High-speed homogenization coupled with microwave-assisted extraction followed by liquid chromatography–tandem mass spectrometry for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria L. hairy root cultures

A new, simple and efficient analysis method for fresh plant in vitro cultures—namely, high-speed homogenization coupled with microwave-assisted extraction (HSH–MAE) followed by liquid chromatography–tandem mass spectrometry (LC–MS/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 HSH–MAE offers the advantages of easy manipulation, higher efficiency, energy saving, and reduced waste. Cytohistological studies were conducted to clarify the mechanism of HSH–MAE at cellular/tissue levels. Moreover, the established LC–MS/MS method showed excellent linearity, precision, repeatability, and reproducibility. The HSH–MAE–LC–MS/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.