Amoxicillin related substance testing
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Amoxicillin related substance testing
1. Experimental method
Cometro high performance liquid chromatography system;
Column: Comasil C18 250 * 4.6mm, 5μm;
Wavelength: 254nm;
Flow rate: 1.0ml / min;
Sample: Amoxicillin raw material sample (about 2mg / ml), control substance (20μg / ml);
Injection volume: 20μl
Mobile phase: A phosphate (pH = 5) -acetonitrile = 99: 1;
B Phosphate (pH = 5) -acetonitrile = 80: 20.
Gradient elution
Time / min
Mobile phase A /%
Mobile phase B /%
0.00
92
8
15.00
92
8
40.00
0
100
55.00
0
100
56.00
92
8
70.00
92
8
2. Experimental results
1. Sample of amoxicillin
Channel A Results
Name
Retention Time
Area
Theoretical plates (USP)
Resolution (USP)
1
2.787
9464
6111
0.00000
2
5.142
27245
9917
13.49134
3
7.080
24450
11532
8.24395
Amoxicillin
11.223
4690129
8036
10.83894
4
17.268
8253
11995
10.68492
5
25.923
13824
160765
19.46456
6
27.470
7857
161474
5.81389
7
30.797
10138
315697
13.50415
8
31.830
15339
372563
4.83051
9
33.063
4103
206428
4.93651
10
34.923
20142
359640
7.09890
11
37.430
3251
64676
6.10147
12
39.792
3050
108857
4.40964
Ladder Peak
41.345
8157
266135
3.86885
Ladder Peak
46.210
31615
205741
13.36384
Ladder Peak
58.990
38043
61357
18.79275
2. Amoxicillin reference substance
Channel A Results
Name
Retention Time
Area
Theoretical plates (USP)
Resolution (USP)
Amoxicillin
12.748
53226
12032
0.00000
Ladder Peak
41.603
6591
367607
78.05457
Ladder Peak
46.417
24679
245449
14.82780
Ladder Peak
58.977
48226
57297
18.46654
3. Gradient blank
Channel A Results
Name
Retention Time
Area
Theoretical plates (USP)
Resolution (USP)
Ladder Peak
41.513
6560
352379
0.00000
Ladder Peak
46.323
24802
236337
14.55634
Ladder Peak
58.977
42866
56800
18.45865
3. Conclusion
1. The first picture focuses on 8 small peaks of impurities, and the second picture focuses on the large peak at 12 minutes. From the third picture, we can judge that the peaks at 41min, 46min, and 58min are cascade peaks, so they are not considered. Secondly, according to the pharmacopoeia, "any test solution is less than 0.05 times the main peak area of ​​the control solution (53226 * 0.05 = 2613.3) The peak of is negligible ", the experimenter has ignored it during the integration.
2. According to the two regulations of related substances, we analyze one by one:
If there is an impurity peak in the chromatogram of the test solution, the area of ​​a single impurity peak shall not be greater than the main peak area of ​​the control solution;
Analysis: The main peak area of ​​the reference substance is 53226, and the impurities 1-12 are smaller than it, which is qualified.
The sum of the peak areas of the impurities should not be greater than three times the main peak of the control solution.
Analysis: The area of ​​the main peak of the reference product tripled was 159678, and the sum of the peak areas of impurities 1-12 was 147116, which was qualified.
3. This sample meets the relevant standards of the relevant substances in the 10th edition of Pharmacopoeia Amoxicillin.