To Develop and Validate RP-HPLC Analytical Technique for the Solid Dosage Forms of Dapagliflozin and Metformin Hydrochloride
Ragini Ghotale, Sameep Sonvane*, Kranti Satpute, Mahesh Birajdar, Balaji Shetkar,
Gopal Lohiya, Prakash Shivnechari
Dayanand Education Society’s, Dayanand College of Pharmacy, Latur, Maharashtra, India.
*Corresponding Author E-mail: sameepsonvane@rediffmail.com
ABSTRACT:
A straightforward, precise, and high-performance reverse phase liquid chromatography technique has been created for the simultaneous measurement of Metformin hydrochloride and Dapagliflozin in solid dose form. The HPLC model was used to make the determination: For chromatographic separation, an Agilent 1260 Infinity II with an Agilent 5 TC-C18 (2) 250 × 4.6mm column was utilized. PDA detector was applied. The mobile phase's flow rate was increased to 1 milliliter per minute and contained 0.02% triethylamine: acetonitrile (50:50 v/v). A 236 nm wavelength and a 50 µl injection volume were chosen for the detection. The retention times for Dapagliflozin and metformin, when using the described approach, were determined to be 5.6 and 2.6 minutes, respectively. The linearity, accuracy, and precision of the procedure have all been confirmed. Dapagliflozin and Metformin's linearity were in between 30 and 70 μg/ml and 0.6 and 1.4 μg/ml, respectively. Metformin and Dapagliflozin were shown to have percentage recoveries of 101.88% and 99.35%, respectively. The analytical processes are deemed suitable for their intended purpose and meet the standards outlined in ICH Q2R2, as evidenced by the results of the validation parameters.
KEYWORDS: Metformin hydrochloride, Dapagliflozin, RP-HPLC, Validation, Robustness.
INTRODUCTION:
The process of choosing a precise assay technique to determine a formulation's composition is known as analytical method development. The process of developing an analytical method is what establishes its suitability for usage in a laboratory setting. The ICH recommendations Q2 (R1) include procedures and acceptance criteria for the development of analytical methods, which must be utilized in GMP and GLP contexts1. The "process of establishing documented evidence" known as "method validation" offers a high degree of assurance that the equipment or product will satisfy the needs of the intended analytical applications2.
A validation analysis involves evaluating several aspects of a technique to ascertain whether, when used robotically, it could yield accurate and meaningful data 3-7. The validation test should contain standard test circumstances, such as product excipients, in order to precisely examine method parameters. As a result, a method validation analysis is product-specific8-12.
Metformin hydrochloride is to treat type 2 diabetes mellitus. It is a member of the biguanide medication class and acts by enhancing insulin sensitivity in adipose and muscle cells and reducing the liver's synthesis of glucose. This aids in lowering blood sugar. Metformin improves glucose absorption by cells by decreasing the liver's ability to produce glucose (gluconeogenesis) and raising insulin sensitivity13-17.
Figure No 1: Metformin HCl
In similar way, Dapagliflozin is to treat type 2 diabetes mellitus. It is a member of the group of medications called inhibitors of the sodium-glucose co-transporter 2 (SGLT2). It functions by preventing the kidneys' SGLT2 protein, which is in charge of reabsorption of glucose. Dapagliflozin lowers blood glucose levels by increasing the excretion of glucose in urine and inhibiting SGLT2 18-20.
Figure No 2: Dapagliflozin
The primary goal of this effort is to create and validate a Reverse Phase- High Performance Liquid Chromatography method that will allow the official guidelines to be followed in determining the solid dosage form of Dapagliflozin and Metformin21-25.
MATERIALS AND METHODS:
Analytical and Software:
Chromatography was performed on, an Agilent 1260 Infinity II with open lab CDS 2.7 as a software and Agilent 5 TC-C18 (2) 250 × 4.6mm column was utilized. PDA detector was applied. The mobile phase's flow rate was increased to 1 milliliter per minute and contained 0.02% triethylamine: acetonitrile (50:50 v/v). A 236nm wavelength and a 50µl injection volume were chosen for the detection.
Reagent and Chemicals:
HPLC grade solvent such as triethylamine and acetonitrile from Research Lab were used for this present work. Active pharmaceutical ingredients were procured from Smruthi Organics Limited, Solapur.
Preparation of Mobile Phase:
In order to prepare the mobile phase, 0.1ml of triethylamine was combined with 500ml of HPLC grade water. Acetonitrile were added in a 50:50 v/v ratio, and the mixture was well mixed. The mobile phase was then filtered using what's man filter paper no. 41 and degassed using sonication for approximately 10 minutes.
Chromatographic Condition:
Agilent 1260 Infinity II with an Agilent 5 TC-C18 (2) 250 × 4.6mm column was utilized for chromatographic resolution a detection of wavelength 236nm.Mobile phase used 0.02% Triethylamine: Acetonitrile (50:50) v/v. The elution was monitored by injecting the 50μl and the flow rate was adjusted to 1ml/min.
Preparation of Standard Solution:
Weigh out 250mg of Metformin hydrochloride and 5mg of Dapagliflozin precisely, and then transfer into a 100 ml different volumetric flask. To this added 50ml of diluent it is Sonicated for 10min and makeup the volume up to (100ml) the mark. Filter the mixture using what’s man filter paper No. 41. Pipetted out 2ml of the previously prepared each solution and placed into a second 100ml volumetric flask. Added water to the volumetric flask until the indicated volume is reached(.
RESULT AND DISCUSSION:
Chromatographic Condition Optimization:
The impact of chromatographic factors such mobile phase ratio, mobile phase composition, and flow rate were examined in order to optimize chromatographic conditions. Chromatographic characteristics including tailing factor, capacity factor, and theoretical plate count were computed after the resultant chromatograms were recorded. Ultimately, a straightforward and economical technique was created by using 0.02% Triethylamine: Acetonitrile (50:50). Table No. 1 contains a list of ideal chromatographic conditions.
Table No 1: chromatographic conditions
|
Method Parameter |
Optimized Value |
|
Column |
Agilent 5 TC-C18 (2) 250 × 4.6mm column |
|
Particle size packing |
5 μ |
|
Stationary phase |
5 TC-C18 (2) |
|
Mobile Phase |
0.02% Triethylamine: Acetonitrile (50:50) v/v |
|
Detection Wavelength |
236nm |
|
Flow rate |
1.0 ml/min |
|
Sample size |
50 μL |
Figure No 3: Chromatogram of Metformin HCl and Dapagliflozin
Method Validation:
Linearity and Range:
The calibration curve was constructed by plotting concentrations versus peak areas, and the regression equations were calculated. The linear regression equation is Y = 239.73x+286.92.
Figure No 4: Calibration Curve for Metformin HCl
The results show that all the system suitability parameters meet the acceptance criteria. The results show that the response is found to be linear. Correlation coefficient is more than 0.99 hence; the method is linear in the given tested range i.e. 30PPM-70PPM for Metformin Hydrochloride and 0.6PPM-1.4PPM for Dapagliflozin.
Accuracy:
The degree of agreement between True value and Found value is expressed by the accuracy of an analytical technique. The accuracy of the procedure is within acceptable bounds, according to the accuracy test findings. As Table No. 2 and 3 shows, the HPLC method is accurate in determining the assay of Metformin Hydrochloride and Dapagliflozin tablet since the mean recovery of these two drugs is 101.88% and 99.35%, respectively, and the percentage RSD of these two drugs is 0.531% and 0.871%, respectively.
Precision:
Six replicate injections of a sample injected. The results' percentage RSD (relative standard deviation, was computed). (table-4).
Table No 2: Data sheet for Accuracy (Metformin HCl)
|
% Level |
Set |
Amount Added (mg) |
Amount Recovery (mg) |
% Recovery |
Mean |
SD |
% RSD |
|
80% |
1 |
204 |
209.305 |
102.6006 |
102.649 |
0.133 |
0.129 |
|
2 |
204.9 |
210.636 |
102.7995 |
||||
|
3 |
206 |
211.2467 |
102.5469 |
||||
|
100%
|
1 |
250 |
253.653 |
101.461 |
100.140 |
1.367 |
1.365 |
|
2 |
250 |
250.577 |
100.230 |
||||
|
3 |
247 |
243.863 |
98.730 |
||||
|
120%
|
1 |
302 |
310.946 |
102.962 |
102.864 |
0.104 |
0.101 |
|
2 |
308 |
316.856 |
102.875 |
||||
|
3 |
308.8 |
317.305 |
102.754 |
Table No 3: Data sheet for Accuracy Dapagliflozin
|
% Level |
Set |
Amount Added (mg) |
Amount Recovery (mg) |
% Recovery |
Mean |
SD |
%RSD |
|
80%
|
1 |
4 |
4.044 |
101.116 |
100.453 |
0.582 |
0.580 |
|
2 |
3.9 |
3.900 |
100.022 |
||||
|
3 |
4.1 |
4.109 |
100.222 |
||||
|
100%
|
1 |
4.8 |
4.781 |
99.606 |
99.141 |
0.755 |
0.761 |
|
2 |
4.8 |
4.716 |
98.270 |
||||
|
3 |
5 |
4.977 |
99.548 |
||||
|
120% |
1 |
5.8 |
5.642 |
97.287 |
98.458 |
1.183 |
1.201 |
|
2 |
5.8 |
5.779 |
99.653 |
||||
|
3 |
6 |
5.906 |
98.434 |
Table No 4: Data sheet for Precision
|
Sample No. |
Metformin Hydrochloride |
Dapagliflozin |
||
|
Sample Area |
%Assay |
Sample Area |
% Assay |
|
|
1. |
11491.865 |
101.89 |
83.311 |
101.91 |
|
2. |
11586.052 |
102.72 |
81.384 |
99.55 |
|
3. |
11162.544 |
100.58 |
78.226 |
97.25 |
|
4. |
11461.449 |
103.09 |
78.345 |
97.22 |
|
5. |
11007.813 |
99.01 |
81.679 |
101.36 |
|
6. |
11727.338 |
103.98 |
79.753 |
97.56 |
|
Average |
- |
101.877 |
- |
98.590 |
|
SD |
- |
2.030 |
- |
1.825 |
|
%RSD |
- |
1.992 |
- |
1.851 |
The % RSD of the method precision for Metformin Hydrochloride is 1.992% and for Dapagliflozin is 1.851% therefore, the HPLC method for the determination of Metformin Hydrochloride and Dapagliflozin is precise.
Specificity:
The Standard and sample solution were prepared as per the procedure and blank and excipient does not have any interference to the standard and sample chromatogram.
|
RT [min] |
Area |
Height |
Area % |
Name |
Tailing Factor |
|
2.719 |
10933.64 |
2102.57 |
99.33 |
Metformin HCl |
0.86191 |
|
5.664 |
73.50 |
9.91 |
0.67 |
Dapagliflozin |
1.90933 |
Figure No 7: Specificity Sample solution chromatogram
Table No 6: Specificity Sample solutions
|
RT [min] |
Area |
Height |
Area % |
Name |
Tailing Factor |
|
2.725 |
10847.10 |
2056.13 |
99.31 |
Metformin HCL |
0.88606 |
|
5.716 |
75.91 |
11.74 |
0.69 |
Dapagliflozin |
1.12013 |
Robustness:
The method robustness was proven by purposefully altering the flow rate of the chromatography. Less than 2% was the average percentage RSD for determining the method robustness.
Table No.7: Data sheet for Robustness (Metformin Hydrochloride)
|
Change in parameter
|
Area of Slandered |
Mean |
SD |
% RSD |
|
|
Change in flow rate (±0.2ml/ min) 1.0ml/min |
0.8ml/ min |
14536.702 |
14551.808 |
42.28
|
0.290 |
|
14519.154 |
|||||
|
14599.57 |
|||||
|
1.2ml/ min |
10113.191 |
10047.274 |
172.9 |
1.720 |
|
|
10177.533 |
|||||
|
9851.099 |
|||||
Table No.8: Data sheet for Robustness Dapagliflozin
|
Change in Parameter |
Area of Standard |
Mean |
SD |
% RSD |
|
|
Changes in Flow rate (± 0.2ml/min) 1.0ml/min |
0.8ml/min |
93.64 |
92.099 |
1.507 |
1.636 |
|
92.03 |
|||||
|
90.628 |
|||||
|
1.2ml/min |
66.799 |
66.555 |
0.467 |
0.702 |
|
|
66.85 |
|||||
|
66.016 |
|||||
After the study of stability study it found that both Standard and sample solutions are stable for 24hrs at room temperature.
Table No.9: Data sheet for Stability of Analytical solutions (Metformin HCl)
S No |
Name |
Area |
Assay |
% Relative Change |
% absolute Value |
1 |
Standard solution-0 hrs |
11217.58 |
NA |
1.4789 |
NA |
2 |
Standard solution -24 hrs |
11383.48 |
NA |
||
3 |
Standard solution-0 hrs |
11162.54 |
100.58 |
NA |
1.17 |
4 |
Standard solution -24 hrs |
11292.07 |
101.75 |
Sr. No |
Name |
Area |
Assay |
% Relative Change |
% absolute Value |
1 |
Standard solution-0 hrs |
76.486 |
NA |
1.1309 |
NA |
2 |
Standard solution -24 hrs |
77.351 |
NA |
||
3 |
Standard solution-0 hrs |
78.226 |
97.25 |
NA |
0.34 |
4 |
Standard solution -24 hrs |
78.5 |
97.59 |
Assay Analysis:
The % Assay of Metformin Hydrochloride and Dapagliflozin from the tablet was 99.11 % and 97.92 % respectively.
DISCUSSION:
RP-HPLC was used to analyze the formulation that contained Dapagliflozin and Metformin hydrochloride. The precision, accuracy, linearity, and range of these techniques serve as validation. These techniques produce precise and accurate outcomes. These approaches are less time consuming, steady, sensitive, robust, accurate, and exact because they require calculation on the part of the analyst. Accuracy, precision, specificity, robustness, linearity, and range are used to validate methods the findings of this procedure are precise and accurate. This method is less time-consuming, exact, and precise.
CONCLUSION:
With a correlation coefficient better than 0.99, the
measured signal was demonstrated to be precise, accurate, and linear over the
concentration range tested (30-70 ug/mL). Furthermore, a more economical and
environmentally friendly chromatographic process is produced by the method.
Metformin hydrochloride and Dapagliflozin had accuracy mean recovery values
that are within the limit, at 101.88% and 99.35%, respectively. A precision RSD
of no more than 2.0% is acceptable. It's found inside the range.
We conclude that the developed RP-HPLC validated method is robust, sensitive,
affordable, accurate, and precise for the quantitative estimation of metformin
hydrochloride and Dapagliflozin in formulation. It is also used for routine
analysis of these drugs in the pharmaceutical industries.
ACKNOWLEDGMENT:
Smruthi Organics Limited, Solapur for providing API.
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Received on 25.07.2024 Revised on 22.11.2024 Accepted on 03.01.2025 Published on 01.07.2025 Available online from July 05, 2025 Research J. Pharmacy and Technology. 2025;18(7):3273-3277. DOI: 10.52711/0974-360X.2025.00472 © RJPT All right reserved
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