Dilute-and-Shoot Liquid Chromatography

Approach for Simple and High-throughput

Analysis of 5-Hydroxymethylfurfural in
Fruit-based Baby Foods

Mateus Henrique Petrarca, Adriana

Dillenburg Meinhart & Helena Teixeira
Godoy

Food Analytical Methods

ISSN 1936-9751
Volume 13
Number 4

Food Anal. Methods (2020) 13:942-951

DOI 10.1007/s12161-020-01713-7

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 Author's personal copy
Food Analytical Methods (2020) 13:942–951
https://doi.org/10.1007/s12161-020-01713-7

Dilute-and-Shoot Liquid Chromatography Approach for Simple

and High-throughput Analysis of 5-Hydroxymethylfurfural
in Fruit-based Baby Foods
Mateus Henrique Petrarca 1 & Adriana Dillenburg Meinhart 2 & Helena Teixeira Godoy 1

Received: 7 August 2019 / Accepted: 21 January 2020 / Published online: 27 January 2020
# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
The present work focuses on a cost-effective and environment-friendly approach for the determination of 5-
hydroxymethylfurfural (HMF) in baby foods, a process-induced food compound of toxicological interest. The proposed method
includes acetonitrile-based extraction followed by dilution of the extract with water in order to minimize the interference from
other co-extracted matrix components and eliminate the demand for further clean-up procedure, and then high-performance
liquid chromatography analysis with diode array detection (HPLC-DAD). Adequate performance characteristics were achieved
including limits of detection and quantification of 0.02 and 0.04 μg g−1, respectively; recoveries from 98 to 102% and precision
(RSD values ≤ 4%) under repeatability and within-laboratory reproducibility conditions. The application of the validated method
to commercial fruit-based baby foods and the detection of HMF in all samples (contents between 2.3 and 195.4 μg g−1)
demonstrated their suitability for baby food control as well as it contributed to the first data on HMF in fruit-based baby foods
available in the Brazilian market.

Keywords Food analysis . HPLC-DAD . Infant foods . Processing contaminant . Sample preparation .
5-Hydroxymethyl-2-furaldehyde

Introduction solvents; replacement of usual solvents by ionic liquids and

In the last years, green analytical chemistry has received grow-
ing interest and efforts have been directed to improve the
classical methodologies in order to obtain more cost-
effective and environment-friendly approaches. The sample
preparation has been considered the most critical step of ana-
lytical process which influences significantly on the quality of
results obtained as well as it contributes to the “greenness” of
the entire procedure (Filippou et al. 2016; Ramos 2012). In
this way, several sample preparation aspects have been ex-
plored including reduction or elimination of use of reagents
and/or hazardous substances; reuse and recovery of organic
* Mateus Henrique Petrarca
petrarcamh@gmail.com
1 2014).
Department of Food Science, School of Food Engineering,
University of Campinas (UNICAMP), Campinas, SP 13083 862,
Brazil
2
Department of Food Science and Agrotechnology, Federal
University of Pelotas (UFPEL), Pelotas, RS 96105-900, Brazil
supercritical fluids; miniaturization, integration and automa-
tion of analytical operations; and application of pressure, mi-
crowave and ultrasound radiation aiming high process effec-
tiveness in a shorter time (Płotka et al. 2013).
Within the green chemistry context, dilute-and-shoot pro-
cedure has been an attractive sample preparation strategy for
routine analysis due to its easy, fast and simple operation
which consists in the dilution of aqueous or liquid matrix with
a suitable solvent followed by mass spectrometry analysis
(Dubascoux et al. 2018; Seraglio et al. 2016; Suzuki et al.
2019). The dilution minimizes the matrix effects and thus
eliminates the need for further clean-up procedure; nonethe-
less, it can result in reduced analyte detectability. Therefore,
the dilute-and-shoot strategy has been used for substances that
show good ionization efficiency and with high detection
levels, particularly, in biomonitoring of human exposure to
different chemicals and analytical toxicology (Deventer et al.
5-Hydroxymethylfurfural (HMF), a heterocyclic aldehyde,
is one of the most important process-induced compound oc-
curring in ascorbic acid– and carbohydrate–rich foods. This
furanic compound is an intermediate product in the Maillard
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Food Anal. Methods (2020) 13:942–951
reaction as well as it can be generated from the dehydration of
reducing sugars under acidic conditions, via 1,2 enolisation
and cyclization reactions, and from the degradation of hexoses
and decomposition of the ascorbic acid. Consequently, the
HMF content has been used as a quality indicator regarding
the severity of thermal treatment applied, deterioration and
storage conditions of food products (Capuano and Fogliano
2011; Morales 2009).
The potential health risk related with the occurrence of
HMF in the foods is its possible biotransformation to
5-[(sulphoxy)methyl]furfural (SMF) through the sulfonation
of 5-allylic hydroxyl group by the sulfotransferases resulting
in a reactive electrophilic allylic carbocation which can inter-
act with important cellular nucleophiles (i.e., DNA, RNA and
proteins) and promote structural damages and genetoxic and
mutagenic effects (Abraham et al. 2011; [EFSA] European
Food Safety Authority 2011). In addition, association between
HMF intake and SMF formation has been observed in both
animal and human studies (de la Cueva et al. 2017).
High-performance liquid chromatography (HPLC) has
been the main analytical technique used for the determination
of HMF in food matrices, coupled to diode array detector
(DAD) or mass spectrometry systems with atmospheric pres-
sure chemical ionization (APCI) operating in positive mode
(Gökmen and Şenyuva 2006; Hu et al. 2013; Oral et al. 2014;
Salvatierra-Virgen et al. 2017; Teixidó et al. 2008; Tomasini
et al. 2012). Other analytical techniques such as capillary zone
electrophoresis (CZE) (Bignardi et al. 2014; Morales and
Jiménez-Pérez 2001) and gas chromatography (GC) have also
been used (Teixidó et al. 2006); however, a derivatization
reaction with N,O-bis-trimethylsilyltrifluoroacetamide
(BSTFA) is required previously to GC analysis. Regarding
the sample preparation, procedures based on acidified aque-
ous extraction and subsequent clean-up with Carrez solutions
and/or solid-phase extraction (SPE) have been commonly
used for HMF analysis (Gökmen and Şenyuva 2006; Hu
et al. 2013; Oral et al. 2014; Salvatierra-Virgen et al. 2017;
Teixidó et al. 2008). As also, headspace solid phase-
microextraction (Kamalabadi et al. 2015) and QuEChERS
(quick, easy, cheap, effective, rugged, and safe) with acetoni-
trile as extraction solvent and primary and secondary amine
(PSA) sorbent in dispersive solid-phase extraction (d-SPE)
clean-up step (Tomasini et al. 2012). As mentioned, miniatur-
ized techniques have been used for HMF analysis; however,
the cost of SPE cartridges and SPME fibers has limited their
application to daily routine analysis.
Although HMF is ubiquitous in the human diet and a
health-based guidance value such as acceptable daily intake
(ADI) has not been established for HMF in foodstuffs, the
occurrence of this compound in baby foods attracts special
attention. Infants and children are commonly presumed to be
potentially more sensitive to some toxicological insults when
compared to adults due to greater daily food intake on the
943
body weight basis and metabolism differences ([EFSA]
European Food Safety Authority 2012). In this context, we
proposed an approach based on dilute-and-shoot sample prep-
aration and HPLC-DAD analysis as a cost-effective and
environment-friendly alternative for the monitoring of HMF
in fruit-based baby foods. The method was in-house validated
and then applied for samples of most popular brands of fruit-
based baby foods available in the Brazilian market.
Materials and Methods
Chemicals and Reagents
Analytical standard of 5-hydroxymethylfurfural (purity of
98%) was purchased from Carbosynth Ltd. (Compton
Berkshire, UK). A stock solution (1710 μg mL−1) was pre-
pared in 10 mL of deionized water and stored at − 18 °C. The
working standard solutions were prepared weekly by diluting
the stock solution with deionized water to obtain concentra-
tions at 10 and 100 μg mL−1 and these solutions were stored at
4 °C in amber glass flasks. Acetonitrile and methanol both
HPLC grade were acquired from J.T. Baker (USA). The de-
ionized water used for the dilution step and chromatographic
analysis was obtained using a Milli-Q system (Millipore,
Milfold, MA, USA). PVDF syringe filters (0.45 μm pore size,
13 mm i.d.) were supplied by Merck Millipore (Barueri, SP,
Brazil).
Baby Food Samples
Samples of ready-to-eat baby foods, all containing fruit purées
in their composition and other ingredients such as cereals,
milk or yogurt, were purchased in the city of Campinas, SP,
located in the Southeastern region of Brazil. A total of 25
samples was randomly collected from three supermarkets
and all samples were maintained in their original packaging,
glass jars (120 g each) or stand up pouch (113 g each), at room
temperature until analysis.
Sample Preparation Method
Five grams of homogenized baby food was weighed into a
conical polypropylene centrifuge tube and 5 mL of acetonitrile
was added, the mixture was vortexed for 2 min and centri-
fuged at 3000 ×g for 15 min. After centrifugation step, the
extract was transferred to a 100 mL volumetric glass flask
and the volume was completed with deionized water. The
obtained aqueous extract was filtered through a 0.45 μm
PVDF syringe filter for subsequent HPLC-DAD analysis.
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Instrumentation and Chromatographic Conditions
A 1260 Infinity high performance liquid chromatography sys-
tem (Agilent Technologies, Germany) equipped with quater-
nary pump, autosampler and diode array detector (DAD) was
employed for the HMF analysis. The chromatographic sepa-
ration was achieved on a C18 analytical column (250 mm ×
4.6 mm i.d., 5 μm particle size; Vydac 218TP54, Grace Vydac
Inc., Worms, Germany) maintained at 25 °C. The mobile
phase consisted of deionized water (A) and acetonitrile (B)
at a flow rate of 1 mL min−1. The gradient elution employed
was: 0–6 min, 5% B; 6.1–9 min, 100% B; 9.1–14 min, 5% B;
resulting in a total run time of 14 min. The injection volume
was 20 μL and the DAD was set at 284 nm.
In-house Validation
The suitability of the proposed method was verified through
in-house validation procedure, including selectivity, limit of
detection (LOD), limit of quantification (LOQ), linearity in
solvent and matrix-matched calibration curves, matrix effects,
recovery and precision, under repeatability and within-
laboratory reproducibility conditions.
Since HMF is generated during thermal processes, a home-
made baby food elaborated with fruits (apple, banana, and
papaya) and freshly squeezed orange juice was employed as
a representative blank matrix (baby food free of HMF) for
validation procedure and to obtain the matrix-matched cali-
bration curves for the quantification of HMF in the commer-
cial samples. This homemade baby food was prepared on the
same day the validation procedure was carried out. For this
purpose, fresh fruits were peeled and then homogenized in a
food processor (Walita RI7761, Philips) and the mixture ob-
tained was not heat treated. Although HMF has been reported
in tropical fruit juices (Lee et al. 2014), HMF was not detected
in the homemade baby food as can be observed in the Fig. 2A.
The moisture content in this baby food matrix was 84.8 ± 5%
(n = 3), which was gravimetrically determinate by loss on dry-
ing at 100–105 °C ([ANVISA] Agência Nacional de
Vigilância Sanitária 2005).
The fortified samples used for evaluation of the extraction
efficiency (recovery and precision experiments) were pre-
pared on the day of analyses. Five grams of blank baby food
was weighed into a 50 mL conical centrifuge tube and then
fortified at three levels (0.04, 2 and 4 μg g−1) with working
standard solutions, followed by vigorous vortex agitation for
1 min. In order to obtain a better interaction between the HMF
standard and the baby food matrix, the fortified samples were
allowed to stand at room temperature for at least 1 h, and then
the extraction procedure was carried out. The HMF content
determinate in the commercial baby food samples was not
corrected for the recovery.
Food Anal. Methods (2020) 13:942–951
Results and Discussion
Dilute-and-Shoot Sample Preparation
As an alternative to usual methods for HMF analysis in food-
stuffs, this work was focused on achieve the following criteria:
(i) minimal use of reagents and waste generation, (ii) simple
and fast operation, and (iii) analytical selectivity and sensitiv-
ity for routine analysis of HMF in baby foods.
Since the most of commercial fruit-based baby foods are
semi-solid matrices composed by fruit purées, cereal flours,
starch, milk and/or yogurt, an extraction step was mandatory
prior to the dilute-and-shoot procedure. In this way, acetoni-
trile, methanol and water were tested as extraction solvents
under constant vortex agitation for 2 min using blank matrix
(homemade baby food) fortified at 4 μg g−1. Taking into ac-
count the high moisture content of commercial fruit-based
baby foods (82 ± 2%, n = 10) and homemade baby food
(84.8 ± 5%, n = 3), a 1:1 sample to extraction solvent ratio
(5 g of sample: 5 mL extraction solvent) was enough to obtain
a consistent homogenization between the matrix and the ex-
traction solvent and adequate reproducibility (RSD values
lower than 5%). Suitable mean recoveries (n = 3) were
achieved for acetonitrile (104.6%), methanol (96.9%) and wa-
ter (97.2%); however, acetonitrile resulted in a less colored
yellowish extract without suspended particulate materials after
the centrifugation step, indicating visually lower content of
co-extracted matrix components when compared with water
or methanol. In addition, an advantage associated with use of
acetonitrile is the precipitation of proteins, important feature
particularly for analysis of milk-based baby foods. Therefore,
acetonitrile was chosen as extraction solvent. Additionally, the
number of extraction cycles using acetonitrile as extraction
solvent was studied employing a fruit-based baby food sample
naturally containing HMF. A slight increase in HMF recovery,
approximately 5%, was verified after the re-extraction of ma-
trix pellet with other 5 mL of acetonitrile, thus only one ex-
traction cycle was fixed for the sample preparation.
As a compromise between free-interference analysis and an
environment-friendly approach, dilute-and-shoot strategy was
adopted after acetonitrile-based extraction with the aim of
minimizing the interference from others co-extracted matrix
components and eliminating the necessity for further clean-up
procedure. Then, the acetonitrile extract obtained was trans-
ferred to a glass volumetric flask and the volume was com-
pleted to 100 mL with deionized water, followed by HPLC-
DAD analysis. Since high content of HMF is expected in fruit-
based baby foods and great detectability has been achieved
using DAD set at 284 nm, the dilution step did not hamper the
determination of HMF at the lowest contents found in the
commercial samples resulting in a good peak shape and re-
duced intensity of co-extracted matrix compounds (tR =
2.5 min), as can be seen in the Fig. 1B.
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Food Anal. Methods (2020) 13:942–951
Fig. 1 HPLC-UV chromatograms (λ = 284 nm) of fruit-based baby food
fortified with HMF (4 μg g−1) obtained from A acetonitrile-based extrac-
tion without dilution step, B acetonitrile-based extraction followed by
In addition to dilute-and-shoot strategy, other two clean-up
procedures were separately tested after acetonitrile-based ex-
traction in order to obtain a cleaner extract, including disper-
sive solid-phase extraction (d-SPE) with PSA (primary and
secondary amine) sorbent and liquid-liquid extraction (LLE)
with hexane. In the d-SPE procedure, 50 mg of PSA was used
to each 1 mL of extract followed by vortex agitation for 1 min
and centrifugation at 3000 ×g for 5 min; PSA is an anion
exchange sorbent used mainly to retain polar matrix compo-
nents such as sugars, organic acids and some pigments as
anthocyanidins (González-Curbelo et al. 2015). In the LLE
procedure, 3 mL of hexane was added to acetonitrile-based
extract, followed by vortex agitation for 1 min and centrifu-
gation at 3000 ×g for 5 min, to remove non-polar matrix com-
ponents. Notwithstanding the cost of the PSA sorbent as also
the use of an additional solvent (hexane) in the sample prep-
aration, neither procedure was efficient for complete removal
of a co-extracted matrix compound at the retention time of
2.5 min (Fig. 1C and D); thus, the green and straightforward
dilute-and-shoot sample preparation was validated and then
applied to fruit-based baby food samples.
Dilute-and-shoot approach has gained visibility in food
analysis in recent years, especially regarding the simplicity
and high sample throughput. The procedure has been adapted
for solid matrices and then successfully applied for analysis of
food contaminants such as mycotoxins and pesticides in ce-
reals, botanical nutraceuticals and other matrices (Malachová
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dilution step, C liquid-liquid extraction (LLE) with hexane, and D dis-
persive solid-phase extraction (d-SPE) with primary secondary amine
(PSA) sorbent
et al. 2014; Martínez-Domínguez et al. 2015; Nathanail et al.
2015; Silva et al. 2019). However, this sample preparation
strategy has been used in combination with mass spectrometry
systems which contribute to high degree of selectivity and
sensitivity of the analytical scope. To best of our knowledge,
this is the first report on the application of dilute-and-shoot
sample preparation combined with HPLC-DAD for analysis
of complex matrices such as baby foods. Since the occurrence
of HMF in foods is several orders of magnitude (μg g−1)
higher than other food contaminants (ng g−1), the detectability
of the compound was not affected by the dilution step; thus,
the approach should be tested with other matrices in the future.
Compared to previous methods for HMF analysis in foods
matrices (Table 1), the proposed approach avoids clean-up
step with Carrez solutions (Arribas-Lorenzo and Morales
2010; Contreras-Calderón et al. 2017; Teixidó et al. 2008)
and time consuming solid-phase extraction which uses SPE
cartridges (Hu et al. 2013; Murkovic and Pichler 2006;
Salvatierra-Virgen et al. 2017; Teixidó et al. 2008), thus min-
imizing time, cost and waste generation involved in the rou-
tine analysis.
Method Performance Characteristics
In HPLC-DAD analysis, the selectivity is a decisive parameter
for the distinction between the target analyte and others com-
pounds from the matrix. For this, the method selectivity was
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Table 1 Comparison of the developed method with other liquid chromatography methods for HMF analysis in different food matrices

Matrix Sample preparation Analytical technique LOD LOQ Reference

Coffee Extraction with water followed by SPE with HPLC-DAD 0.035 μg g−1 0.12 μg g−1 Murkovic and Pichler (2006)
Bond elut C18 cartridges
Orange juice, honey, plum Extraction with acidic water (pH 1) and HPLC-MS/MS 0.141–0.276 μg g−1 Not reported Teixidó et al. (2008)
jam, biscuits, and Carrez solutions followed by SPE with
breakfast cereals ENV+ cartridges
Coffee Extraction with 0.1% formic acid followed by HPLC-DAD Not reported 4 μg g−1 Arribas-Lorenzo and Morales (2010)
clean-up with Carrez solutions
Honey QuEChERS method–based extraction HPLC-MS/MS 0.33 μg g−1 1 μg g−1 Tomasini et al. (2012)
−1 −1
Apple cider and wine SPE with LiChrolut EN cartridges followed HPLC-DAD and HPLC-MS/MS 12–25 μg L 41–82 μg L Hu et al. (2013)
by evaporation and dissolution in methanol
Fruit juices Extraction with ethyl acetate followed by HPLC-DAD 0.46 μg L−1 1.4 μg L−1 Lee et al. (2014)
evaporation under nitrogen stream and
dissolution in acetic acid solution (pH 4)
Palm oil Three extraction cycles with methanol 80% HPLC-DAD 0.02 μg g−1 0.05 μg g−1 Ariffin et al. (2014)
under agitation followed by centrifugation
Corn chips Extraction with water (pH 5.2) followed by HPLC-DAD 0.8 μg g−1 2.2 μg g−1 Salvatierra-Virgen et al. (2017)
SPE with Oasis HLB cartridges
Infant formula Extraction and clean-up with Carrez solutions HPLC-DAD 0.0495 μg g−1 0.17 μg g−1 Contreras-Calderón et al. (2017)
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−1
Beer Sample degassed using an ultrasonic bath HPLC-DAD 80 μg L 200 μg L−1 Viegas et al. (2018)
−1 −1
Fruit-based baby food Extraction with acetonitrile followed by HPLC-DAD 0.02 μg g 0.04 μg g Proposed method
dilution of extract with deionized water

Carrez solutions (potassium ferrocyanide and zinc acetate solutions); DAD: diode array detector; HPLC: high performance liquid chromatography; MS/MS: tandem mass spectrometry; QuEChERS: quick,
easy, cheap, effective, rugged and safe; SPE: solid-phase extraction
Food Anal. Methods (2020) 13:942–951
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Food Anal. Methods (2020) 13:942–951 947

Fig. 2 HPLC-UV chromatograms and corresponding spectrum of the yogurt- and fruit-based baby food containing naturally HMF and addi-
HMF peak at the retention time of 4.9 min. A Blank matrix (homemade tional HMF standard; D commercial plum-based baby food containing
baby food); B HMF standard solution at 0.4 μg mL−1; C commercial naturally HMF at 37.4 μg g−1

qualitatively evaluated based on the analysis of blank baby HMF peak (tR = 4.9 min) from co-extracted matrix compo-
food, fortified baby food and commercial baby food contain- nents was observed and the spectrum profile was indistin-
ing naturally HMF (Fig. 2). An unequivocal separation of the guishable from that of the HMF standard (Fig. 2),
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948 Food Anal. Methods (2020) 13:942–951

demonstrating the capacity of the method to identify and
quantify the HMF without interference from other compo-
nents of the matrix (Vessman et al. 2001).
LOD and LOQ of 0.02 and 0.04 μg g−1 were obtained,
respectively, indicating that the dilution step did not affect
the detection of HMF at low concentration levels. The
LOD was set as the smallest concentration of HMF in
fruit-based baby food that was reliably detectable but not
necessarily quantifiable, and could be distinguished from
noise adopting the signal-to-noise ratio of 3:1. The LOQ
was set as the smallest concentration of HMF that could be
detected (signal-to-noise ratio of 10:1) and quantified with
acceptable trueness (mean recovery of 102 ± 3%, n = 6)
and precision (RSD values ≤ 4%), under repeatability and
within-laboratory reproducibility conditions. Compared to
previous HPLC methods designed for HMF analysis in
different food matrices, the proposed method achieved
lower LOD and LOQ (Table 1).
Linearity was evaluated in solvent (deionized water) and
matrix-matched calibration (blank baby food extract) curves,
which included seven concentration levels each in the range
between 0.04 μg g−1 (LOQ) and 4 μg g−1 of standard equiv-
alent in the sample. Adequate linearity was achieved for sol-
vent and matrix-matched calibration curves with determina-
tion coefficients (R2) of 0.9979 and 0.9981, respectively.
Furthermore, analysis of variance (ANOVA) demonstrated
high significance of the regression (p < 0.00001) and no sig-
nificance of the lack of fit (p > 0.999) in the range studied
(Table 2). The slopes obtained from calibration curves of same
concentration in solvent and matrix extracts were employed to
estimate the matrix-induced effects using the equation: Matrix
Effect (%) = [(matrix slope − solvent slope) / solvent slope] ×
100 (Sapozhnikova and Lehotay 2013). A low signal suppres-
sion (ME = − 2%) was observed, indicating negligible matrix
effect in the HPLC-DAD analysis.
The extraction efficiency of method was verified by recov-
ery and precision experiments. For this, blank matrix (home-
made fruit-based baby food) was spiked at the levels of 0.04
(LOQ), 2 and 4 μg g−1, and then a total of six independent
replicates at each spiked level was analyzed on two different
days resulting in mean recoveries between 98 and 102%
(Table 3). The precision, estimated as relative standard devia-
tions (RSD), was evaluated under repeatability (RSDr %) and
within-laboratory reproducibility (RSDR %) conditions. A to-
tal of five independent replicates at each spiked level was
analyzed on the same day by the same analyst under the same
chromatographic conditions, with RSDr values ranging from 2
to 4%. Under within-laboratory reproducibility conditions, a
total of ten independent replicates at each spiked level was
analyzed on two different days by the same analyst under
the same chromatographic conditions, whose RSDR values
varied between 1 and 4% (Table 3).
5-Hydroxymethylfurfural in Fruit-based Baby Foods
The validated method was applied to 25 commercial samples
of the most popular brands of fruit-based baby food available
in the Brazilian market, and HMF was detected in all samples
analyzed (Table 4). The positive identification of HMF in the
baby food samples was established by the retention time (tR)
equal to that obtained with standard solutions, with a maxi-
mum tolerance of ± 0.1 min, and the acquisition of UV spec-
trum between 200 and 400 nm with maximum absorption at
284 nm.
A wide variation in the mean contents of HMF was ob-
served, between 2.3 and 195.4 μg g−1, with the highest con-
tents detected in baby foods elaborated with plum (Table 4).
Plum-based products have been reported as an important
source of HMF for humans (Abraham et al. 2011). In a study
with dried fruits, the highest HMF content was found in plum

Table 2 Analysis of variance (ANOVA) for solvent and matrix-matched calibration curves

Sum of squares Degree of freedom Mean square F testa p value

Solvent calibrationb
Regression 42,607.78 1 42,607.78 6842.728 < 0.00001
Lack of fit 0.22 4 0.05 0.009 0.999829
Pure error 87.17 14 6.23
Total 42,695.32 20
Matrix-matched calibrationc
Regression 40,819.07 1 40,819.07 7982.816 < 0.00001
Lack of fit 0.4 4 0.1 0.02 0.999145
Pure error 71.59 14 5.11
Total 40,891.48 20
a
F0.05; 1;14 (F tabulated) = 4.6
b
Deionized water
c
Homemade baby food composed by a mixture of fresh fruits (apple, banana, orange and papaya)
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Food Anal. Methods (2020) 13:942–951 949

Table 3 Performance characteristics obtained with the proposed Table 4 Content of HMF in fruit-based baby foods available in the
method using a representative matrix of fruit-based baby fooda Brazilian market

Parameters HMF Sample Principal ingredient HMF (μg g−1)a

LOD (μg g−1) 0.02 1 Apple (brand A) 7.2 ± 0.2

LOQ (μg g−1) 0.04 2 Apple (brand B) 4.2 ± 0.1
Recovery (%, n = 6) 3 Apple (brand C) 6.5 ± 0.1
0.04 μg g−1 102 ± 3 4 Apple (brand D) 13.6 ± 0.2
2 μg g−1 98 ± 2 5 Apple, guava and banana 3.5 ± 0.1
4 μg g−1 101 ± 2 6 Apple, papaya and orange 7.7 ± 0.2
Precision (RSD %) Intra-day, n = 5 (inter-day, n = 10) 7 Apple, pear and peach 12.8 ± 0.1
0.04 μg g−1 4 (4) 8 Apple and yogurt 3.6 ± 0.1
2 μg g−1 3 (2) 9 Banana (brand A) 2.8 ± 0.2
4 μg g−1 2 (1) 10 Banana (brand B) 9.6 ± 0.3
11 Banana and apple 5.5 ± 0.1
LOD limit of detection, LOQ limit of quantification, RSD relative stan-
12 Banana and oat 2.4 ± 0.1
dard deviation
a 13 Banana and yogurt 2.8 ± 0.4
Homemade baby food composed by a mixture of fresh fruits (apple,
banana, orange and papaya) 14 Banana, apple, mango and orange 6.3 ± 0.2
15 Banana, mango, orange and pineapple 3.8 ± 0.1
16 Banana, pineapple and orange (brand A) 2.5 ± 0.2
(2200 μg g−1) when compared to apple (80 μg g−1), pear 17 Banana, pineapple and orange (brand B) 2.3 ± 0.1
(100 μg g−1) and pineapple (280 μg g−1) (Murkovic and 18 Orange and papaya 5.9 ± 0.2
Pichler 2006). High HMF contents were also related in plum 19 Pear and mango 17.1 ± 0.3
jam (1200 μg g−1) (Murkovic and Pichler 2006) and bever- 20 Pear and yogurt 16.4 ± 0.3
ages prepared from plums (707.7 μg g−1) (Abraham et al. 21 Plum (brand A) 37.4 ± 3.6
2011). 22 Plum (brand B) 195.4 ± 11
Our findings support previous studies conducted in other 23 Yogurt and apple 2.5 ± 0.4
countries that HMF is prevalent in baby foods. Contents be- 24 Yogurt, banana, orange and pineapple 3.3 ± 0.1
tween 0.14 and 65 μg g−1 were reported in baby foods pur- 25 Yogurt, apple, banana, papaya and strawberry 2.8 ± 0.2
chased in Spain (Mesías-García et al. 2010; Rada-Mendoza
et al. 2002, 2004) and between 2.1 and 28.9 μg g−1 in fruit-
a
Mean ± standard deviation (n = 3)
based baby foods from Czech Republic (Cizková et al. 2009;
Vorlová et al. 2006). Additionally, HMF was related in milk- behavior was observed in fruit-based baby foods, with initial
and cereal-based baby foods from Turkey with contents vary- HMF content of 1 μg g−1 and after 12 months of storage at
ing from 0.17 to 57.18 μg g−1 (Gökmen and Şenyuva 2006), 35 °C a content of 65 μg g−1; however, lower content
and between 0.26 and 9.62 μg g−1 in cereal-based baby foods (6 μg g−1) was found in the samples stored at 20 °C (Rada-
from Italy (Bignardi et al. 2014). Mendoza et al. 2004). Also, higher HMF contents were ob-
No maximum HMF limit has been stated for baby foods; served in infant formulas stored at 37 °C when compared to
however, maximum levels have been established for HMF those samples stored at 20 °C, after 12 months (Ferrer et al.
in apple juice (50 μg g−1) and honey (40 μg g−1 in general 2002) or 24 months (Ferrer et al. 2005). In addition to the
or 80 μg g−1 for honeys from regions with tropical climate) storage conditions, the control of thermal treatment applied
(Codex Alimentarius 2001; [EC] European Community during the manufacturing process as also the ingredients used
2002). In comparison, the HMF contents detected in the baby in the formulation of these products such as pre-processed
foods elaborated with apple (Table 4) were lower than cereals, dried fruits, heat-treated milk and honey, represent
the maximum HMF level set for apple juice (50 μg g−1). critical points to minimize the content of HMF in baby foods
Although HMF has not been used as a food additive in and consequently to reduce the infant exposure to HMF
baby foods, it rapidly accumulates during the storage of (Capuano and Fogliano 2011; Morales 2009).
carbohydrate- and ascorbic acid–rich products. For instance,
a significant increase in the HMF content was observed in
infant rice cereal samples after 12 months of storage at Conclusions
32 °C or 55 °C in nitrogen atmosphere, with HMF content
at 55 °C (2.3 ± 0.03 μg g−1) two fold higher than at 32 °C Dilute-and-shoot sample preparation combined with HPLC-
(1.06 ± 0.02 μg g−1) (Ramírez-Jiménez et al. 2003). The same DAD analysis proved to be an efficient and green alternative
 Author's personal copy
950
for the determination of HMF in fruit-based baby foods. In
addition to suitable performance characteristics achieved, the
proposed method presents attractive features for daily routine
analysis such as small amount of sample, minimal consump-
tion of chemicals and simple and fast operation. The dilution
step minimizes the interference from other matrix components
and eliminates the demand for further clean-up procedure
without affecting the detection of HMF at the lowest contents
found in the fruit-based baby foods. The application of the
proposed method to commercial fruit-based baby foods and
the detection of HMF in all samples demonstrates their feasi-
bility for baby food control and it contributes to the first data
on HMF in fruit-based baby foods from Brazil. Wide variation
in the HMF content was verified with the highest levels de-
tected in plum-based baby foods indicating that fruit-based
baby foods can be an important source of HMF at early age.
Acknowledgements The authors are grateful to the National Council for
Scientific and Technological Development (CNPq) of Brazil for the
scholarship awarded to the first author and financial support (Process
no. 140271/2013-9).
Funding information This study was funded by National Council for
Scientific and Technological Development – CNPq (grant number
140271/2013–9).
Compliance with Ethical Standards
Conflict of Interest M. H. Petrarca declares that he has no conflict of
interest. A. D. Meinhart declares that she has no conflict of interest. H. T.
Godoy declares that she has no conflict of interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed Consent Not applicable.
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