Rapid extraction of abscisic acid and its metabolites for liquid chromatography–tandem mass spectrometry

Abstract

We have described a simple, reliable and rapid method of extracting and partially purifying the phytohormone (+)-abscisic acid and its catabolites for liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS) analysis. Lyophilized tissue samples were powdered by high-speed agitation with ceramic beads for 5 s. Metabolites were extracted from the tissue powder using acetone–water–acetic acid (80:19:1, v/v) with the addition of deuterated internal standards for quantification. Essentially all endogenous hormones were recovered by three successive tissue extractions. However we demonstrated that, with the use of internal standards, one extraction with vigorous vortexing was sufficient to obtain accurate results (recovery 65–90%). Solvents were optimized for partial purification of abscisic acid and related compounds by solid-phase extraction using Oasis HLB cartridges. The eluted metabolites were then analyzed by LC–MS–MS. To illustrate the applicability of these techniques, we analyzed the levels of abscisic acid and metabolites in seeds and valves of Brassica napus siliques at two stages of development. We detected abscisic acid, phaseic acid, 7′-hydroxyabscisic acid, dihydrophaseic acid and abscisic acid glucose ester. In both tissues, dihydrophaseic acid was the major accumulating product, reaching 97 300 pmol/g dry mass in valves at 24 days after anthesis. The amount of abscisic acid in seeds was high at 24 days after anthesis (23 300 pmol/g dry mass), but low in the other samples (292–447 pmol/g dry mass).