Chemical speciation and fractionation of metals in wine

Krystyna Pyrzynska

Warsaw University, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland

ABSTRACT
The analysis of metals in wines is of great importance for authenticity purposes and
quality control. The elements can influence the wine making process or change its taste and
quality. The research has been mostly focused on the determination of total content of
metals regardless of what species were present. However, there is a considerable evidence
that metal bioavailability and toxicity is mainly governed by their chemical forms. Metals
may exist in wines as free ions, as complexes with organic acids as well as species with
large molecules of pectic polysaccharides, peptides, proteins and polyphenols. This review
presents the main developments in chemical speciation and fractionation analysis of metals
in wine samples.

Keywords: metals, chemical speciation, wine

*
E-mail: kryspyrz@chem.uw.edu.pl
INTRODUCTION

Wine is one of the most widely consumed beverages in the world and the evaluation of
its quality is important for manufacturers, merchants and consumers. It contains a large
number of inorganic as well as organic compounds. The principal dissolved species are
inorganic ions (such as potassium, sodium and calcium), organic acids, polyphenols,
proteins, aminoacids and polysaccharides. White wines are less abundant in dissolved
substances than red wines, particularly with respect to polyphenols. Monomeric
polyphenols, such as catechin, are generally considered to be more bitter, while polymeric
tannins are highly astringent (Ebeler, 2001). Phenolic compounds not only contribute to
their sensory characteristics of wine, such as color, flavors and astringency, but may also act
as antioxidants both free-radical scavenging and metal chelation (Peña-Neira et al., 2000). It
has been proved that a moderate consumption of wine is related to a decrease in the risk of
cardiovascular disease (Soleas et al., 1997)
The growing concern about human exposure to different mineral components provided
the impetus for studying their content in food and beverages. In some countries alcoholic
beverages, such as wine, represent over a dozen percent of the daily intake of beverages.
Thus, they could be an important source of several metal ions. Analysis of certain elements
in wines is of special interest due to their toxicity in case of excessive intake as well as the
effect they seem to have on the organoleptic properties of wine. A typical example is copper,
which is both an essential and a potentially toxic element for humans when in excess.
Several elements, including Cu, Fe, Al and Zn, contribute to haze formation and sometimes
taste effects (Riganakos and Veltsistas, 2003). Determination of other elements, such as Pb,
As or Cd is of considerable importance due to their potential toxic effects. Moreover, the
content of some metals can be used to identify the geographic region in which the grapes
were grown due to the direct relationship with soil composition (Barbaste et al., 2002;
Greenough et al., 2005).
The research in respect to the presence of metals in wine has been mostly focused on
the determination of their total amount regardless of what species were present. However,
there is a considerable evidence that metal bioavailability and toxicity is mainly governed
by their chemical forms. Metals may exist in wines as free ions, as complexes with organic
acids as well as species with large molecules of pectic polysaccharides, peptides, proteins
and polyphenols. The speciation of metals with biological ligands in food samples is a

2
subject of increasing interest since complexation may influence their toxicity and
bioavailability. The aim of this article is to present the main developments in chemical
speciation and fractionation analysis of metals in wine samples.

Metals content in wines

The content of metals in wine can be attributed to the natural sources (the atmospheric
deposition of airborne particulate matter on grapes and transfer of metals from the soil via
the roots to the grapes and finally to wine) and to the contamination during the winemaking
process. The presence of different elements can influence the wine making process or can
change the taste and quality of the final product. As a way to control the metal ions in wine,
some countries have imposed rules restricting their content. The rules must be followed by
the producers to gain the right to export to the markets. For example, the Office International
de la Vigne et du Vin (OIV) has legislated the concentrations to 5 mg/L for zinc, 1 mg/L for
copper, 0.01 mg/L for cadmium, 0.2 mg/L for arsenic and lead ((European Union
Regulation, 1990).
Potassium and calcium are the natural components of the grape and their concentration
in wine is a reflection of the grapevine status in the final stages of berry ripening. High
potassium level could result in the precipitation of potassium hydrogen tartrate. The natural
content of calcium can be affected by the addition of CaCO3 for deacidification and elevated
level of this metal can lead also to the onset of calcium tartrate precipitation. Concentration
of aluminum in both grape juice and wine could be elevated by the use of betonite and, to
the less extent, from contact with aluminum surfaces. Increases from a juice concentration of
around 0.1 mg/L to 1 mg/L in the finished wine have been observed (Laroque et al., 1994.
Copper may be carried over into grape juice from the use of copper-based vineyard
sprays, although the concentration in freshly fermented wine is generally low owing to the
ability of dead yeast cells to take up this metal (Scolary, 1997). The main source of copper
in finished product is a consequence of the practice of CuSO4 addition for the removal of
hydrogen sulfide. High residual copper can be contribute to enhanced rate of oxidative
spoilage, which ultimately results in the browning of the wine, particularly white ones. This
phenomenon constitutes one of the principal enological problems for wine producers and
some metal ions (Cu, Fe, Mn) are the activators of this process. In organoleptic terms, this
phenomenon translates into a process of continuous oxidation, a loss of aromatic freshness

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and, in the final stages, in the appearance of precipitates of condensed phenolic material in
the bottled wine. To minimize the incidence of these problems, it is generally recommended
to maintain copper concentration below 0.3–0.5 mg/L (Scolary, 1997). Benitez et al. (2002)
showed that application of ion-exchange resins is extremely effective in lowering the
content of copper, iron and manganese in white wines. The treated wines exhibited a
reduced susceptibility to undergo browning, however, presented also lower polyphenolic
and aromatic profiles.
A possible source of metals can be attributed to residues of agrochemical products
used as insecticides and fungicides as well as fertilizers, which contain salts of metals. A
significant amount of metals is due to contact with the apparatus used in the wine production
and packaging processes. Long maceration at excessively high temperatures probably causes
a through extraction of metals from must. Technological features (pressing), chemical
factors (alcohol content, must acidity) and physical parameters such as temperature could
also affect metal extraction. On the basis of the analysis of chromium concentrations for
different vintage wines of the same vineyard and winery, Cabrera-Vique et al. (1997) found
that Cr content significantly increased with the age of wine. This could be the result of
contamination during storage by stainless steel or by chromium oxides used for
pigmentation of the bottles.
The content of lead in wine may be explained by the natural sources as well as those
related to the production processes. Elevated Pb levels have been attributed to several
possible contamination sources – leaded gasoline, tin-lead capsules used to cover the bottle
neck and brass alloys (Teissedre et al., 1994; Kaufmann, 1998). However, analysis of lead in
several thousands of French wines from 1951 to 1991 showed that atmospheric pollution
associated with automobile emission was not a significant source of lead (Rosman et al.,
1998), Moreover, it was clearly visible that the average lead concentration in wine has
decreased considerably within recent years, probably due to replacement of the brass faucets
by stainless steel (Medina et al., 2000).
Among several analytical methods for metal determination, techniques of atomic
spectrometry (Aceto et al., 2002; Pyrzynska, 2004; Rivero-Huguet, 2004), ICP-MS (Perez-
Jordan et al., 1998; Castiňeira et al., 2001; Almeida et al., 2002) as well as electrochemical
methods (Vasconcelos, et al., 2000; Brainina et al., 2004) have been commonly used. The
matrix components could cause interferences which influence the signal intensity during
measurements. Sample decomposition prior to analysis is very often necessary, particularly

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when metal contents are close to the method’s detection limit. Wet digestion in the closed
systems with the addition of reagents (nitric acid and hydrogen peroxide) to oxide organic
matrix is often applied for decomposition of wine samples (Lara et al., 2001; Castiňeira et
al., 2001; Galani-Nikdakaki et al., 2002). The long time required to perform a total sample
decomposition has been shortened with the use of microwave energy or UV photolysis
(Almeida et al., 2002; Wierzbicki and Pyrzynska, 2002; Riganakos et al., 2003).
Preconcentration and separation methods are needed when the concentrations of metal ions
in wine samples are too low to be determined directly (Lara et al., 2001; Cvetković et al.,
2002).

Speciation and fractionation studies

Speciation analysis gained significance and has a important role in food analysis as
well as in wine analysis. However, wine is a very complex matrix and the accurate
determination of particular chemical forms of metals is a real analytical challenge. Metals
can exist in wines as free metal ions and complexes with organic ligands. The most
important groups of ligands in wine are large molecules of pectic polysaccharides, peptides
or proteins and polyphenols. The large number of potential complexes make speciation
analysis very difficult. Ultrafiltration has been used for examining the distribution of metals
in different particle size fractions (McKinnon and Scollary, 1997; Murányi and Papp, 1998).
Enologicallly the colloidal fraction is very important, because it destroys the quantity of
wines.
According to the main wine organic compounds a scheme was proposed for
fractionation and determination of Fe, Cu and Zn in different fractions (Karadjova et al.,
2002). The charge of metal species was established using cation and anion-exchangers. The
resin Amberlite XAD-8 was applied for separation of wine polyphenols in complexes with
wine proteins and polysaccharides. Labile species of Fe(II) was separated by sorption of its
complex with 1,10-phenantroline. The analytical procedure used for wine component
fractionation is presented in Fig. 1. The results indicated that almost 30% of iron was
complexed with polyphenols and proteins in untreated wines, while the same fraction equals
to 20% in finished and bottled wines. Less than 5% of iron was found in the polysaccharide
fraction. Approximately 50% of copper was present as labile species. Organically-bound
copper was found mainly in the polyphenol/protein fraction while less than 4-5% in the

5
polysaccharide fraction. More than 60% of zinc in investigated wines was present as a
positively charged labile ion and less than 15% was retained on the XAD-8 sorbent as a
complex with polyphenols.
Regarding polyphenol content, flavonoids, anthocyanins and tannins have shown a
marked complexing activity on metal ions resulting in small changes in wine colour
(Vasconcelos et al., 2000; Esparza et al., 2005). During the first day of vinification when the
polyphenols or anthocyanins concentrations were increased, copper and iron contents
decreased (Esperza et al., 2004). Markis and Rossiter (2000) reported on the browing rates
of quercetin and rutin in the presence of Fe(II) and Cu(II) as these metal ions promote
flavonol oxidation through reactive oxygen species formation. Recently, complexation
studies have been carried out involving copper and zinc with synthetic solutions of selected
polyphenols (catechin, rutin and quercetin) as well as with wine samples, concluding that
both metals chelate with the studied polyphenols (Salinas et al., 2005).
Esparza et al. (2006) studied the disruptions of equilibria responsible for colour of
samples undergoing vinification and on a few commercial red wines at the typical
wavelength upon addition of Fe(III). They concluded that a small quantity of iron in wines
produces an increment of blue colour and in a decrease of red colour. No significant
variation in yellow color intensity was found along this experiment. These finding are
consistent with the fact that o-diphenyl group containing anthocyanins may complex iron
inducing a batochromic displacement to the blue region.

Copper
The combination of potentiometry, anodic stripping voltammetry (ASV) and kinetic
photometry permitted the fractionation of copper in wine for determination of its ionic,
labile and tightly bound species (Wiese and Schwedt, 1997). Electrochemical stripping
procedures discriminate between the labile and inert fraction of copper, whereas the
application of ion selective electrode in potentiometry gives only results for free and
uncomplexed metal ion concentration. The labile metal concentration determined by ASV
includes contribution from free metal ions and its readily dissociated complexes. The tightly
bound fraction could be obtained by the difference between the total and labile content.
Figure 2 shows the distribution of copper species in the examined 16 wine samples (Wiese
and Schwedt, 1997). The content of free copper ions was in the range of 3.3 - 31%, while
the fraction contains its labile species was mostly much higher (33-86%). In case of wines

6
where tightly bound species dominated, the concentration of proteins was high (> 34 mg/L)
but small total Cu concentration (< 0.3 mg/L).

Iron
At low concentration, iron plays an important role in metabolism and fermentation
processes as an enzyme activator, stabilizer and functional component of proteins. Above
trace levels, it has other roles such as altering redox systems of the wine in favor of
oxidation, affecting sensory characteristics and participating in the formation with tannins
and phosphates resulting in instabilities. At concentrations above 10 mg/L iron creates
insoluble suspensions which are known as hazes. 60 – 87% of iron in Hungarian wines was
found in colloid solution as this fraction was removed by filtration through a 0.2 µm-pore-
size membrane filter (Murányi and Papp, 1998). Differentiation between insoluble-
suspended iron and aqueous iron was proposed using a sequential cloud point extraction
procedure (Paleologos et al., 2002). The bound-iron fraction was extracted without any
treatment into the small surfactant-rich phase, while in the remaining aqueous phase free
iron could be determined by AAS. Diagrammatic representation of the overall procedure is
shown in Fig. 3.
When protect from air, wine exhibits reducing characteristics and iron is present
mostly as Fe(II). Fe(III) is present in much lower concentrations and usually complexed
with citric and oxalic acids. Ajlec and Štupar (1989) applied different solid sorbents for
separation of iron compounds in wine. In 0.1 mol/L HNO3 medium ionic species of Fe(II)
and Fe(III) were sorbed on cation-exchange resin Dowex 50Wx8, while uncharged organic
iron was passed through the column. The sorbed species were then eluted with 2.25 mol/L
hydrochloric acid and determined by AAS. For separation of negatively charge citrate
complexes of Fe(III) anion-exchange resin Dowex 1x8 was used and uncharged organic iron
was enriched on Amberlite XAD-2 column. It was found that in bottled wines the content of
Fe(III) was higher (75.8 - 94.2%) than in local wines from small grower (32.0 – 82.6%). The
total iron content in bottled wines were significantly lower, compared with non-bottled ones
(Riganakos and Veltsistas, 2003). This may be attributed to the removal of its part during
wine processing, following the alcoholic fermentation.
For determination of Fe(III) and total iron in Portuguese table wines sequential
injection analysis with atomic absorption spectrometry detection was applied (Campos-
Costa and Araújo, 2001). Fe(III) was extracted as its thiocyanate complex with

7
methylisobutylketone. Detection limit of the proposed procedure was 0.03 mg/L with the
sampling rate of 18.5 samples per hour.

Lead
The speciation analysis of ionic organolead compounds in wines was proposed by
Łobinski et al. (1993; 1994). These compounds may arise in wine during the fermentation
due to the natural biomethylation processes from inorganic lead. The analytes were extracted
as diethyldithiocarbamate complexes into hexane and propylated with a Grignard reagent.
The derivatized extract was analyzed by capillary gas chromatography/microwave induced
plasma optical emission spectrometry. Some wines from southern France showed
concentration of Me3Pb+ in the range of 8.1–112 ng/L. Ethyllead compounds were generally
not found in wines produced in rural areas away from the highway. Wines made from grapes
grown close to industrialized centers showed an elevated content of triethyllead (Fig. 4).
Attempts to determine “electrochemically labile” lead, which is supposed to be
bioavailable and thus potentially toxic, by ASV and potentiometric stripping analysis
showed no or little labile fraction at its natural pH of 3.2-3.4 (Scolary, 1997; Green et al.,
1997; Vasconcelos, 2000; Medina et al., 2000). Fractionations pattern using ultrafiltration
demonstrated that the behavior of Pb is significantly different to the other metals (McKinnon
and Scollary, 1997). In red wine, there was a sudden decrease between 100 000 and 30 000
nominal molecular weight cut off (NMWCO), while in white wine, a gradual decrease in the
lead concentration was observed by 100 000 NMWCO ultrafilter.
More recent work has attempted to identify the ligands primarily responsible for lead
complexation in wines (Szpunar et al., 1998). Application of size-exclusion high
performance liquid chromatography with on-line ICP-MS detection evidenced that this
metal is associated with a dimer of a pectic polysaccharide, rhoamnogalacturonan II (dRG-
II), with molecular mass of the order 10 kDa. Wine and fruit juice may have, depending of
the source, content ranging between 50 and 400 mg/L of dRG-II (Perez et al., 2003). Other
minor species with apparent molecular masses in the range of 500 – 3000 Da were not
identified. The fact that lead can be found in the naturally occurring RG-II isolated from
plant cell walls indicates the potentially vital role of this polysaccharide in the Pb uptake by
plants. The excess of unbounded RG-II in wine (its complexation capacity equals about 2
mg/L) can apparently be used to complex lead ions taken with other foodstuffs. However, in

8
order to claim a detoxification capability of this compound, the stability of the Pb-RG-II
complex in the gastrointestinal system needs to be investigated.

Tin
A survey of butyltin compounds in Chinese alcoholic beverages purchased from retail
markets was carried out by the headspace solid-phase microextraction followed by gas
chromatography coupled with flame photometric detection (Liu and G.-B.Jiang, 2002).
Organotin compounds have been used extensively as polyvinyl chloride stabilizers, wood
preserves and agrochemicals. The levels of monobutyltin (MBT) and dibutyltin(DBT)
ranged from < 0.02 to 5.9 µg/L and from < 0.002 to 33.3 µg/L as Sn, respectively. The
results indicated that dry wines generally contained more DBT than sweet wines. The level
of dibutyltin decreased with time; after 110 days of storage the concentration of DBT was
27% of the original content. It was presumed that butyltin compounds degradate during the
storage period.

Conclusion

The analysis of trace metals in wine is of great importance for quality and authenticity
control of wine, metals bioavailability and toxicity. The presence of these elements can
influence the wine making process or can change the taste and quality of the final product.
The content of metals may provide good differentiation about wine geographical origin
due to the direct relationship with soil composition. Although, it requires a necessary
prerequisite that the concentrations within a group of elements or species depend only or at
least mainly on the composition of the soil in the cultivation region, but will not be
significantly influenced from wine production, transport or storage.
More recently work has attempted to identify the ligands primarily responsible for
metals complexation in wines. Metals bioavailability as well as toxicity depend on the
chemical forms in which they are present at the site of absorption to overcome the intestinal
barrier. However, there is a limited number of studies concerning determination of specific
species of a given metal in wine samples. Mostly the methods of physico-chemical
fractionation have been employed taking into consideration an aspect of significance from a
practical point of view, such a size of species, their charge, the presence of “labile” or
“inert” complexes with biological ligands. Although a direct quantification of metal

9
speciation in wine is not attainable, fractionation studies can still provide much useful
information. Recent impressive progress toward lower detection limits in inductively
coupled plasma mass spectrometry, toward higher resolution in separation techniques,
particularly capillary electrophoresis and electrochromatography, and toward higher
sensitivity in electrospray mass spectrometry for molecule-specific detection at trace levels
in complex matrices allows new frontiers to be crossed. This applies in particular to
identification and/or structural characterization of endogenous species of essential and toxic
elements.

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Wierzbicki, T. and Pyrzyńska, K. 2002, Determination of vanadium content in wine by
GFAAS. Chem. Anal. (Warsaw) 47, 449-454.

14
 Captions to Figures

Figure 1. Analytical procedure for wine component fractionation and determination of Cu,
Fe and Zn in different fractions (Karadjova et al., 2002).

Figure 2. Distribution of copper between ionic, labile and tightly bound species in wine
samples (Wiese and Schwedt, 1997)

Figure 3. Flow chart of the procedure followed for the sequential cloud extraction of
suspended and dissolved iron in wine (Paleologos et al., 2002).

Figure 3. Chromatogram of organolead compounds in a white wine sample. Peaks: Me3Pb+

(48.0 ng L-1), Et3Pb+ (3.6 ng L-1), Et2Pb2+ (6.3 ng L-1) (Łobinski et al., 1994).

15
 Fig. 1

Wine sample filtered through 0.45 µm membrane filter

Total Cu, Zn, Fe

Sorption of polyphenols on Cu. Zn and Fe mainly
Amberlite XAD-8 bound to polyphenols

Precipitation of proteins with

acetone Cu, Zn and Fe bound to
protein fraction

Precipitation of polysaccharides Cu, Zn and Fe bound to

with ethanol polysaccharide fraction

Sorption on Dowex 50x8 Labile species of Cu, Zn,

Fe(III) and Fe(II)
conditioned with 0.2 M HNO3

Sorption on Dowex 50x8 Cu, Zn and Fe

negatively charged
conditioned with tartaric acid
species

Sorption on Amberlite XAD-8

after complexation with Labile species of Fe(II)
1,10-phenantroline

16
 Fig. 2

90 ionic species
labile species
80
tightly boud
70
Copper species, %

60

50

40

30

20

10

0
1 2 3 4 5 6 7 8 9 10
Wine sample

17
Fig. 3

18
 Fig. 4

Me3Pb+ (48 ng/L)

Et2Pb2+
(6.3 ng/L)
Et3Pb+ (3.6 ng/L)

19