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Chromatographic analysis of lignans

Article  in  Journal of Chromatography A · May 2006

DOI: 10.1016/j.chroma.2005.11.054 · Source: PubMed

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 Journal of Chromatography A, 1112 (2006) 64–77

Review

Chromatographic analysis of lignans

S.M. Willför a,∗ , A.I. Smeds b , B.R. Holmbom a
a Process Chemistry Centre, Laboratory of Wood and Paper Chemistry, Åbo Akademi University, Porthansgatan 3, FI-20500, Åbo/Turku, Finland
b Process Chemistry Centre, Department of Organic Chemistry, Åbo Akademi University, Biskopsgatan 8, FI-20500, Åbo/Turku, Finland

Available online 13 December 2005

Abstract
Methods and procedures for analysis of lignans in trees and other plants are reviewed. The importance of cautious sample handling and
pretreatment procedures to avoid contamination, loss of sample, and unwanted chemical reactions is discussed. Sequential extraction with a non-
polar solvent followed by extraction with acetone or ethanol is recommended to separate the lignans from the plant matrix. An additional step of
acid, alkaline, or enzymatic hydrolysis may be necessary for some plant matrixes. Flash chromatography is a convenient method for preparative
separation and isolation of pure lignans from raw extracts. TLC is very suitable for qualitative screening of extracts and for monitoring of lignan
isolation and purification steps. Trimethylsilyl ethers of lignans can be separated and quantified by GC even in the case of complex mixtures of
lignans and other polyphenols, and the lignans can be identified by GC–MS in a routine manner. HPLC on reversed-phase columns is especially
suited for analysis of lignans and their metabolites in biological matrixes. The recent development of HPLC-electrospray ionisation (ESI)-iontrap
MS (MSn ) and corresponding techniques with high sensitivity and selectivity has proven valuable in lignan analysis. Lignan enantiomers can be
separated on chiral HPLC columns.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Extraction; GC; HPLC; Lignans; Purification; TLC

1. Lignans in plants

Lignans constitute a group of important plant phenols that

Abbreviations: 2-D, Two-dimensional; ASE, Accelerated solvent extrac-
tor; APCI, Atmospheric pressure chemical ionisation; cLari, Cyclolari-
ciresinol; CEA, Coulometric electrode array; CEAD, Coulometric electrode
array detector; Coni, Conidendrin; ConiA, Conidendric acid; DAD, Diode-
array detector; DCM, Dichloromethane; EC, Electrochemical; EI, Electron
impact; END, Enterodiol; ENL, Enterolactone; ESI, Electrospray ionisation;
EtOH, Ethanol; FID, Flame ionisation detection; GC, Gas chromatography;
HMGA, Hydroxymethyl glutaric acid; HMR, Hydroxymatairesinol; HPLC,
High-pressure liquid chromatography/high-performance liquid chromatogra-
phy; HPSEC, High-performance size-exclusion chromatography; HPTLC,
High-performance thin-layer chromatography; ID-GC-MS-SIM, Isotope dilu-
tion gas chromatography mass spectrometry selected-ion-monitoring; Lari, Lari-
ciresinol; LC, Liquid chromatography; LT-ELSD, Low-temperature evaporative
light-scattering detector; MPLC, Medium-performance liquid chromatography;
MR, Matairesinol; MRM, Multiple-reaction-monitoring; MS, Mass spectrome-
try; MTBE, Methyl tert-butyl ether; NMR, Nuclear magnetic resonance; NTG,
Nortrachelogenin; PDA, Photodiode-array; Pino, Pinoresinol; RP, Reversed-
phase; Seco, Secoisolariciresinol; SFE, Supercritical fluid extraction; SGD,
Secoisolariciresinol diglucoside; SPE, Solid-phase extraction; TLC, Thin-layer
chromatography; TMS, Trimethylsilyl; UV, Ultraviolet
∗ Corresponding author. Tel.: +358 2 2154729; fax: +358 2 2154868.
E-mail address: swillfor@abo.fi (S.M. Willför).
are structurally characterized by the coupling of two phenyl-
propanoid units by a bond between the ␤-positions in the propane
side chains (Fig. 1). According to recent nomenclature recom-
mendations [1], the units are treated as propylbenzene units,
giving the positions 8 and 8 to these linked carbon atoms. When
the phenylpropane units are linked by another carbon–carbon
bond, the compound class is named as neolignan. The true
dimeric lignans are in trees accompanied by smaller amounts
of related trimers and tetramers commonly called sesquilignans
and dilignans, respectively, or as a class called oligolignans [2].
This review is focussed on the extraction, purification, and chro-
matographic analysis of lignans and oligolignans in trees and
other plants.
Lignans were identified in trees already in the 19th century
[3,4]. The name lignan was proposed by Haworth in 1936 [5].
Today it is well documented that lignans occur widely in vas-
cular plants. Lignans have been found in wooden parts, roots,
leaves, flowers, fruits, and seeds. Several hundreds of lignans
have been identified. They occur either in free form or glyco-
sidically linked to a wide variety of different carbohydrates [6].
The amounts of lignans in various plants and their tissues are not

0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2005.11.054
 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77 65

Fig. 1. Structures of some commonly occurring lignans in trees and other plants, and the two main mammalian lignans enterodiol and enterolactone.
66 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77

Table 1
Amount of lignans (␮g g−1 dry weight) in selected plant and food items, and in tree tissues
Food/Plant tissue Seco MR Lari HMR Pino Ref.

Flaxseed 3700 10.9 [12]

2900 5.5 30.4 33.2 [8]
12600 58.6 [13]
Sesame seed 293 4.8 95 0.7 [8]
Rye bran 1.3 1.7 [14]
Rye flour 7.2 1.7 [13]
Rye bread, dark 0.1 0.1 1.2 1.7 [8]
Curly kale (Brassica) 0.2 0.1 6.0 16.9 [8]
Broccoli 4.1 0.2 [15]
0.4 0 9.7 3.2 [8]
Garlic 0.5 0 2.9 2.0 [8]
Strawberry 15.0 0.8 [16]
0 0 1.2 2.1 [8]
Olive oil 0 0 0 2.4 [8]
Black tea 24.2 3.0 [15]
Green tea 28.9 2.0 [15]
Picea abies heartwood 3–370 10–520 17–370 10–20 [17]
Picea abies knots 1400–6800 1700–5500 1000–2500 36000–88000 tr [17]
Abies alba heartwood 140 250 180 150 20 [18]
Abies alba knots 29000–36000 2500–2600 4600–10000 7000–7800 360–1000 [18]

yet accurately known due to the complicated analysis of different
lignan glycosides, and the data found in the literature are much
varying (Table 1). Secoisolariciresinol (Seco) and matairesinol
(MR) have been determined in many food items. Recently, it has
been found that also other lignans, such as lariciresinol (Lari)
and pinoresinol (Pino) occur commonly in food products [7,8].
The biological role of lignans in plants is still under discus-
sion, but it is generally assumed that they play a role in the
plant defence. Lignans may also play a role in the regulation of
plant growth. Trees live long and therefore need a good chemical
defence. Thus, it is not surprising that trees contain high con-
centrations of lignans and other polyphenols. Lignans and other
polyphenols occur commonly in the heartwood in trees. Soft-
wood species (angiosperms) contain mainly lignans, whereas
hardwood species (gymnosperms) contain mainly flavonoids in
their heartwood. Stilbenes are typical of pines, and occur also in
bark. It has recently been discovered that knots in trees, i.e. the
branch roots inside the stem, contain exceptionally high concen-
tration in lignans. Softwood knots typically have 5–15% (w/w)
of lignans (Table 1). In some extreme cases, knots can contain
even up to 30% (w/w) of lignans.
Much of the interest in lignans is due to their possible appli-
cation in the fields of pharmacy and nutrition. Lignans have
been found to possess a variety of biological properties. Semi-
synthetic derivatives of the lignan podophyllotoxin, naturally
present in Podophyllum plants, have found defined applications
in clinical medicine [9]. Some of the plant lignans are hydrolyzed
and metabolized by the colon microflora to the “enterolignans”
enterolactone (ENL) and enterodiol (END). These enterolig-
nans, also called mammalian lignans, are present in body fluids
of humans and animals. Seco and MR have long been known to
undergo this metabolism. More recently also Lari, Pino, and
sesamin were found to be precursors to mammalian lignans
[7,10]. Hydroxymatairesinol (HMR), the predominant lignan in
spruce wood and knots, is also converted to enterolignans [11].
The great significance of lignans, not only for the plants but
also as bioactive compounds with potential application in phar-
macy and nutrition, demands appropriate analytical techniques
for their extraction, separation, and structural determination.
Extraction and analysis is fairly straight-forward for tree tissues
where lignans occur in free aglycone form. However, analysis of
other plants where lignans are at least partly in glycoside form
is more complicated because the glycosidic linkages should be
cleaved without degradation or alteration of the lignan struc-
tures. Some lignans, such as HMR and Lari are rather sensitive
to either acidic or alkaline conditions.
Plant extracts commonly contain not only lignans but also
flavonoids and stilbenes, which have a similar solubility. The
extracts can contain a large number of components. Analysis
of lignan extracts is nowadays made mainly by gas chromatog-
raphy (GC) or high-pressure liquid chromatography (HPLC).
Thin-layer chromatography (TLC) is used mainly for qualita-
tive screening of a large number of extracts, and for monitoring
isolation procedures.
2. Pretreatment and extraction
2.1. Sample handling and pretreatment
To determine the amount and composition of lignans in plant
materials, it is usually necessary to separate these from the
plant matrix. Traditionally, this involves several, often tedious,
work-up steps, each involving risks for contamination, loss of
sample, and unexpected reactions of the lignans. This problem
was recently addressed by Tura and Robards in a review arti-
cle on sample handling strategies for phenols in food and plants
[19]. Nevertheless, the preparative and analytical methods, and
equipment available, have evolved and nowadays, it is possi-
ble to apply rather safe methods of extraction and pretreatment
of plant samples. A general scheme that is applicable to lignan
 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
Fig. 2. General analytical scheme for analysis and isolation of plant lignans.
analysis of plant materials is shown in Fig. 2. In practice, the
researcher usually has to choose the methods and the apparatus
available in his/her own laboratory, even though these may not
be state-of-art. However, when it is possible, several analytical
techniques should be combined or used in parallel to achieve the
best result.
The sampling procedure, sample storage, and sample pre-
treatment are crucial for the outcome of the lignan analysis.
For example, obtaining a representative sample of wood from a
certain tree species involves questions such as place of growth,
age, characteristics, and physical condition of the sampled tree,
and sampling height. Samples should preferably be placed in a
freezer immediately after sampling to avoid unwanted reactions
such as oxidation, polymerisation, and enzymatic reactions. Oxi-
dation of lipophilic extractives may also cause problems later on
in the analysis of the lignans.
Pretreatments necessary for most plant samples prior to
extraction are drying, and grinding or pulverising. To avoid
oxidation or polymerisation of sensitive compounds, it is advis-
able to use a mild drying technique such as freeze-drying or, if
unavailable, drying at room-temperature. The grinding can also
cause unwanted reactions since the temperature in the mill may
rise too high. Adding some pure dry ice to the mill can eliminate
this problem. Ground or pulverized samples are more suscepti-
ble to oxidation and degradation and should therefore be stored
in a cold, dark, and dry place.
2.2. Solvent and solid-phase extraction
Soxhlet extraction or percolation with a polar solvent are the
traditional methods for extracting lignans from plants. However,
less polar extractives are present in various amounts in most
plant tissues and may disturb the subsequent lignan analysis if
direct extraction with a polar solvent is used. Therefore, it is
advisable to do sequential extraction and first remove lipophilic
compounds with a non-polar organic solvent such as hexane,
pentane, petroleum ether, or dichloromethane [2,20–23]. The
67
hydrophilic extractives, including the lignans, can then sub-
sequently be extracted with a polar solvent such as acetone,
methanol, or ethanol. Adding 5–10% of water to the solvent
will facilitate the penetration of the solvent and promote the
extraction of more polar compounds such as lignan glycosides.
Extraction of polyphenols, including lignans and lignan glyco-
sides, from certain grains may require a solvent mixture with
a larger amount of water, such as 30% aqueous acetone that
recently was used for extraction of polyphenols from sorghum
[24]. However, some lignans are of low or medium polarity and
will be extracted with the non-polar extraction solvent. A com-
plex mixture, or a large amount of lignans, or, for example, resin
acids, can also influence the solubility and selectivity of certain
compounds towards the non-polar solvent [23,25].
Direct extraction with a hot or room-temperature polar sol-
vent has also been used for extraction of plant lignans [26–32].
This may also be the appropriate approach for lignans of low
polarity, containing no or only one free hydroxyl group, since a
selective extraction of lipophilic non-lignan extractives may be
impossible [26,28,33]. However, the subsequent clean-up steps
to separate the lignans from the polar extract are usually time-
consuming and laborious.
An excellent method for solvent extraction of plant lignans
is the accelerated solvent extraction (ASE), which is performed
at elevated temperature and pressure and under inert nitrogen
atmosphere [21,23]. This method enables fast, automated, and
sequential extraction using relatively small solvent volumes.
ASE extraction has recently been applied for extraction of lig-
nans from different tree species. Especially, lignan-rich extracts
were obtained from Norway spruce knots, which can contain up
to 30% of lignans alone [17,18,21,23,25,34].
Lignans in some plant materials may require special pretreat-
ments before extraction and analysis. Seco diglucoside (SDG) in
flaxseed (Linum usitatissimum) is such an example, since SDG is
present in the plant as ester-linked oligomers, or even polymers,
consisting of SDG and hydroxymethyl glutaric acid (HMGA)
[35,36]. This oligomeric/polymeric lignan derivative appears to
be readily soluble in aqueous methanol or ethanol, but alkaline
hydrolysis is subsequently needed to release free SDG. Further-
more, an additional acid hydrolysis step should be added if the
free aglycone Seco is of interest [15,35,37]. Several analytical
procedures, including various steps and combinations of enzy-
matic, acidic, and alkaline hydrolysis, before or after extraction,
have been developed for analysing SDG and Seco in food and
feed [15,38]. Hydrolysis is often also done to simplify the sub-
sequent chromatographic analysis of extracts containing lignan
glycosides.
Especially in technical processes, such as pulping and paper-
making, but also in environmental samples and for some food
applications, the lignans are dissolved into process waters or
aqueous effluents. According to Ekman and Holmbom [22], the
lipophilic extractives of a water sample (fibres filtered away)
from a pulp mill are first extracted with diethyl ether, and then
a portion of the water phase containing the lignans is dried and
analysed by GC [22,39,40]. Although this is a simple method, it
has the drawback of requiring filtration to remove fibres, which
can trap some lignans with high affinity to the fibres or other
68 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
compounds in the fibre mat that will form on the filter. Later, Örså
and Holmbom [39] published an improved sampling method for
extractives in water samples, which includes centrifugation to
remove fibres and particles, adjustment of pH to 3.5 with dilute
sulphuric acid, and extraction with MTBE three times before
final analysis of the pooled MTBE-extracts by GC. This method
allows extraction of >95% of the lignans present in water sam-
ples, but is less effective for sesqui- and dilignans. The authors
stated that ethyl acetate gave even better extraction if only lig-
nans were of interest.
The use of solid-phase extraction (SPE) of lignans is not as
wide-spread as different solvent extraction techniques are. How-
ever, SPE has been applied for analysis of lignans in flaxseed or
biological fluids, where it has been used as a purification step
after alkaline or enzymatic hydrolysis of a raw extract or for
fractionation of oligomers from a non-hydrolysed raw extract
[36,37]. SPE has also been used for extraction of simple phe-
nols and polyphenols from plants and food, but for some reason,
the lignans seem to have been neglected in these applications
[19].
Supercritical fluid extraction (SFE) is an interesting extrac-
tion technique which could have some advantages also in lignan
analysis. SFE extraction with carbon dioxide seems to work
satisfactory for lignans of low to medium polarity such as those
obtained from Schizandra chinensis, but only for extraction from
the seeds and not from the leaves [41,42]. Nonetheless, addition
of methanol to the carbon dioxide can considerably improve the
extraction efficiency of certain lignans [41,43]. However, SFE is
a convenient method for removing lipophilic extractives from,
for example, flaxseed prior to further extraction of lignans with
a hydrophilic solvent [44].
2.3. Preparative separation and isolation of pure lignans
Preparative enrichment and separation of individual lignans
in pure form is often desired for analytical purposes, and is
demanded for structural characterisation of new, unknown lig-
nans. The recent discovery that knotwood of several tree species
contain exceptionally large amounts of free aglycone lignans
has provided the opportunity to isolate pure lignans even in
gram-amounts [2,17,18,21,23,25,34,45,46]. Especially interest-
ing is large-scale isolation of HMR from different spruce species,
since HMR can be used as a starting material for making other
lignans (e.g. MR, 7-hydroxy-Seco, Lari, cLari, ENL, END, 7-
oxo-MR, and iso-HMR) [21,47–51]. The obtained pure lignans
are valuable not only as new standard compounds for analyt-
ical purposes, but also for biotesting. The basic principle for
isolating different knotwood lignans is as follows: Knotwood
is splintered, freeze-dried, and ground [21,23]. After removal
of lipophilic extractives using solvent extraction with hexane,
the hydrophilic extractives are extracted with an acetone–water
mixture or with ethanol. Pure lignans can then be obtained by
flash chromatography on normal-phase silica columns and/or by
crystallisation from a suitable solvent (e.g. dilute 2-propanol,
methanol, or cyclohexane/ethanol). HMR can also be precipi-
tated from an ethanol solution by addition of potassium acetate,
achieving a purity of 90–95% [52].
Flash chromatography is a convenient method for prepara-
tive isolation of selected lignans from raw extracts, since the
sample load can be much larger than with HPLC. However,
the conditions of separation and purification need to be opti-
mized. Dichloromethane and ethanol in different ratios (e.g.
DCM/EtOH 98:2; 95:5) has worked well for free aglycone lig-
nans such as HMR and also for sesquilignans [2,53]. Other
suitable solvent mixtures may be found from experiments with
column chromatography and TLC.
Various combinations and applications of open-column
chromatography, medium-performance liquid chromatography
(MPLC), (semi)preparative HPLC, and preparative TLC have
also been used to isolate lignans. For example, Owen et al. [41]
isolated lignans from olive oil by preparative TLC followed
by semi-preparative HPLC. Willför et al. [2] used combina-
tions of flash chromatography (normal-phase silica columns),
MPLC (normal-phase), and polar reversed-phase (ether-linked
phenyl with polar end-capping) semi-preparative HPLC to
isolate several sesquilignans and lignans from spruce knot-
wood. Kawamura et al. (27( isolated lignans and sesquilignans
from western hemlock wood by preparative TLC and semi-
preparative HPLC on both normal-phase and reversed-phase
silica columns. Takaku et al. [28] isolated several lignans from
shoots and leaves of hinoki cypress using column chromatog-
raphy with different solvent mixtures: DCM/methanol, ethyl
acetate/n-hexane, and acetone/DCM, followed by TLC (same
solvents) and reversed-phase HPLC using isocratic elution with
acetonitrile/water (37:63). Smeds and Hakala [54] separated and
isolated HMR isomers from a crude HMR extract by prepara-
tive reversed-phase HPLC using ethanol or methanol and water
mixtures. Johnsson et al. [37] isolated SDG from an extract
of defatted flaxseed flour using first a reversed-phase column
(methanol) and then a normal-phase silica column (chloro-
form/methanol/water 10:5:1), while Pihlava et al. [44] suggested
the direct use of a reversed-phase column and elution with an
aqueous alcohol.
As seen from the examples above, the choice of col-
umn or TLC material for preparative separation of lignans
appears to be either normal-phase or reversed-phase (RP) C18.
However, experience has shown that RP C8 in some cases
actually gives better separation than C18 [34,51,56]. Prelim-
inary tests with even shorter chain material, such as C5,
did not improve the separation (unpublished results). Suitable
solvent mixtures can be, for example, methanol/water, ace-
tonitrile/water, acetonitrile/methanol/water, ethanol/water, n-
hexane/chloroform/methanol, or n-hexane/ethyl acetate in dif-
ferent ratios. Both isocratic and gradient elution may work,
depending on the complexity of the extract.
2.4. Artefacts and unwanted chemical transformations
Unfortunately, researchers usually do not report on unsuc-
cessful results and artefacts. In doing so, many unnecessary
failures and moments of frustration could be avoided in the
laboratories. Simple measures, such as the use of an inert atmo-
sphere, absence of light, and avoiding high temperatures are
often neglected, which evidently leads to unwanted chemical
 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
reactions such as oxidation, thermal degradation, or polymeri-
sation of sensitive lignans.
HMR is one of the most studied lignans. Freudenberg
and Knof [52] reported that strongly acidic conditions lead
to the formation of ␣-Coni from HMR. Ekman [20] dis-
cussed the possibility of auto-oxidation of HMR to 7-oxo-
MR in non-fresh wood samples. He also suggested that the
presence of ␣-ConiA in the extract of a fresh wood sam-
ple could be a result of hydrolysis of the lactone ring of
␣-Coni during the extraction procedure. Later, Ekman and
Holmbom [22] reported that pH values in the range of 9–12
in water cause base-catalysed transformation of HMR to the
compound (E)-4-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-
3-methoxyphenylmethyl)but-3-enoic acid and transformation to
␣-ConiA due to a build-up of the alkalinity during drying of the
sample [57]. This was also confirmed by Eklund et al. [53].
Recently, Eklund and co-workers [50,51] showed that HMR
undergoes several transformations under both basic and acidic
conditions. Possible products include ␣- and ␤-Coni, ␣- and ␤-
ConiA, HMR acid, and isomers of iso-HMR. HMR also reacts
with various nucleophiles in both acidic and basic conditions,
resulting in products such as MR, and 7-methoxy- and 7-ethoxy-
MR, among others. In fact, HMR is very unstable in solution and
will disappear almost completely during both acidic and alka-
line hydrolysis. Furthermore, transformation to ␣-Coni may be
unnoticed since the solubility of ␣-Coni in water is low and
it may therefore precipitate and disappear from the subsequent
analysis. HMR also undergoes oxidative transformations upon
exposure to light [58]. The two isomers of HMR can equili-
brate and both are oxidised to 7-oxo-MR and further to coloured
oligomers when exposed to irradiation by light.
The presence of free radicals in a solution containing lig-
nans can cause unwanted chemical transformations, since most
lignans are known to be effective radical scavengers [21,59]. Lig-
nans cannot only form different adducts, but can also undergo
dimerisation. Lignan dimers and other adducts can be over-
looked during the analysis, thus making a quantification difficult
and the radical reactions may go unnoticed. Even though the
reaction kinetics is relatively slow, this risk should be kept in
mind during sample treatment and analysis.
Seco can partially transform into Seco-acetonide during col-
umn chromatography with acetone on slightly acidic silica
columns [53]. Similar artefacts, namely the acetonides of Seco,
cLari, and isotaxiresinol were also reported by Yang et al. [60]
upon isolation of phenolic compounds from Taxus mairei. Acidic
conditions can also bring about artefacts such as anhydro-Seco
and anhydro-cLari by elimination of a water molecule from the
diol structure, as was shown for the guaiacylglyceryl ethers of
these lignans [53]. Anhydro-Seco is also an artefact produced
upon acid hydrolysis of SDG [38,12,13].
The lignan 7-hydroxy-Seco, so far reported to occur naturally
only in knotwood of Abies amabilis, can be transformed to Lari
and cLari by an acid-catalysed intramolecular cyclisation reac-
tion [18,47]. Lari will also further rearrange to cLari at stronger
acidic conditions. Anjaeyulu et al. [61] discussed whether the
lignans methyl and ethyl arboreal were natural products from
Gmelina arborea or artefacts being formed during the extrac-
69
tion process. They concluded that these lignans most certainly
were naturally occurring.
Silylation before analysis with GC is usually quite straightfor-
ward, but sterically hindered hydroxyls, such as the 8-OH group
in (−)-NTG, can give rise to only partially silylated compounds,
which can cause confusion. Another possible silylation artefact
is the cyclisation of Lari to cLari by time, which probably is due
to the formation of hydrochloric acid in the silylated sample. It is
recommended that silylated samples should be analysed within
12 h to avoid degradation of the silylated compounds [22,39].
3. Separation and detection techniques
3.1. Thin-layer chromatography
Thin-layer chromatography (TLC) has been used in lignan
research since the 1960s [62–64]. It is a simple and inexpensive
technique and is particularly suitable for screening of a large
number of samples, and for monitoring isolation procedures.
TLC is routinely used for a first qualitative examination of plant
extracts. Quantitative determination can be achieved by den-
sitometric detection. Slanina and Glatz [41] recently reviewed
separation procedures, including TLC, for lignan analysis, espe-
cially in plants of Oriental medicine.
The most used phase has been silica gel. A wide range of
eluents and detection techniques have been applied. Preparative
TLC is much practised for the isolation and purification of small
amounts of lignans and other polyphenols [34]. Isolated TLC
fractions can be identified by subsequent analysis by GC–MS or
HPLC-MS. Lignans all absorb UV light and can thus be detected
using 254 nm UV light. Spraying with a 5% solution of sulphuric
acid in ethanol is also commonly used.
New lignans of western red cedar (Thuja plicata) were sep-
arated by TLC and column chromatography by MacLean and
Murakami [63]. Barton [64] reported Rf values on silica gel
plates for three solvent systems for Coni, MR, HMR, and Pino.
Coni, MR, and Pino had quite similar Rf values, whereas HMR
had a clearly lower Rf value. The trend for lower Rf values for
increasing number of hydroxyl groups was pointed out. Weiss-
man [65] also used TLC on silica gel plates on extracts of
Araucaria angustifolia. He also found that MR, Pino, and Coni
had similar Rf values, but Lari, Seco, and cLari could be sepa-
rated.
2-D techniques with multiple solvent developments have
been used for lignan analysis in seed oils of Sesamum species
[66]. An HPTLC-densitometric method has been developed
for routine analysis of SDG in flaxseed [67]. Opletal et al.
[68] used TLC and HPLC for isolation and determination of
dibenzo[a,c]cyclooctadiene lignans of the genus Schisandra.
In our studies of lignans in trees, we have found TLC valu-
able for fast screening of extracts and for preparative separation
of lignans for further studies by GC and HPLC. For example,
we used TLC on RP-plates (RP-8) for preparative isolation of
lignans occurring in small amounts in extracts of some fir, pine
and spruce species [34]. TLC provides a good overview of the
lignan pattern, although all lignans cannot be separated (Fig. 3).
The separation is governed mainly by the number of hydroxyl
70 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
Fig. 3. TLC of acetone extracts from knots in Parana pine (Araucaria angustifo-
lia), silver fir (Abies alba), and Norway spruce (Picea abies) trees. Silica Gel 60
PF-254 plates (Merck). Eluent: dichloromethane:ethanol 93:7 (v/v). Detection:
spray with sulphuric acid in ethanol, followed by 2 min heating at 150 ◦ C, giving
the following colours for the lignans: Pino, Me-Pino, Me2 -Pino, and lignan A:
violet; MR, Coni, and HMR (HMR and allo-HMR): red or red-brown; Lari and
cLari: dark grey; Seco, Me-Seco, and Todo: blue. All lignans were also extracted
from the plates and identified by GC–MS and comparison to authentic samples.
groups, but lignans with the same number of hydroxyl groups
may also be separated. Two-dimensional TLC can essentially
improve the separation of individual lignans. Identification of
the predominant lignans is facilitated by the specific colours
obtained by spraying with sulphuric acid in ethanol followed by
rapid heating in an oven.
3.2. Gas chromatography
Gas chromatography is a powerful technique with excellent
chromatographic resolution for analysis of lignans. Ayres and
Chater [70] were the first to describe GC for analysis of lig-
nans and they showed that the principal classes and geometrical
isomers of lignans could be distinguished.
Since the majority of plant lignans contain at least one free
hydroxyl group, it is usually necessary to increase the volatility
of the compounds by derivatisation. It is also advantageous if the
derivatisation increases the thermal stability of the lignans and
enhances the selectivity of the detection. For podophyllotoxin-
type lignans, without any free hydroxyl groups, no derivatisation
is necessary [26]. Krahmer et al. [71] analysed lignans from
Tsuga heterophylla wood as their trimethylsilyl ether deriva-
tives (TMS). Later, Ekman [20] published the first mass spectra
of TMS ethers of plant/wood lignans. Silylation has become
the most used method for derivatisation of lignans prior to GC
analysis. Nevertheless, one problem is still that there are only
a few mass spectra of silylated lignans available in commer-
cial databases. Commonly used silylation reagents are mixtures
of bis-(trimethylsilyl)-trifluoroacetamide-trimethylchlorosilane
in pyridine, hexamethyldisilazane-trimethylchlorosilane in pyri-
dine, or variants of the same [21,53,72,73]. The first mentioned
mixture may require somewhat longer silylation times to fully
silylate lignans with partially sterically hindered hydroxyls such
as the 8-OH group in (−)-NTG. It is also possible that the occur-
rence of lignan B, reported by Ekman [20], was actually an
artefact arisen from problems with the silylation. However, the
occurrence of lignan B has also later been reported [17,40].
The choice of GC column for detailed analysis of individual
TMS derivatives of lignans is preferably a standard non-polar
capillary column, coated with cross-linked methyl polysiloxane,
such as HP-1, CP-Sil 5 CB, SPB-1, or similar [21,22,26]. How-
ever, there may be some advances of using a slightly more polar
column such as HP-5 in parallel with the HP-1, especially if the
gas chromatograph is equipped with a dual-channel system. In
that way, it may be possible to correct for some overlapping of
similar compounds, since the separation pattern will be slightly
different on the two columns (Fig. 4).
It is also possible to analyse lignans as a group using a wide-
bore, short (5–7 m), HP-1 thin-film column as described by Örså
and Holmbom [39]. This will not completely separate all indi-
vidual lignans, but enables simultaneous analysis of sesqui- and
dilignans (Fig. 5) [2,21]. Fig. 5 also shows an HPSEC chro-
matogram of the same extract as analysed by GC. For GC–MS
analysis of sesqui- and dilignans, an MXT-65TG column coated
with a 65% diphenyl–35% dimethyl polysiloxane film, which
is stable up to 370 ◦ C, can be used. Another advantage with the
short-column system and the MXT column is that also lignan
glycosides, in addition to sesqui- and dilignans, can be eluted in
the same analysis as normal lignans. However, the ester-linked
SDG-HMGA oligomers [35,36] will not elute in this system,
but requires an alkaline hydrolysis to release the SDG before
analysis.
The final column temperature is usually between 280 and
300 ◦ C for lignans, while the analysis of sesqui- and dilignans
requires temperatures up to 340 ◦ C [2,12,21,22,39,40,72]. A
final column temperature of 240 ◦ C has also been used for true
lignans [73]. Changing the temperature program will change
the order of elution of the lignans only little, but it may help
with some overlapping compounds. The choice of carrier gas
is traditionally helium, but a better separation may be obtained
with hydrogen. The safety risk is naturally higher when using
large volumes of hydrogen gas, but the development of on-line
hydrogen generators has improved that situation.
The most widely used ionisation technique for GC–MS anal-
ysis of lignans is electron impact (EI). However, if reference
compounds are unavailable, care should be taken when inter-
preting the fragmentation pattern of the lignans. For example,
Ekman [20] showed mass spectra of silylated Norway spruce lig-
nans and tentatively identified the tetrahydrofuran lignan liovil,
among others. Recently, two isomers of this lignan were isolated
and their structure determined by NMR spectroscopy and X-ray
analysis [34]. The analyses showed that the lignans in question
actually were 9-epimers of 7-hydroxy divanillyl butyrolactol lig-
nans.
Quantification of lignans, or any compounds, by GC requires
addition of an internal standard substance, preferably added
to the sample already before extraction. The internal standard
should be available in pure form and have a structure and
polarity close to the lignans. One approach is to use isotope
 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77 71

Fig. 4. HP-1 and HP-5 gas chromatograms (retention interval 21.5–26.5 min.) of a lignan/flavonoid extract obtained by mixing hydrophilic extracts from Pinus
sibirica, Pinus contorta, Larix decidua, and Picea abies knotwood. Analytical conditions according to Willför et al. [21].

dilution GC–MS selected-ion-monitoring (ID-GC-MS-SIM),
which originally was developed for urine, plasma, and tissue
samples, but also has been used for plant and food materials
[12,15,38]. This is a powerful method, but it requires pure,
deuterated lignans, which still are not commercially available.
Furthermore, this method is not possible for new, unknown
compounds. Calibration curves for certain lignans, using dif-
ferent amounts of the lignans to be analysed, requires pure
compounds and is therefore not applicable to new, unknown
lignans. Nevertheless, it is today possible to buy several dif-
ferent pure lignans. Another approach is to use one or several
readily-available, pure compounds that are not present in the
sample to be analysed. Several compounds have been used as
internal standards, for example: n-dotriacontane [20], heptade-
canoic acid [22], 5␤-pregnane-3␣,20␣-diol [40], cinchonidin
[26], and betulinol [21,39] for true lignans, while cholesteryl
heptadecanoate and 1,3-dipalmitoyl-2-oleyl glycerol have been
used for sesquilignans and dilignans, respectively [21,39]. Flame
ionisation detection (FID) is the natural choice of detector for
quantitative GC analysis due to its unique ability to monitor
organic compounds over a wide range of concentrations. Still,
the response factor for each compound versus the internal stan-
dard should be determined. For example, the response factor
for wood-derived lignans calculated against betulinol was first
assumed to be 1 [20], but when pure individual lignans later
became available the response factor was corrected to 1.2 [23].
3.3. High-performance liquid chromatography
High-performance liquid chromatography (HPLC) is prob-
ably the most frequently used analytical technique for lignans,
especially in the analysis of lignans in biological matrixes, since
no time-consuming pretreatment of the samples is needed.
3.3.1. Normal-phase silica columns
Normal-phase columns are often used for isolation or
purification of lignans in a preparative or semi-preparative
scale, but for analytical purposes, normal-phase columns have
been used mainly for lipophilic lignans such as lignans
present in Podophyllum species [74–76] or in sesame seed
oils [73]. Three lignans in Podophyllum resin (podophyllo-
toxin, ␣- and ␤-peltatin) were separated using 1.8% ethanol
in chloroform as mobile phase [74]. Seven diastereoiso-
mers of podophyllotoxin were successfully separated using
72 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
Fig. 5. HP-1 short-column GC and HPSEC chromatograms of an Abies lasio-
carpa hydrophilic knotwood extract. Analytical conditions (GC) were according
to Örså and Holmbom [24]. The analytical conditions for the HPSEC anal-
ysis were as follows. TSK guard column HHR-H (40 mm × 6 mm) and two
TSK-gel G3000 HHR (300 mm × 7.8 mm) columns and a universal Sedex
low-temperature evaporative light-scattering detector (LT-ELSD). The solvent
(tetrahydrofuran) flow was 1 ml min−1 . No standard was used in the HPSEC
analysis. (*) = standard.
n-heptane–dichloromethane–methanol (90:10:4) as mobile
phase [75]. Furthermore, a method was developed for quantifi-
cation of podophyllotoxin in different varieties of Podophyllum
resins using n-hexane–methanol–tetrahydrofuran–acetic acid
(85:10:4:1) as mobile phase [76]. Lignans from Podophyl-
lum species can also be successfully separated using reversed-
phase columns; however, for separation of the lipophilic lignans
sesamolin and sesangolin from sesame seed oils, a normal-phase
silica column (using 6% diethyl ether in n-heptane as mobile
phase) was found to be superior to a reversed-phase column
[73].
3.3.2. Reversed-phase columns
As the most studied lignans generally are of medium polarity,
the majority of HPLC separations have been performed by using
reversed-phase columns. Most of these separations are carried
out with gradient elution and an acidic mobile phase because of
the acidity of the phenolic groups. Methanol or acetonitrile have
been extensively used as organic solvent in the mobile phase
(Fig. 6). For the separation of mixtures containing both diastere-
omers and functional-group derivatives, ternary or quaternary
mobile phases are required e.g., mixtures of methanol and ace-
tonitrile or dimethyl sulphoxide [75]. Methanol is required for
the separation of diastereomers, while acetonitrile or dimethyl
sulphoxide are effective for the separation of functional deriva-
tives. The most widely used reversed-phase column is octade-
Fig. 6. HPLC-UV chromatogram (at 280 nm) of a standard solution containing
13 pure reference lignans. Column XTerra MS C8 , 2.1 mm × 150 mm, 3.5 ␮m
particle size (Waters Corp., Milford, MA, USA). Eluent A 0.1% acetic acid,
eluent B methanol:acetonitrile 50:50. Gradient elution from 10% B to 30% B in
6 min, then to 50% B in 10 min, to 80% B in 13 min, which was held for 5 min.
cylsilica (RP-18), but an RP-8 column is more suitable for
separation of more hydrophilic lignans such as HMR isomers
or HMR acid and conidendric acids [50–51,56].
3.3.2.1. Analysis of plant extracts. In plants (except in tree
species) and plant foods, lignans are usually glycosidically
linked to carbohydrates and therefore these samples are usually
treated by acid or enzymatic hydrolysis before or after extraction
of the lignan conjugates or free lignans from the plant matrix.
In plant extracts, UV detection (at a single wavelength) may
offer sufficient selectivity and sensitivity for determination of
lignans. In one work, six major lignans in extracts of Schisan-
dra chinensis (e.g., schisandrins and gomisins) were quantified
by HPLC-UV (at 254 nm) [77]. Eight tetrahydrofuran nucleus
lignans (e.g., sesamin, asarinin, Syri, and phillygenin) were sep-
arated on an RP-18 column using gradient elution or through
altering the flow rate in an isocratic elution, which improved the
resolution [78]. The lignans were detected using a UV detec-
tor at 240 nm. A method for quantification of lignans isolated
from the roots of Asarum species, asarinin, and sesamin, was
developed using the improved chromatographic method.
In more recent years, UV detection is often used only as com-
plementary to other detection techniques, especially after the
development of HPLC-MS techniques, which mostly include
also a UV diode-array detector (DAD) [also called photodiode-
array (PDA) detector] (HPLC-UV-MS technique). An advanced
method for identification of lignans and lignan glucosides
(e.g., Pino, epi-Pino, MR, arctigenin, arctiin, phillygenin, and
phillyrin) and their enantiomeric forms was developed for deter-
mination of lignans in crude methanolic extracts of Forsythia
intermedia [79]. HPLC-UV at 280 nm was coupled to laser
polarimetric detection.
HPLC in combination with mass spectrometry has been used
for lignan analysis since this method became common usage
in the middle of the 1990s. Mostly, the analyses are performed
in the negative mode as the acidic phenol groups occurring in
most lignans are easily deprotonated. In one of the earlier lignan
HPLC-MS applications, HPLC-thermospray-MS coupled with
 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
or in combination with UV detection was used for analysis of
the plant lignans Seco and MR in flaxseed and nordihydrogua-
iaretic acid in chaparral (Larrea tridentata) [80]. This technique
was also used for analysis of some lipophilic lignans (eudesmin,
magnolin, yangambin, and kobusin) in Polygala species [81]
and of three lignans (phylligenin, eudesmin, and epieudesmin)
in leaves of Orophea enneandra [82]. The thermospray tech-
nique was replaced by electrospray ionisation (ESI), which is
the preferred ionisation technique in HPLC-MS lignan analysis
today. HPLC-ESI-MS (coupled or in combination with a PDA
detector) was used for the identification of 15 lignans (schisan-
drins and gomisins) and tentative identification of another nine
lignans in extracts of Schizandra chinensis fruits [83] and for the
identification of arctiin and its aglycone arctigenin in leaves of
burdock (Arctium lappa L.) [84]. HPLC-ESI-MS has also been
used for detection of lignan conjugates in a crude extract of pine
bark [85].
HPLC-quadrupole MS, although a selective and sensitive
method, does not permit complete characterisation of the chem-
ical structure, and is therefore suitable mainly for quantification
of already known compounds available as pure reference stan-
dards. With LC-ion trap MS (MSn ), the possibilities for identifi-
cation of unknown compounds are better. LC-ESI-MSn was used
for deducing the structure of products formed in alkaline solu-
tions of HMR, i.e. iso-HMR, HMR acid, and conidendric acids
[50]. HPLC-atmospheric pressure chemical ionisation (APCI)-
MSn was applied for identification of several lignans and lignan
glucosides in a crude methanolic extract of Forsythia intermedia
[79].
Another method developed in recent years allows complete
structural characterisation of compounds in complex mixtures
at microgram levels: HPLC coupled to 1 H NMR spectroscopy.
This method has also been applied for identification of lignans in
plant extracts. The structures of three lignans in Orophea enne-
andra leaves (phillygenin, eudesmin, and epieudesmin) [82]
and of seven lignans in Torreya jackii needles (trachelogenin,
NTG, arctigenin, thujaplicatin-methyl ether, Pino, methyl-Pino,
and dihydrodehydrodiconiferyl alcohol) [86] were primarily
assigned using this technique. HPLC-NMR can be further cou-
pled to other techniques: an MS may be coupled to the HPLC
parallel with the NMR instrument or SPE cartridges may be
installed between the HPLC column and the detector. On-line
HPLC-NMR-MS was applied for the separation and character-
isation of two diastereomers of SDG from flaxseed [87]. The
installation of SPE cartridges between the HPLC and the NMR
instrument solves problems with interference of the eluted sol-
vent that often prevents proper structural elucidation. Using
HPLC-SPE-NMR, the structures of nine lignans (virgatusin,
three diarylbutanes, and five aryltetralins) present in Phyllan-
thus urinaria were elucidated [88].
3.3.2.2. Analysis of plant foods. Examples of lignan-rich foods
are oilseeds, especially flaxseed which together with sesame
seeds seems to be the most lignan-rich of all foods [8,15,89].
The major lignan component in flaxseed seems to be Seco, but
also the amount of MR is larger than in any other analysed foods
[8,15]. The major lignan component in sesame seeds is sesamin,
73
but they also contain HMR [89]. Sesame seeds contain the largest
amount of Pino and Lari of 83 analysed solid foods and 26 bev-
erages [8]. Considerable amounts of lignans (some or all of the
lignans with guaiacyl moieties: Pino, Lari, Seco, and MR) are
also found in other seeds, nuts, grains, grain products, cereals,
vegetables, legumes, fruits, berries [8,15], and olive oil (con-
tains especially Pino) [8,54,89,90]. All or some of these four
lignans can also be found in beverages such as beer, tea, cof-
fee, juices [15], and wine [8,91,92]. In wines also other lignans
have been analysed, and several wines were found to contain
Syri [92] and cLari, which is the major lignan in several wines
[91,92].
Lignans in olive oil were analysed using HPLC-UV (at
278 nm) [54,90]. Flaxseed lignans (SDG, anhydro-Seco, cLari,
Lari, Pino, and MR) have been determined using HPLC-DAD
[37,93,94], which was also used for analysis of lignans in pump-
kin seeds [94]. Lignans in a crude extract of unroasted defatted
black sesame seeds (sesamin, sesamolin, sesamol, sesaminol,
and sesaminol di- and triglucosides) were quantified using
HPLC-UV at 280 nm [95]. However, also other detectors such as
fluorescence and electrochemical (EC) detectors may be useful
in lignan analysis. In one work, HPLC was used in combina-
tion with radiochemical detection for the study of furanofuran
lignan metabolism in sesame seeds (Sesamum indicum) [96].
The lignans analysed were Pino, piperitol, sesamin, sesamoli-
nol, and sesamolin. Fluorescence detection was found to be
superior to UV or electrochemical detection in analysis of
some lignans (1-acetoxy-Pino and Pino) in olive oil extracts
[97].
Lignans in flaxseed and in chaparral (dietary supplement)
have also been analysed using HPLC-thermospray-MS cou-
pled with or in combination with HPLC-UV [80]. Two isomers
of SDG in a methanolysed dioxan-methanol extract of defat-
ted flaxseed meal were identified by HPLC-ESI-MS/MS and
HPLC coupled to continuous-flow-fast-atom bombardment-MS
[98]. The lignans Syri, arctigenin, Pino, Lari, cLari, Seco, MR,
and anhydro-Seco were quantified in wines using HPLC cou-
pled with a coulometric electrode array detector (CEAD) [92].
This technique is based on multiple electrochemical detectors
in series, maintained at different potentials. HPLC coupled to a
heated nebuliser-APCI-MS/MS (triple quadrupole tandem MS)
in the multiple-reaction-monitoring (MRM) mode was used for
quantification of Seco and MR in 112 food items/groups [99].
More recently, an HPLC-APCI-MS/MS (MRM) method was
developed and validated for quantification of Seco, MR, Lari,
and Pino in different foods [100].
3.3.2.3. Analysis of biological fluids. In biological fluids, the
major part of the lignans are in conjugated form, as glucuronides
and sulphates, and therefore these samples are usually enzymati-
cally hydrolysed using ␤-glucuronidase/sulphatase preparations
before sample extraction and chromatographic analysis. Urine
and blood (serum or plasma) are the most frequently analysed
biological fluids.
HPLC-UV has had a limited use in the determination of
lignans in biological fluids. This method was used for the quan-
tification of Pino and Lari in extracts of bacteria and general
74 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
anaerobic medium broth mixtures (at 280 nm) [101]. HPLC-UV
(at 283 nm) and -UV-DAD were used for the analysis of Seco
and MR metabolites in extracts of Seco or MR incubations with
rat or human liver microsomes [102]. Usually, more sensitive
and selective methods than HPLC-UV are required for reliable
quantification of lignans in biological samples, with low lig-
nan levels in complex matrixes. Examples of such methods are
HPLC with CEA or MS detection, especially HPLC-MS/MS
(MRM). The HPLC-CEAD method is limited to lignans with
free phenolic hydroxyl groups and HPLC-MS is limited to com-
pounds with ionisable groups, but this should not be a problem
with lignans. However, very stable compounds that do not easily
undergo fragmentation in the MS cannot be determined using
MRM, which is more selective than single-ion recording.
HPLC-CEAD has been used for the quantification of END
and ENL in blood plasma and uterine tissue of rats [103] and in
human urine samples [103,104]. This technique was also applied
for developing and validating a method for the quantification of
several lignans, i.e. Seco, MR, Lari, Pino, Syri, and cLari, END,
and ENL in human urine [105]. HPLC-CEAD methods were
also developed and validated for the quantification of Seco, MR,
anhydro-Seco, END, and ENL in plasma [106] and for the same
compounds and Lari, cLari, HMR, Pino, Syri, and medioresinol
in human plasma [107].
Urinary levels of END and ENL in female subjects were
measured using HPLC-heated nebuliser-APCI-MS/MS (MRM)
[108]. This appears to be the first study in which lignans were
quantified in urine or blood samples using the HPLC-MS tech-
nique. After this, the technique has been applied in several
studies and fast, sensitive, and selective methods for quantifi-
cation of several lignans in urine and blood samples have been
developed and validated. In one study, HPLC-heated nebuliser-
APCI-MS/MS (MRM) was applied for development and vali-
dation of a method for quantification of MR, END, and ENL in
human serum and urine [109]. The detection limits were com-
parable to those achieved by using HPLC-CEAD.
Methods for quantification of enterolignans and several plant
lignans with guaiacyl moieties in human plasma or rat urine
were developed using the HPLC-ESI-MS/MS (MRM) technique
[55,56,110]. The plant lignans included HMR, MR, 7-oxo-MR,
␣-Coni, ␣- and ␤-ConiA, iso-HMR, epi-iso-HMR, Lari, cLari,
and Seco and the enterolignans included END, ENL, 7-hydroxy-
ENL, monodemethylated MR, 4,4 -dihydroxy-ENL, and 7-oxo-
ENL. The same technique was applied for quantification of END
and ENL in human serum [111] and human plasma [104]. The
lowest detection limits of lignans are achieved using this tech-
nique.
3.3.3. Chiral columns
Lignans are chiral plant metabolites that mostly occur enan-
tiomerically pure or in enantiomeric excess in plants. Determi-
nation of the enantiomeric form of a lignan occurring in a plant
species has usually involved isolating the lignan and measur-
ing the optical rotation. It is, however, possible to determine the
enantiomeric composition in a mixture by using HPLC cou-
pled to UV/laser polarimetric detection, as described in one
work [79]. The enantiomeric composition in a mixture can also
be determined by separation on a chiral HPLC column. This
requires access to known pure enantiomers as references. Both
normal- and reversed-phase chiral HPLC columns have been
used for separation of lignan enantiomers, mostly normal-phase
columns of 4.6 mm i.d. with cellulose carbamate as packing
material. Chiral columns are usually eluted using isocratic flow.
3.3.3.1. Normal-phase chiral columns. Normal-phase columns
Chiralcel OC [adsorbent cellulose tris(phenyl carbamate)] and
Chiralcel OD [adsorbent cellulose tris(3,5-dimethylphenyl car-
bamate)] have been applied in numerous studies for the
separation of lignan enantiomers. A Chiralcel OC column
(250 mm × 4.6 mm) eluted at 0.5 ml min−1 was used for sep-
aration of wikstromol enantiomers isolated from Wikstroemia
sikokiana [112] and for separation of Lari enantiomers in cell-
free extracts from seeds and petiols of Arctium lappa L. [113]
with a mixture of ethanol and hexane as mobile phase, in a ratio
of 50:50 [112] or 80:20 [113].
Chiralcel OD (250 mm × 4.6 mm) columns have been used
in several studies. MR and Pino enantiomers isolated from W.
sikokiana [112] and Pino and piperitol enantiomers in sesame
seeds [96,114] were separated using radiochemical detection
[96] or UV detection at 280 nm [96,112,114]. As mobile phase,
ethanol:1% acetic acid in hexane 15:85 was used at 1 ml min−1
for MR and ethanol at 0.4 ml min−1 [112] or ethanol:hexane
1:1 (flow rate 0.8 ml min−1 ) for Pino [96,114]. For piperitols,
the mobile phase consisted of ethanol: hexane 1:4 (flow rate
0.8 ml min−1 ) [96] or 1:9 (flow rate 0.5 ml min−1 ) [114]. Pino
and Seco enantiomers in cell-free extracts from seeds and peti-
ols of Arctium lappa L. were separated using a mobile phase of
ethanol at 0.4 ml min−1 for Pino and ethanol-n-hexane–acetic
acid (300:693:7) at 0.8 ml min−1 for Seco [113]. Enantiomers
of Seco, anhydro-Seco, Lari, and MR isolated from flaxseed
(detection with UV-DAD) were separated using a mobile phase
of ethanol and n-hexane (flow rate 0.5 ml min−1 ) [94]. Individual
solvent ratios were used for each lignan (65–85% hexane). Pino
enantiomers were separated using a smaller Chiralcel OD col-
umn (3.5 mm × 50 mm) which was eluted with ethanol:hexane
1:1 at 0.5 ml min−1 and UV detection at 280 nm, radiochemical
monitoring or inline laser polarimetry [79].
In a recent work, semi-micro columns of 1.0–2.0 mm i.d.
were found to provide five- to 20-fold better sensitivity than
conventional analytical columns of 4.6 mm i.d. in chiral lignan
analysis [115]. Enantiomers of Pino, Seco, MR, and Lari were
separated using normal-phase chiral columns (Chiralcel OC, OD
or OD-H) of 1.0, 2.0, and 4.6 mm i.d. with UV detection at
280 nm. The mobile phases used consisted basically of ethanol
and n-hexane. Analyses were also performed using an HPLC-
APCI-MS system.
3.3.3.2. Reversed-phase chiral columns. A Chiralcel OD-R
column (250 mm × 4.6 mm) with MS/MS (MRM) detection
was used for the separation and quantification of ENL enan-
tiomers in rat urine extracts [116]. The mobile phase consisted
of methanol/0.1% acetic acid 70:30 and the flow rate was
0.5 ml min−1 . ENL enantiomers were also separated using a chi-
ral CD-Ph column (250 mm × 4.6 mm) at 0.5 ml min−1 using
 S.M. Willför et al. / J. Chromatogr. A 1112 (2006) 64–77
acetonitrile:water 33:77 as mobile phase and UV detection at
280 nm [117].
4. Conclusions
The sampling procedure, sample storage, and sample pre-
treatment are crucial for the outcome of a lignan analysis, since
unwanted chemical reactions such as oxidation, thermal degra-
dation, or polymerisation of sensitive lignans may occur if care
is not taken. Sequential extraction with a non-polar solvent fol-
lowed by extraction with acetone or ethanol is recommended
to separate the lignans from the plant matrix. Additional steps
of acid, alkaline, or enzymatic hydrolysis may be necessary for
some plant matrixes, but care should be taken to avoid chemi-
cal alteration of certain lignans. Accelerated solvent extraction,
which is performed at elevated temperature and pressure and
under inert nitrogen atmosphere, is an excellent method for
extraction of plant lignans. Flash chromatography, preparative
HPLC, and preparative TLC are convenient methods for isola-
tion of pure lignans from crude extracts for structural character-
isation and testing of unknown lignans.
GC is a powerful technique with excellent chromatographic
resolution for analysis of lignans. FID is the best choice of
detector for quantitative GC analysis due to its unique ability
to monitor organic compounds over a wide range of concen-
trations, while GC–MS is an excellent tool for identification of
individual lignans. However, HPLC is the most frequently used
analytical technique for lignans, since no time-consuming pre-
treatment of the samples generally is needed. Reversed-phase
columns are normally used for analytical purposes, since most
studied lignans are of medium polarity. The evolution of new,
tailor-made stationary phases offers growing means to achieve
excellent chromatographic resolution also for complex mixtures
of compounds. Nowadays, UV detection is often used only as
complementary to other techniques, especially after the develop-
ment of HPLC-MS techniques, which mostly include also a UV
diode-array detector. The potential for identification of unknown
compounds is excellent with LC–MSn , while the recent devel-
opment of HPLC-NMR techniques allows complete structural
characterisation of compounds in complex mixtures at micro-
gram levels. HPLC-MS/MS is a powerful method for quantify-
ing known lignans, but requires pure, deuterated compounds that
still are not commercially available. Nevertheless, it is today pos-
sible to buy several different pure lignans also in large amounts.
Lignan enantiomers can be determined using HPLC coupled to
UV/laser polarimetric detection, or by using a chiral HPLC col-
umn if access to known enantiomers as references is available.
The researcher typically has to choose methods and chromato-
graphic equipment available in his/her own laboratory, even
though these may not be state-of-art. However, when it is pos-
sible, several analytical techniques should be combined or used
in parallel to achieve the best result.
Acknowledgements
This work is part of the activities at the Åbo Akademi Pro-
cess Chemistry Centre within the Finnish Centre of Excellence
75
Programme (2000–2005) by the Academy of Finland. Jarl Hem-
ming, Thomas Holmbom, and Markku Reunanen are acknowl-
edged for their help with the examples and figures.
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