ABSTRACIA rapid, sensitive, and selective method for the determination of betaines is described and discussed. The method entails derivatizing the quaternary ammonium compounds to increas their sensitivity to detection by fast atom bombardment mass spectrometry. Sensitivity of detection increases markedly as the length of the carbon chain of the alcohol used to esterify the betaine carboxylic acid group is inreased (C4 > C3 > C2 > Cl > Ca). The lower limit of detection of glycine betaine as the i-propyl ester is 0.05 nanomole per microliter of glycerol. Betaine aldehyde can be readily derivatized to the di-n-butyl or di-n-propyl acetal derivatives which exhibit lower limits of detection of about 5 picomoles and 10 picomoles per microliter of glycerol respectively. Accurate quantification of these compounds is accomplished by the use of deuterium labeled internal stanards or quaternary ammonium compound homologs of distinct mass. Methods for the synthesis of these internal standards are reported. Some applications of these methods are iflustrated with stable isotope tracer studies on the kinetics of metabolism of choline to betaine aldehyde and glycine betaine in spinach leaf discs, and the identification of several Zea mays genotypes which appear deficient in glycine betaine. Tracer studies with deuterium labeled betaine aldehyde suggest that the deficiency of glycine betaine in one sweet corn hybrid is probably not due to a deficiency in the capacity to oxidize betaine aldehyde.Quaternary ammonium compounds occur widely in nature and may serve important roles as compatible osmotic solutes or osmoprotectants (13,17,22,(28)(29)(30)34). Glycine betaine (N,N, N-trimethyl glycine) is thus of considerable interest in terms of the osmotic stress tolerance of the Chenopodiaceae and Gramineae (7, 15-18, 20, 32, 33). But a key test of the adaptive significance of glycine betaine accumulation in osmoregulation in higher plants must ultimately come from the identification and detailed characterization ofglycine betaine deficient mutants of glycine betaine accumulating species (16,17 In considering screening maize germplasm for genotypic differences in glycine betaine levels, and potential methods for characterizing putative betaine null genotypes, we noted a paucity ofconvenient methods which would lend themselves to both accurate quantification of low glycine betaine levels in the presence of other quaternary ammonium compounds (e.g. choline, phosphoryl-choline, and betaine aldehyde) and stable isotope tracer studies on the betaine biosynthetic pathway. Available methods for betaine determination lack either sensitivity, resolution, and/or amenability to stable isotope tracer work (4, 8, 12-14, 19, 20). We therefore sought sensitive mass spectral methods for quantifying glycine betaine and its stable isotope abundance.Mass spectral analysis of quaternary ammonium compounds of synthetic and natural origin has been approached for the most part by desorption methods. FABMS2 (6), 252Cf fission-fragment and laser desorption (...

Rapid, sensitive, and selective methods for the determination of the '5N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directiy tested in an analysis of the kinetics of I'5NIH4' assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. There is a need for more detailed studies of the flow of N in the plant. Questions remain concerning the relative importance of primary ammonia-assimilation pathways (24), the pathways of utilization of the various N sources arriving in the growing leaf (2), and the mechanisms and significance of amino acid accumulation in response to environmental stress (31). One a,pproach to these questions is through examination of the use of N-labeled precursors (1,2,4,8,12,24,28,29,32,33). However, 15N labeling studies with plant tissues are hindered by the lack of sensitivity inherent in traditional MS and optical emission spectrometry methods (6, 9, 13, 14, 34) and by the complexities of the endogenous pools of amino acids which lead to problems in the interpretation of isotopic labeling kinetics in the absence of mathematical models (24,29). A consequence of these combined constraints is that relatively few '5N experiments have been performed with the precision necessary to yield definitive conclusions regarding precursor-product relationships, rates of synthesis and utilization, and metabolic compartmentation of the components of the pathways of amino acid biosynthesis in plant cells (18). In this paper, we specifically address the problem of limitations of sensitivity in methods for the determination of the 15N abundance of amino acids in isotopic tracer experiments with plant tissues.Since the pioneering work of Rittenberg (25), the mass spectrometric determination of the 15N abundance of amino acids has routinely involved the following: (a) the isolation of individual amino acids by ion-exchange chromatography (29) or preparative high-voltage electrophoresis and paper chromatography (2); (b) quantitative conversion of the amino and amide groups to N2 in evacuated vessels (23, 25); and (c) introduction of N2 to the mass spectrometer for "5N analysis by monitoring the ratios of ions 28, 29, and 30 (25, 29). Careful corrections for traces of residual N2 in the mass spectrometer and for atmospheric N2 as a contaminant of the sample introduced for '5N analysis are required. Consequently, samples of less than 10 ,tg of N cannot be subjected to "N analysis with any degree of accuracy. Quantities of N in excess of 0.3 mg are typically required. These traditional methods are, thus, time-consuming when applied to a large number of amino acids in several independent extracts, and they become technically demanding when dealing with amino acid constituents present at relatively low levels in plant tissues.Optical emission spectrometry has been more recently...