1. Non-anthocyanin polyphenols quantification in Euterpe oleracea fruits by an UHPLC−LTQ-Orbitrap MS method ALS Dias 1, E Rozet 2, L Margalho 3, Y Larondelle 4, Ph Hubert 2, H Rogez 3, J Quetin-Leclercq 1RESULTS ACKNOWLEDGEMENTS 1 Laboratoire de Pharmacognosie, LDRI, UCL, Av. E. Mounier, 72, 1200 Brussels, Belgium. 2 Laboratoire de Chimie Analytique, Département de Pharmacie, CIRM, ULg, CHU, B36, B-4000 Liège, Belgium. 3 Faculdade de Engenharia de Alimentos, UFPA & CVACBA, Av. Perimetral s/n, 66.095-780 Belém-PA, Brazil. 4 Institut des Sciences de la Vie, UCL, Place Croix du Sud 2 bte8, B-1348 Louvain- la-Neuve, Belgium INTRODUCTION EXPERIMENTAL Fig. 2 Sample preparation and analysis Application of the method Fig. 1 Fruits and fruit juice of E. oleracea FFruits were harvested in Para state (Brazil). Sample preparation is showed in Fig. 2. FUHPLC conditions: C18 HSS column, 1.8 µm, 100-2.1 mm, Waters, mobile phase gradient of solvent A: 0.1% HCOOH in H 2 O and B: 0.1% HCOOH in MeOH. Flow rate of 400µL/min. Total run time of only 35 min. FMS conditions: LTQ-Orbitrap XL from the MASSMET platform. ESI in NI mode with the following conditions: a capillary temperature and voltage of 275 °C and −21 V respectively. The source voltage was set at 5 kV and the tube lens voltage at −162 V. N 2 was used as the sheath gas and helium as auxiliary gas with a flow rate of 80 and 30 arbitrary units respectively. FStandard solutions: Standards of 12 flavonoids were prepared without and in the matrix at 5 concentration levels (m=5). Each one was analysed three times (n=3) for 3 series of experiments (k=3). The validation standards were prepared in the same way as the matrix based calibration standards (m=5, n=3, k=3). 7-OH flavone was used as internal standard. FValidation of the method: Three series (k=3) of experiments were carried out and the validation criteria tested (Table 1). Total error was used as decision criterion for the validation process [4]. Fruits Pulp Lyophilised pulp (0.5g) Solubilisation 40% MeOH Speed Vac concentration 35°C Clean-up petroleum ether extraction by sonication 5x30minx5mL Residue MeOH extraction by sonication 5x30minx5mL UHPLC−LTQ-Orbitrap MS CONCLUSION We validated for the first time the quantification of 12 non-anthocyanin flavonoids of E. oleracea fruit juice using an UHPLC−LTQ-Orbitrap MS method. The method was selective and gave good estimators of linearity, accuracy, trueness and precision. Calibrations in the matrix showed better accuracy profiles in comparison to those without matrix. Extraction time was also optimized. The chromatographic separation allowed separation of the major compounds and even of isomers. In addition eriodictyol was identified for the first time in this fruit by HRMS and MS/MS spectra and comparison with a standard sample. We would like to thank Prof. J.-L Habib-Jiwan for his scientific orientation (in memoriam), Pr G. Muccioli, J. Masquelier and Dr. M-F Hérent for their kind help with LC-MS experiments, E. Lautié for her scientific and technical advices, M.–C. Fayt, J.P. Vanhelleputte and R. Colak for their technical support in the LC-MS system. Dr. V. Baeten from CRA-W who kindly donated some commercial standards is acknowledged. ADRI-UCL is acknowledged for financial support to the first author, Belgian National Fund for Scientific Research (FNRS) (FRFC 2.4555.08), the Special Fund for Research (FSR) and the faculty of medicine of UCL for their financial support for the acquisition of the LTQ-Orbitrap-XL, the program PIC of the Coopération universitaire au développement (CUD), and CNPq (Brazil) for financial support on this research. A research grant from the Belgium National Fund for Scientific Research (FRS-FNRS) to E. Rozet is gratefully acknowledged. REFERENCES [1] C.M.G. Bichara et al., in: E.M. Yahia (Ed), vol 2, Woodhead Publishing, Cambridge, UK, 2011, p. 2-10 [2] J. Kang et al., Food Chem. 122 (2010) 610-617 [3] J. Kang et al., Food Chem. 128 (2011) 152-157 [4] Ph. Hubert et al., J. Pharm. Biomed. Anal. 36(2004)579 E. oleracea is a widespread palm of the Amazonian floodplains. Its fruits (Fig. 1) are especially rich in phenolic compounds and were recently acclaimed as “super fruit” due to their extremely high antioxidant capacity and potential anti-inflammatory activities [1-3]. Accurate quantification of these compounds is very important for the post-harvest and food industry. The aim of this work was to quantify major non-anthocyanin polyphenols in fruit juices by a validated UHPLC−LTQ-Orbitrap MS method after having optimized the extraction procedures and the sample preparation. Samples and pretreatment Sample preparation Analysis Fig. 3 Optimization of the time of the MeOH extraction (n = 3). a The extraction yields were calculated according to the total extract mass obtained in each treatment Compounds Dosing range (µg/mL) Accuracy a Linearity (Slope / Intercept / R 2 ) LOD / LOQ (µg/mL) Isoorientin6 − 30 -21.99 − 26.531.003 / -0.2233 / 0.98740.57 / 5.78 Quercetin0.5 − 10 -25.63 − 25.400.9909 / -0.02897 / 0.99570.13 / 0.45 (+)-Dihydrokaempferol0.5 − 14 -23.32 − 18.010.9944 / -0.03389 / 0.99600.12 / 0.5 Isovitexin1− 14 -27.09 − 19.690.9861 / -0.05954 / 0.99340.26 / 0.85 Rutin1− 14 -22.22 − 20.770.9974 / -0.08971 / 0.98780.21 / 0.72 (+)-Catechin0.5 − 14 -24.68 − 26.90.9822 / -0.03031 / 0.99640.15 / 0.5 Eriodictyol0.5 − 10 -17.44 − 19.271.011 / 0.005701 / 0.99600.04 / 0.15 Chrysoeriol0.2 − 10 -19.60 − 23.731.001 / -0.02018 / 0.99570.06 / 0.20 Quercetin 3-glucoside0.5 − 10 -23.68 − 26.551.012 / -0.06427 / 0.97720.14 / 0.47 Kaempferol 3-rutinoside0.5 − 14 -21.03 − 23.260.9837 / 0.005535 / 0.99550.14 / 0.46 Luteolin0.5 − 10 -15.73 − 25.460.9642 / 0.05855 / 0.99750.14 / 0.47 Orientin3 − 18 -28.27 − 16.420.8630 / 0.3059 / 0.99200.81 / 2.69 Table 1 Validation results for quantification method of non-anthocyanin polyphenols of E. oleracea fruits a Accuracy was evaluated by the relative β−expectation tolerance limits Sample preparation Validation of the quantitative procedure a b c b a a b c b a a Concentration estimated in equivalents of Isovitexin, b Rutin and c (+)-Catechin. nq: unquantifiable (<LOQ) Fig 4 Non-anthocyanin flavonoids quantification in E. oleracea fruit juices