Separation and Characterization of New Components and Impurities in Leucomycin by Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Combined with Ion Trap/Time-of-Flight Mass Spectrometry

  • Jian Wang
  • Guijun Liu
  • Yu Xu
  • Bingqi Zhu
  • Zhijian WangEmail author


In this study, nine new components and six impurities in leucomycin were discovered. A method was developed for the separation and characterization of new components and impurities in leucomycin by multiple heart-cutting two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry in both positive and negative electrospray ionization modes. With this method, a non-volatile buffer solution was used as mobile phase in the first-dimensional system for good separation. Eluent of each peak from the first-dimensional system was trapped by a switching valve and sent to the liquid chromatography-mass spectrometry system using a volatile mobile phase. The complete fragmentation patterns of the new components and degradation impurities were deduced based on MSn data. The structures of nine new components in leucomycin were deduced as unsaturated ketone in the 16-membered ring of leucomycin. The structures of six impurities were characterized for the first time, four of which were acid degradation products, and the other two were process impurities. The correlation between impurities and the purification process of leucomycin was also studied. The degradation impurities were produced during purification of leucomycin fermentation broth, which requires a low-pH environment. Based on the characterization of impurities, this study not only revealed the mechanism of impurity production, thus providing guidance to pharmaceutical companies for manufacturing process improvement and impurity reduction, but also provided a scientific basis for further improvement of official monographs in pharmacopoeias.


Kitasamycin Leucomycin Impurity Multiple heart-cutting two-dimensional liquid chromatography Ion trap/time-of-flight mass spectrometry 



This work was supported by Key Technologies and Standards for Drug Consistency Assessment of National Science and Technology Major Project (No. 2017ZX09101001).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10337_2019_3754_MOESM1_ESM.doc (2.7 mb)
Supplementary material 1 (DOC 2724 kb)


  1. 1.
    Liu QQ (2003) Improved preparation of kitasamycin tablets. Chin J Pharm 34:22–23Google Scholar
  2. 2.
    Yang Q, Ma SH, Hu M, Hu CQ (2006) Determination of active ingredients of kitasamycin by high performance liquid chromatography. Chin J Anal Chem 34:95–99Google Scholar
  3. 3.
    Balducci Y, Balducci Y, Bodey GP, Bodey GP (1974) In vitro activity of kitasamycin against gram-positive cocci. J Antibiot 27:516–519CrossRefGoogle Scholar
  4. 4.
    Filadoro F, Cipriani P, Ravagnan L (1968) Antibacterial effect in vitro and in vivo of kitasamycin. Antibiotica 6:5–23Google Scholar
  5. 5.
    ICH Harmonised Tripartite Guideline Q3A(R) (2006) Impurities in new salt substances, The International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use. Accessed 25 Jan 2019
  6. 6.
    Fukutsu N, Kawasaki T, Saito K, Nakazawa H (2006) Application of high-performance liquid chromatography hyphenated techniques for identification of degradation products of cefpodoxime proxetil. J Chromatogr A 1129:153–159CrossRefGoogle Scholar
  7. 7.
    Pan YH, Zhang HY, Xi CG, Huang LL, Xie SY, Chen DM, Tao YF, Liu ZL, Yuan ZH (2018) Simultaneous determination of multicomponent of acetylkitasamycin and kitasamycin by LC-MS/MS in swine plasma and its application in a pharmacokinetic study. Biomed Chromatogr 32:e4268CrossRefGoogle Scholar
  8. 8.
    Chen X (2011) Study on contents determination methodology of kitasamycinum composition in kitasamycin tartrate for injection by HPLC. J Hubei Univ Med 30:588–590Google Scholar
  9. 9.
    Hu M, Hu CQ (2006) Identification of the components and products of hydrolysis in acetyl leucomycin by LC-MS. Acta Pharm Sin 41:476–480Google Scholar
  10. 10.
    Zhu SQ, Niu CQ (2007) LC-MS analysis of components of kitasamycin. Chin J Antibiot 32:478–480Google Scholar
  11. 11.
    Van den Bossche F, Daidone F, Van Schepdael A, Hoogmartens J, Adams E (2013) Characterization of impurities in josamycin using dual liquid chromatography combined with mass spectrometry. J Pharm Biomed Anal 73:66–76CrossRefGoogle Scholar
  12. 12.
    Wang MJ, Wang Y, Li J, Li YP, Hu CQ, Hoogmartens J, Van Schepdael A, Adams E (2013) Characterization of the components of meleumycin by liquid chromatography with photo-diode array detection and electrospray ionization tandem mass spectrometry. J Pharm Biomed Anal 84:69–76CrossRefGoogle Scholar
  13. 13.
    Wang MJ, Hu CQ (2013) Impurity profiling of macrolide antibiotics by liquid chromatography-mass spectrometry. Acta Parm Sin 48:642–647Google Scholar
  14. 14.
    Holm R, Elder DP (2016) Analytical advances in pharmaceutical impurity profiling. Eur J Pharm Sci 87:118–135CrossRefGoogle Scholar
  15. 15.
    Martano C, Ferretti F, Ghiani S, Buonsanti F, Bruno E, Lattuada L, Medana C (2017) Development and validation of a new HPLC-MS method for meglumine impurity profiling. J Pharm Biomed Anal 149:517–524CrossRefGoogle Scholar
  16. 16.
    Shipkova PA, Heimark L, Bartner PL, Chen G, Pramanik BN, Ganguly AK, Cody RB, Kusai A (2000) High-resolution LC/MS for analysis of minor components in complex mixtures: negative ion ESI for identification of impurities and degradation products of a new oligosaccharide antibiotic. J Mass Spectrom 35:1252–1258CrossRefGoogle Scholar
  17. 17.
    Reig MN, Jaumot J, Baglai A, Truyols GV, Schoenmakers PJ, Tauler R (2017) Untargeted comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry analysis of rice metabolome using multivariate curve resolution. Anal Chem 89:7675–7683CrossRefGoogle Scholar
  18. 18.
    Yang Q, Wang ZY, Tang SF (2016) Application of two-dimensional UPLC-QTof MS technology in the study of the impurity profile of bleomycin hydrochloride*. Chin J Pharm Anal 36:1231–1242Google Scholar
  19. 19.
    Long Z, Zhan ZQ, Guo ZM, Li YQ, Li CK, Yao JT, Ji F, Zheng X, Ren B, Huang TH (2019) A novel two-dimensional liquid chromatography-mass spectrometry method for direct drug impurity identification from HPLC eluent containing ion-pairing reagent in mobile phases. Anal Chim Acta 1049:105–114CrossRefGoogle Scholar
  20. 20.
    Wang J, Xu Y, Wen C, Wang Z (2017) Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and characterization of impurities and isomers in cefpiramide. Anal Chim Acta 992:42–54CrossRefGoogle Scholar
  21. 21.
    Petersson P, Haselmann K, Buckenmaier S (2016) Multiple heart-cutting two dimensional liquid chromatography mass spectrometry: towards real time determination of related impurities of bio-pharmaceuticals in salt based separation methods. J Chromatogr A 1468:95–101CrossRefGoogle Scholar
  22. 22.
    Schans MGMVD, Blokland MH, Zoontjes PW, Mulder PPJ, Nielen MWF (2017) Multiple heart-cutting two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry of pyrrolizidine alkaloids. J Chromatogr A 1503:38–48CrossRefGoogle Scholar
  23. 23.
    National Pharmacopoeia Committee (2015) Chinese pharmacopoeia, part 2, 2015th edn. China Medical Science and Technology Press, BeijingGoogle Scholar
  24. 24.
    Editorial Board of Japanese Pharmaceutical (2016) The Japanese pharmacopoeia, Seventeenth edn. Ministry of Health, Labour and WelfareGoogle Scholar
  25. 25.
    Govaerts C, Chepkwony HK, Van Schepdael A, Adams E, Roets E, Hoogmartens J (2004) Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate. J Mass Spectrom 39:437–446CrossRefGoogle Scholar
  26. 26.
    Hu M, Hu CQ (2005) Identification of the components of 16-membered macrolide antibiotics by LC/MS. Anal Chim Acta 535:89–99CrossRefGoogle Scholar
  27. 27.
    Przybylski P, Pyta K, Brzezinski B (2010) Fragmentation pathways of new aza derivatives of 16-membered macrolide antibiotic-analog of Josamycin investigated by ESI and FAB mass spectrometric methods. J Mass Spectrom 44:1395–1401CrossRefGoogle Scholar
  28. 28.
    Zhang X, Li J, Wang C, Song DQ, Hu CQ (2017) Identification of impurities in macrolides by liquid chromatography–mass spectrometric detection and prediction of retention times of impurities by constructing quantitative structure–retention relationship (QSRR). J Pharm Biomed Anal 145:262–272CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Zhejiang University of TechnologyHangzhouChina
  2. 2.Zhejiang Institute for Food and Drug ControlHangzhouChina

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