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Anal Bioanal Chem (2015) 407:48414848 DOI 10.1007/s00216-015-8668-4
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High-speed homogenization coupled with microwave-assisted extraction followed by liquid chromatographytandem mass spectrometry for the direct determination of alkaloidsand flavonoids in fresh Isatis tinctoria L. hairy root cultures
Jiao Jiao1 & Qing-Yan Gai2,3 & Lin Zhang2,3 & Wei Wang2,3 & Meng Luo2,3 &
Yuan-Gang Zu2,3 & Yu-Jie Fu2,3
Received: 6 January 2015 /Revised: 12 March 2015 /Accepted: 27 March 2015 /Published online: 17 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract A new, simple and efficient analysis method for fresh plant in vitro culturesnamely, high-speed homogenization coupled with microwave-assisted extraction (HSH MAE) followed by liquid chromatographytandem mass spectrometry (LCMS/MS)was developed for simultaneous determination of six alkaloids and eight flavonoids in Isatis tinctoria hairy root cultures (ITHRCs). Compared with traditional methods, the proposed HSHMAE offers the advantages of easy manipulation, higher efficiency, energy saving, and reduced waste. Cytohistological studies were conducted to clarify the mechanism of HSHMAE at cellular/ tissue levels. Moreover, the established LCMS/MS method showed excellent linearity, precision, repeatability, and reproducibility. The HSHMAELCMS/MS method was also successfully applied for screening high-productivity ITHRCs. Overall, this study opened up a new avenue for the direct determination of secondary metabolic profiles from fresh plant in vitro cultures, which is valuable for improving quality control of plant cell/organ cultures
and sheds light on the metabolomic analysis of biological samples.
Keywords Isatis tinctoria hairy root cultures . High-speed homogenization . Microwave-assisted extraction . Alkaloids . Flavonoids . Liquid chromatographytandem mass spectrometry
Introduction
Isatis tinctoria L. (Cruciferae) is a biennial herbaceous plant that is widely distributed in China and other Asian countries.I. tinctoria root (Radix isatidis) is used in traditional Chinese medicine for the treatment of inflammatory diseases [1]. Over the past decades, numerous compounds belonging to various structural classes such as alkaloids, flavonoids, fatty acids, porphyrins, lignans, carotenoids, glucosinolates, cyclohexenones, amino acids, and isoprenoids have been identified in I. tinctoria [13]. Moreover, extraction methods such as supercritical fluid extraction and pressurized liquid extraction have been applied to obtain extracts from I. tinctoria leaves for the analysis of chemical compositions [25].I. tinctoria hairy root cultures (ITHRCs) generated from the genetic transformation of Agrobacterium rhizogenes have become feasible to produce active compounds effectively, economically, and in an environmentally friendly way [6]. Therefore, the establishment...