Determination of endogenous and supplied deuterated abscisic acid in plant tissues by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring*1

Abstract

A specific, sensitive, and accurate method for determination of abscisic acid (ABA) in plant tissues is described. The method employs reversed-phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry for multiple reaction monitoring of underivatized ABA and deuterated ABA analogs. Specific analogs were used to study the mechanism of ABA fragmentation, to select appropriate standards, and to identify compounds suitable for metabolic studies involving the supply of differentially labeled ABA. Limits of detection and quantification of 1.9 and 4.7 pg, respectively, were obtained over a linear calibration range of 0–1.5 ng ABA (on-column injected) using 5,8′,8′,8′-d4 ABA as the internal standard. Accuracy and precision were within 15% for routine quality control samples. The method of standard additions, as applied to Arabidopsis thaliana seed extracts, was also used to validate the method for analysis of plant tissue samples. The utility of the method was further demonstrated by determining levels of ABA in western white pine seeds and of ABA and supplied 8′,8′,8′,9′,9′,9′-d6 ABA in Brassica napus tissues, using 5,8′,8′,8′-d4 ABA or 8′,8′,8′-d3 ABA as the internal standard. Limits of quantification as low as 0.89 ng/g were achieved by optimizing the extraction procedure for each type of plant tissue.