The^13 C NMR method is not suitable for routine analysis of a large number of samples because of
lack of sensitivity (a total final concentration of 10 mM is required), lack of resolution (signals from GB
and betaine homologues overlap), and the fact that it is also time consuming. For quantification, care
should be taken to ensure that the intensity of analyte specific signals for a given specific resonance be
directly proportional to analyte concentration. This is pertinent for every method but more relevant for
NMR as a complete relaxation of the resonance used between data acquisition and maximum expression
of the Overhauser effect are required.
MASS SPECTROMETRY Mass spectrometric analysis of QACs of synthetic and actual origin has
been approached by desorption methods. Fast atom bombardment mass spectrometry (FAB-MS), Cf fis-
sion fragment and laser desorption, thermal ionization, field desorption, direct chemical ionization, and
secondary ion mass spectrometry have all been applied to determine ionization and fragmentation com-
mon to QACs. Although all of these techniques produce intact molecular cations, FAB-MS produces
molecular cations of QACs with the greatest relative abundance and allows monitoring of the long-lived
signal derived from stable ion emission.
Rhodes et al. [23] developed an application of FAB-MS in quantitative determination of betaines of
plant extracts. The method entails converting QACs to aliphatic alcohol esters followed by FAB-MS anal-
ysis. Removal of free amino acids is essential because in the derivatization process, amino acids also yield
esters that can complicate the mass spectra. Also, it is important to remove valine, which gives a molec-
ular ion of mass identical to that of GB. The lower limit of detection for GB as the n-propyl ester is 0.05
nmolL^1 glycerol. Accurate quantification of QACs is accomplished by the use of deuterium-labeled
internal standards or QAC homologues of distinct mass. A linear correlation between the molar ratio of
GB to standard and the signal ratio of d 0 (m/z174)/d 9 (m/z183) is maintained over a wide range of 0.01
to 1 (correlation coefficient 1.00). It is by far the most sensitive method (MDL 0.05 nmol) and can
discriminate the same molecules of stable isotope. Because of these attributes, this method is particularly
suited to stable isotope tracer studies of the GB biosynthetic pathway and also can reliably and selectively
quantify low nanomolar amounts of betaines in complex mixtures of QACs in plant extracts. The method
has been applied to quantification of QACs and determination of the stable isotope abundance of QACs.
This method has potential for the identification of genotypes that lack certain QACs [31,35,53].
Plasma desorption–MS (PD-MS) is also developed for quantification of GB and choline [83,106].
However, this method is different from FAB-MS as it does not require prior derivatization of GB and thus
is particularly useful for choline determination. Glycine betaine is quantified from the signal intensity at
m/z118 relative to the stable isotope labeled [d 9 ] glycine betaine internal standard (m/z127). The ion in-
tensity ratio of m/z118:127 is correlated with molar ratio [d 0 ] glycine betaine/[d 9 ] glycine betaine. A use-
ful feature of the PD-MS method is that the signal from choline (m/z104 and 113 for [d 0 ] choline and [d 9 ]
choline internal standard, respectively) is more intense than the signal from GB. This method has advan-
tages over alternative methods such as DCI-MS and FAB-MS as derivatization of the QACs is not re-
quired and choline can be readily detected and quantified even in the presence of large amounts of GB.
- Remarks on Methodology
The relative advantages and disadvantages associated with various analytical methods for GB are sum-
marized in Table 3. There are several other techniques, such as radioisotope dilution in conjunction with
micro-Kjeldahl analysis and methyltransferase-catalyzed reaction [107,108], that are not covered in this
review. Depending on the research objectives and facilities available, researchers need to choose the ap-
propriate analytical techniques that meet their needs. It is highly recommended that when a new plant
species is under investigation, a full evaluation of recovery, reproducibility, and accuracy of the analyti-
cal methodology should be conducted as part of the standardization procedure.
III. BIOSYNTHETIC PATHWAY IN HIGHER PLANTS
A. Biosynthetic Pathway of Glycine Betaine from Choline
The biosynthetic pathway of GB is studied mostly in species of Chenopodiaceae (spinach, sugarbeet), and
to some extent in Amaranthaceae and Gramineae (barley, maize). Glycine betaine is synthesized via a
two-step oxidation of choline catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehy-
GLYCINE BETAINE IN STRESS RESISTANCE 887