3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P50 COMbINED TEChNIQuE LC/MS IN
ANALySES OF ANTIOxIDANT COMPOuNDS
RADKA KOČí, MARIE TRČKOVá, LUKአMüLLER
and IVAnA MáROVá
Department of Food Chemistry and Biotechnology
Faculty of Chemistry,Brno University of Technology
Purkyňova 118, 612 00 Brno, Czech Republic,
koci-r@fch.vutbr.cz
Introduction
Antioxidants constitute a large group of plant and microbial
secondary metabolites (for exemple carotenoids, phenolics,
oligopeptides, saccharidic derivates etc.) with major
interest because of their anti-inflammatory effects and of their
potential anti-atherogenic properities 1,2 . Many foods are good
sources of antioxidants: vegetables, legumes, whole-grain
cereals, tea extracts, honey etc. The best source of these physiologically
active compounds with rapid effects on human
health is represented by some sorts of fruit 1 . Especially citrus
fruit contain high-levels of phenolics and/or ascorbic acid 3 .
Widely consumed apples are rich on polyphenols too with the
most abundant chlorogenic acid 4 .
Phenolics in human plant nurtur play important roles in
sensory properties of food, because of its influence on colour
and/or flavour. Moreover major part of phenolic substances
occure as physiologicaly active components with very hight
antioxidative capability as well as effective protection against
cancer growth and/or other degenerative diseases 3,4 .
Analysis of phenolic substances could be realised using
many different instrumental methods starting with TLC and
ending with GC (some special applications). In the present
the most preffered technique in separation of non-volatile
compounds, such as phenolics are, is modern instrumental
method LC/MS combining perfect separation with universal
detection of individual sample components 5 .
Experimental
Presented work was focused on characterisation of several
substances (phenolics, carotenoids) with antioxidant activity
containned in biologic materials and on their distribution
in individual sample fractions (juice, pulp, peel).
Antioxidants were isolated (i) by ethylacetate-ether
(phenolics) and/or aceton-ether (carotenoids) extraction, (ii)
by SPE method with amide-2 (phenolics) and/or C18 AR
columns (carotenoids).
Separation and detection of phenolics were optimalizated
using followed conditions: Restek C18 Ultra aqueous column
heated on 30 °C, gradient elution using 1% acetic acid:
acetonitril in range 60-45 : 40-55 with flow of mobil phase
0.4 ml min –1 , UV-VIS detection (280 nm and/or 370 nm),
MS tune file on chlorogenic acid (negative ion mode).
Identification of carotenoids was gone on column
Polaris C18 A heated on 45 °C, using isocratic elution with
LC/MS methanol (flow 0.25 ml min –1 ), UV-VIS detection
s682
(450 nm) and/or MS detection (tune file on β-carotene in
positive ion mode).
Standard compounds as well as food samples were determined
by MS full scan and/or MS/MS full scan mode.
Results
Standards of antioxidants were analysed using off-line
and/or on-line HPLC/UV-VIS/ESI-MS. However reported
results were obtained by on-line LC/UV-VIS/MS, because of
low response of off-line method.
Table I
Precursors (MS full scan) and fragments (MS 2 full scan) of
standards
Compound m/z [M-H]– or [M] +• Fragments (m/z)
β-carotene 537 –
epicatechin 289 217, 245, 271
catechin 289 227, 245, 271
quercetin
301
107, 121, 151,
239, 257, 273
morin 301 229, 257, 273
fisetin 285 163, 213, 229
kaempferol 285 151, 229, 257
acid gallic 169 –
acid chlorogenic 353 191, 309, 339
myricetin 317 179, 245, 289, 299
naringenin 271 –
rutin 609 301
procyanidin 577 –
In Table I determined parent ions (MS full scan) and/or
typical fragments obtained by tandem mass spectrometry are
resumed. MS full scan spectra of chosen phenolic standards
are demonstrated on Fig. 1. and Fig. 2.
Fig. 1. MS full scan spectrum of rutin
Fig. 2. MS full scan spectrum of acid chlorogenic