Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
38, Number 10, October 2000, pp. 441-449
Development
and Evaluation of a New Method for the Determination of the Carotenoid Content
in Selected Vegetables by HPLC and HPLC–MS–MS Christian
W. Huck1,*, Michael Popp2, Heimo Scherz3, and G.K. Bonn1 1Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens
University Innsbruck, Innrain 52a, 6020-Innsbruck, Austria; 2Bionorica/Plantamed
GmbH, Kerschensteinerstr. 11-15, 92318-Neumarkt/Oberpfalz, Germany; and 3Deutsche
Forschungsanstalt für Lebensmittelchemie, Lichtenbergerstr. 4, 85748-Garching,
Germany
Epidemologic
studies have shown inverse correlation between the consumption of carotenoid-rich
vegetables and the incidence of cancer. Therefore, analytical techniques for
the quantitative determination of carotenoids in complex sample matrices are
important. The most used method is reversed-phase (RP)-high-performance liquid
chromatography (HPLC). In this study, seventeen mobile-phase systems described
in the literature and six RP-HPLC columns with differences in particle size
and porosity are evaluated. Derived from these results, a new mobile-phase (acetonitrile,
methanol, chloroform, and n-heptane) including solvent modifiers is presented,
which allows an improved and more efficient separation of carotenoids. From
all columns tested, the best chromatographic parameters are found using a silica
C18 column (250 ¥ 2 mm, 5 µm, 100 Å). As was found, absorbance
detection at 450 nm allows the determination of the carotenoids down to the
picogram range with good linearity (R2 > 0.98). For the identification and
quantitation of carotenoids in complex sample matrices (containing additionally
other ultraviolet-absorbing compounds), the optimized RP chromatographic system
is coupled to a mass spectrometer (MS) using an atmospheric pressure ionization
interface. The calibration plots show high linearity (R2 > 0.99), and the
detection limit is found in the lower nanogram range. Furthermore, collision-induced
dissociation in the ion source allows for the identification of carotenoids
by their characteristic fragmentation pathways. In this study, a total of nine
species of vegetables commonly consumed in Central Europe are analyzed for their
contents of carotenoids (namely lutein, zeaxanthin, b-cryptoxanthin, and b-carotene)
by RP-HPLC and RP-HPLC–MS–MS. It is found that good sources for lutein
are spinach, kale, and broccoli, and sources for b-carotene are broccoli, spinach,
kale, carrots, and tomatoes. This new method is an improvement for the identification
and quantitation of carotenoids in complex biological tissues.
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