Isomeric quantification of O-diglycosyl flavonoids by a complex-free kinetic method using ESI/QToF mass spectrometry Kuangcai Chen a , Hua Jin a,c , Yanwei Li b , Junyoung Shin a , Jae-Min Lim a , Yong-Ill Lee a, ⁎ a Anastro Laboratory, Department of Chemistry, Changwon National University, Changwon 641-773, Republic of Korea b Environment Research Institute, Shandong University, Jinan 250100, China c Yanbian University Hospital, Yanji, Jinlin, 133000, China abstract article info Article history: Received 30 May 2014 Accepted 9 June 2014 Available online 17 June 2014 Keywords: Kinetic method Flavonoids Structural isomers QToF The complex-free kinetic method (CFKM), a novel variant of the kinetic method, was developed for direct quantification of isomeric O-diglycosyl flavonoids using electrospray ionization tandem mass spectrometry. Direct isomeric quantification of O-diglycosyl flavonoids by the developed CFKM was presented for the first time. The detailed fragmentation behaviours of these flavonoids, which are tightly related to their structures, were also proposed with the help of molecular modelling. The calibration curves enable the quantification of these isomeric pairs by the linear relationship of lnR vs. molar fractions of hesperidin and eriocitrin with good accuracy and good correlation coefficient(r2). High isomeric selectivity was achieved with Riso = 6.5349 and Riso = 3.5257 for hesperidin/neohesperidin and eriocitrin/neoeriocitrin, respectively by our developed CFKM. The CFKM represents a promising and practical mass spectrometric method for direct stereochemical analysis of flavonoid compounds as well as other multiple chiral center compounds. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Flavonoids are the most common group of polyphenolic phytochemicals as secondary metabolites and are distributed ubiq- uitously in the edible parts of plants. The biological activities and chemopreventive properties of flavonoids make them favourably linked with reduced risks of numerous diseases and thus aroused great interest [1–5]. Glycosylated flavonoids are the main forms of naturally occurring flavonoids. They can be O- and/or C-glycosylated. Two O- diglycosides, rutinos [rhamnosyl-(α1 → 6)-glucose] and neohesperidose [rhamnosyl-(α1 → 2)-glucose] are very common disaccharides found in the flavonoid glycosides, these two compounds differ merely by the interglycosidic linkage. Considerable efforts have been devoted to the structural characterization of the glycosylated flavonoids by mass spectrometry (MS) due to their great sensitivity, specificity, tolerance to impurities and low sample consumption. However, these studies were qualitative [6–11]. To the best of our knowledge, there is no report of attempting quantitative studies based on the glycosides exclusively by MS. The mass spectrometric methods for differentiation and quantifica- tion of chiral compounds are classified into the following five wide types: (a) host-guest diastereomeric adducts formation [12] ; (b) ion/molecule reactions [13]; (c) collision-induced dissociation of diastereomeric adducts [14]; (d) solution-phase kinetic resolution [15]; (e) kinetic method [16]. However, all of them require adding chiral selectors to form the diastereomeric complex ions of interest, which limits the coupling of these strategies with separation techniques. The kinetic method was introduced by Cooks et al. [17–19] for ther- mochemical determinations and then developed for stereochemical analysis and applied to various amino acids [20,21], isomers [22,23], α-hydroxy acids [24], drugs [25–28]. So far, four types of kinetic method including single ratio kinetic method (SRKM) [20], single ratio fixed- ligand kinetic method (SR fixed KM) [26], quotient ratio kinetic method (QRKM) [21] , and quotient ratio fixed-ligand kinetic method (QR fixed KM) [27] were developed by Cooks et al. for stereochemical analysis and were discussed in our recent review paper [29] based on our studies [23,28]. In contrast to the broad investigations of kinetic method in static system, only one report was found attempting to apply it in a flowing system, both by flow injection analysis and a chiral detection after achiral separation by HPLC [30]. The expected worse ac- curacy compared to that in a static system was due to the influence of the experimental conditions such as the chiral selectors (metal ion and chiral reference) properties and concentrations as well as the flow rate. In this work, we propose the complex-free kinetic method (CFKM) without the use of metal ions and reference compounds to offer the potential capacity to combine with other separation techniques. The present study also describes the direct application of the CFKM to the discrimination and quantification of isomeric O-diglycosyl flavonoids implemented on a commercial ESI/QTof Mass Spectrometry. Microchemical Journal 117 (2014) 46–51 ⁎ Corresponding author at: Department of Chemistry, Changwon National University, Changwon 641-773, Republic of Korea. E-mail address: yilee@changwon.ac.kr (Y.-I. Lee). http://dx.doi.org/10.1016/j.microc.2014.06.009 0026-265X/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc