INTRODUCTION:

Metformin hydrochloride is a biguanide hypoglycemic agent and non-insulin-dependent diabetes mellitus. Chemically metformin hydrochloride is a 1,1-Dimethyl biguanide hydrochloride the drug is official in USP¹. Literature survey reveals spectrophotometric along with HPLC^2,3 and HPLC ^4,16 for assay of drug. This proposed work presents simple, accurate and reproducible UV spectroscopy, area under curve method for determination of metformin hydrochloride in tablet dosage form.

MATERIALS AND METHOD:

Instrument and reagents:

Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5 nm with automatic wavelength corrections by using a pair of 10 mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software.

Reference standard of metformin hydrochloride was obtained from reputed firm with certificate of analysis.

Preparation of standard drug solutions:

100 mg standard metformin hydrochloride was weighed accurately and transferred to a 100 ml volumetric flask and sonicated with 30 ml of distilled water for 15 minutes. The volume was made up to the mark with distilled water to give a stock solution of metformin hydrochloride of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with distilled water to give a working standard solution of concentration 100 μg/ml.

Estimation from tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of metformin hydrochloride was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100 μg /ml. Such solution was used for analysis.

Experimental:

Method:

Area under curve (AUC) method

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ₁ and λ₂. The area under curve between λ₁ and λ₂were calculated by UV probe 2.42 software. In this method, 10 μg/ml solution of metformin hydrochloride was scanned in the spectrum mode from 200 nm to 350 nm. From zero order spectrum, the AUC calculation was done. The AUC spectrum was measured between 228 nm to 236 nm (Fig. 1)

Fig. 1. Area under curve spectrum of metformin hydrochloride (10 μg/ml) showing area from 228 nm to 236 nm.

Into series of 10 ml graduated flask, varying amount of standard solutions of metformin hydrochloride was pipette out and volume was adjusted with distilled water. Solutions were scanned between 350 nm to 200 nm in spectrum mode. The AUC calculations were done and the calibration curve for metformin hydrochloride was plotted in the concentration range of 1 to 14 μg/ml (Fig. 2).

Fig. 2. Calibration curve for metformin hydrochloride by area under curve spectroscopy

Results of analysis are given in table 1.

Table 1: Values of results of optical and regression of drug

Parameter	Area under curve (AUC) method
Detection Wavelength (nm)	228-236
Beer Law Limits (µg/ml)	1-14
Correlation coefficient(r²)	0.9998
Regression equation (y=b+ac)
Slope (a)	0.0249
Intercept (b)	0.003

Validation:

Accuracy:

Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2).

Precision:

The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in six replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 3.

Table 3: Precision- method precision

Experiment No.	Weight of metformin hydrochloride taken in mg	Weight of metformin hydrochloride found in mg
1	10	9.959677
2	10	10.04032
3	10	10.04032
4	10	10.04032
5	10	9.959677
6	10	10.000
	Standard deviation	0.03628
	%RSD	0.362588

Inter-day and intra-day precision:

An accurately weighed quantity of tablets powder equivalent to 10 mg of metformin hydrochloride was transferred to 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100 μg /ml. Such solution was used for analysis.

Solution was scanned between 400 nm to 200 nm in spectrum mode. The area under curve of resulting solutions was measured at between 228 nm to 236 nm by using as blank as distilled water. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 228 nm to 236 nm. Similarly area under curve of the same solution was read on 1^st, 2^nd and 5^th day. The amount of metformin hydrochloride was estimated by comparison with standard at 228 nm to 236 nm, table 4.

Table 4: Summary of validation parameter for intra-day and inter-day

Sr. No.	Parameters	Area under curve (AUC) method
(A)	Intra-day precision (n=3) Amount found ± % RSD	99.31% 0.1286
(B)	Inter-day precision (n=3) Amount found ± % RSD	99.1179% 0.653
(c)	Ruggedness Analyst to analyst (n= 3) %RSD	0.239

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-

LOD = 3.3 σ/S and LOQ = 10 σ/S

Where σ is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.

The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability. The values of LOD and LOQ are given in table 5.

Table 5: Values of results of LOD and LOQ

Parameters	Values
Limit of Detection (μg/ml)	0.365818
Limit of Quantification (μg/ml)	1.108539

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of metformin hydrochloride sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of metformin hydrochloride was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.

RESULT AND DISCUSSION:

The area under curve UV-spectroscopic method is useful for routine analysis of metformin hydrochloride in bulk drug and formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. Metformin hydrochloride has the absorbance maxima in the areas were measured between 228 nm to 236 nm. The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1 to 14 μg/ml and given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation. The most striking features of method is its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods. From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of metformin hydrochloride in pharmaceutical formulation.

ACKNOWLEDGMENT:

Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel College.

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Received on 24.05.2019 Modified on 23.06.2019

Asian J. Research Chem. 2019; 12(3):187-190.

DOI: 10.5958/0974-4150.2019.00035.X

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	1.997	99.85	0.0042	5.584
2	2	3.976	99.42	0.1115	2.019
2	4	6.077	101.29	0.0803	1.005
2	6	8.056	100.052	0.0610	0.7714
			Mean =100.153	Mean =0.0643	Mean =2.345