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Determination of Aflatoxins in Plant-based Milk and Dairy Products by Dispersive Liquid–Liquid Microextraction and High-performance Liquid Chromatography with Fluorescence Detection

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  • Faculty of Agriculture, Cairo University

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The consumption of plant-based milk has increased due to their nutritional attributes. However, these products may contain aflatoxins if contaminated raw materials were used, although little concern is present in international regulation regarding this topic. In this work, dispersive liquid–liquid microextraction (DLLME) was used for the determination of the most important aflatoxins (B1, B2, G1, and G2) in oat, rice, coconut, almond, and birdseed plant-based milk and milk-based products enriched with oats, almonds, and walnuts using high-performance liquid chromatography (HPLC) with photochemical derivatization and fluorescence detection. Calibrations in matrix were performed for all of the samples, obtaining satisfactory linearity, with correlation coefficients exceeding 0.994 for all of the aflatoxins. The precision in terms of repeatability and intermediate precision, expressed as the relative standard deviation, was lower than 9.7%, and recoveries ranged between 82 and 104%, fulfilling current legislation for the determination of aflatoxins. In addition, the limits of quantification were 0.5 µg L⁻¹ for the aflatoxins, allowing the determination of these compounds below the maximum levels established by European Commission in these commodities. Finally, 23 commercial products were analyzed to characterize the presence of these toxins.
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Analytical Letters
ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20
Determination of Aflatoxins in Plant-based Milk
and Dairy Products by Dispersive Liquid–Liquid
Microextraction and High-performance Liquid
Chromatography with Fluorescence Detection
Ahmed M. Hamed, Mahmoud Abdel-Hamid, Laura Gámiz-Gracia, Ana M.
García-Campaña & Natalia Arroyo-Manzanares
To cite this article: Ahmed M. Hamed, Mahmoud Abdel-Hamid, Laura Gámiz-Gracia, Ana
M. García-Campaña & Natalia Arroyo-Manzanares (2018): Determination of Aflatoxins in
Plant-based Milk and Dairy Products by Dispersive Liquid–Liquid Microextraction and High-
performance Liquid Chromatography with Fluorescence Detection, Analytical Letters, DOI:
10.1080/00032719.2018.1467434
To link to this article: https://doi.org/10.1080/00032719.2018.1467434
Published online: 26 Jul 2018.
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FOOD ANALYSIS
Determination of Aflatoxins in Plant-based Milk and Dairy
Products by Dispersive LiquidLiquid Microextraction
and High-performance Liquid Chromatography with
Fluorescence Detection
Ahmed M. Hamed
a
, Mahmoud Abdel-Hamid
a
, Laura G
amiz-Gracia
b
,
Ana M. Garc
ıa-Campa~
na
b
, and Natalia Arroyo-Manzanares
b
a
Department of Dairy Science, Faculty of Agriculture, Cairo University, Giza, Egypt;
b
Department of
Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain
ABSTRACT
The consumption of plant-based milk has increased due to their
nutritional attributes. However, these products may contain aflatoxins
if contaminated raw materials were used, although little concern is
present in international regulation regarding this topic. In this work,
dispersive liquidliquid microextraction (DLLME) was used for the
determination of the most important aflatoxins (B
1
,B
2
,G
1
, and G
2
)in
oat, rice, coconut, almond, and birdseed plant-based milk and milk-
based products enriched with oats, almonds, and walnuts using
high-performance liquid chromatography (HPLC) with photochemical
derivatization and fluorescence detection. Calibrations in matrix were
performed for all of the samples, obtaining satisfactory linearity, with
correlation coefficients exceeding 0.994 for all of the aflatoxins. The
precision in terms of repeatability and intermediate precision,
expressed as the relative standard deviation, was lower than 9.7%,
and recoveries ranged between 82 and 104%, fulfilling current
legislation for the determination of aflatoxins. In addition, the limits
of quantification were 0.5 mgL
1
for the aflatoxins, allowing the
determination of these compounds below the maximum levels estab-
lished by European Commission in these commodities. Finally, 23
commercial products were analyzed to characterize the presence of
these toxins.
ARTICLE HISTORY
Received 9 March 2018
Accepted 17 April 2018
KEYWORDS
Aflatoxins; dispersive liquid-
liquid microextraction
(DLLME); high-performance
liquid chromatography
(HPLC) with fluorescence
detection; plant product
enriched milk; vegetable-
based milk
Introduction
Currently, an increasing number of consumers prefer nondairy plant-based milk
substitutes due to a lifestyle choice or medical reasons, including lactose intolerance
(affecting up to 75% world population), cows milk allergies, or the prevalence of
hypercholesterolemia. Much attention has been paid to soy milk as an alternative to
bovine milk. However, other commodities such as cereals, oilseeds, or nuts have been
explored recently for new food uses (including plant-based milks) on the basis of their
functional properties. These beverages are manufactured by extracting the plant material
CONTACT Natalia Arroyo-Manzanares narroyo@ugr.es Department of Analytical Chemistry, Faculty of Sciences,
University of Granada, Campus Fuentenueva s/n, E-18071, Granada, Spain
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lanl.
ß2018 Taylor & Francis
ANALYTICAL LETTERS
https://doi.org/10.1080/00032719.2018.1467434
(nuts, cereals, legumes, seeds, or other products) in water, separating the liquid, and for-
mulating the final product. They resemble cows milk in appearance, but their composi-
tions differ significantly (M
akinen et al. 2016; Sethi, Tyagi, and Anurag 2016).
On the other hand, currently the dairy industry is trying to offer new innovative prod-
ucts with added values. This is the case of enriched milks, which offer to the consumer
the nutritional properties of the milk, as well as other health benefits from the added
ingredients. Among these products are some beverages, which include nuts or cereals in
their composition. Taking into account that plant sources (cereals and legumes) are
accepted as functional food and nutraceuticals because of presence of health promoting
components, their derived products (nondairy plant-based milk and enriched dairy prod-
ucts) can also be considered as functional foods. However, if the raw plant material has
suffered contamination, these derived products are susceptible to be contaminated too.
Mycotoxins are one of the most common natural contaminants of plants, they are toxic
secondary metabolites produced by filamentous fungi. According to the Food and
Agriculture Organization, up to 25% of the crops all over the world are contaminated with
mycotoxins, including the highly toxic aflatoxins B
1
,B
2
,G
1
,andG
2
,producedby
Aspergillus flavus and Aspergillus parasiticus fungi. Aflatoxin contamination can occur dur-
ing the storage and poor processing conditions in a wide variety of commodities including
various cereals, oilseeds, spices, and nuts, as well as their derivative products. Aflatoxins
infect humans by consumption of contaminated foods, being a remarkable public health
problem, as chronic toxicity by aflatoxins can cause liver damage, immunosuppressive and
carcinogenic effects.AFB
1
is the most toxic and classified by the International Agency for
Research on Cancer as a human carcinogen. The high number of recent publications
devoted to mycotoxins, including aflatoxins, is an indicative of the relevance of this prob-
lem (Alshannaq and Yu 2017; Kumar et al. 2017;Sarmaetal.2017).
Considering the above, in order to protect health consumers and to ensure the inter-
national food trade, different countries have set maximum limits for mycotoxins. Thus,
around 100 countries have regulations to control mycotoxin levels in food and most
include maximum permitted or recommended levels for specific commodities (Anukul,
Vangnai, and Mahakarnchanakul 2013; Lawley 2013; Anfossi, Giovannoli, and Baggiani
2016). In this regard, the European Commission has set maximum contents for AFB
1
and for total aflatoxins in nuts, almonds, hazelnuts, dried fruits, rice, cereals, and spices,
ranging from 212 μgkg
1
for AFB
1
and from 415 μgkg
1
for total aflatoxins, depend-
ing upon the commodity (European Commission 2006b,2010).
Quantitative analytical methods for determination of aflatoxins are mainly based on
sample treatments based on liquid extraction, followed by a clean-up step in order to
remove interferents, with subsequent quantification by liquid chromatography with
fluorescence or mass spectrometry (MS) detection. Recent reviews have compiled the
most relevant applications (Bakirdere et al. 2012; Selvaraj et al. 2015; Yao, Hruska, and
Di Mavungu 2015; Anfossi, Giovannoli, and Baggiani 2016; Xie, Chen, and Ying 2016;
Alshannaq and Yu 2017). The bottleneck of all these methods is the sample treatment,
as aflatoxins must be efficiently extracted from the samples in order to reduce matrix
effects, allowing quantification at the very low concentration levels required. Besides
traditional liquid extraction, solid phase extraction (SPE) and the use of immunoaffinity
columns, other alternative extraction methods, such as the well-known quick, easy,
cheap, effective, rugged, and safe (QuEChERS) (Sartori et al. 2015; Zhu et al. 2015;
2A. M. HAMED ET AL.
Geary et al. 2016; Zhao et al. 2016) and dispersive liquidliquid microextraction
(DLLME) (Campone et al. 2011; Afzali et al. 2012; Campone et al. 2013; Hamed et al.
2017b) approaches have been recently applied for determination of aflatoxins in com-
modities. Moreover, both methodologies have been combined in order to achieve
cleaner extracts (Arroyo-Manzanares et al. 2014). However, the effectiveness for the
determination of aflatoxins in plant-based milks has not been demonstrated.
Due to advantages that include simplicity, reduced solvent consumption and high
enrichment factors, DLLME has obtained widespread acceptance in food analysis,
including several applications in milk and dairy products (Saraji and Boroujeni 2014;
Quigley, Cummins, and Connolly 2016). Considering the scarce attention that functional
vegetable milks have received concerning the possible contamination by mycotoxins, as
only few articles are devoted to this aspect (Hamed et al. 2017a; Mir
o-Abella et al.
2017); in this article, DLLME has been proposed combined with HPLC coupled with
photochemical derivatization and fluorescence detection for the determination of the
four main aflatoxins in nondairy plant-based milk products (oat, rice, coconut, almond,
and birdseed milk). Additionally, milk-based products enriched with nuts and cereals
(oats, almonds, and walnuts) have been analyzed in this study.
Materials and methods
Chemicals and reagents
Methanol, acetonitrile, formic acid, and chloroform (all LC-MS grade) were supplied by
VWR International Eurolab, S.L. (Barcelona, Spain). Magnesium sulfate, trisodium cit-
rate, and sodium chloride were purchased from Panreac Qu
ımica (Barcelona, Spain).
Potassium dihydrogen phosphate and disodium hydrogen citrate sesquihydrate were
supplied by Merck (Darmstadt, Germany).
Analytical standards of aflatoxins were supplied by Sigma-Aldrich (St. Louis, MO).
Individual stock standard solutions containing 20 mgL
1
for AFG
2
and AFB
2
and 2.5 mgL
1
for AFB
1
and AFG
1
were used by dissolving accurate volume in acetonitrile. Intermediate
working solutions were prepared in acetonitrile and stored at 20 C and used throughout
this work. Working standard solutions containing all the aflatoxins were daily prepared by
dilution of these solutions with methanol:water (1:1, v/v).
Ultrapure water (18.2 MX/cm, Milli-Q Plus system, Millipore Bedford, MA) was used
throughout all work. Nylon syringe filters, 0.22 mm25 mm (Agela Technologies, New
York) were used for filtration of samples before chromatographic analysis.
Instrumentation
Chromatographic separations were developed in a modular HPLC system that consisted
of a quaternary low pressure gradient pump (Model PU-2089, Jasco, Tokyo, Japan); an
autosampler with a 100-mL loop (Model AS-2055, Jasco); a column oven (X-LC-
3067CO), a ultraviolet derivatization module, consisting on a photochemical reactor
with a 254-nm lamp (LCTech, Dorfen, Germany) placed between the C18 Kinetex sep-
aration column (150 mm 4.6 mm, 2.6 mm from Phenomenex, Torrance, CA), and the
ANALYTICAL LETTERS 3
fluorescence detector (Model FP 2020, Jasco). ChromNAV software (1.18.03 version,
Jasco) was used for data acquisition and processing.
A Universal 320 R centrifuge (HettichZentrifugen, Tuttlingen, Germany), a bench
mixer multitube vortex agitator (model BV1010, Edison, NJ), and a nitrogen evaporator
(System EVA-EC from VLM GmbH, Bielefeld, Germany) were also used in this work.
Sample treatment
In order to select the best extraction methodologies, two approaches previously developed
in our laboratory for determination of mycotoxins (DLLME and QuEChERS) were tested.
DLLME method. A previously reported DLLME procedure for determination of aflatox-
ins in milk and yogurt was adapted (Campone et al. 2013; Hamed et al. 2017b). A 5-mL
sample was placed in a 15-mL falcon tube and 6mL of acetonitrile and 1.5g of NaCl
were added. The solution was vortexed for 30 s and centrifuged (5 min, 6000 rpm) allow-
ing protein precipitation. Then, the upper phase was quantitatively transferred (approxi-
mately 4mL) into a 10-mL vial. In order to carry out the DLLME, the organic phase
containing the extracted analytes and acting as disperser solvent and 1500μLofCHCl
3
(extractant solvent) was injected in 5mL of deionized water. This solution was strongly
shaken for 30 s, providing the formation of a stable cloudy solution. The mixture was
centrifuged (5 min, 6000 rpm) for phase separation and the organic phase was collected
and dried under a gentle nitrogen stream. The final residue was dissolved in 1000 μLof
methanol:water (1:1, v/v) and filtered before HPLC analysis.
QuEChERS method. For the QuEChERS-based procedure, a previously optimized
method for determination of Fusarium toxins in similar matrices was tested (Hamed
et al. 2017a). Briefly, 5 mL of sample and 5 mL of 50 mM potassium dihydrogen phos-
phate at pH 7.0 were placed into a 50-mL tube and vortexed for 10 s. Subsequently,
10 mL of acetonitrile containing 5% formic acid was added and vortexed for 2 min.
Then, 4 g MgSO
4
, 1 g NaCl, 1 g trisodium citrate, and 0.5 g disodium hydrogen citrate
sesquihydrate were added, shaken for 1 min, and centrifuged at 4500 rpm for 5 min.
Next, 2 mL of the upper acetonitrile layer was transferred to a glass vial, evaporated
to dryness under nitrogen, reconstituted with 500 mL of methanol:water (1:1, v/v), and
filtered prior to introduction into the HPLC system.
Chromatographic conditions
Separation was performed using HPLC photochemical derivatization in order to enhance
the native fluorescence of AFB
1
and AFG
1
using conditions described in the literature
(Arroyo-Manzanares et al. 2015). The derivatization reaction is based on the conversion
of AFB
1
and AFG
1
to AFB
2a
and AFG
2a
, respectively, using a photochemical reactor
with a 254 nm lamp. Briefly, a C18 Kinetex separation column (150 mm 4.6 mm,
2.6 mm) was used, while the mobile phase consisted on a mixture of water (eluent A),
acetonitrile (eluent B), and methanol (eluent C) at a flow rate of 0.9 mL min
1
, using a
gradient elution with constant composition of 27% B, 0% C (0.3min), 13% C (20 min)
and 68% C (2123 min), returning to the initial conditions using a 1 min linear gradi-
ent. The column oven was maintained at 30 C, and a volume of 50 mL was injected.
4A. M. HAMED ET AL.
The excitation and emission wavelengths for fluorescence measurements were 365 and
460 nm, respectively, with the detector operating at a gain 100.
Results and discussion
Optimization of the sample pretreatment
Two approaches were characterized for the extraction of aflatoxins from plant-based milks,
using rice milk as a representative matrix: a QuEChERS-based extraction and DLLME.
Theses sample treatments were selected as alternatives to standard methods for aflatoxins
determination in other matrices based on immunoaffinity columns. These columns contain
specific antibodies to the analytes, allowing excellent clean-up and sample purification.
However, this methodology presents some drawbacks such cost, complexity, inefficiency, and
high susceptibility to sample pH, therefore suffering from low recoveries for some aflatoxins.
In order to evaluate the extraction efficiency of both methods, the recovery values were
estimated. First, the QuEChERS-based extraction was tested, but this approach did not
provide good recoveries (lower than 54%). Alternatively, and considering the good results
previously reported for the determination of aflatoxins in yogurt using DLLME (Hamed
et al. 2017b), this sample treatment was tested. Cleaner extracts and high recoveries for all
of the analytes were provided without requiring any further optimization of the significant
variables affecting the extraction efficiency. A chromatogram corresponding to a rice milk
sample analyzed by the proposed DLLME method is shown in Figure 1. No interference
peaks co-eluted with the analytes. Thus, DLLME was selected for sample treatment.
Figure 1. Chromatograms of (a) a spiked rice milk sample at 2.5 mgL
1
for each aflatoxins and (b) a
blank sample. The column was aC18 Kinetex (150 mm 4.6 mm, 2.6 mm) using a mobile phase of
water (eluent A), acetonitrile (eluent B), and methanol (eluent C). The gradient was 27% B, 0% C
(0.3 min), 13% C (20 min), 68% C (2123 min), 0% C (24 min) at a flow rate of 0.9 mL min
1
at 30 C.
The injection volume was 50 mL; the excitation wavelength was 365; the emission wavelength was
460 nm; and the gain was 100.
ANALYTICAL LETTERS 5
Method validation
The method was characterized in oat, rice, coconut, almond, and birdseed milk and
milk products enriched with oats, almonds, and walnuts. The linear dynamic ranges,
limits of quantification, precision, and trueness were evaluated for all of the
studied matrices.
Calibration curves. Spiked blank samples (previously analyzed to ensure that they
were free of aflatoxins) were used to establish the calibration curves at five concentration
levels (0.5, 1, 2.5, 5, and 10 mgL
1
of each aflatoxin). Each level was prepared and
injected in triplicate. The slopes, intercept, and coefficients of determination (R
2
) were
calculated by least-square regression. Table 1 summarizes the performance characteris-
tics of the method. Responses were linear with R
2
higher than 0.99 for all aflatoxins.
Limits of quantification were set as the lowest concentration level (0.5 mgL
1
), showing
the suitability of the method for the quantification of very low concentrations of aflatox-
ins in these matrices.
Precision. The precision was evaluated in terms of repeatability (intraday precision) and
intermediate precision (interday precision) by application of the DLLME method in the
different samples spiked at two concentration levels of aflatoxins (0.5 and 2.5 mgL
1
).
Repeatability was evaluated in one day by analyzing three samples, prepared and injected
Table 1. Statistical and performance characteristics of the method for each sample matrix.
Product Analyte
Linear range
(mgL
1
)
Coefficient of
determination Slope Intercept
Oat milk (14% oat) AFG
2
0.2810 0.997 374661 172.83
AFG
1
0.1810 0.997 183207 12654
AFB
2
0.2210 0.997 241871 19662
AFB
1
0.1810 0.997 205557 24872
Rice milk (15% rice) AFG
2
0.0410 0.996 530816 83658
AFG
1
0.0410 0.997 229517 22902
AFB
2
0.0410 0.996 313917 50936
AFB
1
0.0310 0.996 233732 47481
Coconut milk (5.9% coconut) AFG
2
0.1210 0.997 566738 49141
AFG
1
0.2110 0.997 247770 14270
AFB
2
0.1010 0.996 342899 29909
AFB
1
0.1110 0.997 246650 30222
Almond milk (2% almond) AFG
2
0.1610 0.998 567068 6402.6
AFG
1
0.1210 0.999 246899 4948.9
AFB
2
0.1710 0.998 342759 6679.2
AFB
1
0.1810 0.999 250773 7497.4
Birdseed milk (15% birdseed) AFG
2
0.2610 0.999 551275 63621
AFG
1
0.4810 0.998 242650 210143
AFB
2
0.1210 0.999 332773 5634.4
AFB
1
0.3210 0.999 249578 15624
Milk-based product with oat (0.2% oat) AFG
2
0.1710 0.998 356966 40305
AFG
1
0.2510 0.994 169018 51365
AFB
2
0.1410 0.997 225131 35373
AFB
1
0.2110 0.996 187531 42413
Milk-based product with almond (0.2% almond) AFG
2
0.2110 0.997 607015 132881
AFG
1
0.2510 0.995 272321 25077
AFB
2
0.1310 0.997 371034 44224
AFB
1
0.1410 0.995 267662 22806
Milk-based product with walnut (0.2% walnut) AFG
2
0.2110 0.997 374661 172.83
AFG
1
0.2410 0.997 183207 12654
AFB
2
0.2410 0.997 241871 19662
AFB
1
0.0510 0.997 205557 24872
6A. M. HAMED ET AL.
in triplicate under the same conditions. Intermediate precision was evaluated for 5 days
(one sample per day was prepared and injected in triplicate).
Trueness. Trueness was assessed by recovery experiments in the selected matrices. The
samples were spiked at two levels of each aflatoxins (0.5 and 2.5 mgL
1
). Each sample
was analyzed three times and injected in triplicate. Previously, a blank sample was ana-
lyzed to verify the absence of aflatoxins and co-eluting peaks.
Table 2 shows the results of the precision expressed as the relative standard deviation
of peak area and trueness. Good precision (relative standard deviation lower than 10%)
and recoveries between 82% and 104% were obtained in all cases, in compliance with
regulations for methods describing the official control of the levels of mycotoxins in
food (European Commission 2006a).
Analysis of commercial samples
Commercial samples of functional vegetable milks and milk-based products were pur-
chased at local stores from Granada, Spain. They comprise four oat milk products (14%
oat), five rice milk samples (15% rice), three coconut materials (5.9% coconut), two
almond milk products (2% almond), one birdseed milk sample (15% birdseed), and
Table 2. Recovery and precision as the relative standard deviation for the proposed DLLME-HPLC- fluorescence method.
Recovery % (n¼9) Intraday precision (n¼9) Interday precision (n¼15)
Product Analyte
Level 1
(0.5 mgL
1
)
Level 2
(2.5 mgL
1
)
Level 1
(0.5 mgL
1
)
Level 2
(2.5 mgL
1
)
Level 1
(0.5 mgL
1
)
Level 2
(2.5 mgL
1
)
Oat milk AFG
2
95 101 5.1 0.9 6.7 5.1
AFG
1
96 101 4.6 1.1 8.5 7.7
AFB
2
94 100 3.5 1.1 6.6 5.8
AFB
1
98 98 4.0 1.8 4.0 3.9
Rice milk AFG
2
97 97 5.6 4.4 9.7 6.2
AFG
1
95 98 6.0 4.0 6.9 6.8
AFB
2
98 100 4.9 4.8 7.7 6.9
AFB
1
104 104 5.2 4.8 7.5 4.6
Coco milk AFG
2
92 93 3.9 1.7 5.1 4.7
AFG
1
93 93 5.9 2.3 7.5 6.5
AFB
2
92 93 3.9 2.6 6.8 6.2
AFB
1
94 94 4.6 2.6 7.5 5.2
Almond milk AFG
2
91 94 5.6 4.7 6.6 6.1
AFG
1
93 93 7.4 0.2 7.9 7.8
AFB
2
87 87 6.6 2.5 7.7 6.8
AFB
1
91 95 4.8 0.6 6.8 5.6
Birdseed milk AFG
2
99 100 4.4 1.8 6.6 4.5
AFG
1
97 103 7.3 0.5 8.6 7.5
AFB
2
92 99 5.5 1.8 7.1 5.7
AFB
1
90 100 6.2 2.0 7.9 6.5
Milk-based
product
with oat
AFG
2
86 89 3.7 3.9 4.4 4.1
AFG
1
99 100 8.9 2.5 9.7 5.7
AFB
2
85 87 1.0 2.5 5.0 4.2
AFB
1
92 93 3.6 1.4 6.6 4.0
Milk-based
product
with
almond
AFG
2
92 92 4.1 1.8 9.1 7.4
AFG
1
91 93 4.4 2.0 7.9 4.7
AFB
2
87 91 2.6 2.2 5.4 4.8
AFB
1
91 91 4.1 2.3 5.4 4.8
Milk-based
product
with walnut
AFG
2
84 90 4.4 2.3 6.7 4.7
AFG
1
92 95 0.6 4.3 6.8 5.8
AFB
2
82 94 5.5 1.3 6.1 6.0
AFB
1
88 91 2.7 1.0 8.1 7.0
ANALYTICAL LETTERS 7
milk-based products: three enriched with oats (0.2%), three with almonds (0.2%), and
two with walnuts (0.2%). All of the milk samples were stored at 4 C in their original
1 L packaging prior to use. Fortunately, none of the studied aflatoxins were detected at
concentration higher than limits of quantification.
Conclusions
After characterization of QuEChERS and DLLME as possible alternative sample treat-
ments, the latter was applied for the quantification of AFB
1
, AFB
2
, AFG
1
, and AFG
2
in
plant-based beverages and enriched milk. Moreover, the use of photochemical derivati-
zation for enhancing the fluorescence emission coupled to HPLC avoids the use of deri-
vatization reagents and provides an easy and sensitive alternative to these hazardous
agents. The proposed method provided good results in terms of precision, recovery and
extract cleanliness, allowing the determination of these mycotoxins at very low concen-
trations in infrequently analyzed but increasingly consumed vegetable-based milk and
milk-based products.
Disclosure statement
The authors report no conflicts of interest with this study.
Funding
Financial support was provided from the Project Reference AGL2015-70708-R (MINECO/FEDER,
UE). AMH thanks the Erasmus MundusAl Idrisi II program for a predoctoral grant.
ORCID
Laura G
amiz-Gracia http://orcid.org/0000-0002-8880-5000
Ana M. Garc
ıa-Campa~
na http://orcid.org/0000-0002-3191-3350
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10 A. M. HAMED ET AL.
... Plant-based beverage are complex matrices (proteins, carbohydrates, lipids and fiber) [9]; therefore, pre-treatments and cleanup steps to avoid the matrix effects that might interfere in quantification by analytical instruments are necessary [10]. Several authors developed methods in plant-based beverages by applying salting-out assisted liquid-liquid extraction in the case of Fusarium toxin determination [11,12]; dispersive liquid-liquid microextraction for aflatoxins [13]; or QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction in the case of a multidetermination of mycotoxins [8] to avoid matrix effects. ...
... the number of samples containing these combinations is indicated in parentheses. Recently, works studied mycotoxin presence in oat, soy and rice-based beverage, as they are the most consumed globally [8,[11][12][13]30]. Miró-Abella et al. [8] reported the presence of mycotoxins and found DON, OTA, ZEN and T-2 toxin co-occurrence with AFB 1 , AFB 2 , AFG 1 and AFG 2 , and oat-based beverages were the most frequently contaminated. ...
... The traditional tigernut beverage consumed in Spain and other African countries [13] was studied to evaluate the presence of mycotoxins, and the presence of aflatoxins (AFB 1 , AFB 2 and AFG 2 ) and OTA [32,33] was detected. Rubert et al. [33] reported more mycotoxins contamination in fresh beverage than those concentrated in Spanish tigernut beverages. ...
Article
Full-text available
This study developed and validated an analytical methodology for the determination of aflatoxins, enniatins, beauvericin, zearalenone, ochratoxin-A, alternariols, HT-2 and T-2 toxin in soy, oat, rice and almond beverages, based on solid phase extraction columns (SPE) and analyzed by liquid chromatography coupled to mass spectrometry in tandem. C18 SPE was successfully applied, obtaining recoveries that range from 72 ± 12% (ochratoxin-A) to 99 ± 4% (ENA1) at high level (L1) and 65 ± 8% (T-2) to 128 ± 9% (alternariol monomethyl ether) at low levels (L3). The methodology was validated according to Commission Decision 2002/657/EC, with limits of quantification ranging from 0.3 (AFs in oat beverages) to 18 ng/mL (HT-2 in rice beverage). The analysis of 56 beverage samples purchased from Valencia (Spain) showed at least one mycotoxin occurring in 95% of samples, including carcinogenic aflatoxins, and oat beverage was the most contaminated. This is a newest validated methodology for the quantification of sixty mycotoxins in oat, rice, almond and soy beverages.
... Several works studied the occurrence of different mycotoxins in oat, soy, and rice-based milks, as they are the most consumed worldwide [32][33][34][35]. Miró-Abella et al. [32] reported, for the first time, the presence of mycotoxins in plant-based milks and found DON, OTA, ZEN, and T-2 toxin together with aflatoxins B1, B2, G1, and G2 present in certain samples, with oat-based beverages being the most frequently contaminated. ...
... Due to the increasing importance of plant-based milks, several authors developed new detection methods to detect different mycotoxins in these products by applying different pretreatment methods to avoid matrix effects. These methods are based on salting out assisted liquid-liquid extraction in the case of Fusarium toxin determination [33,34], dispersive liquid-liquid microextraction for aflatoxins [35], or QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction in the case of a multidetermination of 11 mycotoxins [32]. In the case of mycotoxin determinations in tigernut milk, a matrix solid phase dispersion was adapted to eliminate lipidic interferences [38,39]. ...
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Consumer dietary habits have drastically changed in recent decades and functional beverages now have a strong position in the market. The majority of these beverages are produced using simple processes that use raw products, such as cereals, legumes, fruits, and nuts, among others, and these are known to be frequently contaminated with mycotoxins. This review is focused on the occurrence of these toxic compounds in plant-based milks, fruit juices, and herbal teas. The fate of the toxins during processing is discussed to establish the potential risk posed by the consumption of these kind of beverages regarding mycotoxin uptake.
... Limits of quantifications for AFs were found below 50 ng kg −1 (Hamed et al. 2017) providing high sensitivity, accuracy, and no mass spectrometry signal interference allowing for the determination of these mycotoxins at trace levels. However, this method has several drawbacks including high cost, complexity, inadequacy, and high sensitivity to the sample pH, thus low recovery for some AFs (Hamed et al. 2019(Hamed et al. , 2017. ...
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Fermented dairy products are dominant constituents of daily diets around the world due to their desired organoleptic properties , long shelf life, and high nutritional value. Probiotics are often incorporated into these products for their health and technological benefits. However, the safety and possible contamination of fermented dairy products during the manufacturing process could have significant deleterious health and economic impacts. Pathogenic microorganisms and toxins from different sources in fermented dairy products contribute to outbreaks and toxicity cases. Although the health and nutritional benefits of fermented dairy products have been extensively investigated, safety hazards due to contamination are relatively less explored. As a preventive measure, it is crucial to accurately identify and determine the associated microbiota or their toxins. It is noteworthy to highlight the importance of detecting not only the pathogenic microbiota but also their toxic metabolites so that putative outbreaks can thereby be prevented or detected even before they cause harmful effects to human health. In this context, this review focuses on describing techniques designed to detect potential contaminants; also, the advantages and disadvantages of these techniques were summarized. Moreover, this review compiles the most recent and efficient analytical methods for detecting microbial hazards and toxins in different fermented dairy products of different origins. Causative agents behind contamination incidences are also discussed briefly to aid in future prevention measures, as well as detection approaches and technologies employed. Such approach enables the elucidation of the best strategies to control contamination in fermented dairy product manufacturing processes.
... Two methods were published for the determination of AFs by HPLC with fluorescence detection with sample preparation by DLLME. One method focused on AFs in oats, rice, coconut, almond, and birdseed, as well as plant-based milk and milk-based products enriched with oats, almonds, and walnuts (Hamed et al., 2018). Analytes were extracted from the samples with DLLME techniques and were analysed with post column photochemical derivatisation. ...
Article
This review summarises developments on the analysis of various matrices for mycotoxins published in the period from mid-2019 to mid-2020. Notable developments in all aspects of mycotoxin analysis, from sampling and quality assurance/quality control of analytical results, to the various detection and quantitation technologies ranging from single mycotoxin biosensors to comprehensive instrumental methods are presented and discussed. Aside from sampling and quality control, discussion of this past year’s developments is organised by detection and quantitation technology and covers chromatography with targeted or non-targeted high resolution mass spectrometry, tandem mass spectrometry, detection other than mass spectrometry, biosensors, as well as assays that use alternatives to antibodies. This critical review aims to briefly present the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.
... To minimize solvent consumption of conventional liquid-liquid extraction and increase concentration factors, liquid-liquid microextraction or dispersive liquid-liquid microextraction (DLLME) [75] has been increasingly used for the determination of aflatoxins in dairy and oil products [76][77][78][79]. As a miniaturization of liquid-liquid extraction, DLLME allows extracting and preconcentrating target aflatoxins into a very small amount (microvolume) of the extraction phase. ...
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Full-text available
As a class of mycotoxins with regulatory and public health significance, aflatoxins (e.g., aflatoxin B1, B2, G1 and G2) have attracted unparalleled attention from government, academia and industry due to their chronic and acute toxicity. Aflatoxins are secondary metabolites of various Aspergillus species, which are ubiquitous in the environment and can grow on a variety of crops whereby accumulation is impacted by climate influences. Consumption of foods and feeds contaminated by aflatoxins are hazardous to human and animal health, hence the detection and quantification of aflatoxins in foods and feeds is a priority from the viewpoint of food safety. Since the first purification and identification of aflatoxins from feeds in the 1960s, there have been continuous efforts to develop sensitive and rapid methods for the determination of aflatoxins. This review aims to provide a comprehensive overview on advances in aflatoxins analysis and highlights the importance of sample pretreatments, homogenization and various cleanup strategies used in the determination of aflatoxins. The use of liquid-liquid extraction (LLE), supercritical fluid extraction (SFE), solid phase extraction (SPE) and immunoaffinity column clean-up (IAC) and dilute and shoot for enhancing extraction efficiency and clean-up are discussed. Furthermore, the analytical techniques such as gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS), capillary electrophoresis (CE) and thin-layer chromatography (TLC) are compared in terms of identification, quantitation and throughput. Lastly, with the emergence of new techniques, the review culminates with prospects of promising technologies for aflatoxin analysis in the foreseeable future.
... The sample carries along the mobile phase and sorbent, which leads to differential partitions of compounds between stationary and mobile phases in accordance with the moving rate of different components of the sample. The limit of quantification (LOQ) for AFB1, AFB2, AFG1 and AFG2 was reported as 0.5 mg·L −1 using the HPLC method in enriched milk and plant-based beverages, meaning it was lower than the maximum EU level [132]. In a study, the levels of AFG1, AFB1, AFG2 and AFB2 were determined in plant-based beverages and enriched milk samples using the LC-MS/MS and HPLC analysis, the results of which the showed a recovery range of 82-104%, an LOQ value of 0.5 mg·L −1 and a relative standard deviation of <9.7%, suggesting some merits for this method such as a shortened time and reduced cost of data analysis due to ease of use and the need to consume a smaller solvent [133]. ...
Article
Full-text available
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Chapter
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ABSTRAC Main group of contaminants including toxic compounds be formed during food processing and packaging (incidental group) and fungal toxins have been listed as critical challenge for food safety and human health. Because of absorption and transferring of these compounds into the human body and accumulation of them in different organs, several chronic diseases have been observed. The levels of these toxicants have been seriously monitored using analytical techniques. In this review, formation mechanism, toxicological effect and analytical methods of biogenic amines, furfural, hydroxymethylfurfural, polycyclic aromatic hydrocarbon, acrylamide, bisphenol A, nitrosamine and aflatoxin were discussed in different food samples.
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Article
As a new class of green solvents, hydrophobic deep eutectic solvents (DESs) have attracted considerable attention for liquid phase microextraction. In this study, a hydrophobic DES composed of trioctylmethylammonium chloride and oleic acid was successfully applied for the vortex-assisted liquid-liquid microextraction (VA-LLME) of Sudan I in food samples coupled with high performance liquid chromatography with diode array detection (HPLC-DAD). Trioctylmethylammonium chloride and oleic acid at a molar ratio of 1:2 showed higher extraction recovery for Sudan I than other DESs. The deep eutectic solvent was prepared and characterized by Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance spectroscopy (¹H NMR). The parameters affecting extraction recovery were optimized in detail. This hydrophobic DES based VA-LLME technique exhibited a high extraction recovery close to 100% at room temperature within 3 min. Under the optimized conditions, wide linear ranges were acquired with r² equal to 0.9992. The limit of detection was 0.3 µg kg⁻¹. The inter day and intra-day precision values were less than 5.4%. This hydrophobic DES based VA-LLME technique was successfully applied for the determination of Sudan I from duck blood and chili power samples with the satisfactory recoveries from 83.4 to 114.1% and relative standard deviations from 1.5 to 7.6%. In addition, the analytical parameters of this method and other liquid phase microextraction methods were compared.
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Vegetable milks are considered as functional foods due to their physiological benefits. Although the consumption of these products has significantly increased, they have received little attention in legislation, as regard to contaminants. However, they may contain mycotoxins resulting from the use of contaminated raw materials. In this work, ultra-high performance liquid chromatography tandem mass spectrometry has been proposed for the determination of the most relevant Fusarium toxins (fumonisin B1 and B2, HT-2 and T-2 toxins, zearalenone, deoxynivalenol and fusarenon-X) in different functional beverages based on cereals, legumes and seeds. Sample treatment consisted of a simple salting-out-assisted liquid-liquid extraction with no further clean-up. The method provided limits of quantification between 3.2-57.7 µg L(-1), recoveries above 80% and precision with RSD lower than 12%. The method was also applied for studying the occurrence of these mycotoxins in market samples of vegetable functional beverages and deoxynivalenol was found in three oat-based commercial drinks.
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Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi (molds). These low molecular weight compounds (usually less than 1000 Daltons) are naturally occurring and practically unavoidable. They can enter our food chain either directly from plant-based food components contaminated with mycotoxins or by indirect contamination from the growth of toxigenic fungi on food. Mycotoxins can accumulate in maturing corn, cereals, soybeans, sorghum, peanuts, and other food and feed crops in the field and in grain during transportation. Consumption of mycotoxin-contaminated food or feed can cause acute or chronic toxicity in human and animals. In addition to concerns over adverse effects from direct consumption of mycotoxin-contaminated foods and feeds, there is also public health concern over the potential ingestion of animal-derived food products, such as meat, milk, or eggs, containing residues or metabolites of mycotoxins. Members of three fungal genera, Aspergillus, Fusarium, and Penicillium, are the major mycotoxin producers. While over 300 mycotoxins have been identified, six (aflatoxins, trichothecenes, zearalenone, fumonisins, ochratoxins, and patulin) are regularly found in food, posing unpredictable and ongoing food safety problems worldwide. This review summarizes the toxicity of the six mycotoxins, foods commonly contaminated by one or more of them, and the current methods for detection and analysis of these mycotoxins.
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The aflatoxin producing fungi, Aspergillus spp., are widely spread in nature and have severely contaminated food supplies of humans and animals, resulting in health hazards and even death. Therefore, there is great demand for aflatoxins research to develop suitable methods for their quantification, precise detection and control to ensure the safety of consumers’ health. Here, the chemistry and biosynthesis process of the mycotoxins is discussed in brief along with their occurrence, and the health hazards to humans and livestock. This review focuses on resources, production, detection and control measures of aflatoxins to ensure food and feed safety. The review is informative for health-conscious consumers and research experts in the fields. Furthermore, providing knowledge on aflatoxins toxicity will help in ensure food safety and meet the future demands of the increasing population by decreasing the incidence of outbreaks due to aflatoxins.
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Dispersive liquid-liquid microextraction (DLLME) is an extraction technique developed within the last decade, which involves the dispersion of fine droplets of extraction solvent in an aqueous sample. Partitioning of analytes into the extraction phase is instantaneous due to the very high collective surface area of the droplets. This leads to very high enrichment factors and very low solvent consumption, relative to other liquid or solid phase extraction methods. A comprehensive review of the various modes of DLLME in the analysis of organic and inorganic analytes in dairy products (milk, cheese, infant formula, yogurt, and breast milk) is presented here. Dairy products present a complex sample matrix and the removal of interfering matrix components can prove troublesome. This review focuses on sample pretreatment prior to the appropriate DLLME procedure, the extraction and dispersive solvents chosen, derivatisation methods, and analytical figures of merit. Where possible, a critical comparison of DLLME methods has been undertaken. The overall suitability, and limitations, of DLLME as a sample preparation technique for dairy products has been assessed.
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Environmental occurrence of Aspergillus and other fungal spores are hazardous to humans and animals. They cause a broad spectrum of clinical complications. Contamination of aflatoxins in agri-food and feed due to A. flavus and A. parasiticus result in toxicity in humans and animals. Recent advances in aspergillus genomics and aflatoxin management practices are encouraging to tackle the challenges posed by important aspergillus species.
Article
A method was developed for the simultaneous determination of 11 mycotoxins in plant-based beverage matrices, using a QuEChERS extraction followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry detection (UHPLC–(ESI)MS/MS). This multi-mycotoxin method was applied to analyse plant-based beverages such as soy, oat and rice. QuEChERS extraction was applied obtaining suitable extraction recoveries between 80 and 91%, and good repeatability and reproducibility values. Method Quantification Limits were between 0.05 μg L⁻¹ (for aflatoxin G1 and aflatoxin B1) and 15 μg L⁻¹ (for deoxynivalenol and fumonisin B2). This is the first time that plant-based beverages have been analysed, and certain mycotoxins, such as deoxynivalenol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, T-2 toxin and zearalenone, were found in the analysed samples, and some of them quantified between 0.1 μg L⁻¹ and 19 μg L⁻¹.
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A simple and efficient method for determining multiple mycotoxins was developed using a QuEChERS (quick, easy, cheap, effective, rugged and safe) based extraction procedure in vegetable oils. High performance liquid chromatography tandem mass spectrometry (HPLC-MSMS) was used for the quantification and confirmation of 16 chemically diversified mycotoxins. Different extraction procedures were studied and optimized by spiking 16 analytes into blank matrix, and the extraction with 85% MeCN solution and C18 as cleaning sorbent allowed an efficient recovery of 72.8-105.8% with RSDs less than 7%. The limit of detection (LOD) ranged from 0.04 to 2.9 ng/g. The developed method was finally applied to screen mycotoxins in 62 vegetable oil samples. Zearalenone (ZEN), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and α-zearalenol (α-ZOL) were detected, with maximum concentrations of 0.59 (AFG1) – 42.5 (ZEN) ng/g. The method developed has the advantages of high sensitivity, accuracy and selectivity, and it can be applied to the target screening of mycotoxins in real samples.
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Plant-based or non-dairy milk alternative is the fast growing segment in newer food product development category of functional and specialty beverage across the globe. Nowadays, cow milk allergy, lactose intolerance, calorie concern and prevalence of hypercholesterolemia, more preference to vegan diets has influenced consumers towards choosing cow milk alternatives. Plant-based milk alternatives are a rising trend, which can serve as an inexpensive alternate to poor economic group of developing countries and in places, where cow’s milk supply is insufficient. Though numerous types of innovative food beverages from plant sources are being exploited for cow milk alternative, many of these faces some/any type of technological issues; either related to processing or preservation. Majority of these milk alternatives lack nutritional balance when compared to bovine milk, however they contain functionally active components with health promoting properties which attracts health conscious consumers. In case of legume based milk alternatives, sensory acceptability is a major limiting factor for its wide popularity. New and advanced non-thermal processing technologies such as ultra high temperature treatment, ultra high pressure homogenization, pulsed electric field processing are being researched for tackling the problems related to increase of shelf life, emulsion stability, nutritional completeness and sensory acceptability of the final product. Concerted research efforts are required in coming years in functional beverages segment to prepare tailor-made newer products which are palatable as well as nutritionally adequate.
Article
A high-performance liquid chromatography (HPLC) method with fluorescence detection for the determination of aflatoxins B1, B2, G1, G2, and M1 in yogurt using dispersive liquid–liquid microextraction as alternative sample treatment has been developed. To enhance the fluorescence of aflatoxins B1 and G1, a post-column photochemical derivatization has been proposed, avoiding the use of derivatization reagents. The method was validated using natural yogurt as representative matrix, showing a good linearity in the studied range (25–500 ng kg−1 ) and limits of quantification below the maximum level established by European Union in milk for the manufacture of milk-based products. Satisfactory recoveries ranging from 69.4 to 99.7 %, with relative standard deviations lower than 11.2 %, were obtained for all the compounds. The proposed method is simple, rapid, with low solvent consumption, inexpensive, and environmentally friendly.