3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P56 LIGNANS IN FLAxSEED – LC-MS/MS
DETERMINATION
AnnA KRAJČOVá a , VěRA SCHULZOVá a , JAnA
HAJŠLOVá a and MARIE BJELKOVá b
a Department of Food Chemistry and Analysis, Faculty of
Food and Biochemical Technology, ICT Prague, Technická 5,
166 28 Prague 6, Czech Republic,
b Agritec Plant Research s. r. o., Šumperk
anna.krajcova@vscht.cz
Introduction
Lignans constitute a group of important biologically
active plant phenols, structually characterized by the coupling
of two phenylpropanoid units by a bond between β-positions
in propane side chains. Lignans were identified in trees
already in the 19th century. Currently, wide occurrence of lignans
in various vascular plants has been well documented1 .
The interest in lignans and other phytoestrogens has
grown in the recent years because of their putative beneficial
health effects2 . They have been suggested to induce a various
effects, such as antioxidant, antitumor, estrogenic, and
antiestrogenic activities, and the protection against coronary
heart disease3 .
The reachest dietary source of lignans are flaxseeds,
with glycosides of secoisolariciresinol (SECO) (Fig. 1.) and
matairesinol (MAT) (Fig. 2.) as the major representatives of
this group2 . After ingestion, these lignans are deglycosylated
and partly converted to the mammalian enterodiol and enterolactone
by colonic bacteria. These forms are considered as
the biologically active substances responsible for the benefical
effects in humans4 .
In the presented study, a quantitative method (LC MS/
MS) for determination of the plant lignans, secoisolariciresinol
and matairesinol, in flaxseed has been developed. The
study of variation in SECO and MAT content was based on
examination of various cultivars of flaxseed.
HO OH
HO OH
HO
OH
HO
OH
MeO
MeO
Figure 1: SECO
Fig. 1. Figure SECO 1: SECO
HO
HO
MeO
MeO
Fig. 2. MAT
Figure 2: MAT
Figure 2: MAT
O
O
O
O
OMe
OMe
OH
OH
OMe
OMe
s696
Experimental
C h e m i c a l s
The lignan standards, secoisolariciresinol and mataresinol
were purchased from Sigma Aldrich (Germany). The
β-glucuronidase/sulfatase was purchased from Sigma-Aldrich
(Germany).
Ethylacetate, cyclohexane, and acetic acid glacial were
purchased from Sigma-Aldrich (Germany). Sodium hydroxide
was purchased from Penta Chrudim (Czech republic).
The deionised water was prepared using Milli-Q water system
(Millipore, USA).
S a m p l e s
The flaxseeds were obtained from Agritec Plant Research
s.r.o. (Šumperk, Czech republic). The levels of SECO and
MAT were monitored in different cultivars. Cultivars AGT,
Alaska, Astral, Atalante, Bajkal, Baladin, Flanders, Lola,
Oural, Recital represented oil cultivars. Cultivar Venica
represented fibre flax.
S a m p l e P r e t r e a m e n t
Defated flaxseeds were incubated overnight with enzyme
of Helix pomatia β glucuronidase/sulfatase in sodium acetate
buffer at 37 °C. Subsequently the enzymatic hydrolysates
were extracted with ethylacetate/cyclohexane. After
removing the solvent, the residue was transfered into mobile
phase.
A n a l y s i s
LC-MS/MS method was employed for examination of
extracts. The system consisted of Alliance chromatography
separation Module 2695 (Waters, USA) equipped with Discovery
C 18 column (Supelco, Germany) (50 mm × 3.0 mm
i.d., 5 µm), and Quattro Premier XE (Waters, UK) mass
spectrometer detector employing an electrospray ionization
source operating in negative mode.
M e t h o d V a l i d a t i o n
Analytes were quantified by the standard addition
method to compensate the influence of the co-isolated matrix
on the effectivity of the ionisation process. Matrix standards
could not be used due to unavailability of blank flaxseeds.
Repeated measurements were carried out to get performance
characteristic of the method.
Results
Optimization of extraction and hydrolysis. The release
of aglycones (SECO, MAT) from their glycosidic forms prior
to analysis is necessary. Enzymatic hydrolysis was tested,
and optimized. β glucuronidase/sulfatase (Helix pomatia)
in sodium acetate buffer was used for hydrolysis of defated
ground flaxseeds. Subsequent extraction of target analytes
was carried out with ethylacetate/cyclohexane solvent mixture.
Chromatografic and detection conditions. The LC MS/
MS method earlier described by Milder at al. (2004) was