INTRODUCTION:

Metformin hydrochloride is chemically known as 1,1-Dimethylbiguanide hydrochloride. Metformin hydrochloride is a biguanide hypoglycemic agent used in the treatment of non-insulin-dependent diabetes mellitus not responding to dietary modification. Metformin improves glycemic control by improving insulin sensitivity and decreasing intestinal absorption of glucose. Metformin Hydrochloride is the hydrochloride salt of the biguanide metformin with antihyperglycemic and potential antineoplastic activities. The drug is official in USP¹. Literature survey reveals spectrophotometric along with HPLC^2,3and HPLC^4-16 for assay of drug. Such HPLC method can be used for the routine analysis. In the proposed methods optimization and validation of this method are reported.

EXPERIMENTAL:

Instrumentation:

The HPLC system Merck-Hitachi equipped with separation module and UV detector (L-7400) was used. The chromatogram was recorded and peaks are quantified by means EZ Chrom Elite software. A Shimadzu analytical balance with 0.01mg was used.

Materials and reagents:

Reference standard of metformin hydrochloride was obtained from reputed firms with certificate of analysis. Analytical grade of phosphoric acid and tri ethyl amine and HPLC grade of methanol were used from Merck and the HPLC grade water was obtained by using Millipore water system.

Procedures:

Standard stock solution:

About 10mg of standard metformin hydrochloride was weighed accurately and transferred in 10ml volumetric flask. About 5ml of diluent (buffer: methanol (90:10% v/v)) was added and sonicated for 5 minutes. The volume was adjusted to the mark with diluents to give concentration as 1000μg/ml. The working standard solution was prepared by diluting 1ml of 1000μg/ml solution to 10ml with diluent to get concentration 100 μg/ml.

Sample preparation:

Twenty tablets were weighed accurately and average weight of each tablet was determined. The powder equivalent to 10mg of metformin hydrochloride was weighed accurately and transferred in 10ml volumetric flask. About 5ml of diluent (buffer: methanol (90:10% v/v) was added and sonicated for 5 minutes. The volume was adjusted up to mark with diluent to give concentration as 1000μg/ml. The working sample solution was prepared by diluting 1ml of 1000μg/ml solution to 10ml with diluent to give 100μg/ml. A 10µl was injected for analysis.

Method Development:

Chromatographic condition:

Different columns containing octyl and octadecyl silane stationary phase were tried for separation and resolutions. It was found that Hypersil BDS C18 (150mm x 4.6mm x 5μm) column offered more advantage over other columns. The mobile phase was a mixture of buffer and methanol (90:10% v/v). The buffer was 1ml of tri-ethyl amine dissolved in 1000ml of HPLC grade water. The pH 3.5 was adjusted with dilute phosphoric acid. The flow rate of the mobile phase was adjusted to 1ml/min. The detection was carried out at wavelength 230nm. Fig 1.

Figure 1. Spectrum of metformin hydrochloride

The injection volume of the standard and sample solution was set at 10.0µl. The elution and resolution parameters of drug were recorded at the wavelength 260 nm and its response optimization was compared with adequate sensitivity. It produced well shaped peaks for the drug assay. A chromatogram of the drug assayed is depicted in fig. 2.

Figure.2: chromatogram of standard metformin hydrochloride

Method validation:

System suitability:

System performance parameters of developed HPLC method were determined by injecting standard solutions. Parameters such retention time, area, % area and asymmetry were shown in Table-1. It indicated good performance of the system.

Table – 1: System performance parameters. (n = 6).

Retention time	symmetry factor	Area	% Area
2.057 minutes	1.67	15285563	100.00

Linearity:

The linearity of the method was determined for metformin hydrochloride from six concentrations level ranging from 50 to150µg/ml. The calibration curve was constructed by plotting response factor against concentration of the drugs. The regression equation was given as y = 11267 x + 37546. The correlation coefficient (r²) was 0.9999 and concentration range indicated above. The results of the same are tabulated in the table 2.

Table 2: Linearity – regression analysis data

Parameters	Values
Correlation Coefficient (r)	0.9999
Intercept (y)	37546
Slope (m)	11267

Accuracy:

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out and percentage recovery was calculated and presented in Table 3.

Table 3: Accuracy - %Recovery

Level	Test	Weight in mg	Area	Quantity added in μg/ml	Quantity recovered in μg/ml	% Recovery	Mean recovery in percentage
Level	Test	Weight in mg	Area	Quantity added in μg/ml	Quantity recovered in μg/ml	% Recovery	Mean recovery in percentage
80%	1	10.18	12283600	81.68	82.29	100.75	100.53
	2	10.15	12257412	81.68	82.12	100.54
	3	10.28	12229033	81.68	81.93	100.30
100%	1	10.36	15279703	102.1	102.37	100.26	100.23
	2	10.29	15252965	102.1	102.19	100.08
	3	10.28	15292661	102.1	102.45	100.35
150%	1	10.21	22861111	153.15	153.16	100.00	99.90
	2	10.29	22829419	153.15	152.94	99.87
	3	10.24	22820300	153.15	152.88	99.83
					Mean recovery of all level		100.22

* Average of triplicate analysis

Precession:

The method precision was established by carrying out the analysis of metformin hydrochloride. The assay was carried out of the drug using analytical method in six replicates. The value of relative standard deviation lies well with the limits (0.10 %). The results of the same are tabulated in the table 4.

Table 4: Precision – method precision.

Test	Weight of test sample used in mg	Found Area	% Assay
Solution-1	10.21	15285563	99.62
Solution-2	10.25	15258115	99.83
Solution-3	10.26	15290294	100.13
Solution-4	10.23	15244371	99.54
Solution-5	10.27	15218515	99.76
Solution-6	10.31	15226148	100.20
	Mean Assay		99.85
	SD		0.269
	RSD		0.269

Stability of solution:

The stability studies of the solutions under study were established by keeping the solutions at room temperature for 24 hours. The results indicated no significant change in the assay results of the same solutions. It confirmed the stability of the drug in the solvents used for the analysis.

Robustness:

Robustness study of the method was performed by making slight changes in the chromatographic conditions.

In flow rate, variation ± 0.2˚C.

In wavelength, variation ± 5 units

In variation mobile phase composition ± 0.2 unit

The chromatograms demonstrated have no marked changes that developed HPLC method was robust.

Method Application:

The validated high performance liquid chromatographic method was applied for determination of metformin hydrochloride its formulation. Twenty tablets of metformin hydrochloride were used. A portion equivalent to 10mg of metformin hydrochloride was weighed accurately. It was dissolved in 10ml of diluent to obtain final concentration 1000µg/ml. The working sample solution was prepared by diluting 10ml of 1000 μg/ml solution to 100ml with diluent to give 100μg/ml.

10µl of this solution was injected under specified conditions. The analyte peaks were identified by comparison with respective standard and sample chromatogram were recorded. (Fig.3).

Fig 3: chromatogram of metformin hydrochloride (sample)

The assay results expressed as mg/tablets are shown in Table-3. It indicated the amount of each drug in the product meet the requirement.

RESULTS:

In the proposed method, the retention time of metformin hydrochloride was 2.057 min. The linearity was in the range of 50-150μg/ml. The regression equation of the linearity was given as Y= 11267x - 37546 where X is concentration of metformin hydrochloride in μg/ml. and Y is corresponding peak area. The coefficient of co-relation was 0.9999. The result shows that an excellent correlation between peak area and concentration of metformin hydrochloride in the range indicated. The relative standard deviation for method precision was 0.297 (limit % RSD < 2.0%). The mean recovery of the metformin hydrochloride was 100.22%. The high percentage recovery indicates that the proposed method is highly accurate.

The use of 0.1% tri ethyl amine (pH 3.5) and methanol (90:10% (v/v) gave peak with good resolution. The robustness studies indicated that there was no effect other parameters on the drug study. No interfering peaks were found in the chromatogram of the formulation within the run time indicted that excipents used in the formulation did not interfere the estimation of drug.

DISCUSSION:

The reproducibility, repeatability and accuracy of the proposed method were found to be satisfactory which is evidenced by low values of standard deviation 0.269 and percent relative standard deviation 0.269.(Table no.4) The accuracy and reproducibility of the proposed method was confirmed by recovery experiments, performed by adding known amount of the drug to the pre-analyzed active pharmaceutical ingredient and reanalyzing the mixture by proposed method. (Table no.3) The percent recovery obtained indicates non- interference from the excipients used in the formulations. The methods reported in literature method indicate large retention time. Hence more time will be required for validation of drug and its formulation as well it requires more amount of organic solvent.

This can be successfully used for validation of drug as well as for determining stability of drug in various conditions as per ICH guidelines.

Thus the proposed RP-HPLC method is used for validation of metformin hydrochloride from active pharmaceutical ingredient and marketed formulation due to its simplicity and non interference of other peaks and relatively short retention time as 2.057 min. It is more precise, accurate, linear, robust, simple and rapid method. Hence the proposed RP-HPLC method is strongly recommended for the quality control of the raw material, active pharmaceutical ingredient and pharmaceutical formulation per ICH guidelines.

ACKNOWLEDGMENT:

Authors express sincere thanks to the principal of D.G. Ruparel College, Mumbai for encouragement and providing laboratory facilities.

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Received on 15.02.2021 Modified on 16.03.2021

Asian Journal of Research in Chemistry. 2021; 14(4):265-268.

DOI: 10.52711/0974-4150.2021.00045