International Journal of Mass Spectrometry 424 (2018) 27–34

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International Journal of Mass Spectrometry

journal homepage: www.elsevier.com/locate/ijms

Full Length Article

Characterisation of yacon tuberous roots and leaves by DART-TOF/MS

Aleš Rajchl a,∗ , Eloy Fernández Cusimamani b , Jana Prchalová a , Rudolf Ševčík a ,
Helena Čížková a , Jana Žiarovská c , Michaela Hrdličková b
a
Department of Food Preservation, Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Technická 3, 166 28
Prague 6 – Dejvice, Czech Republic
b
Department of Crop Sciences and Agroforestry, Faculty of Tropical AgriSciences, Czech University of Life Sciences in Prague, Kamýcká 129, 165 21 Prague 6
– Suchdol, Czech Republic
c
Department of Genetics and Plant Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76
Nitra, Slovak Republic

a r t i c l e i n f o a b s t r a c t

Article history: Yacon [Smallanthus sonchifolius (Poepp. et Endl.) H. Robinson] is a plant grown worldwide originating
Received 30 March 2017 in the Andes region. Yacon is grown for its sweet tuberous roots and leaves used for the preparation of
Received in revised form 7 November 2017 herbal infusions. Twenty-six yacon landraces’ leaves and roots (both peeled and unpeeled) have been
Accepted 7 November 2017
analysed by DART-TOF/MS. The method has been optimised and the fingerprints of the mass spectra
Available online 22 November 2017
have been statistically processed by PCA and LDA statistical analysis. The DART method has succeeded
in differentiating between the yacon landraces according to their genotype and geographical origin.
Keywords:
© 2017 Elsevier B.V. All rights reserved.
Smallanthus sonchifolius
Statistical analysis
PCA
LDA
DART

1. Introduction
Yacon [Smallanthus sonchifolius (Poepp. et Endl.) H. Robinson]
from the Asteraceae botanical family is a native plant of the Andes
region, where it is cultivated for its sweet tuberous roots. The roots
contain fructooligosaccharides (FOS) and inulin, which are best-
known as sources of prebiotics with a positive effect on human
health [1,2]. The yacon leaves and tuberous roots are rich in
polyphenols [3]. The polyphenolic component of the yacon leaves
have a strong antioxidant effect; yacon leaves can, therefore, be
used in the human diet to prevent chronic diseases [4]. Yacon is
eaten raw as a fruit and is prized for its sweetness, mainly by chil-
dren. In the Andean region, yacon is cultivated from Colombia to
Argentina, mostly in small areas and for the farmer’s own consump-
tion. Peru, is the country with the largest biodiversity of yacon. In
Peru, more than 200 accessions have been found, while in Bolivia
and Ecuador, 40 and 32 accessions have been observed, respectively
[5]. Due to its attributes, yacon has expanded to other regions out-
side the Andes. Currently, it is grown in Brazil, the Czech Republic,
China, Japan, New Zealand, the Philippines, Russia, South Korea,
the U.S.A and other countries [6–10]. The Czech Republic has 26
accessions; it is the largest collection outside of the Andean region.
There is huge morphological, chemical and molecular variability
in yacon [11]. The yacon tuberous root flesh colour can be white,
cream, white with purple striations, purple, pink or yellow. Their
skin can be brown, pink, purple, cream, or ivory white, and contains
resin tubes filled with yellow crystals. These properties are typi-
cal for all genotypes [12,13]. The total saccharide content in the
yacon tuberous roots (80.62–224.7 g/kg dm.) [1], the FOS content
(2.1–70.8 g/100 g dm.) [14] and the content of the total phenols in
the leaves and tuberous roots are influenced by the genotype [3,15].
DART (Direct Analysis in Real Time) is a novel ionisation tech-
nique of mass spectrometry. DART principles and advantages have
been well described in literature [16–18]. This technique is used
for the inspection of food and raw food materials. Possible applica-
tions of the DART-TOF/MS technique are summarised in literature
[16,19]. The aim of this work was to characterise the tuberous
roots and leaves of 26 yacon accessions with the DART-TOF/MS
technique.

∗ Corresponding author.
E-mail address: ales.rajchl@vscht.cz (A. Rajchl).

https://doi.org/10.1016/j.ijms.2017.11.005
1387-3806/© 2017 Elsevier B.V. All rights reserved.
28 A. Rajchl et al. / International Journal of Mass Spectrometry 424 (2018) 27–34

Table 1
Description of the yacon accessions.

Codes Qualitative characteristics Year of introduction to Chromosome

the Czech Republic number (2n)

Colour of root skin Colour of root flesh Colour of leaves

PER01 greyish orange yellow white greenish yellow 2005 58

PER02 greyish orange white greenish yellow 2005 58
PER03 purplish red orange yellow dark green 2005 58
PER04 purplish grey yellow white greenish yellow 2005 58
PER05 purplish red yellow white dark green 2005 87
PER06 purplish grey white dark green 2005 58
PER07 greyish orange yellow white greenish yellow 2005 58
PER08 purplish grey white greenish yellow 2005 58
PER09 greyish orange orange yellow dark green 2005 58
PER10 white orange yellow dark green 2005 58
PER11 greyish orange yellow white greenish yellow 2005 87
PER12 purplish grey white dark green 2005 87
PER13 white white greenish yellow 2005 87
PER14 greyish orange yellow white dark green 2005 87
a
PER15 greyish orange yellow white greenish yellow 2008
BOL20 purplish red yellow white dark green 1995 58
BOL21 greyish orange yellow white greenish yellow 2007 58
BOL22 purplish red yellow white greenish yellow 2007 58
BOL23 purplish grey white with purple red strains dark green 2007 58
BOL24 greyish orange yellow greenish yellow 2007 58
ECU40 purplish red yellow white dark green 1994 58
NZL51 white orange yellow greenish yellow 1993 58
NZL52 purplish grey white dark green 1993 58
NZL53 purplish grey orange dark green 2007 116
NZL54 purplish grey orange dark green 2007 116
GER30 purplish grey yellow white greenish yellow 1994 58
a
Not measured.

2. Materials and methods
2.1. Plant material
For this study, 26 yacon accessions cultivated in the climatic
conditions of the Czech Republic were analysed. The colour of the
leaves and tuberous roots-root skin and flesh (assessed a year after
the introduction to the Czech Republic), along with a chromosome
number for each of the accessions, are shown in Table 1. The yacon
accessions came from different climatic regions: Bolivia (BOL 20,
BOL 21, BOL 22, BOL 23, and BOL 24), Ecuador (ECU 40), Germany
(GER 30), Peru (PER 01, PER 02, PER 03, PER 04, PER 05, PER 06,
PER 07, PER 08, PER 09, PER 10, PER 11, PER 12, PER 13, PER 14,
and PER 15), and New Zealand (NZL 51, NZL 52). Accessions NZL
53 and NZL 54 were obtained by in vitro induced polyploidisation
from accession NZL 51 [20]. The yacon collection is kept by the
Faculty of Tropical AgriScience, the Czech University of Life Sci-
ences Prague. The plant material was cultivated in field conditions
on experimental plots of the Faculty of Tropical AgriSciences, the
Czech University of Life Sciences Prague. The plots lie at an average
altitude of 286 m above sea level (50◦ 04 north latitude and 14◦ 26
east longitude), and have loamy soils. The yacon leaves and tuber-
ous roots were harvested in October 2014 after 146 and 157 days of
cultivation, respectively. The samples (leaves and tuberous roots)
for the analyses were taken randomly from 2 to 4 plants of each
clone.
The leaves were removed from the fourth top node of the main
stem of each plant. The weight and size of the tuberous roots used
in the analyses fluctuated between 100 and 300 g and 12–20 cm,
respectively, depending on which landraces were used.
2.2. DART/TOF-MS method
The parameters of the DART ion source for the measurements
were as follows: a positive ionisation mode, a grid voltage of 350 V,
an autosampler velocity of 1 mm/s, and an ionisation gas temper-
ature of 300 ◦ C. A DART SVP 100 ion source (IonSense, Saugus, MA,
USA) was used. DART consists of a DART controller and a total ion-
isation source. For the measurements, helium gas (SIAD, Prague,
Czech Republic; purity: 4.8) was used, whereas nitrogen gas was
used in the standby mode. Nitrogen was generated by a nitro-
gen generator (Peak Scientific, NM32LA at an operating pressure
of 6.0 bar) and purified by an RMSN-4 Agilent universal trap. The
pressure of helium and nitrogen on the default input of the DART
source was set at 5.5 bar using reduction valves. The DART source
was coupled with a TOF-MS by a vacuum interface (IonSense).
At the interface, an 83 mm long and a 3.18 mm diameter ceramic
tube (for the DART-100 ion source) was used. Evacuation of He
from the space of the interface (to prevent a low vacuum being
indicated by the TOF-MS measurement accessories calibrated for
nitrogen) was performed using an MZ 2NT Vacuum Pump (Vac-
uubrand, Wertheim, Germany). Samples were analysed using a
®
12 DIP-it autosampler. For the measurements, DIP-it (IonSense,
Saugus, MA, USA) sampling rods were used. The DART ion source
can operate at temperatures between 50 and 500 ◦ C. The veloc-
ity of the autosampler movement ranges from 0.2 to 10.0 mm/s.
The default flow of the reaction gas (helium) was set at 3.5 l/min.
The DART source was controlled by the DART – SVP 3.0.3b soft-
ware (IonSense, Saugus, MA, USA). The mass spectrometer was
operated in both positive and negative ion modes; the fragmen-
tor voltage was 175 V and the skimmer voltage was 65 V. For data
acquisition and processing, an Agilent MassHunter Workstation
Acquisition B.04.00 (Agilent Technologies, Santa Clara, USA) and
an Agilent MassHunter Workstation Software Qualitative B.04.00
were used. For mass spectral studies, the total ion current (TIC)
chronogram was registered in the range of m/z 100–1500. A TOF
LC/MS 6224 mass detector (Agilent Technologies, Santa Clara, USA)
was used. A rough vacuum in the TOF-MS was created by an
Edwards E2M28 vacuum pump (Grawley, West Sussex). Tuning of
TOF-MS was carried out before each set of samples using an API-
TOF Reference Mass Solution Kit (Agilent Technologies, Santa Clara,
USA).
 A. Rajchl et al. / International Journal of Mass Spectrometry 424 (2018) 27–34
Fig. 1. The spectra of yacon (ECU 40) leaves in positive (1A) and negative (1B) mode, the yacon tubers in positive (1C) and negative (1D) mode, the peeled yacon roots in
positive (1E) and negative (1F) mode, ionisation temperature 400 ◦ C.
2.3. Sample preparation
Before the analyses, the tubers were carefully washed by drink-
ing water. The tubers were analysed both unpeeled and peeled
with a kitchen peeler. The yacon leaves (2.5 g) and the yacon tubers
(20 g) were weighed and then homogenised with 20 ml and 30 ml
of methanol, respectively, using an ultra-turrax IKA T18 basic labo-
ratory homogeniser. All of the samples were analysed immediately
after preparation. Glass rods (DIP-it) were dipped into the sample
solution and then transferred into the autosampler. The samples
thus prepared were used for the qualitative analyses by DART-
TOF/MS.
2.4. Statistical analysis
Tests were triplicated for each of the samples and the mean val-
ues ± SD (standard deviation) are reported. Differences at p < 0.05
were regarded as significant. All statistical analyses were per-
formed using an Unscrambler X 10.1 (CAMO Software releases,
Oslo, Norway), an XLStat (Addinsoft, Paris, France) and Excel 2010
(Microsoft, Prague, Czech Republic). The Mass Hunter software
enables the export of mass data and spectra into Excel. In Excel, a
macro was created which enabled the sorting of the data according
to m/z for a given abundance threshold. The data were normalised
before the statistical analysis.
29
3. Results and discussion
The tuberous roots (peeled and unpeeled) and leaves have been
used for the characterisation of the yacon plant material. Firstly,
the accessions were qualitatively and quantitatively characterised
according to the colour of the root’s skin and flesh, the colour
of the leaves and the number of chromosomes (Table 1). Table 1
also includes information about the year of the introduction of
the yacon accessions to the Czech Republic: the yacon plants were
introduced between 1993 and 2008. The qualitative characterisa-
tion showed that the most common root skin colours were greyish
orange (34.6%) and purplish grey (34.6%). The most frequent root
flesh colour was yellow white (42.3%). Half (50%) of the accessions
had a greenish yellow leaf. The chromosome number of the yacon
accessions (2n) was typically 58 (octoploid) and 87 (dodecaploid).
Two samples had 116 chromosomes (hexadecaploid).
3.1. Characterisation of the yacon leaves
Preparation of the yacon leaf samples for DART-TOF/MS has
been evaluated. The sample weight, the solvent (methanol, water,
and toluene) and the sample homogenisation have been optimised.
The best signal to noise ratio was observed using a mixture of 2.5 g
of the leaves homogenised well with 20 ml of methanol. Examples
of the DART-TOF/MS spectra of the yacon (ECU 40) leaves in posi-
30 A. Rajchl et al. / International Journal of Mass Spectrometry 424 (2018) 27–34

Fig. 2. PCA analysis of the mass spectra of the measured samples of the yacon leaves, A, C – positive mode, B, D – negative mode, ionisation temperature 400 ◦ C.

Table 2 botanical difference. GER 30 is the oldest grown yacon accession

Identified compounds in the mass spectra.
in the climatic conditions of Central Europe (probably since 1941)
Compound Observed ions Elemental Experimental [25]. Accession ECU 40 was the only one in the analysed sample set
composition mass (m/z) which originated from Ecuador. The LDA plots made from the mass
Dihexose [M+NH4 ]+ C6 H16 NO6 198.10 spectra (A – positive, B – negative) of the yacon leaves are given
Dihexose [2M+NH4 ]+ C12 H28 NO12 378.11 in Fig. 3. The results show that the separation of accessions is not
PLO [M+NH4 ]+ C55 H106 NO6 874.46 satisfactory.
POS [M+NH4 ]+ C57 H108 NO6 878.82
LLS/LOO [M+NH4 ]+ C57 H106 NO6 900.80
LOS/OOO [M+NH4 ]+ C57 H108 NO6 902.82 3.2. Characterisation of the yacon tuberous roots
SSO [M+NH4 ]+ C57 H112 NO6 906.85

P: palmitic acid (C16:0), S: stearic acid (C18:0), O: oleic acid (C18:1), L: linoleic acid
(C18:2).
tive and negative modes are shown in Fig. 1 (part 1A and 1B). The
relative abundance observed in the positive and negative ion spec-
tra of the yacon leaves is given in Tables S1–S3. The interpretation
of the DART-TOF/MS mass spectra is generally difficult, however,
the ions of saccharides and triacylglycerols were observed in pos-
itive mode. The interpretation of the observed ions of saccharides
and triacylglycerols are given in Table 2 [21,22]. The formation of
[M+NH4 ]+ ions is caused by the presence of a negligible concentra-
tion of from breath exhaled ammonia in the laboratory atmosphere
[23,24].
The measured mass spectra was processed by principal com-
ponent analysis (PCA) and linear discriminant analysis (LDA). The
2D and 3D PCA plots based on the mass spectra acquired in the
positive-ion mode of all the yacon accession’s leaves are given in
Fig. 2A and C, respectively. The 2D and 3D PCA plots based on the
mass spectra acquired in the negative-ion mode on all the yacon
accession’s leaves are given in Fig. 2B and D, respectively. The PCA
showed that differences between the yacon leaves were not sig-
nificant. The majority of samples were gathered within a relatively
small cluster and only a few samples were outlying. The outlying
of accession GER 30 was rather expected due to the considerable
Preparation of the yacon tuberous roots for the DART-TOF/MS
analysis was evaluated. The sample weight, the solvent (methanol,
water, and toluene) and the sample homogenisation were opti-
mised. The best results (the best signal to noise ratio) were observed
in the mixture of 20 g of the tubers homogenised with 30 ml of
methanol to make a paste. Examples of the DART-TOF/MS spec-
tra of the yacon (ECU 40) tubers in the positive- and negative-ion
modes are shown in Fig. 1 (part 1C and 1F). The relative abun-
dances observed in the positive and negative ion spectra of the
yacon tubers are given in Tables S1–S3. The identified compounds
of the yacon leaves and tubers are given in Table 2.
The measured mass spectra were processed by PCA (see
Figs. 4 and 5). Yacon accession GER 30 was well separated from
the other accessions in the PCA plots made from the mass spectra
of the unpeeled tubers measured in the positive mode (see Fig. 4C).
From Fig. 4A and B, it can be seen that the yacon accessions cluster
according to the yacon’s geographical origin. The yacon accessions
from Bolivia, Germany and Ecuador are well separated from the
accession from Peru and New Zealand in the PCA plots made from
the mass spectra of the unpeeled tubers measured in the nega-
tive mode (see Fig. 4B and D). Clustering trends similar to those
observed in the unpeeled tubers can be seen in the PCA plots made
from mass spectra of the peeled tubers measured in the positive
and negative mode (see Fig. 5). The accessions from each country
of origin are partially separated. The LDA plots (A – positive mode,
 A. Rajchl et al. / International Journal of Mass Spectrometry 424 (2018) 27–34 31

Fig. 3. LDA analysis of the mass spectra of the measured samples of the yacon leaves, A – positive mode, B – negative mode, ionisation temperature 400 ◦ C.

Fig. 4. PCA analysis of the mass spectra of the measured samples of the unpeeled yacon tubers, A, C – positive mode, B, D – negative mode, ionisation temperature 400 ◦ C.

B – negative mode) derived from the mass spectra of the unpeeled In the positive mode, clustering of the accessions from Peru was be
tubers are given in Fig. 6. In the negative mode, the accessions from seen. The LDA plots (A – positive mode, B – negative mode) made
Bolivia are separated from accessions from Peru and New Zealand. from the mass spectra of the peeled tubers are given in Fig. 7. In the
32 A. Rajchl et al. / International Journal of Mass Spectrometry 424 (2018) 27–34

Fig. 5. PCA analysis of the mass spectra of the measured samples of the peeled yacon tubers, A, C – positive mode, B, D – negative mode, ionisation temperature 400 ◦ C.

Fig. 6. LDA analysis of the mass spectra of the measured samples of the unpeeled yacon tubers, A – positive mode, B – negative mode, ionisation temperature 400 ◦ C.

positive mode and particularly in the negative mode, the acces- suitable for the characterisation of the geographical origin. The pre-
sions were differentiated according to the country of origin. Based sented metabolomics approach can be widely used to assess the
on the recorded results, the DART-TOF/MS technique seems to be taxonomical characteristics of plants.
 A. Rajchl et al. / International Journal of Mass Spectrometry 424 (2018) 27–34
Fig. 7. LDA analysis of the mass spectra of the measured samples of the peeled yacon tubers, A – positive mode, B – negative mode, ionisation temperature 400 ◦ C.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.ijms.2017.11.005.
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