Advertisement

Liquid chromatography–drift tube ion mobility–mass spectrometry as a new challenging tool for the separation and characterization of silymarin flavonolignans

Abstract

Silymarin, milk thistle (Silybum marianum) extract, contains a mixture of mostly isomeric bioactive flavonoids and flavonolignans that are extensively studied, especially for their possible liver-protective and anticancer effects. Because of the differing bioactivities of individual isomeric compounds, characterization of their proportion in a mixture is highly important for predicting its effect on health. However, because of silymarin’s complexity, this is hardly feasible by common analytical techniques. In this work, ultraperformance liquid chromatography coupled with drift tube ion mobility spectrometry and quadrupole time-of-flight mass spectrometry was used. Eleven target silymarin compounds (taxifolin, isosilychristin, silychristins A and B, silydianin, silybins A and B, 2,3-cis-silybin B, isosilybins A and B and 2,3-dehydrosilybin) and five unknown flavonolignan isomers detected in the milk thistle extract were fully separated in a 14.5-min analysis run. All the compounds were characterized on the basis of their accurate mass, retention time, drift time, collision cross section and fragmentation spectra. The quantitative approach based on evaluation of the ion mobility data demonstrated lower detection limits, an extended linear range and total separation of interferences from the compounds of interest compared with the traditional approach based on evaluation of liquid chromatography–quadrupole time-of-flight mass spectrometry data. The following analysis of a batch of milk thistle-based food supplements revealed significant variability in the silymarin pattern, especially in the content of silychristin A and silybins A and B. This newly developed method might have high application potential, especially for the characterization of materials intended for bioactivity studies in which information on the exact silymarin composition plays a crucial role.

Graphical Abstract

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 157

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Csupor D, Csorba A, Hohmann J. Recent advances in the analysis of flavonolignans of Silybum marianum. J Pharm Biomed Anal. 2016;130:301–17.

  2. 2.

    Abenavoli L, Izzo AA, Milić N, Cicala C, Santini A, Capasso R. Milk thistle (Silybum marianum): a concise overview on its chemistry, pharmacological, and nutraceutical uses in liver diseases. Phytother Res. 2018;32:2202–13.

  3. 3.

    Federico A, Dallio M, Loguercio C. Silymarin/silybin and chronic liver disease: A marriage of many years. Molecules. 2017;22:191.

  4. 4.

    Šimánek V, Kren V, Ulrichová J, Vicar J, Cvak L. Silymarin: what is in the name…? An appeal for a change of editorial policy. Hepatology. 2000;32:442–4.

  5. 5.

    Chambers CS, Holečková V, Petrásková L, Biedermann D, Valentová K, Buchta M, et al. The silymarin composition… and why does it matter??? Food Res Int. 2017;100:339–53.

  6. 6.

    Pelter A, Hänsel R. The structure of silybin (silybum substance E6), the first flavonolignan. Tetrahedron Lett. 1968;9:2911–6.

  7. 7.

    Poppe L, Petersen M. Variation in the flavonolignan composition of fruits from different Silybum marianum chemotypes and suspension cultures derived therefrom. Phytochemistry. 2016;131:68–75.

  8. 8.

    Novotná M, Gažák R, Biedermann D, Di Meo F, Marhol P, Kuzma M, et al. cistrans isomerization of silybins A and B. Beilstein J Org Chem. 2014;10:1047–63.

  9. 9.

    Sy-Cordero A, Graf TN, Nakanishi Y, Wani MC, Agarwal R, Kroll DJ, et al. Large-scale isolation of flavonolignans from Silybum marianum (milk thistle) extract affords new minor constituents and preliminary structure-activity relationships. Planta Med. 2010;76:644–7.

  10. 10.

    Pyszková M, Biler M, Biedermann D, Valentová K, Kuzma M, Vrba J, et al. Flavonolignan 2,3-dehydroderivatives: preparation, antiradical and cytoprotective activity. Free Radic Biol Med. 2016;90:114–25.

  11. 11.

    Biedermann D, Vavříková E, Cvak L, Křen V. Chemistry of silybin. Nat Prod Rep. 2014;31:1138–57.

  12. 12.

    Filippopoulou K, Papaevgeniou N, Lefaki M, Paraskevopoulou A, Biedermann D, Křen V, et al. 2,3-Dehydrosilybin A/B as a pro-longevity and anti-aggregation compound. Free Radic Biol Med. 2017;103:256–67.

  13. 13.

    Šuk J, Jašprová J, Biedermann D, Petrásková L, Valentová K, Křen V, et al. Isolated silymarin flavonoids increase systemic and hepatic bilirubin concentrations and lower lipoperoxidation in mice. Oxid Med Cell Longev. 2019. https://doi.org/10.1155/2019/6026902.

  14. 14.

    Liu H, Du Z, Yuan Q. A novel rapid method for simultaneous determination of eight active compounds in silymarin using a reversed-phase UPLC-UV detector. J Chromatogr B. 2009;877:4159–63.

  15. 15.

    Lee JI, Narayan M, Barrett JS. Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography–electrospray ionization mass spectrometry. J Chromatogr B. 2007;845:95–103.

  16. 16.

    Pendry BA, Kemp V, Hughes MJ, Freeman J, Nuhu HK, Sanchez-Medina A, et al. Silymarin content in Silybum marianum extracts as a biomarker for the quality of commercial tinctures. J Herb Med. 2017;10:31–6.

  17. 17.

    Fibigr J, Šatínský D, Solich P. A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract using UHPLC core-shell column with F5 stationary phase. J Pharm Biomed Anal. 2017;134:203–13.

  18. 18.

    Graf TN, Cech NB, Polyak SJ, Oberlies NH. A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts. J Pharm Biomed Anal. 2016;126:26–33.

  19. 19.

    Smith WA, Lauren DR, Burgess EJ, Perry NB, Martin RJ. A silychristin isomer and variation of flavonolignan levels in milk thistle (Silybum marianum) fruits. Planta Med. 2005;71:877–80.

  20. 20.

    Biedermann D, Buchta M, Holečková V, Sedlák D, Valentová K, Cvačka J, et al. Silychristin: skeletal alterations and biological activities. J Nat Prod. 2016;79:3086–92.

  21. 21.

    Kuki Á, Nagy L, Deák G, Nagy M, Zsuga M, Kéki S. Identification of silymarin constituents: an improved HPLC–MS method. Chromatographia. 2012;75:175–80.

  22. 22.

    Kanu AB, Dwivedi P, Tam M, Matz L, Hill HH. Ion mobility–mass spectrometry. J Mass Spectrom. 2008;43:1–22.

  23. 23.

    Cumeras R, Figueras E, Davis CE, Baumbach JI, Gràcia I. Review on ion mobility spectrometry. Part 1: current instrumentation. Analyst. 2015;140:1376–90.

  24. 24.

    Lee JW, Lee HHL, Davidson KL, Bush MF, Kim HI. Structural characterization of small molecular ions by ion mobility mass spectrometry in nitrogen drift gas: improving the accuracy of trajectory method calculations. Analyst. 2018;143:1786–96.

  25. 25.

    Gabelica V, Marklund E. Fundamentals of ion mobility spectrometry. Curr Opin Chem Biol. 2018;42:51–9.

  26. 26.

    Gabelica V, Shvartsburg AA, Afonso C, Barran P, Benesch JLP, Bleiholder C, et al. Recommendations for reporting ion mobility mass spectrometry measurements. Mass Spectrom Rev. 2019;9999:1–30.

  27. 27.

    Borsdorf H, Eiceman GA. Ion mobility spectrometry: principles and applications. Appl Spectrosc Rev. 2006;41:323–75.

  28. 28.

    Kliman M, May JC, McLean JA. Lipid analysis and lipidomics by structurally selective ion mobility-mass spectrometry. Biochim Biophys Acta. 2011;1811:935–45.

  29. 29.

    Causon TJ, Hann S. Theoretical evaluation of peak capacity improvements by use of liquid chromatography combined with drift tube ion mobility-mass spectrometry. J Chromatogr A. 2015;1416:47–56.

  30. 30.

    Righetti L, Bergmann A, Galaverna G, Rolfsson O, Paglia G, Dall’Asta C. Ion mobility-derived collision cross section database: application to mycotoxin analysis. Anal Chim Acta. 2018;1014:50–7.

  31. 31.

    Paglia G, Angel P, Williams JP, Richardson K, Olivos HJ, Thompson JW, et al. Ion mobility-derived collision cross section as an additional measure for lipid fingerprinting and identification. Anal Chem. 2015;87:1137–44.

  32. 32.

    Stow SM, Causon TJ, Zheng X, Kurulugama RT, Mairinger T, May JC, et al. An interlaboratory evaluation of drift tube ion mobility–mass spectrometry collision cross section measurements. Anal Chem. 2017;89:9048–55.

  33. 33.

    Mairinger T, Causon TJ, Hann S. The potential of ion mobility–mass spectrometry for non-targeted metabolomics. Curr Opin Chem Biol. 2018;42:9–15.

  34. 34.

    Zhang X, Kew K, Reisdorph R, Sartain M, Powell R, Armstrong M, et al. Performance of a high-pressure liquid chromatography-ion mobility-mass spectrometry system for metabolic profiling. Anal Chem. 2017;89:6384–91.

  35. 35.

    Cumeras R, Figueras E, Davis CE, Baumbach JI, Gràcia I. Review on ion mobility spectrometry. Part 2: hyphenated methods and effects of experimental parameters. Analyst. 2015;140:1391–410.

  36. 36.

    May JC, McLean JA. Ion mobility-mass spectrometry: time-dispersive instrumentation. Anal Chem. 2015;87:1422–36.

  37. 37.

    Ouyang H, Bo T, Zhang Z, Guo X, He M, Li J, et al. Ion mobility mass spectrometry with molecular modelling to reveal bioactive isomer conformations and underlying relationship with isomerization. Rapid Commun Mass Spectrom. 2018;32:1931–40.

  38. 38.

    Righetti L, Fenclova M, Dellafiora L, Hajslova J, Stranska-Zachariasova M, Dall’Asta C. High resolution-ion mobility mass spectrometry as an additional powerful tool for structural characterization of mycotoxin metabolites. Food Chem. 2018;245:768–74.

  39. 39.

    Xu Z, Li J, Chen A, Ma X, Yang S. A new retrospective, multi-evidence veterinary drug screening method using drift tube ion mobility mass spectrometry. Rapid Commun Mass Spectrom. 2018;32:1141–8.

  40. 40.

    Fan R-J, Zhang F, Chen X-P, Qi W-S, Guan Q, Sun T-Q, et al. High-throughput screening and quantitation of guanidino and ureido compounds using liquid chromatography-drift tube ion mobility spectrometry-mass spectrometry. Anal Chim Acta. 2017;961:82–90.

  41. 41.

    Ma X, Liu J, Zhang Z, Bo T, Bai Y, Liu H. Drift tube ion mobility and four-dimensional molecular feature extraction enable data-independent tandem mass spectrometric ‘omics’ analysis without quadrupole selection. Rapid Commun Mass Spectrom. 2017;31:33–8.

  42. 42.

    Causon TJ, Ivanova-Petropulos V, Petrusheva D, Bogeva E, Hann S. Fingerprinting of traditionally produced red wines using liquid chromatography combined with drift tube ion mobility-mass spectrometry. Anal Chim Acta. 2019;1052:179–89.

  43. 43.

    Křenek K, Marhol P, Peikerová Ž, Křen V, Biedermann D. Preparatory separation of the silymarin flavonolignans by Sephadex LH-20 gel. Food Res Int. 2014;65:115–20.

  44. 44.

    Fenclova M, Novakova A, Viktorova J, Jonatova P, Dzuman Z, Ruml T, et al. Poor chemical and microbiological quality of the commercial milk thistle-based dietary supplements may account for their reported unsatisfactory and non-reproducible clinical outcomes. Sci Rep. 2019;9:1–12.

  45. 45.

    Mordehai A, Kurulugama R, Darland E, Stafford G, Fjeldsted J (2015) Single field direct drift time to CCS calibration for a linear drift tube ion mobility mass spectrometer. St Louis, p 4

  46. 46.

    Prost SA, Crowell KL, Baker ES, Ibrahim YM, Clowers BH, Monroe ME, et al. Detecting and removing data artifacts in Hadamard transform ion mobility-mass spectrometry measurements. J Am Soc Mass Spectrom. 2014;25:2020–7.

  47. 47.

    Boschmans J, Jacobs S, Williams JP, Palmer M, Richardson K, Giles K, et al. Combining density functional theory (DFT) and collision cross-section (CCS) calculations to analyze the gas-phase behaviour of small molecules and their protonation site isomers. Analyst. 2016;141:4044–54.

  48. 48.

    Galaverna RS, Bataglion GA, Heerdt G, de Sa GF, Daroda R, Cunha VS, et al. Are benzoic acids always more acidic than phenols? The case of ortho-, meta-, and para-hydroxybenzoic acids. Eur J Org Chem. 2015;2015:2189–96.

  49. 49.

    Hinnenkamp V, Klein J, Meckelmann SW, Balsaa P, Schmidt TC, Schmitz OJ. Comparison of CCS values determined by traveling wave ion mobility mass spectrometry and drift tube ion mobility mass spectrometry. Anal Chem. 2018;90:12042–50.

  50. 50.

    Yang P, Xu F, Li H-F, Wang Y, Li F-C, Shang M-Y, et al. Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn. Molecules. 2016;21:1209.

  51. 51.

    Yang Y, Sun X, Liu J, Kang L, Chen S, Ma B, et al. Quantitative and qualitative analysis of flavonoids and phenolic acids in snow chrysanthemum (Coreopsis tinctoria Nutt.) by HPLC-DAD and UPLC-ESI-QTOF-MS. Molecules. 2016;21:1307.

  52. 52.

    Hvattum E. Determination of phenolic compounds in rose hip (Rosa canina) using liquid chromatography coupled to electrospray ionisation tandem mass spectrometry and diode-array detection. Rapid Commun Mass Spectrom. 2002;16:655–62.

  53. 53.

    Nagy L, Kuki Á, Nagy T, Mándi A, Deák G, Nagy M, et al. Collision-induced dissociation study of isosilychristin, a constituent of silymarin. Rapid Commun Mass Spectrom. 2013;27:1413–6.

  54. 54.

    Lee JI, Hsu BH, Wu D, Barrett JS. Separation and characterization of silybin, isosilybin, silydianin and silychristin in milk thistle extract by liquid chromatography–electrospray tandem mass spectrometry. J Chromatogr A. 2006;1116:57–68.

  55. 55.

    Seeff LB, Bonkovsky HL, Navarro VJ, Wang G. Herbal products and the liver: a review of adverse effects and mechanisms. Gastroenterology. 2015;148:517–532.e3.

  56. 56.

    de Avelar CR, Pereira EM, de Farias Costa PR, de Jesus RP, de Oliveira LPM. Effect of silymarin on biochemical indicators in patients with liver disease: systematic review with meta-analysis. World J Gastroenterol. 2017;23:5004–17.

Download references

Acknowledgements

Financial support from the Czech Science Foundation project no. 16-06008S is gratefully acknowledged. This work also received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 692195 (MultiCoop) and was also supported by the METROFOOD-CZ research infrastructure project (MEYS grant no. LM2018100) including access to its facilities, by the Operational Programme Prague – Competitiveness (CZ.2.16/3.1.00/21537 and CZ.2.16/3.1.00/24503) and by the “National Program of Sustainability I” - NPU I (LO1601 - No.: MSMT- 43760/2015), as well as by AZV grant nos 16-27317A and RVO-VFN64165/2017 from the Czech Ministry of Health.

Author information

Correspondence to Milena Stranska-Zachariasova.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 489 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fenclova, M., Stranska-Zachariasova, M., Benes, F. et al. Liquid chromatography–drift tube ion mobility–mass spectrometry as a new challenging tool for the separation and characterization of silymarin flavonolignans. Anal Bioanal Chem (2020) doi:10.1007/s00216-019-02274-3

Download citation

Keywords

  • Silymarin
  • Silybum marianum
  • Milk thistle
  • Ultraperformance liquid chromatography
  • Drift tube ion mobility high-resolution mass spectrometry
  • Collision cross section