INTRODUCTION:

Metformin HCl (1,1-dimethylbiguanide HCl), first developed in 1957, is one of the most commonly used oral anti-hyperglycemic agents for the treatment of Type II diabetes mellitus¹. In 1995, the FDA approved metformin HCl for use in the United States, which led to a significant increase in clinical use.²It is currently recommended as first-line therapy in overweight or obese patients with this condition.³Many methods are available for quantitation of metformin in biological samples. Regarding sample preparation from biological fluids, extraction and clean-up of the sample is a critical first step in bioanalysis and requires high selectively for the removal of interfering substances, while ensuring minimal analyte loss.^4,5The extraction of metformin from biological matrices is somewhat complicated by the highly polar nature of the molecule.⁶ This factor, in addition to the drugs widespread clinical use, may explain the relative abundance of different methods for analyzing metformin in biologicalspecimens.^7,8 Many of the current methods utilizevarying techniques for sample cleanup, such as simple protein precipitation, solid phase extraction, ultrafiltration with column switching, chemical derivatization and liquid-liquid extraction.⁹The present investigation was carried out in the view of establishing a simple, rapid, accurate, economic, precise and robust UV method for estimation Metformin HCl in bulk and tablet dosage form using water as the solvent in urine sample.

MATERIAL AND METHOD:

Drug:

The standard sample Sitagliptin was obtained as gift sample Sun Pharma Vadodara (Gujrat) India.

Instrument:

UV Spectrophotometer, Shimadzu, Model 1800.

Preparation of standard solution:

Standard solution of Metformin HCL was prepared by taking 10mg in 100ml volumetric flask containing distilled water and the volume was made up to the mark with distilled water. Then from this 0.1ml was pipetted out and transferred into another 10ml volumetric flask and the volume was made up to the mark with distilled water to give 10μg/ml solution .

Selection of Wavelength:

Solutions of 10μg/ml of Metformin HCL was prepared and the solution was scanned in the spectrum mode from 200nm to 400nm. The maximum absorbance of Bumetanide was observed at 234nm

Figure 1: UV spectrum of standard solution (10μg/ml)

Preparation of Sample:

The dilute and shoot method is employed for preparation of sample in this method 900 ml of distilled water was added to 100ml of urine (1:10 dilution. The Suitable amount of metformin HCL was added to diluted urine was added in urine and was scanned for the maximum absorption of drug solution using UV-Visible spectrophotometer within the wavelength region of

200–400nm and its absorbance was measured at 234nm.

Method validation:

The developed method was validated according to ICH guidelines. The proposed method was validated in terms of specificity, linearity, precision, accuracy, robustness, ruggedness, LOD (Limit of detection) and LOQ (Limit of Quantification).⁵

1. Specificity:

The ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components.⁵

Figure 2: Specificity of Sitagliptin

2. Linearity:

Ability to obtain test results which are directly proportional to the concentration of analyte in the sample. ⁵The proposed spectroscopic method was found to be linear in the range of 1-17μg/ml with correlation coefficient was 0.9991 (figure 4), slope 0.0031 and intercept 0.0529 was shown in table 1.

3. Precision:

Degree of agreement between a series of measurement obtained from multiple sampling of same homogenous sample. The precision of the proposed method was estimated in terms of inter-day and intra-day precision wherein the method was repeated for 6 times respectively. The results shown in table 2 indicating %RSD of less than 2% each level clearly indicate that the proposed method was precise enough for the analysis of drug. ⁵

%RSD = SD of measurement/Mean value of measurement X 100

4. Accuracy:

Closeness of test results obtained by that procedure to the true values.⁵The accuracy of the method was determined by performing recovery studies by spiking standard solution to that of sample solution at three different levels i.e., 50%, 100%, 150% or 80%, 100%, 120%. Values of %recovery greater than 95-105% indicate that the proposed method was accurate for the analysis of drug and the results were reported in table 3.

% Recovery =Amount found/Amount added X 100

5. Robustness:

Deliberate changes in the method are made such as wavelength.⁵The ±nm from the fixed wavelength. The robustness of the proposed method was evaluated by changing wavelength. The %RSD was calculated. The low values of %RSD obtained after small deliberate changes in method indicates that the method was robust and the results were presented in table 4.

6. Ruggedness:

The degree of reproducibility of the results obtained by the analysis of the sample under a variety of conditions such as different analyst and different instrument.⁵ The %RSD was calculated. The low values of %RSD obtained by changing the conditions indicates that the method was rugged and the results were presented in table 5.

7. Limit of Detection:

It is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated under the stated experimental condition.

LOD = 3.3 X SD/Slope

8. Limit of Quantitation:

LOQ = 10 X SD/Slope

9. Assay:

Preparation of standard solution: 10mg of Sitagliptin drug was accurately weighed and transferred into 10ml of volumetric flask and the volume was made up to the mark with diluted urine to get concentration of 1000μg/ml. From this 0.1ml was pipetted out and transferred into 10ml of volumetric flask and the volume was made up to the mark to get 10μg/ml solution and its absorbance was measured at 234nm.

Preparation of test solution: 10tablets were weighed and powdered. Powdered tablet equivalent to 10mg of Sitagliptin was weighed accurately and it was taken into 10ml volumetric flask then volume was made up to the mark with diluted urine. From the above solution 0.1ml of solution was pipetted out and taken in 10ml volumetric flask. The volume was made up to 10ml to get 10μg/ml solution and its absorbance was measured at 234nm.

The % Assay is calculated by using the following formula:

% Assay= Absorbance of the sample/Absorbance of the standard X Concentration of the standard/ Concentration of the sample X 100

RESULT AND DISCUSSION:

1. Linearity:

Figure 3: Linearity Study of Sitagliptin

Table 1: Linearity Study of Metformin HCL

Sr. No	Parameters	Results
1	Absorbance maximum(nm)	222
2	Linearity and range(μg/ml)	1-18μg/ml
3	Slope	0.1168
4	Correlation coefficient	0.9991
5	Y-intercept	0.0529

2. Precision:

Table 2: Precision study of Sitagliptin

Concentration	Intraday precision (%RSD)	Inter-day precision (%RSD)
		Day 1	Day 2
5μg/ml	0.129%	0.1329%	0.2528%

3. Accuracy:

Table 3: Accuracy study of Metformin HCL

Level	Amount of standard added (μg/ml)	Pre-analysed sample (μg/ml)	% Recovery
50%	1	5	97.14%
100%	2	5	98.76%
150%	3	5	99%

4.Robustness:

Table 3: Robustness study of Sitagliptin

S. NO	Concentration	Analyst	%RSD	Instrument	SD
1	5μg/ml	Analyst 1	0.7144%	Instrument 1	0.001123
2	5μg/ml	Analyst 2	0.1744%	Instrument 2	0.001123

5. LOQ and LOQ:

The LOD and LOQ of the proposed method was found to be 0.0234μg/ml, 0.07054μg/ml respectively.

6. Assay:

The % Assay of tablet sample was found to be 94.85%

CONCLUSION:

A simple rapid method with cost effectively and less economically method was developed and validated by using UV-Visible spectrophotometry

REFERENCE:

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Received on 14.09.2022 Modified on 17.03.2023

Research J. Science and Tech. 2024; 16(1):19-23.

DOI: 10.52711/2349-2988.2024.00004