Food Research International 38 (2005) 943–948

www.elsevier.com/locate/foodres

Anthocyanins from pigmented potato (Solanum tuberosum L.)

varieties
S. Eichhorn, P. Winterhalter *

Institute of Food Chemistry, Technical University of Braunschweig, Schleinitzstr. 20, 38106 Braunschweig, Germany

Received 8 June 2004; accepted 17 March 2005

Abstract

Pigmented potato (Solanum tuberosum L.) varieties are a rich source of anthocyanins, in particular acylated derivatives. From
potato cultivars ‘‘Hermanns Blaue’’, ‘‘Highland Burgundy Red’’, ‘‘Shetland Black’’, and ‘‘Vitelotte’’ the major anthocyanins were
isolated and characterised. Sliced potatoes were blanched to minimise enzymatic reactions which cause degradation of anthocyanins.
By means of solid phase extraction, countercurrent chromatography and preparative HPLC, it was possible to separate coumaric
acid derivatives (i.e. 3-p-coumaroylrutinoside-5-glucosides of petunidin, pelargonidin, peonidin and malvidin) from non-acylated
anthocyanins as well as chlorogenic acids. Identity of the isolated compounds was determined by combination of HPLC-DAD
and LC-ESI-MS2 measurements.
Petunidin derivatives were detected in all varieties except Highland Burgundy Red, where pelargonidin was found to be the only
anthocyanidin. Malvidin was the predominant aglycon of the variety Vitelotte. Of the four selected cultivars, Shetland Black was the
only one containing minor amounts of peonidin derivatives.

2005 Elsevier Ltd. All rights reserved.

Keywords: Potato; Solanum tuberosum L.; Acylated anthocyanins; Anthocyanidin; Countercurrent chromatography; HPLC–MS

1. Introduction Nowadays, a return to diversity can be observed.

Potato (Solanum tuberosum L.) is among wheat, rice,
and maize one of the most important food crops of the
world. In 2003, the world production was more then
310,000,000 metric tons (FAOSTAT data, 2004). Pota-
toes can provide humans with carbohydrates, high value
protein, and vitamin C; potato starch can as well be used
for industrial purposes. The per capita potato consump-
tion is highest in Europe where it exceeds 80 kg/year.
The first potato tubers were brought to Europe from
South America in the late 16th century by Spanish
explorers. Since then, breeding of high-yielding, easy-
to-peel and tasty varieties led to the loss of original, in
particular pigmented varieties.
* composition was already carried out by Harborne
Corresponding author. Tel.: +49 531 391 7200; fax: +49 531 391
7230.
E-mail address: p.winterhalter@tu-bs.de (P. Winterhalter).
Cultivators and researchers as well are interested in
the forgotten cultivars, not at least because of frequent
discussions about health benefits of polyphenolic com-
pounds like anthocyanins. As a consequence, ‘‘old’’ po-
tato varieties are collected around the world to save
them in gene pools.
Anthocyanins are well-known plant colorants
responsible for a spectrum of colour impressions: blue,
purple, red, and orange hues of all plant organs can
be generated by them. A detailed overview about the
structure and occurrence of anthocyanins in fruits,
vegetables, and grains is given by Mazza and Miniati
(1993).
Investigation of Solanum tuberosum L. anthocyanin
(1960) and continued by Andersen, Opheim, Aksnes,
and Frøystein (1991), Rodrı́guez-Saona, Giusti, and

0963-9969/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2005.03.011
944 S. Eichhorn, P. Winterhalter / Food Research International 38 (2005) 943–948

Wrolstad (1998), Lewis, Walker, Lancaster, and Sutton
(1998), Naito et al. (1998) and Fossen and Andersen
(2000). In all studies, the predominance of acylated
anthocyanins (cf. Fig. 1) in pigmented potato varieties
is reported. These anthocyanins are attractive for food
colouring purposes because they show increased pH sta-
bility. For example, red-fleshed potato anthocyanins are
discussed as alternatives to synthetic dyes (Rodrı́guez-
Saona, Giusti, & Wrolstad, 1999).
Because anthocyanins exhibit a range of biological
activities, the contribution of these substances to
health-promoting effects is under current investigations
in many studies. For these purposes, large amounts of
purified anthocyanins are required to carry out in vitro-
and in vivo experiments.
In recent years, countercurrent chromatography
(CCC) has proven to be an effective and versatile tool
for the preparative isolation of anthocyanins and other
bioactive compounds. CCC is an automated, easy-
to-handle method of liquid–liquid chromatography.
Schwarz, Hillebrand, Habben, Degenhardt, and Winter-
halter (2003) give a detailed overview of the mechanism
of CCC as well as several interesting applications for
anthocyanin separations from purple corn, blackberry,
elderberry, and red wine.
In the present study, we report the isolation of
anthocyanins from the pigmented potato varieties
‘‘Hermanns Blaue’’, ‘‘Highland Burgundy Red’’,
‘‘Shetland Black’’, and ‘‘Vitelotte’’ by application of
chromatographic methods, in particular countercur-
rent chromatography.
2. Materials and methods
2.1. Raw material
All potato varieties were cultivated under the same
conditions in 2003 by K. Ellenberg, Bioland Bauernhof,
Barum, Northern Germany. Tubers each were harvested
at full maturity. Ten kilogram of each of the cultivars
‘‘Hermanns Blaue’’, ‘‘Highland Burgundy Red’’, ‘‘Shet-
land Black’’, and ‘‘Vitelotte’’ were used.
2.2. Extraction
Potatoes were washed with tap water and cut in slices
with a Kronen vegetable cutter. After blanching for
5 min with 10 L of water, 10 L of a mixture of water–
hydrochloric acid (19:1 v/v) were added and the suspen-
sion was cooled at 20 C for 3 h. The suspension was
stored at room temperature overnight.
Potato pieces were separated from the extract with a
sieve and discarded. The extract was decanted from sed-
imenting starch and filtrated over glass wool.
2.3. Solid phase extraction
The clear filtrate was directly applied onto an Amber-
lite XAD-7 column (45 · 5 cm). Sugars, organic acids,
proteins, and salts were removed by washing the column
with water. Anthocyanins were retained by the resin and
after all eluted with a mixture of methanol–acetic acid
(19:1 v/v). The eluate was concentrated in vacuo, diluted

R1
OH
+
HO O
R2
OH 3 OH
HO OH
5 O O
O
HO O CH2

HO O
OH
H3C O
R3
HO
OH
O
R3 = OH for non-acylated compounds
R3 = coumaric acid for acylated anthocyanins = HO OH
2
= cleavage position during LC-ESI-MS

aglycon R1 R2
petunidin OCH3 OH
pelargonidin H H
peonidin OCH3 H
malvidin OCH3 OCH3

Fig. 1. Structure of isolated potato anthocyanins.

 S. Eichhorn, P. Winterhalter / Food Research International 38 (2005) 943–948
with water and freeze dried to give the anthocyanin-en-
riched XAD-7-extract.
2.4. Countercurrent chromatography
A CCC-1000 high-speed countercurrent chromato-
graph (triple coil, ID 2.6 mm, total volume 850 mL,
800 rpm, Pharma-Tech, USA) was used. MTBE/n-buta-
nol/acetonitrile/water 2:2:1:5 v/v/v/v acidified with 0.1%
trifluoracetic acid was used as solvent system. The lower
layer was the mobile phase and elution mode was head-
to-tail. Flow rate was set at 4 mL/min. Detection was
carried out at k = 320 nm (Knauer Variable Wavelength
Monitor). Three hundred milligram of the respective
freeze-dried XAD-7 extract was separated in each run.
2.5. Analytical HPLC
Separations of CCC fractions were carried out on a
Synergi MAX-RP column (Phenomenex, 250 ·
4.6 mm) with guard column. A binary gradient of a mix-
ture of water–acetonitrile–formic acid was used: A: 87/
3/10; B: 40/50/10 (v/v/v); %A: 0 min 94%, 20 min 80%,
35 min 60%, 40 min 40%, 45 min 10%, 50 min 10%,
55 min 94%. Flow rate was set at 0.5 mL/min. Detection
was either by DAD (Jasco MD-1510) or ESI-MS2 (Bru-
ker, Esquire) in the positive ion mode. Data about the pur-
ity of anthocyanin fractions is based on the DAD-
chromatogram using k = 520 nm as detection wavelength.
2.6. Preparative HPLC
These separations were performed isocratically on a
Luna C18(2) column (Phenomenex 250*10 mm) with
guard column. Flow rate was 6 mL/min. For the separa-
tion of the Shetland Black fraction, the eluent composi-
tion was acetonitrile, formic acid and water (16.5/5/80
v/v/v) and detection wavelength was set at k = 320 nm
(Knauer Variable Wavelength Monitor). Vitelotte
anthocyanin separations were monitored at
k = 520 nm and eluent composition was adjusted to
15/5/78.5 (v/v/v). The respective anthocyanin mixture
was dissolved in the eluent and aliquots containing
5 mg of the mixture were separated consecutively. The
fractions were collected and freeze dried.
3. Results and discussion
3.1. Extraction and solid phase extraction
‘‘Hermanns Blaue, ‘‘Shetland Black’’ and ‘‘Vitelotte’’
are purple coloured varieties with the latter one the most
intensely coloured cultivar. The flesh of ‘‘Highland Bur-
gundy Red’’ exhibits an intense orange hue which can be
945
regarded as a first hint on the presence of pelargonidin
derivatives.
Thermal treatment (i.e., blanching) may cause partial
anthocyanin degradation. Still it was necessary in order
to inhibit polyphenol oxidase catalysed reactions. A sec-
ond extraction of the potato pieces was omitted to keep
the starch content of the liquid extract low. The subse-
quent decanting step also resulted in a loss of anthocya-
nins. Thus, the yield of anthocyanin enriched extract
was only in the range of 6.5 g and 11.5 g per 10 kg.
3.2. Countercurrent chromatography
CCC liquid–liquid chromatography is a method
which works without solid stationary phase. Separation
is achieved by a two-phase solvent system. In the so-
called Droplet Countercurrent Chromatography
(DCCC), one of the phases is filled in glass tubes which
are connected in series. The other layer passes dropwise
through the glass tubes. Thus, distribution of the respec-
tive sample between the two layers and finally separa-
tion occurs. The use of DCCC for the isolation of
potato anthocyanins was reported by Andersen et al.
(1991). Unfortunately, no detailed separation results
were presented.
A CCC method with higher separation power is high-
speed countercurrent chromatography (HSCCC). In
HSCCC the solvents are filled in a teflon tube which is
wound around a coil. These coils can be connected in
series. A planetary rotation of the coils creates an alter-
nating force field in the tube and mixing and settling
zones are generated. Thus, the sample is continuously
distributed between the two phases and separation oc-
curs. The advantages of this technique with regard to
DCCC are replacement of the fragile glass tube con-
struction by coils and shorter separation times.
For the selection of suitable solvent mixtures, antho-
cyanins should be evenly distributed between the two
phases. This can be observed visually by mixing an ali-
quot of the sample with the two phases in a test tube.
For the potato anthocyanin enriched extract, the appro-
priate solvent system was MTBE/n-butanole/acetoni-
trile/water in the ratio 2/2/1/5 acidified with 0.1%
trifluoracetic acid.
Separation conditions, in particular the amount of
sample load, were identical for all varieties to ensure
comparability of the separations.
The CCC chromatograms (cf. Fig. 2) were recorded
at a wavelength of k = 320 nm in order to also monitor
chlorogenic acids. Separation was finished after 7 h.
Four major fractions were observed in the CCC-separa-
tions of all cultivars. Anthocyanins and 3-chlorogenic
acid always eluted in fractions 1–3, while fraction 4 so-
lely contained 5-chlorogenic acid. All fractions were
analysed by HPLC-DAD and LC–MS2.
946 S. Eichhorn, P. Winterhalter / Food Research International 38 (2005) 943–948
Fig. 2. CCC-separations of potato extracts.
3.3. General aspects of anthocyanin identification
Under the chromatographic conditions employed
for analytical HPLC in this work, acylated anthocya-
nins elute at retention times between 30 and 40 min.
Hence, retention time gives a first hint for the pres-
ence of acylated derivatives. Because the anthocyani-
din moiety is permanently positively charged,
anthocyanins and their fragments can reliably be de-
tected by LC-ESI-MSn in the positive ion mode. Fur-
ther structure elucidation is possible through
interpretation of full mass and fragmentation pattern
of MSn-data as applied by Alcalde-Eon, Saavedra,
de Pascual-Teresa, and Rivas-Gonzalo (2004). Briefly,
the molecular ion M+ is detected in the full mass
scan. The fragments after split-off of the substituent
in C3- or C5-position (Fig. 1) as well as the anthocy-
anidin can be detected in the MS2-mode.
Andersen et al. (1991) elucidated the structure of po-
tato anthocyanins by 2D-NMR techniques. Based on
their work, identity and binding sites of the sugar moie-
ties and the aromatic acid were assigned.
Table 1 gives an overview of analytical data, tentative
structure, amount and purity of anthocyanins isolated in
this study.
3.4. Anthocyanin identification and purification
3.4.1. Hermanns Blaue
Fraction 1 (30 mg), the so-called breakthrough, eluted
after 2.5 h (cf. Fig. 2) and yielded 30 mg of lyophilisate.
The retention time of the major component is 13.2 min
(cf. Table 1). The mass of the molecule ion is m/z =
787 U. On the one hand, fragmentation results in m/z =
625 U, a fragment which is in line with the loss of an
anhydroglucose-unit (m/z = 787 162 U = 625 U) at
C5-position. On the other hand, a fragment with m/z =
479 U (787 308 U) was detected. Loss of 308 U is
characteristic for the split-off of anhydrorutinose (anhy-
droglucose (162 U) and anhydrorhamnose (146 U)).
Thus, it can be deduced that this fragment results from
the loss of a rutinose-unit at C3-position. In addition,
the aglycon petunidin with m/z = 317 after cleavage of
both substituents was detectable. The major component
of this fraction (69%) was tentatively identified as petuni-
din-3-rutinoside-5-glucoside (cf. Fig. 1).
Fraction 2 was the major anthocyanin fraction and
gave 38 mg of a dark red powder. The retention time of
the major compound (molecular ion m/z = 933 U, purity
91%) was 34 min. Fragmentation resulted in a fragment
with m/z = 771 U, according to the loss of an anhydroglu-
cose unit (m/z = 933 162 U = 771 U) at C5-position.
Cleavage of an anhydrocoumaric acid–anhydroruti-
nose-unit (454 U) at C3-position resulted in the signal
with m/z = 479 U (933 454 U). The aglycon petunidin
with m/z = 317 U could be detected as well. Based on
these data, the compound was identified as petunidin-3-
p-coumaroylrutinoside-5-glucoside (cf. Fig. 1). This
anthocyanin is also known under the trivial name petanin
(Harborne, 1960; Andersen et al., 1991). In fraction 3 of
this separation 3-chlorogenic acid was detected.
3.4.2. Highland Burgundy Red
The major component of the breakthrough fraction
(purity 94%) exhibits a retention time of 15.5 min in
analytical HPLC. Hence, it is a non-acylated anthocya-
nin. Fragmentation pattern is the same as for petunidin-
3-rutinoside-5-glucoside, but shifted downwards by
46 U because the aglycon moiety consists of pelargoni-
din. In the second fraction 3-chlorogenic acid was iden-
tified. As expected, the fragmentation pattern of the
major anthocyanin peak in fraction 3 is the same as
for petanin. Again, the difference in molecular masses
of 46 U indicated the presence of pelargonidin-3-p-cou-
maroylrutinoside-5-glucoside, also known as pelanin
(Harborne, 1960; Naito et al., 1998; Rodrı́guez-Saona
et al., 1998). Fifty-two milligram of the substance with
a purity of 84% could be isolated from 300 mg of the
XAD-7-extract in a single run.
The variety Highland Burgundy Red contains the
same anthocyanins as Hermanns Blaue, with the excep-
tion that in this case the aglycon is pelargonidin. There-
 S. Eichhorn, P. Winterhalter / Food Research International 38 (2005) 943–948 947

Table 1
Analytical data of anthocyanin fractions
Variety Fraction Rt (min) molecule ion Fragment ions Aglycon Tentative structure Amount (mg) Purityb (%)
M+ (m/z) (MS2)
Hermanns Blaue 1 13.2 787 625 pet pet-3-rut-5-glc 30 69
479
317
2 34.0 933 771 pet pet-3-coum-rut-5-glc 38 91
479
317
Highland Burgundy Red 1 15.5 741 579 pel pel-3-rut-5-glc 35 94
433
271
3 37.0 887 725 pel pel-3-coum-rut-5-glc 52 84
433
271
Shetland Black 1 17.1 771 609 peo peo-3-rut-5-glc 46 38
463
301
2 34.0 933 771 pet pet-3-coum-rut-5-glc 31 83
479
317
3a 34.0 933 771 pet pet-3-coum-rut-5-glc 5c 94
479
317
37.8 917 755 peo peo-3-coum-rut-5-glc 0.6c 93c
463
301
Vitelotte 1 17.5 801 639 mal mal-3-rut-5-glc 57 66
493
331
2 34.0 933 771 pet pet-3-coum-rut-5-glc 5c 95c
479
317
37.3 947 785 mal mal-3-coum-rut-5-glc 11c 94c
493
331
Abbreviations. pet, petunidin; pel, pelargonidin; peo, peonidin; mal, malvidin; coum, coumaroyl; rut, rutinoside; glc, glucoside.
a
Major component of this fraction was 3-chlorogenic acid, but because of the occurrence of an acylated peonidin-derivative this fraction was
listed, too.
b
Based on HPLC-chromatogram, wavelength k = 520 nm.
c
After preparative HPLC.

fore, the absorption maxima of these pigments are
shifted to the range of k = 500–504 nm which corre-
sponds with the orange hue of Highland Burgundy
Red potatoes.
3.4.3. Shetland Black
Fraction 1 is a mixture of non-acylated anthocyanins.
The major compound was tentatively identified as
peonidin-3-rutinoside-5-glucoside (38%). The major
acylated anthocyanin of Shetland Black eluted after 3 h
and was identical with the major anthocyanin of Her-
manns Blaue, i.e., petunidin-3-p-coumaroylrutinoside-
5-glucoside (cf. Fig. 2 and Table 1).
Major component of fraction 3 is 3-chlorogenic acid,
but HPLC-DAD revealed that two acylated anthocya-
nins coelute in this fraction. Analysis in the positive
ion mode by LC–MS2 revealed that these anthocyanins
are petunidin- and peonidin-3-p-coumaroylrutinoside-5-
glucoside, respectively. Because the acylated peonidin-
derivative was not detectable in all other varieties, the
fraction was subjected to preparative HPLC. The chro-
matogram of this separation was recorded at
k = 320 nm to enable detection of the anthocyanins as
well as 3-chlorogenic acid and is displayed in Fig. 3.
Approximately 1 mg of peonidin-3-p-coumaroylrutino-
side-5-glucoside could be isolated so far. The substance
is also known as peonanin and was inter alia isolated
from the sprouts of a Norwegian potato cultivar by Fos-
sen, Øvstedal, Slimestad, and Andersen (2003).
3.4.4. Vitelotte
From the four selected cultivars, Vitelotte is the only
one which contains malvidin derivatives (cf. Table 1).
According to this, the major component (66%) in the
948 S. Eichhorn, P. Winterhalter / Food Research International 38 (2005) 943–948
Fig. 3. Preparative HPLC separations of 3-p-coumaroyl-rutinoside-5-
glucosides, anthocyanin peaks are named with the respective aglycon; (a)
Shetland Black fraction 3, k = 320 nm (b) Vitelotte fraction 2,
k = 520 nm.
first peak was determined as malvidin-3-rutinoside-5-
glucoside.
CCC fraction 2 is a mixture of petunidin- and malvi-
din-3-p-coumaroylrutinoside-5-glucoside, which could
be separated by subsequent preparative HPLC (Fig. 3).
Because analytical HPLC of this fraction with diode array
detection had revealed no compounds except anthocya-
nins, the preparative separation was monitored at
k = 520 nm. For base line separation, the acetonitrile con-
tent had to be reduced to 15%. In this way, 5 mg of petanin
and 11 mg of malvidin-3-p-coumaroylrutinoside-5-gluco-
side with high purity could be isolated. The latter com-
pound is also referred to as negretein by Harborne
(1960) and was recently the objective of a study about
new insights into the mechanism of intramolecular copig-
mentation of anthocanins (George et al., 2001). Major
component of fraction 3 is 3-chlorogenic acid.
4. Conclusions
By a combination of solid phase extraction, counter-
current chromatography and preparative HPLC, antho-
cyanins of Solanum tuberosum L. varieties were isolated.
To our knowledge, this is the first application of
HSCCC to the separation of potato pigments. Structure
identification was achieved by HPLC-DAD and LC-
ESI-MS2. The 3-p-coumaroylrutinoside-5-glucoside of
petunidin, pelargonidin, peonidin, and malvidin as well
as the respective non-coumaroylated anthocyanins were
enriched or even obtained in high purity. Petunidin
derivatives were detected in all varieties except Highland
Burgundy Red, where pelargonidin was found to be the
only anthocyanidin. The predominant aglycon of the
variety Vitelotte was malvidin. Of the four selected cul-
tivars, Shetland Black was the only one providing minor
amounts of peonidin derivatives.
Acknowledgements
The potato cultivars were donated by K. Ellenberg
(Barum, Germany). The assistance of M. Messerer, M.
Wichoczek and J. Rodziewicz is gratefully acknowl-
edged. M. Schwarz is thanked for helpful discussions.
This study was supported by a Grant of Deutsche For-
schungsgemeinschaft (Wi 901/8-1).
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<http://apps.fao.org/faostat/default.jsp> (last accessed May 2004).