Xtimate C8 - Vitamin B12 and its impurities analysis method development report

Test Report

Chromatography conditions:

Column

Xtimate C8（3.0×250mm，3μm）

Mobile Phase

0.028mol/L disodium hydrogen phosphate solution (pH=3.1): methanol = 82:18

Column Temperature

40℃

Detector

Injection Volume

20μL

Flow Rate

0.6 ml/min

Detect wavelength

361 nm

Elution procedure

Time (min)	0.028mol/L disodium hydrogen phosphate solution (pH=3.1): methanol = 82:18
0	100%
45	100%

Note

Chromatogram and Data:

1) Analysis chart of vitamin B12 impurity spiked test solution:

Serial number	Peak name	Retention time (min)	*Peak Area(mAUmin)**	S/N	Relative peak area(%)	Resolution(EP)	Number of plates(EP)	Asymmetry(EP)
1	Impurity H	3.738	0.885	4.7	0.20	24.19	10112	1.33
2	Vitamin B12	12.353	413.471	1052.9	92.03	11.53	7632	1.60
3	Impurity A	19.318	6.741	87.7	1.50	2.47	14318	0.90
4	Impurity C	21.038	15.247	171.1	3.39	4.86	12556	0.93
5	Impurity D	24.795	5.473	218.4	1.22	1.50	15403	0.88
6	Impurity E	25.968	0.447	19.6	0.10	4.11	17983	0.97
7	Impurity B	29.480	7.003	144.0	1.56	N.a.	15814	1.06
Sum			449.267		100.0	48.65

2) Analysis result reproducibility verification superposition spectrum:

Serial number	Peak name	Retention time (min)	*Peak Area(mAUmin)**	S/N	Relative peak area(%)	Resolution(EP)	Number of plates(EP)	Asymmetry(EP)
1	Impurity H	3.738	0.885	4.7	0.20	24.19	10112	1.33
2	Vitamin B12	12.353	413.471	1052.9	92.03	11.53	7632	1.60
3	Impurity A	19.318	6.741	87.7	1.50	2.47	14318	0.90
4	Impurity C	21.038	15.247	171.1	3.39	4.86	12556	0.93
5	Impurity D	24.795	5.473	218.4	1.22	1.50	15403	0.88
6	Impurity E	25.968	0.447	19.6	0.10	4.11	17983	0.97
7	Impurity B	29.480	7.003	144.0	1.56	N.a.	15814	1.06
Sum			449.267		100.0	48.65

3) Multi-needle analysis of pressure stability overlay:

Conclusion:

Vitamin B12 and its impurities were analyzed under these chromatographic conditions using a Welch column Xtimate C8 (3.0×250mm, 3μm). Vitamin B12 and impurities A, C, D, E and B eluted in sequence with a separation degree of no less than 1.5. After multiple injections of the spiked solution of the test sample, the results showed good reproducibility and stable pressure, which met the analysis requirements.