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Improving Resolution of Isomeric Flavonoids and Their Glycosides Using Two-Dimensional Liquid Chromatography Coupled With High-Resolution Mass Spectrometry

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Abstract

The separation of isomers is one of the key issues in analysing complex natural matrices, thus commanding a higher separating power from the applied analytical system. In this study, 42 common flavonoid glycosides from three key subclasses (flavanones, flavones, flavonols) were analysed in three steps to maximise chromatographic and spectrometric resolution via: (1) C18 liquid chromatography (LC) coupled to a quadrupole time-of-flight mass spectrometer (QTOF/MS), (2) tandem mass spectrometry (MS/MS), and (3) two-dimensional LC with heart-cutting for specific regions of co-elution. While the LC-QTOF/MS step was found to separate the majority of flavonoid glycoside isomers, some isomers with identical accurate mass were not resolved. Following this, increased spectrometric resolution was achieved with MS/MS; however, this technique was not suitable for particularly challenging isomers that were also identical in aglycone and saccharide structure. Furthermore, a heart-cutting method was developed using a three-level full-factorial design to separate such challenging isomers, and satisfactory resolution was obtained for the targeted isomers (R > 2.00) with reasonable efficiency (total run time < 25 min) and repeatability (RSD < 2%). In conclusion, the developed strategy highlights the significance of combining high-resolution mass spectrometry with chromatographic separation for added confidence in the identification of isomers.

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Abbreviations

1D:

First dimension

2D:

Second dimension

2D-LC:

Two-dimensional liquid chromatography

AM:

Accurate mass

CE:

Collision energy

CID:

Collision-induced dissociation

ESI:

Electrospray-ionisation

HRMS:

High-resolution mass spectrometry

LC:

Liquid chromatography

LC-QTOF/MS:

Liquid chromatography quadrupole time-of-flight mass spectrometry

MDLC:

Multi-dimensional liquid chromatography

MS/MS:

Tandem mass spectrometry

PFP:

Pentafluorophenyl

UHPLC:

Ultra-high-performance liquid chromatography

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Acknowledgements

The authors are grateful to Mane SEA Pte Ltd for providing technical assistance and funding for this study.

Funding

This study was funded by Mane SEA Pte Ltd (3 Biopolis Drive, #07-17/18/19 Synapse, Singapore 138623), with support from the Department of Food Science and Technology, National University of Singapore (S14 Level 5, Science Drive 2, Singapore 117542).

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Authors

Contributions

Conceptualization: AP, RMVG, K-HE, BY; Methodology: RMVG, AP, K-HE; Formal analysis and investigation: RMVG, AP, K-HE; Writing—original draft preparation: AP, RMVG, K-HE, YH, BY; Writing—review and editing: AP, RMVG, K-HE, YH, BY, SQL, BL; Funding acquisition: BL, SQL; Resources: BL, SQL; Supervision: K-HE, BY, BL, SQL.

Corresponding authors

Correspondence to Shao Quan Liu or Bin Yu.

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The authors have no relevant competing financial and non-financial interests to disclose.

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Pua, A., Goh, R.M.V., Ee, KH. et al. Improving Resolution of Isomeric Flavonoids and Their Glycosides Using Two-Dimensional Liquid Chromatography Coupled With High-Resolution Mass Spectrometry. Chromatographia 84, 507–515 (2021). https://doi.org/10.1007/s10337-021-04027-w

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  • DOI: https://doi.org/10.1007/s10337-021-04027-w

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