UHPLC-Q-Orbitrap/MS技术鉴定阿哌沙班片的有关物质

吕广云; 姚奕然; 徐丹洋; 王舒; 肖蓓

doi:10.11665/j.issn.1000-5048.2024081401

UHPLC-Q-Orbitrap/MS技术鉴定阿哌沙班片的有关物质

南通市食品药品监督检验中心, 南通 226000

基金项目:

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国家药品监督管理局化学药品杂质谱研究重点实验室开放课题（NMPA-KLIPCD-2023-03）

详细信息

通讯作者:
肖蓓: Tel：0513-51001919　E-mail：xbybh@126.com

中图分类号: R917
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- 文章访问数: 18
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- PDF下载量: 5
出版历程
- 收稿日期: 2024-08-13
- 修回日期: 2024-09-08
- 录用日期: 2024-09-24
- 刊出日期: 2025-06-24

Identification of related substances in apixaban tablets by UHPLC-Q-Orbitrap/MS

Nantong Food and Drug Control Center, Nantong 226000, China

Funds:

This study was supported by the Open Project of the National Medical Products Administration Key Laboratory for Impurity Profile of Chemical Drugs (NMPA-KLIPCD-2023-03)

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摘要

摘要:
采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UHPLC-Q-Orbitrap/MS）技术研究阿哌沙班片中的有关物质。采用Waters Xbridge C₁₈（250 mm×4.6 mm，5 μm）色谱柱，以30 mmol/L醋酸铵缓冲液（pH4.50）-乙腈为流动相梯度洗脱，对阿哌沙班片有关物质进行分离；电喷雾正离子化-四极杆-静电场轨道阱串联质谱法测定各有关物质的母离子及碎片离子的准确质荷比和元素组成，并解析鉴定有关物质的结构。在所建立的条件下，阿哌沙班与其有关物质分离良好，检测并鉴定出阿哌沙班片及其强制降解试验样品中30个主要有关物质，其中11个为已知杂质，其余19个为新鉴定的未知有关物质。研究结果可为阿哌沙班的生产工艺控制和质量保障提供参考依据。
- 阿哌沙班 /
- 有关物质 /
- 结构鉴定 /
- UHPLC-Q-Orbitrap/MS
Abstract:
A UHPLC-Q-Orbitrap/MS method was developed to identify the related substances in apixaban tablets. Complete separation was accomplished with a Waters Xbridge C₁₈ (250 mm×4.6 mm, 5 μm) column by linear gradient elution using a mobile phase consisting of 30 mmol/L ammonium acetate buffer solution (pH 4.50) and acetonitrile. The related substances were successfully characterized through the accurate mass and elemental composition of the parent ions and their product ions determined by electrospray positive ionization high-resolution Q-Orbitrap/MS methods. Under the established analytical condition, apixaban and its related substances were well separated, and 30 related substances were detected and identified by hyphenated techniques in apixaban tablets and their stressed samples. Among them, 11 were known impurities and the rest 19 were unknown related substances identified for the first time in this study. The results obtained are valuable for apixaban manufacturing process optimization and quality control.
- apixaban /
- related substances /
- structural identification /
- UHPLC-Q-Orbitrap/MS

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Figure 1. Chemical structure of apixaban

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Figure 2. HPLC-UV chromatograms of representative samples of apixaban tablets

a: Producer g; b: Producer h; c: Producer j; d: Producer k; e: Producer q; f: Producer Pfizer

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Figure 3. HPLC-UV chromatograms of apixaban stress solutions

a: Normal; b: 0.1% Reference; c: Alkaline; d: Oxidation; e: Acid; f: Wet heat; g: Dry heat; h: Dry light; i: Wet light; j: Blank

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Figure 4. Chemical structures of apixaban and its related substances 1−30

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Figure 5. MS/MS fragmentation pathways of [M+H]⁺ ions of apixaban

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Figure 6. MS/MS fragmentation pathways of [M+H]⁺ ion of related substances 12, 9 and 13

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Figure 7. Generation of the related substance in apixaban tablets

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Table 1 Related substances identified in apixaban tablets and its stressed samples by UHPLC-Q-Orbitrap/MS

No.	t_R/min	Parent ion(m/z)	Ion formula	Dif.(×10^-6)	Product ions(m/z)	Origins
1	4.957	354.15570	C₁₈H₂₀N₅O₃⁺	−1.03	337.13354,309.13425,281.12778,253.13300,217.09698,203.11769,191.11775,130.06525,93.04529,79.01844	Dr
2	5.695	478.20804	C₂₅H₂₈N₅O₅⁺	−0.96	461.18130,417.19214,288.09775,271.07101,244.10800,215.08163,199.08658,184.06310	Dr
3	5.872	287.11365	C₁₄H₁₅N₄O₃⁺	−0.76	270.08600,242.09224,227.06659,215.08138,199.08670,184.06326,172.06281,125.05952,95.04979	Dr
4	7.283	479.19235	C₂₅H₂₇N₄O₆⁺	−0.33	461.18173,435.20178,417.15460,405.15543,373.16574,361.16571,238.09680,199.08669,184.06293	Dr
5/A	7.993	479.19217	C₂₅H₂₇N₄O₆⁺	−0.71	461.18179,435.20258,417.15460,405.15564,379.14029,373.16577,361.16562,330.14725,238.09718,199.08675,184.06303	Pr/Dr
6/B	8.658	461.18112	C₂₅H₂₅N₄O₅⁺	−1.79	443.16852,417.19180,374.17343,282.12360,241.06036,199.08653,184.06300,144.06816	Pr/Dr
7/C	9.222	446.18121	C₂₄H₂₄N₅O₄⁺	−2.39	429.15308,401.16098,282.12350,227.04492,185.07085,171.05521,121.03982	Pr/Dr
8	10.022	560.25038	C₃₀H₃₄N₅O₆⁺	0.03	505.21857,487.20856,470.18341,416.17093,387.18143,348.12958,254.12752,199.08675,82.06573	Pr
9	11.188	458.18210	C₂₅H₂₄N₅O₄⁺	−0.40	430.18930,268.07141,242.09233,227.06895,199.07393,109.04006	Pr
10	11.327	476.19275	C₂₅H₂₆N₅O₅⁺	−0.20	459.16357,430.18747,413.16397,371.15039,298.11911,252.11378,241.06096,224.11874,199.08667,185.07103,173.10721,135.05548,125.05988,95.04975	Pr
11/D	11.527	477.22354	C₂₅H₂₉N₆O₄⁺	−1.97	460.19757,432.20313,416.17123,404.17154,299.15009,282.12308,238.09738,219.13611,199.08649,185.07111,130.06505,100.07608	Pr
12	11.902	492.18680	C₂₅H₂₆N₅O₆⁺	−1.95	475.16025,447.19019,418.15030,404.13452,377.14792,241.06052,199.08667,185.07072	Dr
13	12.400	515.20319	C₂₇H₂₇N₆O₅⁺	−1.08	488.19284,404.17267,309.13464,241.06091,199.08658,185.07127,125.06004,84.04510	Pr
14	13.348	492.18741	C₂₅H₂₆N₅O₆⁺	−0.71	475.16023,447.19057,418.15032,404.13442,377.14790,241.06049,199.08659,185.07112	Dr
15	13.825	492.18732	C₂₅H₂₆N₅O₆⁺	−0.90	475.16064,447.19012,418.15032,404.13452,377.14788,241.06052,199.08647,185.07079	Dr
16	13.897	430.18738	C₂₄H₂₄N₅O₃⁺	0.03	413.15930,385.16495,357.17020,282.12396,254.12938,211.05048,169.07608,155.06036,105.04514,95.04968	Pr
17/E	14.178	478.20786	C₂₅H₂₈N₅O₅⁺	−1.34	460.19757,432.20282,416.17139,404.17142,399.14438,377.14865,333.13385,300.13397,238.09715,199.08658,185.07085,101.06010	Pr/Dr
API	14.723	460.19699	C₂₅H₂₆N₅O₄⁺	−2.04	443.16870,415.17426,374.17432,282.12344,241.06059,199.08643,185.07082,135.05528,95.04959	−
18/F	15.263	458.18124	C₂₅H₂₄N₅O₄⁺	−2.26	441.15524,415.17575,385.16565,372.15762,341.13919,318.08701,277.10870,249.11378,221.08209	Pr
19	16.295	507.19867	C₂₅H₂₇N₆O₆⁺	0.02	477.20041,404.17062,390.15579,373.12817,241.06075,199.08659,185.07086,95.04922	Dr
20	16.297	460.19754	C₂₅H₂₆N₅O₄⁺	−0.85	443.16888,415.17413,374.17529,282.12350,241.06078,199.08655,185.07097,135.05547,95.04964	Pr
21/G	17.203	474.21265	C₂₆H₂₈N₅O₄⁺	−1.97	457.18423,429.19089,296.13904,241.06065,199.08646,185.07086,156.06802,125.05978,95.04958	Pr
22	18.140	474.21304	C₂₆H₂₈N₅O₄⁺	−1.13	457.18332,429.19083,296.13912,241.06070,199.08650,185.07088,156.06813,125.05976,95.04971	Pr
23/H	18.470	474.21252	C₂₆H₂₈N₅O₄⁺	−2.23	457.18393,429.19186,296.13913,241.06062,199.08644,185.07079,156.06807,125.05978,95.04958	Pr
24	18.692	474.21338	C₂₆H₂₈N₅O₄⁺	−0.43	457.18344,429.18976,296.13916,241.06065,199.08646,185.07080,156.06816,125.05990,95.04955	Pr
25/I	18.790	464.14771	C₂₄H₂₃ClN₅O₃⁺	−1.48	447.12000,419.12436,282.12338,245.01115,203.03696,189.02141,168.06822,139.00574	Pr
26	19.500	512.16968	C₂₅H₂₇ClN₅O₅⁺	0.30	494.15887,466.16422,450.13263,414.15585,391.13983,334.09528,272.05835,225.06609,199.08661,185.07097,101.06019	Dr
27/K	21.378	475.19714	C₂₆H₂₇N₄O₅⁺	−0.95	461.18185,443.16876,415.17664,282.12375,241.06076,199.08664,185.07101,172.07568,135.05542,125.05988,95.04966	Pr
28	21.835	546.13098	C₂₅H₂₆Cl₂N₅O₅⁺	0.79	528.12018,500.12521,484.09381,446.07825,389.08011,268.00394,225.06563,199.08675,185.07100,101.06020	Dr
29/L	24.480	489.21268	C₂₇H₂₉N₄O₅⁺	−1.16	461.18170,443.16855,417.19278,374.17368,282.12366,241.06078,227.11781,199.08656,185.07088,156.06808,135.05539,125.05988,95.04965	Pr
30	25.358	387.15508	C₂₀H₂₃N₂O₆⁺	0.02	279.64178,212.84285,147.06519,129.05489,119.04932,105.07024	Pr
Pr: Process related substance; Dr: Degradation related substance

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