Food Chemistry 338 (2021) 127924

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Multivariate modelling techniques applied to metabolomic, elemental and T

isotopic fingerprints for the verification of regional geographical origin of
Austrian carrots
Zora Jandrica, , Anastassiya Tchaikovskyb,1, Andreas Ziteka,b, Tim Causona, Vaclav Stursac,
⁎

Thomas Prohaskad, Stephan Hanna,b

a
Institute of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
b
FFoQSI - Austrian Competence Centre for Feed and Food Quality, Safety & Innovation, Technopark 1C, 3430 Tulln, Austria
c
Brno University of Technology, Faculty of Chemistry, Department of Food Chemistry and Biotechnology, Purkynova 118, 612 00 Brno, Czech Republic
d
Chair General and Analytical Chemistry, Montanuniversität Leoben, Franz Josef - Strasse 18, 8700 Leoben, Austria

ARTICLE INFO ABSTRACT

Keywords: An exploratory study for verifying regional geographical origin of carrots from specific production regions in
Carrots Austria (“Genussregionen”) was performed by combining chemical fingerprinting methods, namely n(86Sr)/n
Fingerprinting (87Sr) isotope amount ratios, multi-elemental and metabolomic pattern. Chemometric classification models were
Metabolomics built on individual and combined datasets using (data-driven) soft independent modelling of class analogies and
Multi-element
(orthogonal) projections to latent structures-discriminant analysis to characterise and differentiate carrots grown
Origin
in five regions in Austria. A predictive ability of 97% or better (depending on the classification technique) was
Strontium isotopes
Chemometric classification obtained using combined Sr isotope amount ratios and multi-elemental data. The use of data fusion strategies, in
particular the mid-level option (fusion of selected variables from the different analytical platforms), allowed
highly efficient (99–100%, except soft independent modelling of class analogy with 97%) and correct classifi-
cation of carrot samples.

1. Introduction
Increasing consumer interest in high quality food products with
reliable geographical origins has emerged due to globalization of food
supply chains as well as the variety and improved availability of pro-
ducts from other countries. Moreover, increasingly regionalized labels
of origin are used to highlight specific production regions and also to
strengthen the regional economy. Consumers rely that information
provided on product labels is genuine. However, mislabelling and
adulteration of food products are still two major worldwide problems
(Camin et al., 2017). Fraudulent labelling of agricultural and food
products with respect to geographical origin is detrimental to con-
sumers and legitimate producers (Hiraoka, Morita, Izawa, Aoyama,
Shin, & Nakano, 2016). Thus, verifying the authenticity and origin of
foodstuffs is important for the integrity of the food supply chain and
everyone involved.
In Europe, origin is one of the main authenticity issues concerning
food. Therefore, the European Union has established legislation to
protect the reputation of the regional food and to promote good prac-
tices in rural and agricultural areas using labels such as PDO (Protected
Designation of Origin), PGI (Protected Geographical Indication), TSG
(Traditional Specialties Guaranteed), or optional quality terms (OQT)
such as “mountain product” and “product of island farming”)
(European Commission, 1992; European Commission, 2012). In Aus-
tria, gourmet regions (“Genussregionen”) were introduced by the Aus-
trian Federal Ministry of Sustainability and Tourism (BMNT) and
AgrarMarkt Austria marketing agency (AMA) with the aim to support
local producers and promote Austrian traditional food. This project is
also supported by the EU in the framework of agriculture and rural
development. The gourmet regions label guarantees a traceable origin,
absolute safety and high quality of food across Austria. Most domestic
agricultural products in Austria (labelled as “Genussregionen”) are
often more expensive than imported, possibly because of higher pro-
duction and/or labour costs, administrative burdens, Austria's strict
environmental legislation (BMLFUW, 2015), high confidence in the
quality and safety of these products or specific culinary or organoleptic

⁎
Corresponding author.
E-mail address: zora.jandric@boku.ac.at (Z. Jandric).
1
Present address: University of Vienna, Institute of Analytical Chemistry, Währingerstraße 38, 1090 Vienna, Austria.

https://doi.org/10.1016/j.foodchem.2020.127924
Received 24 January 2020; Received in revised form 30 July 2020; Accepted 22 August 2020
Available online 27 August 2020
0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
Z. Jandric, et al.
qualities associated with regional products. As these products may yield
a higher price on the market, it is possible that some producers and/or
retailers supply agricultural products with false labelling of geo-
graphical origin to obtain illicit profits. Therefore, it is necessary to find
reliable identification tools for Austrian primary agricultural products
which allow tracing them back to their geographical origin. One of the
most economically important vegetables grown in these specific regions
(“Genussregionen”) in Austria (AMA, 2018) and worldwide (FAO,
2016) are carrots (Daucus carota L.). In Austria, the amount of domestic
carrot production and importation in 2017 were 97813 t and 10443 t,
respectively (AMA, 2018). For these reasons, carrots were chosen as a
model test commodity in this study involving a range of advanced
analytical methods.
Recently, there has been an increase in the number of targeted
analytical methods applied for tracing the geographical provenance of
agricultural and food products. The n(87Sr)/n(86Sr) isotope amount
ratio has proven to be an important chemical marker of the local
geology (rocks, soils). This isotopic information is then transferred to
water, soil and plants and can be used as an indicator for the geo-
chemical origin of food (Hiraoka et al., 2016; Brunner, Katona,
Stefanka, & Prohaska, 2010). In order to improve origin determination,
Sr isotopes were also combined with multi-elemental (Di Paola-Naranjo
et al., 2011) or other stable isotopes (e.g. carbon, nitrogen) fingerprints
(Rummel, Hoelzl, Horn, Rossmann, & Schlicht, 2010). In case that the
geochemical or soil information is too similar between regions, other
parameters on the level of the molecular composition of food might be
also helpful for further discrimination, for example via the analysis of
flavonoids (Tomas-Barberan, Ferreres, Garcia-Viguera, & Tomas-
Lorente, 1993), phenols (Jaitz et al., 2010) or specific metabolites
(Sobolev, Circi, Capitani, Ingallina, & Mannina, 2017). Non-targeted
mass spectrometric methods have been applied mainly to study food
adulteration and/or differences in organic vs. conventional production
systems for various food commodities (Cubero-Leon, Rudder, &
Maquet, 2018; Jandric, Islam, Singh, & Cannavan, 2017; Kessler et al.,
2015; Medina, Pereira, Silva, Perestrelo, & Câmara, 2019). The main
advantage is the non-targeted nature which allows unknown changes in
the metabolite profile (e.g. addition of new adulterants) to be detected
without the need of an a priori hypothesis, which offers a great ad-
vantage to stay ahead of food fraud activities (Cubero-Leon et al.,
2018).
A combination of methods analysing different types of food com-
pounds seems to be the most promising approach to establish the
geographical origin of various food products (Luykx & Ruth, 2008).
This requires synergetic data fusion strategies, which have already been
applied to a wide range of food and beverages to authenticate origin
and assess quality (Apetrei C., Apetrei I.M., Villanueva, de Saja, Gu-
tierrez-Rosales, & Rodríguez-Mendez, 2014; Biancolillo, Bucci, Magrì,
Magrì, & Marini, 2014; Borras, Ferre, Boque, Mestres, Acena, & Busto,
2015; Jandric et al., 2015; Monakhova et al., 2014). Fundamentally, the
data from different techniques (e.g. spectroscopy, mass spectrometry,
sensor technology, separation techniques) have been combined to ex-
plore the synergetic and complementary information and enhance the
classification and prediction abilities of models (Borras et al., 2015).
There are three strategies reported so far in the literature for integrating
data blocks depending on the level of the analytical flow at which data
fusion occurs: low-level, mid-level, and high-level (Borras et al., 2015).
In low-level data fusion, data blocks from different sources are con-
catenated and processed by the desired chemometric technique. In the
mid-level strategy, relevant variables are extracted from each data
block and concatenated into a single matrix that is used for multivariate
analysis. In high-level fusion, the fusion occurs at the decision level.
Separate models are built for the different data blocks, and the final
results of the individual models (e.g. their predictions) are integrated
into a single final response.
In order to deal with datasets from such multifactorial approaches,
chemometric analysis of the data is needed. Typically, unsupervised
2
Food Chemistry 338 (2021) 127924
analysis (most commonly principal component analysis (PCA)) is ap-
plied for preliminary evaluation of the data structure, followed by su-
pervised techniques such as canonical discriminant analysis (CDA),
linear discriminant analysis (LDA), orthogonal projections to latent
structures-discriminant analysis ((O)PLS-DA), or classification methods
such as soft independent modelling of class analogy (SIMCA) and data-
driven SIMCA (DD-SIMCA) (Di Paola-Naranjo et al., 2011; Bong, Song,
Gautam, Jang, An, & Lee, 2013; Cubero-Leon et al., 2018; Fidelis,
Santos, Coelho, Rodionova, Pomerantsev, & Granato, 2017; Hiraoka
et al., 2016; Nacarato, Furia, Sindona, & Tagarelli, 2016). Although
promising results have been obtained in food authentication and
quality assessment, the data fusion is not a straightforward approach
and still represents an important challenge for chemometricians (Borras
et al., 2015).
To the best of our knowledge, most of the studies to date have
employed a single or combined targeted techniques (e.g. isotopic,
multi-elemental, molecular, etc.) applied to various food commodities
(e.g. cabbage, onions, wine, chilli pepper, asparagus, etc.) for the ver-
ification of the geographical provenance (Bong et al., 2013; Hiraoka
et al., 2016; Nacarato et al., 2016), but so far no studies have been
published on the use of combined targeted (isotopic and multi-element)
and non-targeted (metabolomics) methods for the verification of the
geographical origin of agricultural products. The aim of the current
study was to explore the potential of applying multivariate data ana-
lysis to individual and combined n(86Sr)/n(87Sr) isotope amount ratios
(note: this is the adequate term according to IUPAC and referred to as Sr
isotope ratios in the following text), multi-elemental, and non-targeted
metabolomic data to provide a unique fingerprint of authentic carrots
received directly from producers from five regions in Austria. Dis-
criminant and class modelling approaches (PLS-DA, OPLS-DA, SIMCA,
and data-driven SIMCA (DD-SIMCA)) were used to determine which
analytical technique or combination of techniques provides the best
classification and prediction abilities.
2. Materials and methods
2.1. Field samples
Carrot samples (Daucus carota L.) of Pariser Markt 4, Napoli, Circeo
von Clause and Dordogne varieties were obtained from five federal
states in Austria: Upper Austria, Salzburg, Tyrol, Vorarlberg and Vienna
(Table S1, Supplementary material). At each sampling site, 15 carrots of
similar size were collected from different parts of the field and packed
in sample zip-bags. All samples were frozen immediately after collec-
tion using a portable freezer at −20° (WAECO CoolFreeze CF – 32UP,
Dometic, Solna, Sweden) and kept frozen until further sample pre-
paration in the laboratory.
Using a Pürckhauer sampler, approximately 250 g of soil sample per
site was collected at a soil depth from 0–60 cm. The soil samples were
put on a clean surface to allow it to dry in air. Once the soil samples
were dried, they underwent a sieving step, using a sieve with 2 mm
width (Retsch, Haan, Germany).
2.2. Reagents and chemicals
Preparatory laboratory work for the elemental and strontium iso-
tope ratio analysis was performed in an ISO class 8 clean room ac-
cording to ISO 14644–1.
Pro analysi (p.a) grade HNO3 (w = 65%) and H2O2 (w = 30%) were
obtained from Merck (Darmstadt, Germany) and NH4NO3 (≥98.5%)
from VWR International GmbH (Vienna, Austria). Laboratory water
type I (18 MΩ cm, F + L GmbH, Vienna, Austria) was further purified
using a sub-boiling distillation system (Milestone-MLS GmbH,
Leutkirch, Germany). Nitric acid was double sub-boiled in a DST-1000
sub-boiling distillation system (Savillex Corporation, Eden Prairie, MN,
USA). All laboratory consumables used for the elemental and strontium
Z. Jandric, et al.
isotope ratio analysis (polyethylene tubes, pipette tips, etc.) were
double acid washed (HNO3, w = 10% and w = 1%) and rinsed with
ultra-pure water before use. The certified reference material SRM 987
(NIST, Gaithersburg, MD, USA) was used for calibrating the instru-
mental isotopic fractionation factor. The accuracy of elemental analysis
was confirmed using a certified reference material BCR 679 (cabbage
powder) (IRMM, Brussels, Belgium). All chemicals used for metabo-
lomics analysis were LC-MS grade. Water, methanol, acetonitrile and
formic acid were purchased from Sigma-Aldrich (St. Louis, USA).
Chloroform was supplied by Merck (Darmstadt, Germany).
2.3. Sample preparation for elemental and n(86Sr)/n(87Sr) isotope amount
ratio analysis
After delivery to the laboratory, the frozen carrot samples were
thawed at room temperature, washed with laboratory water type I
water (PURIST®Ultrapure Water Systems, Rephile, USA), dried using
cleanroom wipes and cut into small pieces using a ceramic knife in
order to avoid metal contamination. An aliquot of 5 g of carrots (wet
weight) was transferred into polypropylene beaker and covered with
parafilm (Parafilm®M, Pecheney Plastics Packaging, MI, USA), which
was tapped with the ceramic knife to produce small holes in order to
allow liquid evaporation. The beakers were additionally covered with a
clean room wipe and put in a freezer at −80 °C (Snijders Labs, VF 1000-
86, Belgium) for at least twelve hours prior to freeze-drying.
Subsequently, the samples were transferred into a freeze-drying cabinet
(CHRIST Alpha 1–2 LD, Ostereode am Harz, Germany) and dried to
constant weight (less than ± 0.1 mg). 0.5 g of the freeze dried carrot
samples were homogenized using a ceramic mortar and mineralized by
microwave assisted acid digestion (Anton Paar, Multiwave 3000, rotor
HF 100, Graz, Austria) using a reaction mixture of 5 mL of sub-boiled
HNO3, w = 65% and 1 mL of H2O2, w = 30%. Digested carrot samples
were diluted with sub-boiled water to HNO3, w = 2%.
The soil extraction procedure followed DIN ISO 19730 (DIN ISO
19730, 2009). Briefly, dried and sieved soil (20 g) samples were ex-
tracted using 50 mL of 1 mol L-1 ammonium nitrate to obtain the labile
fraction. After two hours shaking at 20 rpm in an overhead shaker (GFL
Gesellschaft für Labortechnik GmbH, Burgwedel, Germany), the sam-
ples were filtered using a fluted filter (Grade 14/N, 150 mm, 80 g/m2;
Munktell Filter AB, Falun, Sweden). Afterwards, the extracts were
acidified to HNO3, w = 2% using conc. HNO3, w = 65%. Carrot digests
and soil extracts underwent Sr/matrix separation using a strontium-
specific extraction resin (Triskem, Bruz, France) according to
Tchaikovsky, Irrgeher, Zitek, & Prohaska (2017) prior to strontium
isotopic measurements.
2.4. Elemental and n(86Sr)/n(87Sr) isotope amount ratio analysis
B, Na, Mg, K, Al, Mn, Cu, Rb, Sr, Mo, Cd and Ba were selected as
target elements since a significant natural variation can be expected for
the investigated samples and thus provide a high potential for dis-
criminating food samples according to their geographic origin
(Tchaikovsky et al., 2019). The elemental composition of the selected
elements of carrots was analysed using an inductively coupled plasma
quadrupole mass spectrometer (ICP-QMS, NexION 350 D, PerkinElmer,
Waltham, USA) using a PFA (perfluoroalkoxy) nebulizer (Microflow ST
Nebulizer, Elemental Scientific Inc., Nebraska, USA) combined with a
cyclonic spray chamber (PerkinElmer, Waltham, USA). The elemental
content was determined following blank correction using method
blanks, normalization to 115In, which was used as internal standard
(single element ICP-MS standard, CertiPur, Merck, Darmstadt, Ger-
many) and external calibration using a 5–point calibration (multi-ele-
ment ICP-MS standard VI, CertiPur, Merck, Darmstadt, Germany).
Samples were diluted as required for the working range of the method.
Table S2 (Supplementary material) summarizes the used ICP-MS con-
ditions. Instrumental blanks (HNO3, w = 2%) and in-house quality
3
Food Chemistry 338 (2021) 127924
control standards were analysed repeatedly during each measurement
run to monitor possible contamination of the instrument and determine
the accuracy and repeatability. In addition, the certified reference
material BCR 679 cabbage powder was processed in the same way as
the samples and used to determine the accuracy of the entire analytical
protocol as well as for the estimation of the measurement uncertainty of
the elemental content. The measurement precision was determined as
the relative standard deviation of the elemental signals of the certified
reference material BCR 679 measured by ICP-QMS. It ranged between
1% and 6% RSD depending on the analyte of interest. (Note: The
measurement precision was used as input variable for the calculation of
the measurement uncertainty; see paragraph 2.6 Measurement un-
certainty below). The limit of quantification (LOQ) was calculated as
equivalent to ten times the standard deviation of the method blanks
taking into account dilution factors and the initial weight of the sam-
ples, respectively.
The n(87Sr)/n(86Sr) isotope amount ratio measurements were per-
formed on a multi-collector inductively coupled plasma mass spectro-
meter (MC ICP-MS, Nu Plasma HR, Nu Instruments Ltd., Wrexham, UK).
The samples were introduced using a membrane desolvation system
(Aridus II, Cetac Technologies, Omaha, Nebraska, USA). Strontium
isotope ratios were corrected for blank, residual 87Rb and instrumental
isotopic fractionation according to the protocol of Horsky, Irrgeher, &
Prohaska (2016). Instrumental isotopic fractionation of the Sr isotope
ratio was corrected using an external intra-elemental approach (stan-
dard-sample-bracketing) using NIST SRM 987 (Irrgeher, Vogel, Santner,
& Prohaska, 2015). The reference material BCR 679 followed the same
preparation procedure as the samples. In addition, the SRM 987 un-
derwent Sr/matrix separation for quality control of the separation
method. Table S3 (Supplementary material) summarizes the used MC
ICP-MS conditions for strontium isotopes analysis. Table S4
(Supplementary material) shows the comparison of the measured va-
lues to the reference values of the certified reference materials SRM 987
and BCR 679.
2.5. Sample preparation for metabolomic analysis
The sample preparation procedure was adapted from previous work
(Cubero-Leon et al., 2018). Freeze-dried carrot powder (0.1 g) was
mixed with cooled (4 °C) solvents (0.5 mL of chloroform, 0.3 mL of
methanol and 0.2 mL of water) and vortexed for 5 s. Subsequently, the
samples were shaken for 1 h at 150 rpm (IKA VIBRAX VXR, SPL-Lab,
Staufen, Germany). 0.5 mL of 20% aqueous methanol was added to the
extract and whole mixture was shaken for additional 30 min at
150 rpm. To separate the methanol/water and chloroform fractions, the
samples were centrifuged for 15 min at 2200 rpm using a GeneVac EZ 2
(SP Scientific, Pennsylvania, USA). The methanol/water fraction was
filtered into glass vials through 0.45 µm nylon membrane filters (Su-
pelco, Bellefonte, PA, USA).
Pooled quality control (QC) samples were prepared by combining
equal volumes (50 µL) of all samples. Two different types of QCs were
prepared as follows: regional QCs (pooled samples from each region)
and an overall QC (all carrot samples collected). QCs were measured
after every seven samples (i.e. alternating throughout the sequence).
These injections were used for monitoring the performance of the LC-
TOFMS system. All samples were injected in a randomised sequence
order (randomization was performed via Microsoft Excel). To assess the
performance of the extraction method, five replicates of two carrots
from two different regions were extracted and analysed. Repeatability
of the extraction was calculated as the percentage relative standard
deviation (RSD) of the peak intensity of detected features from five
independent extractions for each carrot. The average repeatability
standard deviation of the extraction method was < 6%. Therefore, each
sample was extracted and measured once.
Z. Jandric, et al.
2.6. Non-targeted metabolomic analysis
Metabolomics analysis was performed using an Agilent model 1290
Infinity II LC system coupled to a 6230b Time-of-Flight mass spectro-
meter equipped with a Dual Jetsream ESI interface (Agilent
Technologies, Santa Clara, USA). The chromatographic method was
adapted from previous work (Jaitz et al., 2010; Causon, Ivanova-
Petropulos, Petrusheva, Bogeva, & Hann, 2019). Separation was per-
formed using a Zorbax SB C18 column (2.1 × 100 mm, 1.8 µm dp,
Agilent Technologies, Santa Clara, USA). Separation was carried out
using gradient elution at a flow rate of 0.2 mL/min. Mobile phase A
consisted of water/acetonitrile (99/1; v/v) with 0.1% formic acid and
mobile phase B contained acetonitrile/water (99/1; v/v) with the same
acid concentration. Initial gradients conditions (95% A) were main-
tained for 2 min, then decreased to 50% A in the next 10 min, and to
20% A for one more minute. Following a cleaning step and suitable re-
equilibration time (5.9 min), the total analysis time was 18 min per
injection. The injection volume was 5 µL, and the column temperature
was set to 40 °C.
Mass spectra between 50 and 1700 m/z were recorded in negative
polarity mode with the following settings for TOFMS detection: drying
gas temperature 180 °C, drying gas (nitrogen) flow 10 L min−1,
nebulizer pressure 40 psi, sheath gas temperature 350 °C, sheath gas
flow 12 L min−1, capillary voltage 3500 V, and fragmentor voltage
150 V. The 2 GHz extended dynamic range detection mode with an
acquisition rate of three TOF spectra/s (each consisting of 3300 TOF -
transients) was used for all measurements.
2.7. Measurement uncertainty
The measurement uncertainty states the quality of the analytical
result. It accounts for all relevant sources of error of the entire analy-
tical protocol (Ellison & Williams, 2012). In this work, the uncertainties
of the elemental and isotopic composition of carrots and soil were de-
termined in a Kragten approach (Kragten, 1994) following EURACHEM
guidelines (Ellison & Williams, 2012). The combined standard un-
certainty of the n(87Sr)/n(86Sr) isotope ratio was calculated based on
the protocol of Horsky et al. (2016). The uncertainty of the elemental
mass fractions was estimated using the CRM BCR 679. It combined the
standard deviations of the blank, the internal standard, the calibration
curve, the measurement precision, the dilution factor, the initial weight
and the heterogeneity of the sample. All combined uncertainties in this
work were subsequently multiplied by a factor of 2, which corresponds
to the expanded uncertainty (U, k = 2, confidence interval of 95%).
The heterogeneity of the samples, which is the variation of the Sr iso-
tope ratio and elemental mass fraction of sample replicates, represented
the major source to the uncertainty. Table S5 (Supplementary material)
shows the elemental mass fractions and respective uncertainties de-
termined for one carrot from the region of Vorarlberg.
2.8. Non-targeted data processing
Various software packages associated with accurate mass data from
LC-TOFMS were used for data acquisition and processing. Data were
acquired using Agilent Mass Hunter Data Acquisition Workstation
(Agilent Technologies, Santa Clara, USA) using online reference mass
calibration. Processing of non-targeted metabolomics raw data was
evaluated using two different software packages (with different peak-
picking algorithms): batch recursive feature extraction (BRE) workflow
in Mass Hunter Profinder B.08.00 (Agilent Technologies, Santa Clara,
USA) and Progenesis QI 2.4 (Nonlinear Dynamics Ltd., Newcastle upon
Tyne, UK).
BRE includes two stages: molecular feature extraction (MFE) and
batch find by ion feature extraction (FbI). MFE performs chromato-
graphic deconvolution to find the features in the samples (peak
picking), and summarizes all software-extracted features in each sample
4
Food Chemistry 338 (2021) 127924
considering accuracy of mass measurements, the grouping of ions re-
lated to the charge-state, isotopologue pattern, and the presence of
adducts or dimers. Subsequently, these molecular features are aligned
across all of the selected samples using mass and retention time (RT).
After calculating the median mass, median RT, and composite spectrum
from the aligned features found using MFE, FbI uses the median values
to perform a targeted extraction to improve the reliability of finding in
complex datasets. For BRE, samples were grouped, aligned, and
checked for software extraction quality based on RT and mass-to-charge
windows ( ± 0.15 min and 20 ppm ± 2.0 mDa, respectively).
Extracted ion chromatograms with symmetric extraction windows (m/
z ± 35 ppm and expected retention time of ± 0.75 min) were then
extracted for all compounds. In order to be considered as a compound
of interest for further evaluation in a differential analysis workflow,
molecular feature appearance in all of the samples with an MFE score
and target score > 70 were required. The total compound abundance
was then calculated as a sum of areas originating from all relevant
single ion features.
Raw data were also processed using automatic settings in Progenesis
QI allowing retention time alignment across all runs, followed by peak
picking, and the automatic deconvolution of the compound’s adducts.
The applied peak picking algorithm was not limited to sensitivity levels,
chromatographic peak width, or retention time windows. Retention
times were aligned to most suitable QC sample, while all runs were
normalized via the calculated scaling factor. In Progenesis QI, features
were chosen whose abundance complied with the following conditions:
minimum 2-fold change between the groups, a maximum standard
deviation of 30% within the groups, one-way analysis of variance
(ANOVA) with a probability (p) of > 95%, and a q-value describing the
chance of false positives below 0.1%.
Using the settings described above, a large number of features was
detected by each software. To be able to compare the software, data
filtering was performed and 207 features were extracted by each soft-
ware and used for multivariate data analysis by PCA. There were no
significant differences observed between discrimination of sample
groups (data not shown) using the two different peak-picking algo-
rithms. Therefore, the data processed by MassHunter Profinder were
further used for chemometric analysis and generation of classification
models. Based peak ion chromatogram (BPC) and extracted ion chro-
matogram (EIC) of all detected features are shown in Fig. S1
(Supplementary material).
2.9. Statistical analysis
Multivariate statistical analysis (PCA-X, PLS-DA, OPLS-DA, SIMCA
and DD-SIMCA) was performed using SIMCA 15.0.2 software
(Umetrics, Umeå, Sweden) and Microsoft Excel with Chemometrics
Add-In (Pomerantsev, 2014). Prior to modelling, the data were pre-
processed using scaling to unit variance (UV-scaling) and pareto scaling
(gives the variable a variance equal to its standard deviation instead of
unit variance). The quality of the models was described by R2 (goodness
of fit) and Q2 (predictability) statistics. PCA-X, PLS-DA and OPLS-DA
models were validated using 7-fold cross-validation (Eriksson, Byrne,
Johansson, Trygg, & Vikström, 2013). Cross-validation is an internal
predictive validation method for determining the number of significant
components by calculating the total amount of explained X-variance
(R2X), Y-variance (R2Y), and cross-validated predictive ability (Q2Y).
To test the robustness and compare performance characteristics
(efficiency) of classification models, the original dataset was randomly
divided into a training set (80%) and a validation test set (20%), and
this procedure was repeated five times so that each sample had equal
chances to be once modelled and once predicted. The training set
samples were used for generation of classification models and the va-
lidation test samples for prediction. Predictions using the validation test
sets allowed the calculation of sensitivity and specificity of the models.
In this context, sensitivity is defined as the percentage of objects
Z. Jandric, et al.
belonging to a given class, which are correctly identified by the model
(true positives), while specificity is the percentage of objects foreign to
the modelled class (belonging to the other category) that are classified
as foreign (true negatives) (Riedl, Esslinger, & Fauhl-Hasssek, 2015).
The efficiency was computed as a balance (geometric mean) between
sensitivity and specificity. Hence, they are calculated according to the
following formula:
Sensitivity = TP/(TP + FN)
Specificity = TN/(TN + FP)
TP: true positive; FN: false negative; TN: true negative; FP: false
positive.
In this study, low- and mid-level data fusion were evaluated. In the
low-level data fusion, the Sr isotope ratio, multi-elemental and meta-
bolomic data were normalized to the same scale (auto-scaling), and
then concatenated to the single matrix. In the mid-level data fusion, two
different approaches were tested: fusion of data from different blocks (n
(87Sr)/n(86Sr) isotope ratios, multi-element, and metabolomics) after
variable selection, and fusion of scores from PCA and OPLS-DA, ob-
tained independently for each data block (hierarchical base models).
After fitting a model, the scores of hierarchical base models were used
as variables in hierarchical top models.
Predictive modelling was performed on single (i.e. strontium iso-
tope ratios/multi-element and metabolomics) and combined (i.e.
strontium isotope ratios/multi-element/metabolomics) datasets using
two different classification methods: (i) discriminant classification
technique that define delimiters between established classes so that
unknown samples are always assigned to one of the classes (PLS-DA and
OPLS-DA), and (ii) class modelling techniques that calculate a separate
model for each established class, so that an unknown sample can be
either assigned to that class or rejected (SIMCA and DD-SIMCA). The
evaluation of the classification-modelling performances was based on
sensitivity, specificity, and efficiency.
3. Results and discussion
3.1. Isotopic and multi-elemental fingerprinting
Fig. 1 shows a box and whisker plot of the Sr isotope ratios for the
carrot samples. The highest values (0.70942 ± 0.00070 (U, k = 2;
n = 15)) were observed in samples form Salzburg and the lowest
Fig. 1. Box and whisker plot of n(87Sr)/n(86Sr) isotope amount ratios of carrots
according to their geographical origin (Vorarlberg, Salzburg, Tyrol, Upper
Austria, and Vienna). The horizontal black line represents the median values of
the data.
5
Food Chemistry 338 (2021) 127924
(0.70736 ± 0.00042 (U, k = 2; n = 15)) in samples from Tyrol.
Strontium isotope ratios of carrots from Vorarlberg, Upper Austria and
Vienna ranged between 0.70791 ± 0.00029–0.70810 ± 0.00074 (U,
k = 2; n = 15). Fig. 1 shows that it is possible to differentiate carrots
grown in Salzburg and Tyrol from samples originating from Vorarlberg,
Upper Austria, and Vienna. The total strontium content against the
strontium isotope ratio plot further supported this observation (Fig. S2)
(Supplementary material).
Recent studies have demonstrated that plants incorporate locally
specific chemical information, which can be used to link primary
agricultural products to their geographical origin (Bong et al., 2013;
Hiraoka et al., 2016). The Sr isotope ratios of agricultural products
reflect the bioavailable strontium of soil in the cultivation area (Yokoo
& Nakano, 2001). Therefore, we were interested in evaluating Sr iso-
tope values in soils corresponding to carrot fields used during this study
as well as the association between Sr isotope ratio profiles of soils and
carrots. The isotope ratio of carrots and soils show no significant dif-
ference since they overlap within the limits of measurement uncertainty
in all investigated agricultural regions (Fig. S3) (Supplementary mate-
rial). The geology of Austria is very diverse and consists of Precambrian
rocks and minerals together with younger marine sedimentary rocks
uplifted by the Alpine orogeny (Janoschek & Matura, 1980). However,
most of the agricultural production in Austria takes place in areas un-
derlined by tertiary, quaternary and the Bohemian mass. The carrot
production regions and geological map of Austria are shown in Fig. S4
(Supplementary material). The Sr isotope ratios of soil samples from
Salzburg, Upper Austria, and Vienna) were generally higher ranging
between 0.70845 ± 0.00056 and 0.70859 ± 0.00023 (U, k = 2;
n = 15) than the strontium isotope ratios
(0.70712 ± 0.00018–0.70792 ± 0.00039) (U, k = 2; n = 15) of soil
from Vorarlberg and Tyrol. The soil in Salzburg region consists of
several different type of soils as the sampling area was a former gravel
pit, which was filled afterwards with soil from different places. This
could be a reason for the observed higher variation of the n(87Sr)/n
(86Sr) values of carrot and soil samples from Salzburg as well as agri-
cultural practices such as the use of mineral fertilizers (Fig. S3)
(Supplementary material).
Strontium isotope ratios alone did not provide sufficient differ-
entiation between carrots grown in all investigated regions within
Austria. Thus, Sr isotope ratios and the mass fractions of B, Na, Mg, K,
Al, Mn, Cu, Rb, Sr, Mo, Cd, and Ba were combined (Table S6)
(Supplementary material) and evaluated using PCA and OPLS-DA. The
PCA 2D scores plot (Fig. S5A) (Supplementary material) shows the se-
paration into four distinctive clusters representing carrots grown in
Salzburg, Vienna, Vorarlberg and Tyrol/Upper Austria, while full dif-
ferentiation of carrots from all regions can be observed on the PCA 3D
plot (Fig. S5B) (Supplementary material). The first three principal
components (PCs) explain the majority of the variation with PC1, PC2,
and PC3 explaining 31.7%, 23.2%, and 17.2%, respectively (Fig. S5 A/
B) (Supplementary material).
By using a supervised technique (OPLS-DA) and the combined da-
tasets, the carrot samples were successfully distinguished between all
production regions with 86.9% of the explained variation
(R2X = 0.869; R2Y = 0.809) and high predictive ability
(Q2cum = 0.694) (Fig. 2A). A loadings plot was also generated to elu-
cidate the most reliable class-discriminating variables for each region
(Fig. 2B). The loadings plot (a summary of the relationships among the
variables) is a means to interpret the patterns seen in the scores plot (a
summary of the relationships among the observations). The results
obtained indicate that the elemental content of B, Sr, Cd and Cu were
the major contributors to the clustering of carrots from Tyrol, while the
elemental content of Ba, Na and Rb was significant for Vorarlberg
carrots, n(87Sr)/n(86Sr) isotope ratios and Al elemental content for
Salzburg carrots, Mn and Mo elemental content for Vienna carrots, and
Mg and K elemental content for Upper Austria carrots, respectively
(Fig. 2 A/B). In order to understand how each variable contributes to an
Z. Jandric, et al. Food Chemistry 338 (2021) 127924

Fig. 2. The OPLS-DA scores plots showing clustering of carrots grown in Vorarlberg, Tyrol, Salzburg, Upper Austria, and Vienna (A), and loadings plot of the most
influential variables responsible for carrots clustering (B).

observed clustering, we used contribution plotting. Fig. S6
(Supplementary material) shows the contribution of the strontium
isotope ratios to discrimination of Salzburg carrots with elevated levels
compared to the average value across all samples. A similar pattern is
observed in Fig. 1. Additionally, a Variable Importance in the Projection
(VIP) values were generated to evaluate the overall variable impact on
sample group differentiation. VIP scores analysis reflects the im-
portance of the X variables in the model with respect to Y (predictive
component of the model). Variables with VIP values > 1 are the most
influential for the model, between 1 and 0.5 the importance level de-
pends on the dataset, while value lower than 0.5 indicate unimportant
X-variables (Eriksson et al., 2013). VIP values > 1 were obtained for Sr
isotope ratio and Mo, Sr, B, Na, Ba, Cd, Rb, K variables, while for Mg,
Al, Mn and Cu variables, VIP values ranged from 0.7 to 0.9.
3.2. Non-targeted metabolomic fingerprinting
PCA analysis of non-targeted metabolomics data was performed on
the pooled QC samples to assess the stability of the LC-TOFMS system
and analytical method across the sequence. Fig. 3A shows a tight
clustering of all QC samples including regional QCs (pooled samples
from each region) and the global QC (pool of all carrot samples col-
lected), indicating instrumental stability during the sequence. Ad-
ditionally, five representative peaks from the QC samples covering a
range of intensities and retention times were selected to evaluate the
shift in retention time, mass error and peak areas. The retention time
shift was 0.01 min, the mass error ranged from 0.38 to 0.90 mDa and
the relative standard deviations of peak areas ranged from 3.0 to 8.1%
verifying the stability of the whole analysis (Table S7) (Supplementary
material). The PCA analysis indicated clustering of carrots based on
their geographical origin with samples from Tyrol, Upper Austria and
Vorarlberg clustered closer to each other (Fig. 3A). The three first
principal components explained 40% of data variation. Thus, in order
to help understand the inter-class separation, identify potential char-
acteristic markers for each class, and to build classification models, data
was further subjected to supervised OPLS-DA analysis. Fig. 3B shows
clustering of samples according to geographical region with improved
discrimination (comparing to PCA) between carrots grown in Salzburg,
Vienna, Upper Austria and Tyrol/Vorarlberg (still overlapping)
(R2X = 0.608; R2Y = 0.920; Q2cum = 0.829).
Supervised modelling with high number of variables often carries
the risk of overfitting, where large number of variables from the dataset
are likely to be random variation and do not contribute to the dis-
tinction between sample groups. Overfitting takes place if the model
learns the idiosyncrasy of the data (meaning that the noise is modelled
as well) and the model loses its generalization ability (Berrueta, Alonso-
Salces, & Héberger, 2007). Therefore, to reduce the random variation
and to select the variables with highest contribution to sample group
differentiation, VIP scoring was used. Forty-six metabolomic variables
with VIP scores > 1 were selected and subjected to further MVA data
analysis in combination with n(87Sr)/n(86Sr) isotope ratio and multi-
elemental (K, Sr, Mg, Na, Cd, Rb, Ba, Al, and B) data.

Fig. 3. The PCA 2D (A) and OPLS-DA 3D (B) scores plot of carrots (Vorarlberg, Tyrol, Salzburg, Upper Austria, and Vienna) generated using metabolomic data.
Quality control (QC) clusters are highlighted within the ellipses big pool (overall QC, pooled all carrot samples collected), regional QCs (pooled samples from each
region).

6
Z. Jandric, et al.
Fig. 4. The OPLS-DA scores plot of carrots (Vorarlberg, Tyrol, Salzburg, Upper Austria, and Vienna) generated using fused strontium isotopic, multi-elemental, and
metabolomic data: low-level (220 variables) (A), and mid-level (selected 55 variables (B); PCA-scores, 10 variables (C); Y-predictive scores, 10 variables (D)).
3.3. Chemometric analysis of fused strontium isotopic, multi-elemental and
metabolomic data
The advantage of using combined fingerprint data (strontium iso-
tope ratio and multi-element) with OPLS-DA compared to using only
one or two marker components has been demonstrated above, whereby
improved discrimination of carrot samples according to their geo-
graphical origin was obtained (Fig. 1/S1/2). For the analysis of fused
data, preprocessing is very important to prevent one block of data being
dominant, especially in our case where the results from non-targeted
analysis assessed by accurate molecular mass spectrometry, strontium
isotope ratios obtained via MC ICP-MS, and multi-elemental data from
ICP-MS are combined (high fold intensity differences between vari-
ables). Thus, two different scaling methods, i.e. scaling to unit variance
(the base weight is computed as 1/sj, where sj is the standard deviation
of variable j computed around the mean), and pareto (the square root of
the standard deviation is used as the scaling factor) were tested. We
observed that the best classification models were obtained when scaling
to unit variance was used. Therefore, the variables in all datasets were
centered and scaled to unit variance. OPLS-DA models were generated
for all fused datasets and the results are shown in Fig. 4. Comparing all
data fusion approaches, we can see that the low-level data fusion
strategy did not effectively enhance discrimination between carrot
samples from different agricultural regions (Fig. 4A), while, in contrast,
full discrimination was obtained for all three mid-level data fusion
strategies (Fig. 4B/C/D). The main drawback of the low-level data fu-
sion strategy is that the fused data matrix contains a very high number
7
Food Chemistry 338 (2021) 127924
of variables and irrelevant or spurious variance is modelled. However,
using selected variables, PCA and Y-predictive scores (mid-level, lower
number of variables) only the relevant variation from each data block
(each analytical technique) is captured and integrated into a combined
model.
3.4. Classification
The strontium isotope, multi-elemental, non-targeted metabolomics
and fused data were analyzed by different multivariate methods to
evaluate the ability of the selected variables to classify the samples
regarding their membership (carrots grown in Vorarlberg, Tyrol,
Salzburg, Upper Austria, and Vienna). Examining the classification re-
sults in Table 1, it can be seen that satisfactory results were obtained for
all classification methods (PLS-DA, OPLS-DA, SIMCA, and DD-SIMCA)
and datasets (efficiency > 93%). The sensitivity values were generally
lower than specificity (Table 1). Considering the analytical techniques
individually, the best classification results were obtained using Sr iso-
tope ratios and multi-elemental content of carrots. As expected (based
on PCA and OPLS-DA results), it is clear that combined strontium iso-
tope ratio and multi-elemental data have slightly more discrimination
power (efficiency > 97%) than metabolomic data (efficiency > 94%)
for discriminating carrots according to origin. However, combining
information generated by three different mass spectrometric techniques
on mid-level data fusion improves the overall classification results
(99–100%, efficiency) in comparison to low-level data fusion
(93–100%, efficiency). As evident from Table 1, when discriminant
Z. Jandric, et al. Food Chemistry 338 (2021) 127924

Table 1
Classification results for strontium isotope ratio, multi-elemental, metabolomics and fused data of carrots grown in Vorarlberg, Tyrol, Salzburg, Upper Austria, and
Vienna. Sensitivity, specificity and efficiency values (%, average values for all groups) are reported for test set validation (approximately one fifth of the initial
dataset, average of five random splitting).
Data set PLS-DA OPLS-DA SIMCA DD-SIMCA

87 86
n( Sr)/n( Sr) + Multi-elements Sensitivity (%) 96 97 100 99
(13 variables) Specificity (%) 99 99 100 100
Efficiency (%) 97 98 100 100
Metabolomics (207 variables) Sensitivity (%) 97 97 91 100
Specificity (%) 99 99 98 100
Efficiency (%) 98 98 94 100

Data fusion
Low-level (220 variables) Sensitivity (%) 99 99 88 100
Specificity (%) 99 100 99 99
Efficiency (%) 99 99 93 100
Mid-level 1 (55 variables) Sensitivity (%) 99 99 99 100
Specificity (%) 100 100 100 100
Efficiency (%) 99 99 99 100
Mid-level 2 (PCA scores; 10 variables) Sensitivity (%) 99 97 96 100
Specificity (%) 100 99 99 99
Efficiency (%) 99 98 97 100
Mid-level 3 (Y-predictive; 10 variables) Sensitivity (%) 100 100 100 100
Specificity (%) 100 100 100 99
Efficiency (%) 100 100 100 100

models were applied to the data (PLS-DA and OPLS-DA), high efficiency
was obtained for all datasets (97–100%). However, the main drawback
of discriminant methods is their inability to provide proper classifica-
tion of new samples, which do not belong to any of the predefined
classes and this would limit detection of potentially fraudulent samples.
In comparison, the advantage of class modeling methods (SIMCA and
DD-SIMCA) is that they do not know anything about existence of al-
ternative classes or samples. Instead they develop the acceptance area
around the target class and, thus, delimit the target objects from any
other objects and classes. Using class modelling techniques, good per-
formance characteristics of carrot classification models were obtained,
where DD-SIMCA (99–100%, efficiency) outperformed SIMCA models
(93–100%, efficiency) for all datasets.
Although data fusion has been successfully applied for a better
discrimination of origin and the authenticity determination of various
food commodities (e.g. wine, olive oil, beer, etc.) (Apetrei et al., 2014;
Biancolillo et al., 2014; Monakhova et al., 2014), it has been also re-
ported that data fusion did not always improve results and, in some
cases, negatively affected the classification and performance of pre-
diction models (Borras et al., 2015). Moreover, as data fusion is mainly
a data-driven approach, it should be carefully explored and the use of
more than a single technique should be justified in terms of cost-benefit
considerations. In our study using strontium isotope ratios and multi-
elemental content of carrots in order to discriminate samples according
to their region of origin, good prediction abilities for all classification
models (97–100%, efficiency) were obtained. However, higher con-
fidence in predictive modelling can be obtained adding other variables
(e.g. selected metabolites, mid-level data fusion, 55 variables) and the
robustness of the models could be enhanced. In principle, the most
powerful methods are generally those that combine as many of the
multi-chemical signatures as possible into a multi-dimensional au-
thenticity matrix where natural variation is more readily constrained
and detection of mis-description is difficult to subvert (Kelly, Heaton, &
Hoogeweiff, 2005). In addition to the classification results in Table 1,
Coomans’ plots were also used to display the SIMCA results (Fig. S7)
(Supplementary materials).
Additionally, the DD-SIMCA technique was also used to build
models for different regions using carrot samples from each region
(combined dataset, selected 55 variables from all techniques), and
evaluate if it is possible to predict whether the samples from all other
regions could be excluded as foreign samples. In the first step, PCA was
applied to the training data from the target class consisting of the
authentic carrot data from each region. In the second step, the training
set of authentic carrots was used to calculate the SD and OD distances of
the samples to the score space and to develop an acceptance area de-
cision rule for a given type I error (α error). The green boundary line in
the Fig. S7A-E (Supplementary material) corresponds to α = 0.05,
where all objects located inside the area (green circles) are considered
to be members of the target class (authentic carrot samples from dif-
ferent regions). The red boundary line corresponds to the outlier
border, γ = 0.01, which specifies the probability that at least one
regular sample could potentially be erroneously considered as an out-
lier. None of the carrot samples fell outside this 99% tolerance level for
all regions (Fig. S7A-E) (Supplementary material).
In the second step, the carrot samples from all other regions were
predicted. The acceptance plots in Fig. 5A-E show that carrots origi-
nating from other regions fell outside of acceptance area (green) of
authentic region model and are correctly classified as outliers. The
sensitivity and specificity values of 100% were obtained for all models
for each region. Taking into account that the number of carrot samples
from each region is relatively small and in order to avoid model over-
fitting and result instability, the DD-SIMCA model with 4 PCs was used
for each region.
Although the number of samples used to build the classification
models was relatively low, the high sensitivity/specificity values (DD-
SIMCA, 99–100%) obtained for all regions (Salzburg, Vienna,
Vorarlberg, Upper Austria, and Tyrol), show that there is potential for
the prediction of geographical origin of carrots based on n(87Sr)/n(86Sr)
isotope ratio, multi-elemental, and metabolomic data. The second im-
portant parameter (after geographical origin) to be considered is carrot
variety. In some studies, it was discussed that different plant varieties
grown in different production regions can be distinguished by isotopic,
elemental and/or metabolomic fingerprints (Di Paola-Naranjo et al.,
2011; Kelly et al., 2005; Monakhova et al., 2014). It can be assumed
that different carrot varieties, which grow in the same region, could
show different elemental and metabolomics composition. (Note: The n
(87Sr)/n(86Sr) isotope ratio is not significantly fractionated during up-
take into plants (Flockhart, Kyser, Chipley, Miller, & Norris, 2015)).
However, this does not represent a concern for our verification ap-
proach. In fact, the chemical pattern determined in carrots from the
investigated fields represents a local and variety-specific chemical fin-
gerprint. This chemical information represents a reference value to
which samples of unknown origin can be compared. This first step in
building a database of the chemical composition of authentic carrots

8
Z. Jandric, et al.
Fig. 5. DD-SIMCA acceptance plots for the carrots classification: (A) Salzburg vs. others; (B) Vienna vs. others; (C) Vorarlberg vs. others; (D) Upper Austria vs. others;
(E) Tyrol vs. others (4 PCs; the 95% confidence interval (α = 0.05)).
from Austria can be used for the verification of the origin of carrots in
the future. Additionally, our sample set contained one variety (Dor-
dogne) originating from two different regions, i.e. Salzburg and Vor-
arlberg. As all analytical approaches allowed a clear differentiation
between these two sample groups, we conclude that, in the present
case, the sample variety does not significantly influence the strontium
isotopic, multi-elemental, and non-targeted metabolomics results.
In the future, additional experiments with more authentic (e.g.
different production years, different varieties from the same production
region, authentic carrot samples from different regions and countries,
etc.) can help to build a strontium isotopic, elemental and metabo-
lomics database. This will allow obtaining of more robust classification/
prediction models and the possibility to investigate their ability to re-
liably classify unknown samples. Moreover, the influence of different
varieties especially on the metabolic fingerprint needs further ex-
ploration. The identification of the metabolites responsible for the
classification was not the aim of this study, thus effort will be made to
get deeper insight into the metabolic differences and comprehensive
structural characterization of these key metabolites will be performed
using LC-QTOFMS to provide high-resolution MS2-level information
amenable for library-based identity confirmation.
4. Conclusions
87
The potential of multi-chemical fingerprinting using n( Sr)/n( Sr)
isotope amount ratio, multi-elemental, and metabolomic data to dif-
ferentiate carrots grown in five different Austrian regions (Salzburg,
Vienna, Vorarlberg, Upper Austria, and Tyrol) was evaluated. In par-
ticular, it was shown that the differentiation of geographical origin of
carrots can be carried out with a good efficiency rate (93–100%) using
any of the recorded fingerprints and statistical techniques. Moreover,
when considering the outcomes from each individual technique, Sr
isotope amount ratios and multi-elemental data provided better
Food Chemistry 338 (2021) 127924
discrimination power than the non-targeted metabolomics data.
However, the use of a mid-level data fusion strategy (combined com-
plementary chemical information) slightly (> 99%, efficiency) im-
proved the predictive ability and robustness of all classification models
tested (PLS-DA, OPLS-DA, SIMCA, and DD-SIMCA). Although the dis-
criminant classification techniques demonstrated good model perfor-
mance, a one-class classification modelling approach based on the use
of the DD-SIMCA algorithm was more suitable for carrot geographical
origin authentication due its capability to model each class individually
and obtained high efficiency (> 99%). The results of this study strongly
indicate that DD-SIMCA model of fused strontium isotopic, elemental
and metabolomic data could provide a suitable method for the dis-
crimination of carrots of different geographical origins, thus allow
controlling geographical origin claims.
CRediT authorship contribution statement
Zora Jandric: Conceptualization, Methodology, Statistics, Writing -
original draft. Anastassiya Tchaikovsky: Data curation. Andreas
Zitek: Supervision, Study conceptualization, Sample collection. Tim
Causon: Supervision, Methodology. Vaclav Stursa: Data curation.
Thomas Prohaska: Supervision. Stephan Hann: Supervision.
Declaration of Competing Interest
86
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We acknowledge the support of the project by the COMET-K1
competence center FFoQSI. The COMET-K1 competence center FFoQSI
9
Z. Jandric, et al.
is funded by the Austrian ministries BMVIT, BMDW and the Austrian
provinces Niederösterreich, Upper Austria and Vienna within the scope
of COMET- Competence Centers for Excellent Technologies. The pro-
gram COMET is handled by the Austrian Research Promotion Agency
FFG. We also want to thank the carrot producers (Gärtnerei Ganger -
Vienna, Gemüsehof Niedermaier - Upper Austria, Family Plank - Tyrol,
Meusburger Jürgen - Vorarlberg, Gemüsebau Reiter - Salzburg) for their
kind collaboration and the provision of the samples. We also greatly
acknowledge Martin Wells for giving us opportunity to use a trial ver-
sion of Progenesis QI 2.4 and Frank Kuhlmann for his support and
advice on the use of Mass Hunter Profinder B.08.00.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2020.127924.
References
AgrarMarkt Austria. Marktbericht: OBST UND GEMÜSE. (2018). https://www.ama.at/
Marktinformationen/Obst-und-Gemuse/Marktbericht. Accessed on 10 April 2019.
Berrueta, L. A., Alonso-Salces, R. M., & Héberger, K. (2007). Supervised pattern re-
cognition in food analysis. Journal of Chromatography, 1158, 196–214.
Biancolillo, A., Bucci, R., Magrì, A. L., Magrì, A. D., & Marini, F. (2014). Data-fusion for
multiplatform characterization of an italian craft beer aimed at its authentication.
Analytica Chimica Acta, 820, 23–31.
Bong, Y. S., Song, B. Y., Gautam, M. K., Jang, C. S., An, H. J., & Lee, K. S. (2013).
Discrimination of the geographic origin of cabbages. Food Control, 30, 626–630.
Borras, E., Ferre, J., Boque, R., Mestres, M., Acena, L., & Busto, O. (2015). Data fusion
methodologies for food and beverage authentication and quality assessment: A re-
view. Analytica Chimica Acta, 891, 1–14.
Brunner, M., Katona, R., Stefanka, Z., & Prohaska, T. (2010). Determination of the geo-
graphical origin of processed spice using multielement and isotopic pattern on the
example of Szegedi paprika. European Food Research and Technology, 231, 623–634.
Camin, F., Boner, M., Bontempo, L., Fauhl-Hassek, C., Kelly, S. D., Riedl, J., & Rossmann,
A. (2017). Stable isotope techniques for verifying the declared geographical origin of
food in legal cases. Trends in Food Science & Technology, 61, 176–187.
Causon, T. J., Ivanova-Petropulos, V., Petrusheva, D., Bogeva, E., & Hann, S. (2019).
Fingerprinting of traditionally produced red wines using liquid chromatography
combined with drift tube ion mobility-mass spectrometry. Analytica Chimica Acta,
1052, 179–189.
Cubero-Leon, E., Rudder, O. D., & Maquet, A. (2018). Metabolomics for organic food
authentication: Results from a long-term field study in carrots. Food Chemistry, 239,
760–770.
BMLFUW. (2015). Agri-enviromental program ÖPUL 2015 Agriculture, Environment and
Nature. https://www.bmlrt.gv.at/english/agriculture/Rural-development/-
pul2015until2020.html. Accessed on 10 July 2020.
Di Paola-Naranjo, R. D., Baroni, M. V., Podio, N. S., Rubinstein, H. R., Fabani, M. P.,
Badini, R. G., ... Wunderlin, D. A. (2011). Fingerprints for main varieties of
Argentinean wines: Terroir differentiation by inorganic, organic, and stable isotopic
analyses coupled to chemometrics. Journal of Agriculture and Food Chemistry, 59(14),
7854–7865.
DIN ISO 19730 (2009): Soil quality – Extraction of trace elements from soil using am-
monium nitrate solution. Beuth, Berlin.
Ellison, S.L.R., & Williams, A. (2012). EURACHEM/CITAC Guide: Quantifying
Uncertainty in Analytical Measurement. (3rd ed.).
Eriksson, L., Byrne, T., Johansson, E., Trygg, J., & Vikström, C. (2013). Multi- and
megavariate data analysis, basic principles and applications. (Third revised edition by
MKS Umetrics AB).
European Commission (1992). Council Regulation (EEC) No 2081/92 of 14 July 1992 on
the protection of geographical indications and designations of origin for agricultural
products and foodstuffs. Official Journal of the European Communities, L208, 1–8.
European Commission (2012). Regulation (EC) No 1151/2012 of 21 November 2012 on
quality schemes for agricultural products and foodstuffs. Official Journal of the
10
Food Chemistry 338 (2021) 127924
European Communities, L343, 1–29.
Flockhart, D. T. T., Kyser, T. K., Chipley, D., Miller, N. G., & Norris, D. R. (2015).
Experimental evidence shows no fractionation of strontium isotopes (87Sr/86Sr)
among soil, plants, and herbivores: Implications for tracking wildlife and forensic
science. Isotopes in Environmental and Health Studies, 51(3), 372–381.
FAO, FAOSTAT (Food and Agriculture Organization of United Nations Statistics Division).
(2016). htpp://faostat3.fao.org/. Accessed on 12 Jun 2019.
Horsky, M., Irrgeher, J., & Prohaska, T. (2016). Evaluation strategies and uncertainty
calculation of isotope amount ratios measured by MC ICP-MS on the example of Sr.
Analytical and Bioanalytical Chemistry, 408(2), 351–367.
Hiraoka, H., Morita, S., Izawa, A., Aoyama, K., Shin, K. C., & Nakano, T. (2016). Tracing
the origin of onions by strontium isotope ratio and strontium content. Analytical
Sciences, 32, 781–788.
Irrgeher, J., Vogel, J., Santner, J., & Prohaska, T. (2015). Meausurment strategies. In T.
Prohaska, J. Irrgeher, A. Zitek, & N. Jakubowski (Eds.). Sector field mass spectrometry
for elemental and isotopic analysis (pp. 126–151). The Royal Society of Chemistry.
Jaitz, L., Siegl, K., Eder, R., Rak, G., Abranko, L., Koellensperger, G., & Hann, S. (2010).
LC–MS/MS analysis of phenols for classification of red wine according to geographic
origin, grape variety and vintage. Food Chemistry, 122(1), 366–372.
Jandric, Z., Haughey, S. A., Frew, R. D., McComb, K., Galvin-King, P., Elliott, C. T., &
Cannavan, A. (2015). Discrimination of honey of different floral origins by a com-
bination of various chemical parameters. Food Chemistry, 189, 52–59.
Jandric, Z., Islam, M., Singh, D. K., & Cannavan, A. (2017). Authentication of Indian citrus
fruit/fruit juices by untargeted and targeted metabolomics. Food Control, 72,
181–188.
Janoschek, W. R., & Matura, A. (1980). Outline of the Geology of Austria. Abhandlungen
der Geologischen Bundesanstalt 34.www.geoleogie.ac.at/ Accessed 14 Jun 2019.
Kelly, S., Heaton, K., & Hoogeweiff, J. (2005). Tracing the geographical origin of food:
The application of multi-element and multi-isotope analysis. Trends in Food Science &
Technology, 16, 555–567.
Kessler, N., Bonte, A., Albaum, S. P., Mäder, P., Messmer, M., Goesmann, A., &
Nattkemper, T. M. (2015). Learning to classify organic and conventional wheat - a
machine learning driven approach using the MeltDB 2.0 metabolomics analysis
platform. Frontiers in Bioengineering and Biotechnology, 3, 1–9.
Kragten, J. (1994). Tutorial review. Calculating standard deviations and confidence in-
tervals with a universally applicable spreadsheet technique. Analyst, 119(10),
2161–2165.
Luykx, D. M. A. M., & Ruth, S. M. (2018). An overview of analytical methods for de-
termining the geographical origin of food products. Food Chemistry, 107, 897–911.
Medina, S., Pereira, J. A., Silva, P., Perestrelo, R., & Câmara, J. S. (2019). Food finger-
prints - A valuable tool to monitor food authenticity and safety. Food Chemistry, 278,
144–162.
Monakhova, Y. B., Godelmann, R., Hermann, A., Kuballa, T., Cannet, C., Schäfer, H., ...
Rutledge, D. N. (2014). Synergistic effect of the simultaneous chemometric analysis
of 1H NMR spectroscopic and stable isotope (SNIF-NMR, 18O, 13C) data: Application
to wine analysis. Analytica Chimica Acta, 833, 29–39.
Pomerantsev, A. L. (2014). Chemometrics in excel (1st ed.). John Wiley & Sons.
Rummel, S., Hoelzl, S., Horn, P., Rossmann, A., & Schlicht, C. (2010). The combination of
stable isotope abundance ratios of H, C, N and S with 87Sr/86Sr for geographical
origin assignment of orange juices. Food Chemistry, 118(4), 890–900.
Sobolev, A. P., Circi, S., Capitani, D., Ingallina, C., & Mannina, L. (2017). Molecular
fingerprinting of food authenticity. Current Opinion in Food Science, 16, 59–66.
Tomas-Barberan, F. A., Ferreres, F., Garcıa-Viguera, C., & Tomas-Lorente, F. (1993).
Flavonoids in honey of different geographical origin. Zeitschrift für Lebensmittel-
Untersuchung und -Forschung, 196, 38–44.
Yokoo, Y., & Nakano, T. (2001). Sequential leaching of volcanic soil to determine plant-
available cations and the provence of soil minerals using Sr isotopes. Water, Air, and
Soil Pollution, 130, 1583–1588.
Riedl, J., Esslinger, S., & Fauhl-Hasssek, C. (2015). Review of validation and reporting of
non-targeted fingerprinting approaches for food authentication. Analytica Chimica
Acta, 885, 17–32.
Tchaikovsky, A., Irrgeher, J., Zitek, A., & Prohaska, T. (2017). Isotope pattern deconvo-
lution of different sources of stable strontium isotopes in natural systems. Journal of
Analytical Atomic Spectrometry, 32, 2300–2307.
Tchaikovsky, A., Zitek, A., Irrgeher, J., Opper, C., Scheiber, R., Moder, K., ... Prohaska, T.
(2019). Chemometric tools for determining site-specific elemental and strontium
isotopic fingerprints in raw and salted sturgeon caviar. European Food Research and
Technology, 11, 2515–2528.