ORIGINAL Determination of Metabolism of Neohesperidin by Human Intestinal Bacteria by UPLC-Q-TOF/MS Wei Zhang • Shu Jiang • Dawei Qian • Er-xin Shang • Jin-ao Duan Received: 5 November 2013 / Revised: 11 December 2013 / Accepted: 16 December 2013 / Published online: 17 January 2014 Ó Springer-Verlag Berlin Heidelberg 2014 Abstract Neohesperidin as the major isoflavonoid in Aurantii Fructus has been investigated intensively. How- ever, the route and metabolites of neohesperidin by human intestinal bacteria are not well understood and its metabo- lites may accumulate and exert physiological effects. In this work, 5 strains including Clostridium sp.8, Bacteroides sp.15, Bacillus sp.46, and Enterobacter sp.41-1 and sp.73 were isolated and their abilities to convert neohesperidin were studied. Human fecal microflora were prepared from a healthy Chinese woman and then anaerobically incubated with neohesperidin sample at 37 °C for 48 h. A rapid and simple liquid–liquid extraction method was used for sample pretreatment. A highly sensitive and specific ultra perfor- mance liquid chromatography/quadrupole time-of-flight mass spectrometry with MetaboLynx TM method has been developed for the analysis of neohesperidin and related metabolites in the reaction samples. The chromatographic separation was performed on a 1.7 lm particle size Sync- ronis C 18 column using gradient elution system. The com- ponents in the extract were identified and confirmed according to the mass spectrometric fragmention mecha- nisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of five metabolites were detected, the results indicated that hydrolysis, dehydroxylation, demethylation and acetylation were the major metabolism of neohesperidin. The present study provided important information about the metabolism of neohesperidin which will be helpful for fully under- standing the impact of the intestinal bacteria on this active component. Furthermore, this work demonstrated the potential of the ultra performance liquid chromatography/ quadrupole time-of-flight mass spectrometry approach using MetaboLynx TM for quite rapid, simple, reliable and automated identification of metabolites of natural product. Keywords UPLC-Q-TOF/MS Á Neohesperidin Á Fecal bacteria Á Metabolism Introduction Aurantii Fructus (Zhiqiao), the dried, immature fruit of Citrus aurantium L., is a well-known traditional Chinese medicine (TCM) with antioxidant, anti-tumor, antihyper- tension, anti-shock, etc. activities. Flavonoids are considered to be the major bioactive constituents of Aurantii Fructus [1]. The flavanone glycoside neohesperidin which composed of the flavanone hesperetin and the disaccharide neohesperi- doside is a plentiful and inexpensive by-product of Aurantii Fructus [2]. Neohesperidin has been investigated intensively and has been found to possess a wide range of pharmaco- logical properties, such as anti-proliferative effect on human hepatoma cell lines [3], the suppression of infection-induced endotoxin shock in mice [4], anti-allergic, antioxidant, anti- estrogen, neuroprotective activity [5], and the inhibition of tumor promotion [6]. It has free radical scavenging activity [7], as well as protective effects on potential gastric diseases W. Zhang (&) Á S. Jiang Á D. Qian Á E. Shang Á J. Duan Laboratory for High Technology Research of TCM Formulae, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210046, People’s Republic of China e-mail: zhangwei880518@163.com D. Qian e-mail: qiandwnj@126.com J. Duan e-mail: dja@njutcm.edu.cn 123 Chromatographia (2014) 77:439–445 DOI 10.1007/s10337-014-2625-9