Food Lipids: Chemistry, Nutrition, and Biotechnology

ionized molecule has the highest m/z ratio, but may not be always present, depending
on the volatility and thermal stability of the compound. Thus, lipids containing polar
groups, such as phospholipids, with low thermal stability and volatility and high
molecular weight cannot be analyzed by conventional electron impact (EI) mass
spectrometry. Therefore, fast atom bombardment (FAB), chemical ionization (CI),
field desorption (FD), or secondary ion (SI) mass spectrometry is required for such
lipid analysis [95].
MS is very useful in identifying structural modification of chain length such
as branching or the presence of rings for saturated species. In this regard, matrixassisted
laser desorption ionization and time-of-flight mass spectrometry (MALDI-
TOF-MS) have several advantages. It does not require prior derivatization of sample
to enhance the volatility of the lipids. The extent of fragmentation of MALDI-TOF-
MS is low; thus, detection of molecular ion is possible in most cases. Schiller et al.
[155] have successfully used a matrix of 2,5-dihydroxybenzoic acid to identify phosphatidylcholine
and different phospholipids as their molecular ions (M � 1). Diacylglycerols
were mainly detected as their corresponding sodium or potassium adducts
but not as their protonated form. MALDI-TOF-MS can be used for direct
investigation of lipid mixtures occurring, e.g., in cell membranes due to its high
sensitivity (up to picomolar concentrations).
MS in combination with GC and HPLC is also useful in structural determination
of the individual lipid molecules. Le Quéré et al. [156] have developed an
on-line hydrogenation method that allows selective hydrogenation of all the unsaturated
species after chromatographic separation for deducing structural information
such as carbon skeleton and double-bond equivalents. Le Quéré [157] has reviewed
this methodology and the use of GC-MS and tandem MS for analysis of structural
features of fatty acids.
D. Enzymatic Methods
Higgins [158] has described an enzymatic method for determining TAG content of
samples. This involves reaction of the TAG with lipase in order to obtain glycerol
and FFAs. The glycerol so produced is then converted to �-glycerophosphate using
glycerol kinase. The �-glycerophosphate dehydrogenase is then used to reduce nicotinamide
adenine diphosphate (NAD) to NADH. The resultant NADH is then
measured by a colorimetric reaction.
Cholesterol oxidase is used for determining cholesterol concentration in blood
plasma. Polyunsaturated fatty acids (PUFA) with cis-methylene groups between their
double bonds (e.g., linoleic, linolenic, and arachidonic acids) can be quantitatively
measured by reading the UV absorbance of conjugated diene hydroperoxides produced
via lipoxygenase (lipoxidase)–catalyzed oxidation. The extinction coefficient
of diene hydroperoxides at 234 nm is the same for all PUFAs. Fatty acid esters have
to be saponified prior to such analysis. The phosphatidylcholine or lecithin content
of foods (e.g., as a measure of the egg content of foods) can be made by catalyzing
conversion of lecithin to phosphatidic acid and choline by lecithinase (phospholipase
D) [5].
The method of stereospecific analysis of TAG described by Brockerhoff and
Yurkowski [159] uses pancreatic lipase that eventually removes fatty acids from the
sn-1 and sn-3 positions of the TAG. This procedure has recently been employed to
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