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Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22

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Abstract

The rapid and accurate quantification of lipopeptide families in biological samples are challenging. We present the development and validation of a method for simultaneous quantification of three families of lipopeptides (iturins, fengycins, and surfactins) and their isoforms, as well as the homologous series. The method was optimized in UPLC-MS for a column temperature at 65 °C, injection volume of 5 μL, and sample temperature of 10 °C. The SIM mode was used for detection and quantification of lipopeptides exhibiting ions [M + H]+ and [M + 2H]2+. Since the maximum mass detection threshold of the equipment is 1250 Da and the fengycins have ions between 1435 and 1505 Da, the ions [M + 2H]2+ were chosen for fengycin identification. The monitored ions were as follows: m/z 1043.5, 1057.5, 1071.5, 718.3, 725.4, 739.4, 732.4, 746.4, 753.4, 1008.6, 1022.6, and 1036.6. The compounds were separated by reverse-phase chromatography using a C18 analytical column in a total time of 19 min. Standard curves were linear with rw 0.99 for all analytes. Intra- and inter-day precision for samples (50, 250, and 750 μg L−1) were within recommended limits. The proposed analytical method was capable of simultaneously quantifying 12 isoforms and homologous series of lipopeptide families in biological samples, thus making it an important industrial tool in the evaluation of lipopeptide production processes.

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Abbreviations

CV:

Coefficient of variation

ICH:

International Conference Harmonization

MAPA:

Brazilian Ministry of Agriculture Livestock and Food Supply

SIM:

Single ion monitoring

UPLC-QDA:

Ultra-performance liquid chromatography with quadrupole detector

UPLC-QToF:

Ultra-performance liquid chromatography with quadrupole time of flight detector

References

  1. Cantrell CL, Dayan FE, Duke SO. Natural products as sources for new pesticides. J Nat Prod. 2012;75:1231–42.

    Article  CAS  PubMed  Google Scholar 

  2. Wetzel S, Bon RS, Kumar K, Waldmann H. Biology-oriented synthesis. Angew Chem Int Ed. 2011;50:10800–26.

    Article  CAS  Google Scholar 

  3. Meena KR, Kanwar SS. Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. Biomed Res Int. 2015;2015. https://doi.org/10.1155/2015/473050.

  4. Pathak KV, Keharia H, Gupta K, Thakur SS, Balaram P. Lipopeptides from the banyan endophyte, Bacillus subtilis K1: mass spectrometric characterization of a library of fengycins. J Am Soc Mass Spectrom. 2012;23:1716–28.

    Article  CAS  PubMed  Google Scholar 

  5. Lanna Filho R, Ferro HM, De Pinho RSC. Biological control mediated by Bacillus subtilis. Rev Trop Cienc Agr Biol. 2010;4:12–20.

    Google Scholar 

  6. Jacques P. Biosurfactants. 2011.

  7. Behary N, Perwuelz A, Campagne C, Lecouturier D, Dhulster P, Mamede AS. Adsorption of surfactin produced from Bacillus subtilis using nonwoven PET (polyethylene terephthalate) fibrous membranes functionalized with chitosan. Colloids Surf B Biointerfaces. 2012;90:137–43.

    Article  CAS  PubMed  Google Scholar 

  8. Kawagoe Y, Shiraishi S, Kondo H, Yamamoto S, Aoki Y, Suzuki S. Cyclic lipopeptide iturin A structure-dependently induces defense response in Arabidopsis plants by activating SA and JA signaling pathways. Biochem Biophys Res Commun. 2015;460:1015–20.

    Article  CAS  PubMed  Google Scholar 

  9. Rangarajan V, Dhanarajan G, Sen R. Bioprocess design for selective enhancement of fengycin production by a marine isolate Bacillus megaterium. Biochem Eng J. 2015;99:147–55.

    Article  CAS  Google Scholar 

  10. Yuan J, Raza W, Huang Q, Shen Q. Quantification of the antifungal lipopeptide iturin A by high performance liquid chromatography coupled with aqueous two-phase extraction. J Chromatogr B Anal Technol Biomed Life Sci. 2011;879:2746–50.

    Article  CAS  Google Scholar 

  11. Meng Y, Liu J-F, Yang S-Z, Ye R-Q, Mu B-Z. Quantification of lipopeptides using high-performance liquid chromatography with fluorescence detection after derivatization. Anal Sci. 2015;31:377–82.

    Article  CAS  PubMed  Google Scholar 

  12. Geissler M, Oellig C, Moss K, Schwack W, Henkel M, Hausmann R. High-performance thin-layer chromatography (HPTLC) for the simultaneous quantification of the cyclic lipopeptides surfactin, iturin A and fengycin in culture samples of Bacillus species. J Chromatogr B Anal Technol Biomed Life Sci. 2017;1044–1045:214–24.

    Article  CAS  Google Scholar 

  13. Biniarz P, Łukaszewicz M. Direct quantification of lipopeptide biosurfactants in biological samples via HPLC and UPLC-MS requires sample modification with an organic solvent. Appl Microbiol Biotechnol. 2017;101:4747–59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Deng Q, Wang W, Sun L, Wang Y, Liao J, Xu D, et al. A sensitive method for simultaneous quantitative determination of surfactin and iturin by LC-MS/MS. Anal Bioanal Chem. 2017;409:179–91.

    Article  CAS  PubMed  Google Scholar 

  15. Yao C, Yang W, Si W, Pan H, Qiu S, Wu J, et al. A strategy for establishment of practical identification methods for Chinese patent medicine from systematic multi-component characterization to selective ion monitoring of chemical markers: Shuxiong tablet as a case study. RSC Adv. 2016;6:65055–66.

    Article  CAS  Google Scholar 

  16. Yang H, Li X, Li X, Yu H, Shen Z. Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC. Anal Bioanal Chem. 2015;407:2529–42.

    Article  CAS  PubMed  Google Scholar 

  17. Ye YF, Li QQ, Fu G, Yuan GQ, Miao JH, Lin W. Identification of antifungal substance (iturin A2) produced by Bacillus subtilis B47 and its effect on southern corn leaf blight. J Integr Agric. 2012;11:90–9.

    Article  CAS  Google Scholar 

  18. Zhao X, Zhou ZJ, Han Y, Wang ZZ, Fan J, Xiao HZ. Isolation and identification of antifungal peptides from bacillus BH072, a novel bacterium isolated from honey. Microbiol Res. 2013;168:598–606.

    Article  CAS  PubMed  Google Scholar 

  19. Bie X, Lu Z, Lu F. Identification of fengycin homologues from Bacillus subtilis with ESI-MS/CID. J Microbiol Methods. 2009;79:272–8.

    Article  CAS  PubMed  Google Scholar 

  20. Wang J, Liu J, Wang X, Yao J, Yu Z. Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis. Lett Appl Microbiol. 2004;39:98–102.

    Article  CAS  PubMed  Google Scholar 

  21. Ma Z, Hu J. Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A. Appl Biochem Biotechnol. 2015;177:1520–9.

    Article  CAS  PubMed  Google Scholar 

  22. Pathak KV, Bose A, Keharia H. Characterization of novel lipopeptides produced by Bacillus tequilensis P15 using liquid chromatography coupled electron spray ionization tandem mass spectrometry (LC-ESI-MS/MS). Int J Pept Res Ther. 2014;20:133–43.

    Article  CAS  Google Scholar 

  23. TECHNICAL, I. C. O. H. O., USE, R. F. R. O. P. F. H. Ich Harmonised tripartite guideline text on validation of analytical procedures. Statew Agric L Use Baseline 2015 2014, 1, 18.

  24. Brasil, Da Qualidade Analítica. Brasília-BR. 2011.

  25. Barbosa PGA, Martins FICC, Lima LK, Milhome MAL, Cavalcante RM, do Nascimento RF. Statistical analysis for quality adjustment of the analytical curve for determination of pesticide multiresidue in pineapple samples. Food Anal Methods. 2018;11:466–78.

    Article  Google Scholar 

  26. Martins FICC, Barbosa PGA, Zocolo GJ, Nascimento RF. Method validation using normal and weighted linear regression models for quantiication of pesticides in mango (Mangifera indica L.) samples. Chromatographia. 2018;49:12.

    Google Scholar 

  27. Chen WC, Juang RS, Wei YH. Applications of a lipopeptide biosurfactant, surfactin, produced by microorganisms. Biochem Eng J. 2015;103:158–69.

    Article  CAS  Google Scholar 

  28. Inès M, Dhouha G. Lipopeptide surfactants: production, recovery and pore forming capacity. Peptides. 2015;71:100–12.

    Article  CAS  PubMed  Google Scholar 

  29. Shih IL, Lin CY, Wu JY, Hsieh C. Production of antifungal lipopeptide from Bacillus subtilis in submerged fermentation using shake flask and fermentor. Korean J Chem Eng. 2009;26:1652–61.

    Article  CAS  Google Scholar 

  30. Huang X, Liu J, Wang Y, Liu J, Lu L. The positive effects of Mn 2+ on nitrogen use and surfactin production by Bacillus subtilis ATCC 21332. Biotechnol Biotechnol Equip. 2015;29:381–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Monaci L, Quintieri L, Caputo L, Visconti A, Baruzzi F. Rapid profiling of antimicrobial compounds characterising B. subtilis TR50 cell-free filtrate by high-performance liquid chromatography coupled to high-resolution OrbitrapTM mass spectrometry. Rapid Commun Mass Spectrom. 2016; 30:45–53. https://doi.org/10.1002/rcm.7408.

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Funding

This study had financial support from the CNPq (447498/2014-2).

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Authors and Affiliations

  1. Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, 60455-760, Brazil

    Caroline Gondim de Souza

  2. Embrapa Agroindústria Tropical, Rua Doutora Sara Mesquita, 2270, Pici, Fortaleza, CE, 60511-110, Brazil

    Caroline Gondim de Souza, Fátima Itana Chaves Custódio Martins, Guilherme Julião Zocolo, Kirley Marques Canuto & Edy Sousa de Brito

  3. Departamento de Química, Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, 60451-970, Brazil

    Fátima Itana Chaves Custódio Martins

  4. Embrapa Milho e Sorgo, Rodovia MG-424, Km 45, Sete Lagoas, MG, 35701-970, Brazil

    José Edson Fontes Figueiredo

Authors
  1. Caroline Gondim de Souza
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  2. Fátima Itana Chaves Custódio Martins
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  3. Guilherme Julião Zocolo
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  4. José Edson Fontes Figueiredo
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  5. Kirley Marques Canuto
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  6. Edy Sousa de Brito
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Correspondence to Edy Sousa de Brito.

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de Souza, C.G., Martins, F.I.C.C., Zocolo, G.J. et al. Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22. Anal Bioanal Chem 410, 6827–6836 (2018). https://doi.org/10.1007/s00216-018-1281-6

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  • DOI: https://doi.org/10.1007/s00216-018-1281-6

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