Rapid Determination of Endogenous 20-Hydroxyecdysone in Plants on MALDI-TOF/TOF Mass Spectrometry via Chemical Labeling Based on Boronate Affinity

20-Hydroxyecdysone (20E) derived from plants has a wide range of physiological and pharmacological effects on animals and humans, and rapid and sensitive methods for screening of the endogenous 20E in plants are thus required. Herein, a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) method is described for rapid and sensitive determination of endogenous 20E in plants. It is based on the use of the (3-(acridin-9-ylamino) phenyl) boronic acid (AYPBA) as the mass tag to assist the MS and tandem MS (MS n ) analysis of 20E on MALDI-TOF/TOF MS. Good linearity was obtained with a determination coefficient ( R 2 ) larger than 0.99 in the range of 0.025–2.5 μΜ. The limit of detection (LOD) was 2.4 fmol. Acceptable precision and accuracy were gained by intra- and inter-day analysis with relative standard deviations less than 19.5% and relative recoveries ranging from 85.7 to 105.2%. In addition, the AYPBA labeled 20E produced abundant characteristic fragment ions under the high energy collision-induced dissociation, which facilitated the identification of 20E by MS 2 analysis on MALDI-TOF/TOF MS. Using the method, we enabled the identification and quantification of endogenous 20E in four herbs including Cyanotis arachoidea , Achyranthes bidentata , Spinacia oleracea and Chenopodium quinoa willd., demonstrating the feasibility of the proposed method for screening of the endogenous 20E in plants.