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

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.