Ultisil AQ-C18-Diosmin Analytical Method Development Report
Test Report
Chromatography conditions:
Column |
Welch Utisil AQ-C18（4.6×100mm,3μm） |
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Mobile Phase |
Acetonitrile: glacial acetic acid: methanol: water = 2:6:28:66 |
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Column Temperature |
25℃ |
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Detector |
UV |
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Injection Volume |
10μL |
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Flow Rate |
1.5 ml/min |
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Detect wavelength |
275 nm |
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Elution procedure |
|
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Note |
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Chromatogram and Data:
- 1. Analysis spectrum of blank solvent dimethyl sulfoxide:
- 2. Impurity B control solution analysis spectrum:
Serial number |
Peak name |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution |
Number of plates(EP) |
Asymmetry |
1 |
ImpurityB |
3.628 |
88.964 |
62.4 |
100.00 |
N.a. |
4299 |
1.05 |
Sum |
88.964 |
|
100.00 |
0.00 |
|
- 3. Impurity C reference solution analysis spectrum:
Serial number |
Peak name |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution |
Number of plates(EP) |
Asymmetry |
1 |
Impurity C |
4.438 |
36.907 |
355.7 |
100.00 |
N.a. |
5994 |
1.08 |
Sum |
88.964 |
|
100.00 |
0.00 |
|
|
- 4. EP system solution analysis spectrum:
Serial number |
Peak name |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution |
Number of plates(EP) |
Asymmetry(EP) |
1 |
Impurity A |
2.230 |
0.284 |
0.2 |
0.26 |
7.90 |
5463 |
0.99 |
2 |
Impurity B |
3.635 |
1.167 |
7.2 |
1.05 |
3.47 |
3789 |
1.16 |
3 |
Impurity C |
4.452 |
1.672 |
97.7 |
1.51 |
3.77 |
5717 |
1.02 |
4 |
API Diosmin |
5.445 |
104.662 |
507.3 |
94.41 |
18.88 |
5534 |
1.10 |
5 |
Impurity D |
13.980 |
2.123 |
27.5 |
1.92 |
14.50 |
8301 |
0.98 |
6 |
Impurity F |
25.400 |
0.947 |
8.6 |
0.85 |
N.a. |
11074 |
1.01 |
Sum |
|
110.855 |
|
100.00 |
48.52 |
|
|
- 5. Analysis spectrum of test solution:
Serial number |
Peak name |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution |
Number of plates(EP) |
Asymmetry(EP) |
1 |
Impurity A |
2.228 |
0.133 |
0.1 |
0.09 |
7.32 |
4644 |
1.35 |
2 |
Impurity B |
3.642 |
1.787 |
90.7 |
1.16 |
3.26 |
3230 |
1.10 |
3 |
Impurity C |
4.473 |
3.963 |
184.2 |
2.58 |
3.55 |
4898 |
1.08 |
4 |
API Diosmin |
5.485 |
145.020 |
4669.8 |
94.49 |
18.06 |
4804 |
1.18 |
5 |
Impurity D |
14.128 |
0.548 |
6.9 |
0.36 |
13.96 |
7718 |
1.03 |
6 |
Impurity F |
25.618 |
2.034 |
29.5 |
1.33 |
N.a. |
10354 |
1.05 |
Sum |
|
153.484 |
|
100.00 |
46.15 |
|
|
3. Conclusion
Using Welch column Ultisil AQ-C18 (4.6×100mm, 3μm) to analyze the system suitability solution under this chromatographic condition, the separation between the main substance and the known impurities was greater than 1.5, and the separation between impurity B and impurity C was 3.5, which met the analysis requirements.