Metformin HCL is an Antidiabetic drug. It is used to reduce the blood glucose level. A drug used to treat diabetes mellitus. Metformin hydrochloride decreases the amount of glucose (a type of sugar) released into the bloodstream. It is a type of antidiabetic agent. Another well-known benefit of this drug is modest weight loss. Metformin is the drug of choice for obese type II diabetes patients^2-5. Chemical Formula:C₄H₁₁N₅, Molecular weight: 129.1636 g/mol. IUPAC Name: 1-carbamimidamido-N, N-dimethylmethanimidam. Structure of Metformin Hydrochloride is given in figure 1.

Voglibose is an alpha-glucosidase inhibitor used for lowering post-prandial blood glucose levels in people with diabetes mellitus. Voglibose is a synthetic compound with potent and enduring therapeutic efficacies against disorders of sensory, motor and autonomic nerve systems due to diabetes mellitus^1,22.

It is made in India by Ranbaxy Labs and sold under the trade name Volix. Chemical Formula: C₁₀H₂₁NO₇, Molecular weight: 267.2762g/mol.

Structure of Voglibose is given in figure 2.

Figure 1

Figure 2

MATERIALS AND METHODS:

Chemicals pharmaceutically pure sample of Metformin HCL was obtained from Wanbury Ltd. Vashi Navi Mumbai India and Voglibose was obtained from Nanoceut Therapeutics Pvt. Ltd. Puducherry Strides Shasun Ltd. (Puducherry) as gift samples. The commercial tablet Metvoglithinq (Metformin HCL 500 mg and Voglibose 0.2mg) was procured from the local drug market. All the chemicals and reagents were of analytical grade.

Instrument:

Double beam UV-visible spectrophotometer model Jasco V-530 using spectra manger software. The spectra were recorded over range 200-400nm against solvent in 1 cm quarts cells.

Selection of Solvents:

On the basis of solubility study methanol was selected as the solvent for dissolving Metformin Hydrochloride and Voglibose.

Standard solution preparations:

Accurately weighed 10mg of Metformin Hydrochloride and Voglibose were transferred into volumetric flasks separately and then volume was made up to 10ml with methanol to get a concentration of 1000µg/ml for all two drugs. Standard stock solution (1000µg/ml) was further diluted with methanol to obtain 8-16µg/ml and 4-24 µg/ml for Metformin HCL and Voglibose.

Figure 3 A: UV-Spectra of Metformin HCL

Figure 3 B: UV-Spectra of Voglibose.

Figure 3 C : Overlain spectra of Metformin HCL and Voglibose.

Study of spectra and selection of wavelength:

From the overlain spectrum of Metformin HCL and Voglibose, two wavelengths were selected one at 248 nm (λmax of Metformin HCL) and other at 276nm (Isoabsorptive point). The method employed Q -values, and the concentrations of drugs in sample solutions were determined using the following equations^8,13,15:

Qₘ – Qᵧ A₁

C₁ = ______ × ___……….………………equation 1.

Qₓ –Qᵧ ax₁

Qₘ – Qₓ A₂

C₂ = ______ × ___.............………………equation 2.

Qᵧ –Qₓ ay₁

Where, A₁ and A₂ are the absorbances of mixture at 248 and 276nm, Qₘ = A₂ / A₁, Qᵧ = ay₂ / ay₁ and Q x = ax₂ /ax₁, ax₁ (0.1471), ax₂ (1.1582), ay₁ (0.7102) and ay₂ (2.4281) are absorptivities (1%, 1cm) of Metformin HCL and Voglibose at 248 and 276nm (Figure 3 A, 3 B and 3 C).

Preparation for analysis of tablet formulation:

Twenty tablets were accurately weighed and crushed to fine powder. The tablet powder equivalent to 100mg of MET was accurately weighed, transferred to 100ml volumetric flask, dissolved in small quantity of double distilled water and finally make up to mark with double distilled water. This solution was filtered through Whatmann filter paper No. 41. The filtrate was further diluted with double distilled water to get concentration of 6µg/ml of MET. The sample solution was scanned over the range of 325nm to 190nm in multi component mode and concentration of each component was estimated by analysis of spectral data of sample solution with respect to that of mixed standards by the instrument. Results of tablet analysis are reported in Table 1.

VALIDATION OF METHOD:

As per ICH guideline the method is validated and following parameters were evaluated^9,12,14.

Linearity:

Its ability (with in a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The calibration curve was constructed between concentration verses absorbance.

Precision:

Precision was determined by repeatability, Interday precision of all two drugs. Repeatability indicates the precision under the same operating condition over short interval time. The Interday precision study is expressed within laboratory variation on different days and analyst to analyst variation by different analyst.

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

Sensitivity of the method was determined with respect to limit of detection (LOD) and limit of quantitation. According to ICH guidelines, the limit of detection is the lowest amount of analyte in a sample that can be detected and the limit of quantitation is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

Accuracy (% recovery):

For carrying out the accuracy of the proposed method recovery studies were employed by the standard addition method. This was carried out by adding known amounts of standard combination of VGB and MET at three different levels of 80%, 100%, and 120% to the sample.

Robustness:

As per ICH norms, small, but deliberate variations by altering the pH and / or concentration of the solvent were made to check the methods capacity to remain unchanged.

RESULTS AND DISCUSSION:

The Absorbance ratio method for estimation of Metformin HCL and Voglibosein tablet dosage form was found to be simple, precise, accurate and reproducible. The solvent used was 100% methanol and do not shows any significant interference in the spectrophotometric assay of all two drugs.

Linearity:

The proposed method was found to be linear in the range of 0.4-2 and 0.8-1.6μg/ml with correlation coefficient 0.9988 and 0.9925 for Metformin HCL and Voglibose respectively.

The calibration curve was constructed between concentration verses absorbance. It is shown into the Figure 4 and 5 resp. and result of linearity study shown in Table 2.

Figure 4: Calibration curve for Metformin HCL

Figure 5: Calibration curve for Voglibose

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

The standard deviation of y-intercept of regression line were determined and substituted in the following equation for the determination of detection of limit and quantification limits.

Detection limit= 3.3 σ/s

Quantification limit= 10 σ/s

Where, σ is the standard deviation of y-intercept of regression line and s is the slope of the calibration curve.

The limit of detection (LOD) and limit of quantification (LOQ) data are given in Table 2.

Accuracy:

Precision:

Precision was determined by repeatability and Interday precision of all two drugs.

I) Repeatability:

The repeatability was performed for six concentrations in linearity range 0.4, 0.8, 1.2, 1.6, 2 and 2.4µg/ml for Metformin HCL and 0.8, 1, 1.2, 1.4, 1.6 and 1.8µg/ml for Voglibose indicates the precision under the same operating condition over short interval time.

II) Interday precision:

Interday precision was also performed within laboratory variation on different days for all two drugs simultaneously in three replicate at three concentrations. Result of precision shown in Table 4.

Robustness:

Standard stock solution of 1000µg/ml of Metformin HCL and Voglibose were prepared using methanol as a solvent. From standard stock solution, sub stock solution of Metformin HCL and Voglibose were prepared separately. In this present work the change was made in the ratio of solvent and absorption maxima.

Table 1: Result of Tablet formulation.

Sr. No.	Drug Name	Labelled Amount (mg)	S.D.	% COV
1	Metformin HCL	500	0.7102	100%
2	Voglibose	0.2	0.0019	97.55%

(Where, S.D. = Standard Deviation, %COV= % Recovery)

Instead of 100%, 95% methanol was used as solvent. And the absorption maxima were decreased and increased 2 nm and carried out the process. The RSD% was calculated. Results of robustness shown in Table 5.

Table 2: Linear regression parameters for Metformin HCL and Voglibose by both proposed methods.

Sr. No.	Parameter	Metformin HCL	Voglibose
1.	Wavelength (nm)	248	287
2.	Calibration range(µg/ml)	0.4-2	0.8-1.6
3.	Correlation coefficient (r²)	0.9988	0.9925
4.	Slope(m)	1.146	0.2515
5.	Intercept(c)	0.7580	0.2813
6.	Limit of detection (µg/ml)	9.09	12.5
7.	Limit of Quantitation (µg/ml)	27.57	41.46

Table 3: Recovery study at three concentration levels for Metformin HCL and Voglibose by both proposed methods.

Sr. No.	Drug	Concentration of standard added	S.D.	% RSD	% Recovery
1	Metformin HCL	80%	0.0067	1.73	100.07%
		100%	0.0359	1.84	104.65%
		120%	0.0068	0.30	99.07%
2	Voglibose	80%	0.0018	0.19	97.40%
		100%	0.0067	0.78	100.83%
		120%	0.0087	1.10	100.91%

Table 4: Precision study for Metformin HCL and Voglibose by both proposed methods.

Sr. No	Parameter	S.D.		% RSD
1.	Drug	MET	VOG	MET	VOG
2.	Repeatability	0.0069	0.0011	0.38	0.28
3.	Interday Precision	0.0085	0.5978	0.48	0.57

(Where, MET= Metformin HCL, VOG= Voglibose)

Table 5: Robustness study at three concentration levels for Metformin HCL and Voglibose by both proposed methods.

Sr. No.	Drug	Wavelength (nm)	S.D.	% RSD
1.	Metformin HCL	241	0.0051	0.58
1.	Metformin HCL	245	0.0011	0.22
2	Voglibose	281	0.0005	0.16
2	Voglibose	289	0.0015	0.55

CONCLUSION:

In this work, a new UV-Spectrophotometric method has developed and validated for Metformin HCL and Voglibose by Absorbance ratio method. The results of present study indicate that the proposed UV-Spectrophotometric method is simple, rapid, precise and accurate. The developed UV-Spectrophotometric method was found suitable for determination of Metformin HCL and Voglibose in tablet dosage form. They can be easily applied in quality control laboratory tests in the dosage form.

ACKNOWLEDGEMENTS:

The authors would like to acknowledge Principal Dr. R. V. Shete, Rajgad Dnyanpeeth’s College of Pharmacy Bhor, for providing laboratory facilities and encouragement.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 20.05.2020 Modified on 29.06.2020

Research J. Pharm. and Tech. 2021; 14(6):3179-3183.

DOI: 10.52711/0974-360X.2021.00554