Methods to determine the extent of lipid oxidation in foods - Oxidation in Foods and Beverages and Antioxidant Applications - page 187

8.4 Chromatographic methods
Chromatographic methods are useful for the specific identification and
quantification of individual oxidation products, which will allow understanding
of reaction mechanisms and follow-up of certain products with undesirable
sensory or nutritional effects. Chromatographic analysis of lipid oxidation in
samples of unknown history will also allow certain predictions about sample
initial composition and storage and environmental condition. Despite these
advantages, the analysis of specific compounds is cumbersome and time
consuming and relies on the correctness of estimations due to lack of standards.
8.4.1 High performance liquid chromatography (HPLC) methods
HPLC coupled to electron spin resonance spectroscopy (HPLC-ESR) and high
performance liquid chromatography-electron spin resonance-mass spectrometry
(HPLC/EPR/MS) have been used for detection and identification of the radical
adducts resulting from lipid oxidation, for example, as -(4-Pyridyl-1-oxide)-N-
tert-butylnitrone (4-POBN) (Iwahashi et al., 1991a,b, 1992, 1996) or DMPO
adducts (Guo et al., 2003).
Various lipid hydroperoxides can be separated from reaction mixtures using
normal-phase or reversed-phase HPLC coupled to UV, post-column derivatiza-
tion-UV/visible, chemiluminescence, electrochemical or mass spectrometry
detectors. Post-column derivatization can be performed using luminol chemi-
luminescence (Yamamoto, 1994), ferrous (II)/xylenol orange (FeXO) reagent
(592 nm) (Sugina, 1999). As hydroperoxides are electro-active, they can be
detected with high sensitivity and specificity, e.g. using HPLC with reductive
mode electrochemical detection on a mercury drop cathode (Korytowski et al.,
1999).
HPLC with UV detection can be used to separate and detect MDA based on
their reaction with 2,4-dinitrophenylhydrazine (DNPH). The DNPH derivatives
are strongly absorbing in the region of 300±380 nm but derivatization
procedures are complicated (Korchazhkina et al., 2003).
8.4.2 High performance size exclusion spectroscopy (HPSEC) methods
At late stages of lipid oxidation and under thermo-oxidation conditions, the
monomeric molecular species of lipids undergo various degrees of dimerization,
oligomerization and polymerization. HPSEC provides a valuable tool to assess
the degree of such alterations and is the most useful technique to study oxidative
polymerization at high temperatures, e.g. in frying oils (Dobarganes, 1998).
Separations (100±20 000 Da) are performed on co-polymers of styrene divinyl
benzenes of pore sizes 50, 100 and 500 Ê using a single solvent, commonly
tetrahydrofuran. Detection is performed on non-selective mass sensitive
detectors such as refractive index (RI) and evaporative light scattering detector
(ELSD). HPSEC separations can be performed for the oxidation products of
fatty acid methyl esters as well as triacylglycerols, either on the overall