Methods for the quantitation of abscisic acid and its precursors from plant tissues

Methods are given for the quantitation of the plant stress hormone, abscisi c acid (ABA), and its two metabolic precursors, ketone and enolate, that ar e applicable to all species tested so far. The plant extract is homogenized at neutral pH, hexane-soluble neutrals are extracted and discarded, and th en the free ABA and other organic acids are extracted as ion pairs. The rem aining aqueous phase is acidified, allowed to stand, neutralized, and extra cted to ave the ABA ex ketone fraction and then the aqueous phase is treate d with base and again extracted to give the ABA ex enolate fraction. Each o f these three fractions, free ABA, ABA ex ketone, and ABA ex enolate, along with a deuteriated internal standard, [side-chain-H-2(4)]ABA, is then deri vatized with pentafluorobenzyl bromide and purified on an automated sample preparation system. The resulting pentafluorobenzyl abscisate samples are t hen quantified using electron capture negative ionization mass spectrometry with methane as the reagent gas. Using these procedures free ABA, and ABA from its precursors, can be quantified at the level of 100 fg on column. If a large volume injector is used so that the total sample is injected it sh ould be possible to quantify ABA and its precursors in the parts per billio n range on a few milligrams of plant tissue. (C) 2001 Academic Press.