目的 建立一种二维高效液相色谱-串联质谱方法,对注射用头孢美唑钠在含不挥发性流动相的一维色谱中检出的未知杂质进行结构解析。方法 一维色谱的流动相为磷酸二氢铵溶液(取磷酸二氢铵5.75 g,加水730 mL使溶解,加10 %四丁基氢氧化铵19.2 mL)-四氢呋喃-甲醇混合液(730∶12.5∶300)(用磷酸调pH值至4.5),色谱柱采用Shim-pack GIS C18(4.6 mm×250 mm,5 μm)柱,流速1.0 mL·min-1。二维色谱流动相为1%甲酸水溶液(A相)/乙腈(B相),梯度洗脱,采用Shim-pack GIS C18(4.6 mm×150 mm, 5 μm)色谱柱,流速1.0 mL·min-1,柱温为25 ℃。采用50 μL定量环收集一维色谱分离到的未知杂质成分。质谱采用电喷雾离子源(ESI)源进行离子化,氩气做碰撞气,全扫描-数据相关分析模式(DDA)进行检测。基于未知杂质的母离子和子离子的精确分子量、头孢美唑的质谱裂解规律和同位素分布等信息,推断出可能的分子结构。结果 对注射用头孢美唑钠中的3种未知杂质进行了结构鉴定,杂质1和杂质3为头孢美唑钠的同分异构体,为热降解杂质,杂质2为7-甲巯基头孢美唑,是头孢美唑钠的工艺杂质。结论 本方法采用二维色谱对注射用头孢美唑钠中3种超过鉴别限的未知杂质进行了结构分析,为采用不挥发性流动相分离得到的β-内酰胺类抗生素中未知杂质的定性研究提供了解决方案。
Abstract
OBJECTIVE To develop a two-dimensional high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (2D-LC-QTOF MS) to identify impurities in thermally degraded cefmetazole sodium for injection. METHODS For the 1st dimensional chromatography, the mobile phase was a mixture of ammonium dihydrogen phosphate/tetrahydrofuran/methanol (730∶12.5∶300). Ammonium dihydrogen phosphate solution was prepared by dissolving 5.75 g salts in 730 mL water, then 19.2 mL tetrabutylammonium hydroxide (10%) was added in the above-mentioned solution. Finally, the solution was adjusted to pH 4.5 with phosphoric acid. The column used was Shim-pack GIS C18 (4.6 mm×250 mm, 5 μm), maintained at 25 ℃. The flow rate was 1 mL·min-1. For the 2nddimensional chromatography, the mobile phase was 1% formic acid aqueous solution (A)/acetonitrile (B), and gradient elution was conducted at a flow rate of 1.0 mL·min-1. The column was Shim-pack GIS C18(4.6 mm×150 mm, 5 μm) and installed in a 25 ℃ column oven. 50 μL quantitative ring was used to collect impurities from the 1st dimension. ESI source was used for ionization with both full scan and data dependent analysis mode (DDA). The collision gas was argon. Based on the accurate molecular weight of impurities and their products, isotope distribution, and fragmentation pathway for cefmetazole, the most reasonable molecular structures were proposed for unknown impurities. RESULTS Three unknown impurities were identified.Impurities 1 and 3 were isomers of cefmetazole, which were thermally degraded impurities of cefmetazole,meanwhile impurity 2 was 7-methylthiocefmetazole, which was from the production process. CONCLUSION The unknown impurities above identification threshold of ICH in cefmetazole sodium for injection were analyzed with 2D-LC-QTOF system, which was also suitable for the identification of unknown impurities in other β-lactam antibiotics using mobile phase containing non-volatile salts.
关键词
二维色谱 /
注射用头孢美唑钠 /
串联质谱法 /
结构鉴定 /
高分辨率质谱
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Key words
two-dimensional chromatography /
cefmetazole sodiumforinjection /
LC-tandem quadrupole time-of-flight mass spectrometry /
impurity characterization /
high resolution mass spectrometry
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中图分类号：
R917
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参考文献
[1] QIN F, DING Y, QIU Y, et al. Polymer impurities in cefotiam hydrochloride by online 2D-GFC-RPLC-TOFMS[J]. Chin Pharm J (中国药学杂志), 2016, 51(9):742-750.
[2] HAO R, WANG Y, CHEN Y, et al. 2D-HPGFC-RPLC-MS method for the characterization of polymer impurities in azlocillin sodium for injection[J]. Chin Pharm J (中国药学杂志), 2017, 52(14):1273-1279.
[3] LI J, YAO S C, HU C Q. Characterization of impurity profiles in latamoxef sodium raw material by LC/MSn[J]. Chin J Antibio (中国抗生素杂志), 2019, 44(7):820-833.
[4] LI J, YAO S C, HU C Q. Rapid characterization of impurities in cefmetazole sodium for injection by online column-switching liquid chromatography tandem mass spectrometry[J]. Chin New Drug J(中国新药杂志), 2019, 28(8):984-995.
[5] XU Y, WANG D, TANG L, et al. Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC×LC-IT-TOF MS[J]. J Pharm Biomed Anal, 2017, 145:742-750.
[6] XU Y, WANG D, ZHU B, et al. Separation and characterization of allergenic polymerized impurities from cephalosporin for injection by trap free two-dimensional high-performance size exclusion chromatography × reversed phase liquid chromatography coupled with ion trap time-of-flight mass [J]. J Pharm Biomed Anal, 2018, 154:425-432.
[7] XU Y, WANG F, LI J, et al. Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-IT-TOF MS and study of their negative-ion fragmentation regularities[J]. J Pharm Biomed Anal, 2017, 140:81-90.
[8] XU Y, WANG D, TANG L, et al. Separation and characterization of unknown impurities and isomers in cefminox sodium and study of the forming mechanisms of impurities by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry[J]. J Chromatogr Sci, 2018, 57(3):204-212.
[9] HAN B, PANG W Z, GAO Y X. Identification of polymer impurities in cefmetazole sodium for injection[J]. China Pharm(中国药业), 2019, 28(24):31-34.
[10] MA F, WU Q, WEI Q. LC-MS method for the isolation and identification of isomeric impurities by thermal degradation in cefodizime[J]. Chin J Pharm Anal (药物分析杂志), 2011, 31(9):1833-1836.
[11] CHEN X, ZHENG X, ZHOU J, et al. Characterization of related substances in cefmetazole sodium by UPLC-Q-TOF-MS[J]. Chin J Antibio(中国抗生素杂志), 2020,45(10):1017-1026.
[12] LI S, WEI W, HAN Z, et al. Improvement on the synthesis of methoxy cephalosporin′s intermediate 7-MAC[J]. Chem Res Appl (化学研究与应用), 2013, 25(4):547-549.
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脚注
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基金
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项目资助(NMPA-KLIPCD-2020-06);北京市科委、中关村管委会专项课题资助(Z211100003421042)
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