Development of a Validated UHPLC-ESI (-)-HRMS Methodology for the Simultaneous Quantitative Determination of Hesperidin, Hesperetin, Naringin, and Naringenin in Chicken Plasma

  • Eirini Baira
  • Ioanna Dagla
  • Eleni Siapi
  • Panagiotis Zoumpoulakis
  • Anthony Tsarbopoulos
  • Panagiotis Simitzis
  • Michael Goliomytis
  • Stelios G Deligeorgis
  • Alexios-Leandros Skaltsounis
  • Evagelos GikasEmail author


A highly sensitive and specific methodology has been developed for the simultaneous determination of the flavonoids hesperidin, hesperetin, naringin, and naringenin in chicken plasma employing UHPLC-HRMS (Orbitrap Velos). Plasma samples were preprocessed by protein precipitation with cold acetone. Analysis was carried out on an INTERCHIM UHPLC C18 column using aq. 0.1% glacial acetic acid, acetonitrile, and isopropanol/acetonitrile/acetone (58/40/2, v/v) as the mobile phase. Detection was performed by means of electrospray ionization (ESI) in the negative ion. All calibration curves exhibited good linearity (r2 > 0.990) over the concentration range of 0.005 to 1 μg/mL with a lower limit of quantification (LLOQ) of 0.005 μg/mL for the four analytes. The repeatability and precision were within %RSD < 20 and accuracy within %Error < 20. No matrix effect or carry over was observed for the proposed methodology. Furthermore, as the quantitation procedures employing an Orbitrap analyzer are yet not well established, the impact of various parameters of MS/MS-based quantitation has been examined in order to achieve analytically solid results. The methodology was applied in plasma samples after dietary supplementation with 0.75 g/kg of feed (low-dose treatment) and 1.5 g/kg of feed (high-dose treatment) of hesperidin and naringin in Ross 308 broiler chickens.


Ion trap FTMS Quantitation Naringin Hesperidin Validation Bioavailability Plasma 



This work was supported by the Hellenic State and European Union under Grand MIS 380231, Project “Thalis—The effects of antioxidant’s dietary supplementation on animal product quality.”

Compliance with Ethical Standards

Conflict of Interest

Eirini Baira declares that she has no conflict of interest. Ioanna Dagla declares that she has no conflict of interest. Eleni Siapi declares that she has no conflict of interest. Panagiotis Zoumpoulakis declares that he has no conflict of interest. Anthony Tsarbopoulos declares that he has no conflict of interest. Panagiotis Simitzis declares that he has no conflict of interest. Michael Goliomytis declares that he has no conflict of interest. Stelios G Deligeorgis declares that he has no conflict of interest. Alexios-Leandros Skaltsounis declares that he has no conflict of interest. Evagelos Gikas declares that he has no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All experimentation was carried out in strict accordance with the guidelines of “Council Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes.” The protocol was approved by the Bioethical Committee of the Agricultural University of Athens (Permit Number: 20/20032013). This article does not contain any studies with human participants performed by any of the author.

Informed Consent

Not applicable.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Eirini Baira
    • 1
  • Ioanna Dagla
    • 1
  • Eleni Siapi
    • 2
  • Panagiotis Zoumpoulakis
    • 2
  • Anthony Tsarbopoulos
    • 3
  • Panagiotis Simitzis
    • 4
  • Michael Goliomytis
    • 4
  • Stelios G Deligeorgis
    • 4
  • Alexios-Leandros Skaltsounis
    • 5
  • Evagelos Gikas
    • 1
    Email author
  1. 1.Department of Pharmaceutical Chemistry, Faculty of Pharmacy, School of Health SciencesNational and Kapodistrian University of AthensAthensGreece
  2. 2.Institute of Biology, Medicinal Chemistry and BiotechnologyNational Hellenic Research FoundationAthensGreece
  3. 3.Department of Pharmacology, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  4. 4.Department of Animal Breeding and Husbandry, Faculty of Animal Science and AquacultureAgricultural University of AthensAthensGreece
  5. 5.Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, School of Health SciencesNational and Kapodistrian University of AthensAthensGreece

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