that UV-Visible spectrophotometric method is suitable for validation of drug as per ICH Q2(R1) guideline (Behera, Ghanty, Ahmad, Santra, & Banerjee, 2012). Mahadik et al. described and developed suggested that UV spectroscopic method is simple, sensitive, specific, accurate, cost effective and precise method for bulk estimation of metformin in tablet formulation (Mahadik et al., 2012). The significance of the study was planned to assess the further stability of the drug beyond its expiration date and to instigate the manufactures to register for shelf-life extension of this drug from Central Drugs Standard Control Organisation (CDSCO). This study outcome might help to refine our prescription drug expiration process and to alleviate the economic burden of procuring the medicines frequently by the consumers. Based on the background our aim is to evaluate and compare the expired and extant metformin drug using UV-Visible spectrophotometry.

Materials and Methods

Drugs used for validation

Metformin Hydrochloride tablets - 500mg (Melmet-500) from micro labs was used in this study. The ten years expired metformin drug (EXP: June 2012, B.No. MEAD0033) and the extant metformin drug currently in usage (EXP: June 2022, B.No. MEAS0033) was utilized for comparative analysis and validation of study parameters. Analytical grade methanol and water were utilised for the experiments. Methanol and Water (15:85, v/v) used as diluent to dissolve and prepare the required concentration of the drug. 10mg drug was dissolved in 10ml of diluent to attain 1000 ppm. From this 10 microliter make up to 1000 microliter to get 10 ppm solution.

Method validation parameters

The method validation of ICH guidelines was followed for evaluation and comparison of expired and extant metformin drugs. Validation of drugs was done using UV-VIS Spectrophotometer (Hitachi – UH5300-3J1-0015). The drug validation parameters are selection of wavelength, specificity, linearity, precision, accuracy, solution stability, robustness, system suitability, limit of detection and limit of quantification.

Results and Discussion

Selection of analytical wavelength and absorption maxima

Drug solutions were measured in the wavelength range of 200-400 nm in UV double beam spectrophotometry. Instrument set at zero and the baseline correction was done (Figure 1). Then the diluent (Figure 2), expired drug (Figure 3) and extant drug (Figure 4) was measured at 200-400 nm to assess the exact absorption maxima. The zero order absorption spectra obtained was selected for the analysis of the drugs. The absorption maximum was found at 233 nm, which is used for further analysis.

Specificity

The resolution of the analyte peak was measured from the nearest peak obtained by repetitive analysis of the analyte using the same solution and settings.

The 10 ppm concentration of the drug was measured multiple times in the range of 200-400nm. We observed the peak falls in the same position at 233nm for both expired (Figure 5) and extant (Figure 6) metformin drug. This indicates that the resolution of the analyte peak is specific at 233nm for metformin.

Linearity

The absorbance was measured at 233nm with the concentration range from 1 – 50 ppm for both the expired and extant metformin drug (Table 1). Calibration curve was obtained and the response of the drug was found to be linear in the investigated concentrations for both the expired and extant metformin. The linear regression equation for expired drug was y=0.0561x+0.0315 with correlation coefficient 0.9989 and for extant drug was y=0.0567x+0.0588 with correlation coefficient 0.9976 (Figure 8; Figure 7).

Precision

The precision of the analytical method was carried out using 10 ppm concentration of the drug and repeated the measurement at 233nm six times intraday and interday for both the expired and extant metformin. The mean value, standard deviation and percentage of relative standard deviation were calculated. We observed the developed method was found to be precise as %RSD of repeatability and intermediate values for both the drug is zero (Table 2).

Accuracy

The accuracy of the proposed method was determined by adding different amounts of drug like 50%, 100% and 150% of metformin within linearity range and added to the pre-analysed constant concentration and percentage recovery was calculated. The accuracy was obtained for zero order spectroscopy was found to be present within the range (Table 4; Table 3).

Solution stability

The solution stability was measured at different time points. It was found that the drug solution prepared for analysis is stable at room temperature and the result was not decreased below the minimum measurable levels. This indicates the drug solution used for the method validation was stable during the experiment at room temperature (Table 5).

Robustness

The robustness evaluation of the drug should be considered for reliability of analysis with respect to the deliberate variations in the developed method. We observed only very subtle considerable difference in the mean values, whereas the standard deviation and %RSD was found to be similar as zero for both the tested drugs. The robustness of the drug solution during all variations were not affected much and it was measured the actual values (Table 6)

System suitability

The system suitability test of spectrophotometric system should be performed before each validation to assess the suitability of the instrument and the method. The analyte was measured at 233nm for six times and the %RSD was calculated which should not be more than 2.0% for acceptance of system suitability. We observed the %RSD for both the metformin drug is zero, indicating that the system suitability is satisfactory and full filled all the parameters of method validation (Table 7).

Limit of detection

The limit of detection of an analytical procedure indicates the lowest amount of an analyte in a sample that can be detected by the instrument and developed method. The detection limit was determined by preparing different concentration range from 1-50 ppm of drug of the absorbance was measured at 233nm in spectrophotometry. The lowest concentration absorbance of expired drug was 0.045 and extant drug was 0.015. We observed for both the drugs the lowest concentration about 1ppm can be detected and quantified as an exact value (Table 8).

Limit of quantification

The limit of quantification is the reliable concentration quantitated with specified accuracy and precision. The quantification limit calculated using a formula with a standard deviation of drug response and the slope of the calibration curve. Mostly the limit of detection is equal to the limit of quantification. We observed the accuracy and precision validation data was perfect for the proposed method. With these observations, the limit of quantification in both the drugs was found to be 1 ppm or 1.2 μg/ml in spectrophotometric method.

Conclusion

We designed our study with the concern of avoiding disposal of enormous expired drugs and to evaluate the shelf life of the drug to extend its usage. Our study results conclude that there is no major difference in the validation between 10 years expired and extant metformin drug. This indicates that the shelf life of the expired drugs is extended to usage even after 10 years specific for metformin drug. We also observed that UV-VIS spectrometry is simple, specific, sensitive, linear, accurate and precise technique for the method validation and evaluation of the drugs. Our results will be helpful for consideration of extension of the expiry date of metformin drug by the regulatory bodies. This will be highly beneficial for the society as frequency of purchase, economical, extensive usage and effective drug for long term users of metformin.

Acknowledgement

We gratefully acknowledge the support of pharmacology department for providing ten years expired metformin drug from their pharmaceutical museum and translational medicine department for the utilization of UV-Vis spectrophotometry.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that there is no conflict of interest for this study.