Analysis of the isoprenoid pathway intermediates, dimethylallyl diphosphate and isopentenyl diphosphate, from crude plant extracts by liquid chromatography tandem mass spectrometry

Objective We sought to develop a sensitive and accurate analytical method for the detection and quantification of IDP and DMADP as well as their monophosphate derivatives in crude plant extracts. Methods A liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) was established to measure the amounts of IDP and DMADP down to low picogram levels, which was linear over at least three orders of magnitude. Extracts were enriched using an anion exchanger, and chromatographic separation was achieved using a beta-cyclodextrin column. A S-thiolodiphosphate analog of DMADP was employed as an internal standard. Results Dilution series of authentic compounds were used to determine the limits of detection and quantification for IDP, DMADP and their corresponding monophosphates. A survey of plant species producing varying amounts of isoprenoids showed a corresponding variation in IDP and DMADP with the ratio of DMADP/IDP ranging from 4:1 to 2:1. Trace levels of isopentenyl monophosphate (IP) and dimethylallyl monophosphate (DMAP) were also detected. Conclusion The LC-MS/MS method described enables absolute quantification of in planta levels of IDP and DMADP for the first time. The method is also suitable for analysing bacterial and animal samples as well as enzyme assays.