Improvements in methodology for liquid chromatographylcontinuous flow secondary-ion mass spectrometry (LC/CFSIMS) are described. Practical procedures have been developed for the preparation of packed fusedsilica capillary liquid chromatography columns and the coupling of these columns to the CFSIMS probe. A modified probe tip is described that significantly reduces ion beam instability arising from acceleration voltage discharge, changes in mobile phase composition during gradient elution, and uneven tip wettability. Interfering matrix background ions in LC/CFSIMS experiments are eliminated by reaction monitoring: the detection limit of the plant hormone metabolite, abscisic acid glucose ester, ABAGE, is below lOOpg (23Sfmol) with this technique. Reproducibility for quantitation is achieved by parallel reaction monitoring of the analyte and an added internal standard, ABAGE-d6. The dddo ABAGE response ratio for the monitored transitions is linear over the range 100 pg to 10 ng of injected ABAGE.The plant hormone abscisic acid (ABA) and its metabolites are present in plants and plant cells at levels in the range to 1 0 -8~. ' Abscisic acid glucose ester (ABAGE) the formula of which is shown below, is a common conjugate of ABA. It is difficult to characterize and quantify as it is prone to decomposition or isomerization during analysis.'.Most previous determinations of ABAGE and other ABA-related conjugates have been performed by detecting the products after removal of the sugar moiety by alkaline hydrolysis, a procedure which does not allow characterization of the specific ~o n j u g a t e .~'~ Direct chemical ionization (DCI) mass spectrometry has been used6 to characterize these types of compounds after they have been isolated from plants and purified. However, to our knowledge, there have been no reports of on-line gas chromatography separations combined with CI mass spectrometry as the conjugates are not amenable to gas chromatography without derivatization.High-performance liquid chromatography (HPLC) has been of value for the analysis of ABA and ABA metabolite^.^, This technique provides the advantage of permitting separation and quantitation without prior derivatization. There is, however, a need for more sensitive methods for the detection and characterization of the components of the LC eluates. Until now, detection of the metabolites has been based on UV absorption, or, in cases in which radioisotopicallyAuthor to whom correspondence should be addressed.labelled ABA has been supplied to metabolically-active plant material, radioactivity detection. This laboratory has reported the application of plasmaspray and ionspray LC/MS techniques to the detection of several ABA metabolite^.^ As part of our continuing effort to develop better methods for the separation, identification and quantification of conjugated ABA metabolites, we have investigated the use of packed fused-silica capillary column LC coupled with continuous flow secondary ion mass spectroscopy (LCICFSIMS). LC/CFSIMS has been shown to be very use...

Racemic abscisic acid (ABA), the cis and trans l', 4'-diols (ABA diols) derived from ABA by reduction of the 4' ketone, and the corresponding 4'-0-acetates were converted into various acetosugar esters by reaction of their cesium salts with the I-chloroacetosugars derived from glucose, galactose, lactose, and maltose. Analytical separations of the acetosugar esters using high-performance liquid chromatography (LC) on reverse-phase columns were developed. Continuous flow secondary ion mass spectra (CFSIMS) of the various acetosugar esters were obtained and an LCICFSIMS protocol employing multiple reaction monitoring was used to detect ABA acetoglucose ester in an acetylated extract obtained from plant cells that had been treated with ABA.Key words: abscisic acid, acetosugar esters, synthesis, chromatography, mass spectrometry. Fig. 1) is a plant growth regulator that has also been implicated in a variety of stress-related responses (1). Several metabolites of endogenous and exogenously added ABA have been isolated and identified, including the reduction products, cis and trnrzs ABA l', 4'-diols (2-5); the oxidized derivative, phaseic acid (6-8); and the glucose esters (P-D-glucose, C-1-linked) of ABA and the aformentioned compounds (Fig. 1) (9-12). There has also been a report of the detection of a maltose ester of ABA (13).We were interested in following ABA metabolism, and in developing analytical methods to facilitate our studies; in particular, we sought to extend the utility of liquid chromatography-continuous flow secondary ion mass spectrometry (LC/ CFSIMS), which we had earlier used to detect ABA glucose ester in a plant extract (14), to other ABA-derived conjugates, which we felt were potential metabolites. To achieve these objectives, samples of potential metabolites or suitable derivatives thereof were required as standards.Previously, ABA glucose ester (P anomer) had been prepared by reaction of ABA (cesium or triethyl ammonium salt) with a-bromoacetoglucose followed by deacetylation of the intermediate acetoglucose ester with an enzyme preparation derived from sunflower seeds (Helianthus arirzus) (15, 16). ABA diols had been prepared by sodium borohydride reduction of ABA methyl ester, which yielded a mixture rich in both the 1', 4'-cis and -trans diols (17).To further extend earlier synthetic efforts as well as the use of LCICFSIMS in analysis of metabolite mixtures, we report here the preparation of various acetosugar derivatives of ABA (and deuterated ABA) and the diols, and acetylated diols of ABA, and the development of LC and LCICFSIMS protocols using these derivatives. The acetosugar derivatives were easily prepared and possessed good stability. Some deuterated analogs were prepared as an aid in assigning fragments in the mass spectrum and for potential use as internal standards. As an example of the utility of the developed protocol, a sample plant extract was analyzed.