Rapid profiling of potential antitumor polymethoxylated flavonoids in natural products by integrating cell biospecific extraction with neutral loss/diagnostic ion filtering-based high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry

Introduction Citri Reticulatae Pericarpium Viride (CRPV, Qing Pi in Chinese) has been widely used in traditional Chinese medicine. Polymethoxylated flavonoids (PMFs), which are a special group of flavonoids with strong antitumor activity, are broadly distributed in citrus peels. However, systematic investigation of antitumor PMFs in CRPV has received little attention to date. Objectives An MCF-7 cell biospecific extraction method integrated with neutral loss/diagnostic ion filtering-based HPLC-QTOF-MS/MS strategy was developed for rapid and specific profiling of antitumor PMFs and systematic identification of PMFs in CRPV. Methodology By incubating MCF-7 cells with CRPV extract, potential antitumor PMFs specifically bound to cells and were isolated. Then, by systematic investigation of fragmentation pathways, neutral loss and diagnostic ion filtering strategies were proposed to comprehensively and accurately identify PMFs. Results Sixteen antitumor PMFs were unambiguously or tentatively identified. Among them, minor compound 15 (5-hydroxy-6,7,8,3 ',4 '-pentamethoxyflavone with a free hydroxyl group at C-5) exhibited excellent antitumor activity, with an IC50 value of 2.81 +/- 0.76 mu g/mL, which is lower than that of 5-fluorouracil (IC50, 4.92 +/- 0.83 mu g/mL). Nobiletin (12) and tangeretin (16), two major PMFs, presented moderate antitumor activities with IC50 values of 13.06 +/- 1.85 and 17.07 +/- 1.18 mu g/mL, respectively, and their contents were sensitively and precisely determined. Conclusions To the best of our knowledge, this is the first report on the systematic investigation of antitumor PMFs in CRPV. The study will lay a foundation for the quality control and clinical application of CRPV.