Bharat Institute of Technology, Mangalpally, Ibrahimpatnam, Rangareddy Telangana India.
In the present work, an attempt was made to provide newer, sensitive, simple, accurate and low cost HPLC methods. It is successfully applied for the determination of Glatiramer acetate in pharmaceutical preparations without the interferences of other constituents in the formulations new RP-HPLC method was developed for both bulk drug and formulation. These proposed methods give reliable assay results with short analysis time (<10.0 min) using the mobile phase of (Acetonitrile: Trifluoracetic acid) with 1.0 ml/min flow rate is quite robust. The optimum wavelength for detection was 220 nm at which better detector response for drugs was obtained. The wavelength was found to be 220nm for Glatiramer acetate. The average retention time for Glatiramer acetate was found to be 9.039min respectively. System suitability tests are an integral part of chromatographic method. They are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions. The HPLC calibration was linear in concentration range of 80-120%with regression 1.000 for Glatiramer acetate. The low values of % R.S.D. indicate that method is precise and accurate. The mean recoveries were found in the range of 99.74-100.02 %for HPLC. These results show the accuracy and reproducibility of the assay. Ruggedness of the proposed methods was determined by analysis of aliquots from homogeneous slot by different analysts, using similar operational and environmental conditions; the % R.S.D. reported was found to be less than 2 %. The proposed method was validated and the results of all methods were very close to each other as well as to the label value of commercial pharmaceutical formulation.
High Pressure Liquid Chromatography
High Performance Liquid Chromatography (HPLC) is a process of separating components in a liquid mixture. A liquid sample is injected into a stream of solvent (mobile phase) flowing through a column packed with a separation medium (stationary phase). Sample components separate from one another by a process of differential migration as they flow through the column.
MATERIALS AND METHOD:
Preparation of mobile phases
Mobile phase A: Mix 1ml of trifluoroaceticacid acid in 1000ml of purified water, filter through 0.45µm membrane filter and degas.
Mobile phase B: Mix 1ml of trifloroaceticacid acid and 1000ml of acetonitrile, filter through 0.45µm membrane filter and degas.
Preparation of Glatiramer acetatestandard stock solution: Accurately weigh and transfer about 104 mg of Glatiramer acetate into 100ml volumetric flask add 50ml purified water sonicate to dissolve and dilute to volume with purified water.
Preparation of sample stock solution: Accurately weigh 5ml of sample into 100ml volumetric flask shake it well dissolve and dilute to volume with purified water.
RESULTS AND DISCUSSION:
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-8.png" target="_blank">
<img alt="Optimized Chromatogram.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-8.png" width="150">
</a>
Fig.1: Optimized Chromatogram
Validation Parameters:
System suitability: The standard Stock solution of Glatiramer acetate was injected six times into HPLC system as per test procedure. The system suitability parameters were evaluated from standard Chromatograms obtained, by calculating the % RSD of retention times, tailing factor, theoretical plates and peak areas from six replicate injections.
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-7.png" target="_blank">
<img alt="Chromatogram of Standard solution.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-7.png" width="150">
</a>
Fig: 2 Chromatogram of Standard solution
Specificity: Solutions of Standard and Sample were prepared as per test procedure and injected into the HPLC system.
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-6.png" target="_blank">
<img alt="Chromatogram of specificity sample solution.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-6.png" width="150">
</a>
Fig: 3. Chromatogram of specificity sample solution
Precision
Repeatability/ Method precision: Six sample solutions were prepared and injected into the HPLC system as per test procedure.
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-5.png" target="_blank">
<img alt="Chromatogram of Precision sample solution.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-5.png" width="150">
</a>
Fig: 4. Chromatogram of Precision sample solution
|
S. No |
Peak Name |
RT |
Area |
%Area |
|
1 |
Glatiramer Acetate |
9.017 |
29161039 |
100.00 |
|
2 |
Glatiramer Acetate |
9.045 |
29267819 |
100.00 |
|
3 |
Glatiramer Acetate |
9.029 |
29269385 |
100.00 |
|
4 |
Glatiramer Acetate |
9.005 |
29162172 |
100.00 |
|
5 |
Glatiramer Acetate |
9.027 |
29240159 |
100.00 |
|
6 |
Glatiramer Acetate |
9.011 |
29215121 |
100.00 |
|
Mean |
|
9.02 |
29219283 |
|
|
Std.Dev |
|
0.01 |
48935 |
|
|
%RSD |
|
0.16 |
0.17 |
|
Linearity: 100 mg of glatiramer acetate was dissolved in 20ml of diluent (water). The concentration of the solution was 5000µg/ml from this parent solution, 10 ml was diluted to 100ml to get a concentration of 500 µg/ml from this solution, solutions of various concentrations such as 80,90,100,110,120µg/ml of glatiramer acetate were prepared using water ( purified water) as diluent. 50 µl of each of the solutions was injected into the HPLC system and the run time was 30 minutes for each injection.
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-4.png" target="_blank">
<img alt="Chromatogram of Linearity sample solution.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-4.png" width="150">
</a>
Fig: 5. Chromatogram of Linearity sample solution
Linearity/Calibration Graph of Glatiramer Acetate
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-3.png" target="_blank">
<img alt="Graph for Linearity Data.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-3.png" width="150">
</a>
Figure: 6 Graph for Linearity Data
Accuracy: Assay was performed in triplicate for various concentrations of Glatiramer acetateequivalent to 80, 100 and 120 % of the standard amount was injected into the HPLC system as per the test procedure.
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-2.png" target="_blank">
<img alt="Chromatogram of Accuracy sample solution.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-2.png" width="150">
</a>
Fig: 7. Chromatogram of Accuracy sample solution
Accuracy data
|
Sample No. |
Spike Level |
Amount (mg) added |
Amount (mg) found |
% Recovery |
% Mean Recovery |
SD |
%RSD |
|
1 |
80 % |
0.81 |
0.809 |
100.11 |
100.02 |
0.16 |
0.160 |
|
80 % |
0.81 |
0.808 |
100.03 |
||||
|
80 % |
0.81 |
0.807 |
99.91 |
||||
|
2 |
100 % |
1.01 |
1.007 |
99.78 |
99.74 |
0.17 |
0.170 |
|
100 % |
1.01 |
1.007 |
99.71 |
||||
|
100 % |
1.01 |
1.007 |
99.73 |
||||
|
3 |
150 % |
1.21 |
1.209 |
99.76 |
99.77 |
0.17 |
0.170 |
|
150 % |
1.21 |
1.209 |
99.76 |
||||
|
150 % |
1.21 |
1.209 |
99.80 |
Robustness: The robustness of the proposed method was determined by analysis of aliquots from homogenous lots by differing physical parameters like flow rate, organic content, wavelength and mobile phase composition which may differ but the responses were still within the specified limits of the assay.
Standard Chromatogram for Robustness (Temperature)
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-1.png" target="_blank">
<img alt="8.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-1.png" width="150">
</a>
Sample Chromatogram for Robustness (Temperature)
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-0.png" target="_blank">
<img alt="9.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250612141016-0.png" width="150">
</a>
CONCLUSION:
All the above methods do not suffer from any interference due to common excipients. Therefore, it was shown that the proposed methods could be successfully applied to estimate Glatiramer acetate. Thus, the above studies and findings will enable the quantification of the drug for future investigation in the field of analytical chemistry.
REFERENCES
Dr. Namratha Sunkara, Pasupulati Haritha, P. Twila Pushpa, Rudra Sandhya, Dr. Swathi Boddupally*, Development of a Novel by RP – HPLC Method for The Estimation of Glatiramer Acetate, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 2373-2379. https://doi.org/10.5281/zenodo.15622038
10.5281/zenodo.15622038
