Abstract
Purpose: To develop and validate a chromatographic method for the simultaneous determination of plasma levels of rutin, avicularin and quercitrin using UHPLC-Q-Exactive-Orbitrap-MS.
 Methods: A sensitive, selective, and reliable UHPLC-Q-Exactive-Orbitrap-MS method was developed and validated for simultaneous determination of the three flavonoids, with puerarin as internal standard (IS). Plasma samples were first treated with methanol, and then acidified using hydrochloric acid (HCl) prior to liquid-liquid extraction with ethyl acetate. The flavonoids were separated on a Syncronis C18 column (100×2.1mm, 1.7 µm) using an elution gradient of acetonitrile and 0.1 % formic acid at a flow rate of 0.3 mL/min.
 Results: A linear correlation was obtained for the three flavonoids over the investigated concentration range, with correlation coefficients > 0.9954. The values of validated lower limit of quantification (LLOQ) were 0.68, 1.42 and 2.54 ng/mL for rutin, avicularin and quercitrin, respectively. Intra- and inter-day precision (RSD) were < 10 %, while accuracy (RE) ranged from −3.76 to 4.04 %.
 Conclusion: The proposed method has been successfully validated and is suitable for studying the pharmacokinetics of the three analytes in rats treated with parasitic loranthus extract (PLE).
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