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THE DETERMINATION OF BISPHENOLS AND RELATED COMPOUNDS IN HONEY AND THEIR MIGRATION FROM SELECTED FOOD CONTACT MATERIALS Marjeta #esen, Dimitra Lambropoulou, Maria Laimou-Geraniou, Tina Kosjek, Urška Blaznik, David Heath, and Ester Heath J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03924 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on November 5, 2016
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Journal of Agricultural and Food Chemistry
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THE DETERMINATION OF BISPHENOLS AND RELATED COMPOUNDS IN HONEY AND THEIR MIGRATION FROM SELECTED FOOD CONTACT MATERIALS
Marjeta Česena,b, Dimitra Lambropoulouc, Maria Laimou-Geraniouc, Tina Kosjeka,b, Urška Blaznikd, David Heatha, Ester Heatha,b* a
Department of Environmental Sciences, Jožef Stefan Institute, Jamova cesta 39, 1000
Ljubljana, Slovenia b
c
Jožef Stefan International Postgraduate School, Jamova cesta 39, 1000 Ljubljana, Slovenia,
Department of Chemistry, Aristotle University of Thessaloniki, University Campus, 54124
Thessaloniki, Greece d
National Institute of Public Health, Trubarjeva cesta 2, 1000 Ljubljana, Slovenia
*Corresponding author: Prof. Dr. Ester Heath, email:
[email protected]; telephone: +386 1 477 3194
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ABSTRACT
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This study reports the analysis of nine bisphenols (BPA, BPAF, BPAP, BPB, BPC, BPE, BPF,
3
BPS, BPZ) and related compounds (4-cumylphenol and dihydroxybenzophenone) in honey
4
and food simulant. After sample pre-concentration with Oasis HLB cartridges, analytes were
5
silylated and analysed by GC-MS. The validated methods with LODs in sub ng g-1 were
6
applied to 36 honey samples from European and non-European countries and food simulant
7
stored in selected corresponding containers. Honey samples contained BPA, BPAF, BPE,
8
BPF, BPS and BPZ in amounts up to 107 ng g-1, 53.5 ng g-1, 12.8 ng g-1, 31.6 ng g-1,
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302 ng g-1 and 28.4 ng g-1, respectively. Under simulating conditions, BPA and BPAF were
10
detected in food simulant up to 42.2 ng mL-1 and 19.8 ng mL-1, respectively. In certain cases,
11
the detected bisphenols in honey probably derive from a source other than the final packaging.
12
13
Keywords: honey, bisphenol, analogue, BPA, BPAF, BPAP, BPB, BPC, BPE, BPF, BPS,
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BPZ, 4-cumylphenol, dihydroxybenzophenone, food contact material, migration
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INTRODUCTION
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The migration of endocrine disrupting compounds (EDC) from food contact materials (FCM)
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into various foodstuffs represents a global health issue as the human population is inevitably
19
exposed to them. A FCM is any material that might transfer its constituents into food under
20
the intended conditions of use, including the raw material packaging, the processing lines, the
21
food packaging (with direct and indirect contact), the auxiliary items, parts of vending
22
machines and food dispensers1. Bisphenol A (BPA) is a monomer used in the production of
23
polycarbonate (PC) plastics, which are widely used as FCMs. It is also a raw material for the
24
synthesis of epoxy resins that are employed in the production of epoxy-based lacquers used as
25
linings in cans, bottle tops and lids to prevent the contents becoming tainted by being in direct
26
contact with metals. BPA migration from plastics or coatings is of health concern since its
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high doses adversely affect the kidneys and liver and because it may cause effects on the
28
mammary gland in animals2. Recently, the safe level (the “tolerable daily intake” or TDI) was
29
reduced from 50 µg kg bw-1 day-1 to 4 µg kg bw-1 day-1 3. The European Union has also
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prohibited its use in the production of baby bottles4, 5 and set a specific migration limit (SML)
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of 0.6 µg g-1 from any FCM into food6. All this has led to "BPA free" products containing
32
BPA alternatives (analogues) that share similar structures and are currently under surveillance
33
for endocrine disrupting potential7. Even though SMLs have also been established in
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Commission Regulation (EU) No. 10/2011 for bisphenol S (BPS), 4-cumylphenol (HPP) and
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2,4-dihydroxybenzophenone (DHBP), and have been set at 0.05, 0.05 and 60 µg g-1,
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respectively, the risks to public health has been so far assessed only for BPA exposure6.
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Research dedicated to the migration of BPA into various foods and food simulants has been
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ongoing for over two decades8-13, whereas BPA analogues have attracted the attention of the
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scientific community only recently7, 14-18.
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Honey is a popular foodstuff, often prized for it naturalness and purity19. Regardless, as with
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any foodstuff, honey could contain undesirable contaminants because of compounds
42
migrating from its packaging and other FCM. Among published studies that address various
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organic contaminants in honey (e.g., antibiotics, pesticides, polycyclic aromatic hydrocarbons
44
and polychlorinated biphenyls20-22, only a study by da Silva et al. reports the migration
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kinetics of diphenylbutadiene, an optical brightener, from a FCM in various foodstuffs,
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including honey23. Moreover, only Inoue and co-workers report the concentrations of two
47
bisphenols, BPA and bisphenol F (BPF) in honey24.
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The aim of this study was to develop and validate a robust analytical method to
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simultaneously determine BPA and its related compounds: bisphenol AF (BPAF), bisphenol
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AP (BPAP), bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), BPF, BPS,
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bisphenol Z (BPZ), HPP and DHBP in various honeys. In addition, the analytical method was
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optimized for testing their migration into an appropriate food simulant using different types of
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packaging intended for honey storage. Although HPP and DHBP are not bisphenol
54
derivatives, all tested compounds within this study are referred to as bisphenols. The
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performance of the optimized methods was evaluated in terms of sensitivity, linearity, limits
56
of detection (LOD) and quantification (LOQ), precision and trueness.
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MATERIALS AND METHODS
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Reagents and chemicals
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Analytical standard of BPA (> 97 %) was purchased from Merck (Darmstadt, Germany),
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while BPAF (> 99 %), BPAP (> 99 %), BPC (> 99 %), BPE (> 98 %), BPF (> 98 %), BPP
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(> 99 %), BPS (> 98 %), BPZ (> 98 %), HPP (> 99 %) and DHBP (> 99 %) were purchased
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from Sigma-Aldrich (Steinheim, Germany). BPB (> 99 %) was purchased from LGC Labor
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GmbH (Augsburg, Germany). Chemical structures, IUPAC names, molecular weights and
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applications are given in Supplementary Information (SI-1). Deuterated bisphenol A (BPA-
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d16), obtained from Sigma Aldrich (Steinheim, Germany), was used as a surrogate standard.
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The derivatizing agent N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA,
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97 % purity) was purchased from Acros Organics (New Jersey, USA), while N-methyl-N-
68
(trimethylsilyl)trifluoroacetamide (MSTFA, 98.5 %) and N-(tert-butyldimethylsilyl)-N-
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methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane (MTBSTFA with 1 %
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TBDMCS, 95 %) were purchased from Sigma Aldrich (Steinheim, Germany). Acetonitrile
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(ACN), ethanol (EtOH), ethyl acetate (EtAc), methanol (MeOH) and purified water were
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purchased from J. T. Baker (Deventer, Netherlands). All solvents were of analytical grade
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purity. Stock solutions (1 mg mL-1) of each compound were prepared in MeOH. Calibration
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standards were prepared by serial dilution of the stock.
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Sample collection
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Thirty-six honey samples were collected at supermarkets from various European and non-
77
European countries as well as directly from Slovenian honey producers in 2015 and 2016 (SI-
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2.1 and SI-2.2). In the SI-2.1 also the type and container material (identification code) are
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given. Moreover, 35 corresponding empty honey containers comprising the following: plastic
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containers, laminated polymer/foil sachets and glass jars with polymer lined metal lids), from
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three Slovene honey suppliers are described in SI-2.3 in details. Honey samples (labelled as
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“H”: H5, H6, H8, H23, H28-S31, H34) and empty containers (labelled as “S”: S5, S6, S8, S23,
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S28-S31, S34) were collected from the same supplier.
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Migration tests with food simulant
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The Commission Regulation (EU) No. 10/2011 proposes 10 % EtOH (v/v; simulant A) as the
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food simulant for food category 03.03 B (Molasses, sugar syrups, and honey)6. For a food that
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is intended to be in contact with the FCM for a period exceeding 6 months at room
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temperature (honey can have shelf-life of over a year if unopened) the regulation states: “The
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specimen shall be tested in an accelerated test at elevated temperature for a maximum of 10
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days at 60 °C”6. Therefore, all 35 collected containers were filled with 10 % EtOH and placed
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into an incubator (IKA KS 4000 i control, Germany) for 10 days at 60 °C. The appropriate
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volume of food simulant was calculated using the Law of Conservation of Mass and the
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density of honey25 (≈ 1,420 mg mL-1; SI-2.3). The empty containers were filled with simulant
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to give a final mass equivalent to the mass of honey. Glass jars with coated metal lids were
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incubated in an inverted position according to ISO standard EN 13130-126. Lids are often
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reused, therefore migration experiments (S6, S29 – S31, S34) were performed in three
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consecutive tests on a single item, each time using fresh simulant as proposed in Commission
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Regulation (EU) No. 10/2011 (Articles for repeated use)6.
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Sample preparation
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a) Honey
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Honey samples were prepared according to Kujawski et al.27. Crystallised samples were
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heated in a water bath (T < 40 °C) and all samples were homogenized by stirring for at least 3
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min27. Known weights (10 g) of honey were then diluted in 100 mL of purified water and
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filtered through glass-microfibre filters (Machery Nagel, Düeren, Germany) and cellulose
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nitrate filters (0.45 µm, Sartorius Stedim Biotech, Göttingen, Germany). Samples were then
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extracted using SPE cartridges containing a divinylbenzene-N-vinylpyrrolidone copolymer
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sorbent (60 mg, 3 mL; Oasis HLB Waters, Massachusetts, USA). Each cartridge was
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conditioned with 3 mL of EtAc, 3 mL of ACN, 15 mL of MeOH and equilibrated with 3 mL
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of purified water. The samples were extracted at a flow-rate of 3 mL min−1 using a Supelco
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Vacuum Manifold (Bellefonte, USA). After loading, the sorbents were washed with 6 mL of a
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20 % MeOH solution and dried under vacuum (- 1.33 kPa, 20 min). The elution step was
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performed using 0.5 mL ACN and 3 mL of EtAc (3 × 1 mL). The remaining solvent was
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removed under a gentle stream of nitrogen. Derivatization involved reacting the extracts with
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30 µL of MTBSTFA with 1% TBDMCS in 220 µL EtAc. The samples were derivatized for
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16 h at 60 °C. All samples were prepared in triplicate.
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b) Food simulant
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A 10-mL aliquot of food simulant was collected from each of the test containers. The sample
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was diluted to 50 mL with purified water to reduce the amount of EtOH in a sample, which
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can affect SPE efficiency. Extraction was then performed as per the honey samples but
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excluding the washing step. The samples were then derivatized as described for the honey
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samples. All samples were prepared in triplicate.
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GC-MS analysis
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Honey and food simulant extracts were analysed using an Agilent 7890B series gas
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chromatograph with a 5977A single quadrupole mass spectrometer (Agilent, USA).
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Separation was achieved using a DB-5 MS capillary column (30 m × 0.25 mm × 0.25 µm;
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Agilent, USA) with helium as the carrier gas. One µL of the sample extract was injected in
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splitless mode at 250 °C. For optimal chromatographic separation the temperature program
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was as follows: an initial temperature 120 °C was ramped at 20 °C min-1 to 200 °C (held for 2
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min), then at 10 °C min-1 to 280 °C (held for 5 min) and finally at 20 °C min-1 to 310 °C (held
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for 3 min). Total GC-MS runtime was 23.5 min. The mass spectrometer was operated in EI
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mode at 70 eV. Selected compounds were qualitatively and quantitatively determined using
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selective ion monitoring (SIM mode). The retention times and fragment ions of derivatized
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compounds that were used for identification and quantification are presented in Table 1. Data
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was processed using MassHunter Workstation - Quantitative Analysis software (Agilent
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Technologies).
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Table 1
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Method validation
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Methods performances were assessed in terms of linearity, accuracy, limits of detection,
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limits of quantification, sensitivity and precision expressed as method and instrumental
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repeatability. For honey and food simulant samples, a 6-point calibration curve was
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constructed using a least square linear regression analysis of standard mixtures of the analytes
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at different concentrations. The range of 0.25 ng g-1 to 30 ng g-1 and 0.25 ng mL-1 to 30 ng
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mL-1 was used for most of the compounds for honey and food simulant, respectively. For
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compounds with lower or higher LOQ than 0.25 ng g-1 or 0.25 ng mL-1, their LOQ is
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considered as the lowest calibration standard. The coefficient of determination (R2) was used
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to express linearity. Sensitivity was expressed as the slope of the calibration curves. Since the
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initial analysis of several honey samples revealed concentrations of several compounds above
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the calibration range, an additional calibration from 5 ng g –1 to 100 ng g -1 was performed.
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Where necessary, dilution of samples was also performed. Method accuracy was expressed as
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[(experimental value - spiked value)/ spiked value] (n=3; at two concentrations). The LODs
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and LOQs were calculated as 3-times and 10-times the standard deviation (SD) of the baseline
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of the blanks, respectively, divided by the slopes of their calibration curves. The blanks for
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determination of LODs and LOQs were blank honey samples (n = 6) and blank food simulant
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samples (n = 6). Analytical method repeatability was calculated as the relative standard
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deviation (RSD) of three replicates (at two concentrations), while instrumental repeatability
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was determined as the RSD of three consecutive injections of the same sample (at 10 ng g-1).
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Literature reports that BPA recovery can be affected by filtration due to adsorption on the
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filters28. For this reason, the possible loss of all bisphenols during filtration was assessed by
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comparing filtered and non-filtered samples spiked at 10 ng g-1 in purified water (n = 3).
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Uncertainty measurement
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Uncertainty associated with the concentrations of target compounds in both honey and FCM
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samples was determined by using a “bottom-up” approach adopted by EURACHEM and
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Eurolab29, 30. It was based on the identification, estimation and combination of all sources
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associated with the result, enabling the interpretation and evaluation of individual
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contributions and the detection of the most significant ones. Six main sources of uncertainty
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were considered: urec: uncertainty associated with recovery (trueness; U1); uRw: uncertainty
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associated with within-lab-reproducibility (U2); ucal: uncertainty associated with calibration
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(U3); uw: uncertainty associated with balance calibration and weighing uSt (U4); uv:
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uncertainty associated with volume (U5); uncertainty associated with the preparation of Stock
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and Standard solution (weighing/volumetric flask/pipette/purity; U6).
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Combined uncertainty (uˊ) was calculated according to Guide to the Expression of
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Uncertainty in Measurement (GUM) guidelines31, following the law of propagation of
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uncertainty (Eq. 1):
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ݑ′ = ඥ(ݑ )ଶ + (ݑோ௪ )ଶ + (ݑ )ଶ + (ݑௐ )ଶ + (ݑ )ଶ + (ݑ௦௧ )ଶ
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Finally, the expanded uncertainty (U) was calculated by using the coverage factor k = 2, at the
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confidence level of 95 %.
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Background contamination – sources and removal
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It is difficult to avoid contamination with bisphenols since they have wide application (SI-1)32,
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33
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various matrices, whereas to the authors’ knowledge no contamination with other bisphenols
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has been reported34, 35. Regardless, besides BPA also BPAF was observed as contaminant in
(1)
. Studies report the background contamination of samples with BPA, BPF and BPS in
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this study. To determine their source of contamination and minimise it, the following blank
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samples were prepared:
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a) Background contamination blanks: Several blank samples were prepared to identify and
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eliminate sources of contamination. Among the studied bisphenols, only BPA and BPAF
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were present as contaminants in the blanks. The details are given in SI-3. Briefly, to
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reduce their contamination, all glassware was heated to 400 °C for 4 h and the SPE
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cartridges were rinsed with 15 mL of MeOH within the conditioning step.
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b) Carry over check blanks: Ethyl acetate, used for derivatization, was analyzed within each
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sample batch i.e., every 10th sample was a solvent blank to prevent potential carryover
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between samples.
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c) Procedural blanks (n = 6): A blank honey and food simulant sample were spiked with
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BPA-d16 (at 5 ng g-1) and used to quantitatively asses any remaining contamination. All
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data were then blank corrected. BPA contamination was almost negligible (honey: 0.08
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ng g-1; food simulant: 0.02 ng mL-1), while BPAF presence was slightly higher in matrix
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blanks (honey: 0.48 ng g-1; food simulant: 0.05 ng mL-1).
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RESULTS AND DISCUSSION
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Method optimization
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Derivatization process
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Different derivatization agents, solvents, reaction times and temperatures were tested in order
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to improve the sensitivity of the analytical method. First, 30 µL of each agent (MSTFA,
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MTBSTFA and MTBSTFA with 1 % TBDMCS) and 220 µL of EtAc was used and compared
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under constant conditions (1 h at 60 °C). MTBSTFA with 1 % TBDMCS gave the highest
204
abundances of the derivatized species. Various reaction times (1 h, 2 h, 4 h, 16 h and
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temperatures (60 °C and 80 °C) were also tested. Temperature had little effect, whereas the
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prolongation of the derivatization time (16 h) yielded a significant improvement (SI-4) and
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the use of ethyl acetate as a derivatization solvent gave consistently better yields than ACN
208
(SI-4).
209
Solid-phase extraction of honey was studied under various experimental conditions on
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cartridges containing a divinylbenzene-N-vinylpyrrolidone copolymer sorbent (Oasis HLB;
211
3cc, 60 mg). Initially, 10 g of a “blank” honey, diluted in 100 mL of purified water, was
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spiked with all of the compounds of interest at five working concentrations (0.25 – 30 ng g-1
213
honey). After conditioning (EtAc, ACN and MeOH) and sample loading, the dried sorbent
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was eluted with 0.5 mL ACN and 3 × 1 mL EtAc. The final extracts were reduced to dryness
215
(N2), derivatized and analysed by GC-MS. This standard procedure was compared with the
216
following variations: i) acidification of the samples with 1 mL of 1 M HCl prior to SPE, ii)
217
washing the SPE sorbents with 20 % MeOH solution and iii) washing with 80 % MeOH.
218
Acidification resulted in similar responses for most compounds compared to the standard
219
procedure. The exceptions were HPP and BPC, which were poorly extracted from the
220
acidified samples. For this reason, acidification was omitted. A washing step (6 mL of either
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20 % or 80 % of MeOH) was introduced to remove sample matrix interferences and to
222
improve chromatography. A loss of all analytes was observed with 80 % of MeOH solution,
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whereas using 20 % MeOH solution for the wash step resulted in overall improved responses
224
and peak shapes. The exceptions were BPAP and BPS, the responses of which remained
225
similar as for the standard SPE procedure. Solid-phase extraction efficiency using a 20 %
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MeOH wash step was then determined as a quotient of the peak area of analytes spiked prior
227
to SPE and the peak area obtained by spiking the same amount of analytes added post SPE to
228
extracted matrix blank at two concentrations: 2.5 and 20 ng g-1 honey (n = 3). The results are
229
presented in Table 2. With the exception of BPS, all recoveries were > 78 % and > 89 % at
230
lower and higher concentration, respectively. BPS is the most acidic compound (pKa = 7.42–
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8.03) from the target bisphenols, hence, its low recovery may be due to the loss of this
232
compound during sample loading since it is partially dissociated in the neutral conditions.
233
Despite the poor SPE efficiency for BPS, the described SPE procedure was adopted for honey
234
samples as our goal was to unify the analysis of all tested bisphenols.
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For food simulant, no washing step was necessary and recoveries were all > 71 % (Table 2)
236
with the exception of BPS (19.5 – 22.3 %). The low recovery of BPS from food simulant
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confirms that matrix complexity does not play an important role in SPE of this compound in
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honey samples. Table 2
239
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Validation parameters
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Method validation parameters are given in Tables 3 and 4. Instrumental repeatability in terms
242
of RSD was between 2.25 % (for BPB) and 9.86 % (for HPP). The two R2 values for both
243
calibration ranges: 0.25 – 30 ng g-1 and 5 – 100 ng g-1 are reported in Table 3. All other
244
reported parameters refer to the lower concentration range. All compounds have R2 > 0.97
245
showing good linearity over the two concentration ranges. As expected method accuracy was
246
higher at 30 ng g-1 than that at 2.5 ng g-1, regardless of matrix type (Tables 3 and 4). Except
247
for BPS in food simulant, method repeatability was < 20 % for food simulant and < 17 % for
248
honey. Instrumental repeatability was < 10 % for all compounds (Table 3) and all the
249
determined LODs and LOQs were in the sub ng g-1 (ng mL-1) to pg g-1 (ng mL-1) range
250
(Tables 3 and 4). Filtering the samples did not affect recovery (Table 3).
251
The expanded measurement uncertainties for all the compounds were between 6.99 % and
252
49.88% for honey samples and between 7.48 % and 63.57 % for food simulant. According to
253
the results (SI-5), “within-lab reproducibility” (U1), “trueness” (recovery of the method)
254
(U2) and “calibration” (U3) were the largest contribution parameters in the uncertainty
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budget. On the other hand, other sources including mass, volumetric equipment and purity of
256
standards provided only a small contribution in the uncertainty values.
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Clearly, at low concentration level the relative standard uncertainty is higher in honey and
258
food stimulant. In addition, the importance of the calibration curve uncertainty (U3) increases
259
at the lower concentration level (2.5 ng g-1). In fact, in food stimulant, for most of the studied
260
compounds, U3 accounts for more than 80 % of the global uncertainty (SI-5). Tables 3 and 4
261 262
Honey analysis
263
Table 5 gives the results of honey analysis. Among the 36 tested honeys, only two had levels
264
of all investigated compounds < LODs (H29 and H30). Of the 11 target compounds BPA,
265
BPAF, BPE, BPF, BPS and BPZ were found in levels up to 107 ng g-1, 53.5 ng g-1, 12.8 ng g-1,
266
31.6 ng g-1, 302 ng g-1 and 28.4 ng g-1, respectively. The most frequently detected was BPA
267
(27 samples), followed by BPAF (23 samples), BPF and BPE (6 samples), BPZ (5 samples)
268
and BPS (1 sample). To the best of our knowledge, this is the first study in which, besides
269
BPA, the presence of BPAF, BPE, BPF, BPS and BPZ at concentration levels > LODs in
270
honey samples has been confirmed. For instance, a residual amount of BPA was previously
271
reported by Inoue et al. in 107 honey samples collected from stores in Japan with the declared
272
origin from all around the world24. In Inoue’s case low levels of BPA (from < LOD (2.0 ng g-1)
273
up to 33.3 ng g-1) were found in 17 out of 107 samples, whereas BPF was not detected. The
274
higher number of BPA positive hits (27 out of 36) observed within our study may be
275
attributed to the lower LOD (0.128 ng g-1) compared to the one reported by Inoue et al.
276
(2 ng g-1)24.
277
Comparison with other studies conducted in other food products and packaging materials
278
revealed similar concentration ranges for BPA and BPF (Tables 5 and 6). Regarding BPAF
279
and BPZ, higher concentration levels were detected in honey samples compared to those
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reported for various foodstuffs, which are generally in the low ng g-1 levels. Bisphenol S was
281
detected in the highest concentration of all bisphenols studied, although it was detected only
282
in one sample (H22: 302 ng g-1). Low concentration levels were in general reported for this
283
compound by other authors (< LOD to 0.609 ng g-1) with the exception of a study by Viñas et
284
al., who reported BPS concentration in canned food and supernatant of canned food at
285
34.1 ng g-1 and 170 ng g-1, respectively. (Table 6)36. As far as BPE is concerned, it is
286
interesting to note that although it was already included in several studies addressing the food
287
contamination with various bisphenols, its presence was below the LOD (Table 6). To the
288
best of our knowledge, our findings constitute the first data on the presence of BPE in
289
foodstuff samples. In particular, the presence of BPE in samples H7, H9, H10, H23, H31 and
290
H33 suggests it is being used for the production of FCMs.
291
Tables 5 and 6
292
The levels of detected compounds in the samples varied significantly, e.g. BPA was detected
293
in concentrations from 0.364 ng g-1 to 107 ng g-1 honey (Table 5). These variations could
294
derive from different FCMs manufacturers, honey suppliers, FCM type (lids, irregularly-
295
shaped plastic containers etc.) and the surface that is in contact with honey, storage conditions
296
and varying masses of honey (SI-2.1). No correlation was observed between the mass in a
297
certain container and the detected concentration. For example, BPAF concentrations in 10 –
298
40 g packaging were < LOD – 53.5 ng g-1, whereas in packaging > 500 g, they were < LOD –
299
47.2 ng g-1 (Table 5). Similar results were observed in the case of BPA (packaging up to 40 g:
300
< LOD – 107.2 ng g-1; above 500 g: < LOD – 97.7 ng g-1).
301
The regulatory SMLs for BPA and BPS migration from FCM are 600 ng g-1 and 50 ng g-1,
302
respectively6. Table 5 shows that none of the honey samples contained BPA at concentration
303
higher than the SML. On the contrary, BPS, which as mentioned before detected in the
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highest concentration among all investigated compounds (302 ng g-1 honey), was measured in
305
the sample H22 at a concentration level approximately 6-times higher than the SML making
306
this honey inappropriate for consumption6. To check whether BPS was migrating from the
307
container of sample H22, the remaining honey was removed and the food simulant was
308
introduced into the container. Afterwards, the migration test was performed as described in
309
section “Sample preparation – b)”. The analysis (n =3) revealed the presence of BPS < LOD,
310
suggesting that its presence in H22 probably derives from other FCM than the final container.
311
This is in accordance with the stated Resin Identification Code of container of sample H22 in
312
SI-2.1 (PET1), as BPA or its analogues are not used for the synthesis of this type of material.
313
The analysis also revealed the presence of BPAF in food simulant at 0.462 ± 0.130 ng g-1.
314
BPAF migrated from the tested FCM during the test as it was not detected in the honey
315
sample. This can be attributed to more extreme testing conditions (higher temperature and
316
ethanol) when using food simulant compared to actual storage conditions.
317
BPA and BPAF were detected in measurable concentrations in the honey samples from all
318
tested types of containers, i.e. plastic, foil sachet, plastic/paper composite container and glass
319
jar with coated lids (Table 5). In addition, BPF and BPZ were also present in honey from
320
plastic and foil sachet containers, while BPE was detected only in honey from plastic
321
containers and glass jars with lids. Only BPS was detected solely in a honey from one plastic
322
container (H22). A comparison of FCMs in terms of the abundance of investigated
323
compounds was omitted due to the varying number of honeys tested from plastic (23), glass
324
jars with lids (10), foil sachet (2) and paper/plastic composite (1) containers. Regardless, the
325
highest concentrations of BPA (H13), BPAF (H21), BPF (H8), BPS (H22) and BPZ (H23)
326
were detected in honey from plastic containers, while BPE was the most abundant in one
327
tested honey from glass jars with coated lids (H31).
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It is also difficult to compare honey samples based on their declared origin as varying number
329
of samples from each country was collected and since 14 had undeclared origin or not
330
specifically reported country of origin (SI-2.1). Regardless, we observed that among all tested
331
samples only two Slovene honeys contained all the compounds below LODs (H29 and H30)
332
whereas two samples from Greece (H9 and H10) contained comparable concentrations of
333
BPA, BPAF and BPE. The majority of samples (14) had unspecified origin and contained the
334
highest number of targeted compounds, i.e. BPA, BPAF, BPE, BPF and BPZ (Figure 1).
335
Among the remaining samples, four samples from Chile and Mexico also contained the same
336
compounds (Figure 1), while sample from Italy (H6) contained only BPA at low amount
337
(0.475 ng g-1). The sample from Hungary and Ukraine (H22) was contaminated with BPS at a
338
concentration of 302 ng g-1, the highest level of any bisphenol determined. Samples from
339
Chile and Mexico contained the highest BPA (H13 = 107 ng g-1) and BPZ (H23 = 28.4 ng g-1)
340
levels, while BPE and BPF were the most abundant in Slovene samples (H31 = 12.8 ng g-1
341
and H8 = 31.6 ng g-1). Finally, the highest concentration of BPAF was detected in a sample
342
from Germany (H21 = 53.5 ng g-1). Figure 1
343
344
Food simulant analysis
345
The analysis of food simulant revealed measurable concentrations of only BPA and BPAF,
346
from < LODs up to 42.2 ng mL-1 and 19.8 ng mL-1 food simulant, respectively (Table 7, First
347
test). Both compounds were detected in samples from all three types of tested containers
348
(plastic, glass jars with lids and foil sachets), however three tested plastic containers (S8a-d;
349
S23a,b; S33a-f) contained all of the target compounds below LODs.
350
The variations between the same containers were observed on several occasions. For example,
351
sample taken from the container S5a contained 0.395 ng mL-1 of BPA, while samples from
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S5b, S5c and S5d contained it < LOD. Similarly, only the sample from S5b contained
353
measurable levels of BPAF. Another example is laminated foil sachets (S28a), which had
354
approximately 4-times higher BPA concentration that other tested parallel foil sachet
355
containers (Table 7). Regardless, as only one parallel was analyzed for these samples (S28a-
356
d), the actual variation might be lower. The highest absolute differences among the tested
357
containers were observed in the case of samples S30a, S30b and S30c (BPA: 30.9 –
358
42.2 ng mL-1; BPAF: 0.714 – 19.8 ng mL-1) and S31a, S31b, S31c and S31d (BPA: 3.95 –
359
32.5 ng mL-1; Table 7). Since the lids were analysed under identical conditions, these
360
variations originate from the different levels of BPA and BPAF residues in the tested lids.
361
Literature review revealed a study by Fasano et al.38, who report the migration of BPA from a
362
marmalade jar lids (n = 3) using water as a food simulant, which was exposed for 10 days at
363
40 °C. The levels of BPA found were < LOD (21 pg mL-1)38. Other reported BPA migration
364
tests were performed on e.g. baby bottles, cans, yogurt packaging, tetrapack and plastic dishes,
365
while no data exists for single serving plastic or foil sachet containers. Moreover, little data is
366
available for BPA analogues in terms of their migration from FCMs12, 38-40. Cacho et al.14
367
reports the migration of biphenyl, BPA, BPF and BPZ from cans using 3 % (v/v) acetic acid
368
solution as food simulant, which was in contact with tested cans for 10 days at 40 °C. The
369
reported concentrations were up to 0.04 ng mL-1, 0.04 ng mL-1, 0.35 ng mL-1 and < LOD,
370
respectively14. There is a lack of data for the other compounds tested, hence, the detection of
371
BPAF under the simulating conditions in the present study constitute the first report on the
372
migration of this compound from FCM into food simulant.
373
Table 7 shows the results of consecutive tests on jars with coated metal lids (Second and
374
Third test). With the exception of S6b and S30c (for BPAF) and S34b (for BPA),
375
concentrations of BPA and BPAF were lower in the samples collected from Second and Third
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test. This suggests that the majority of the BPA and BPAF migrated into the simulant within
377
the first 10 days. Table 7
378
379
Honey vs. food simulant
380
The analysis of honey samples revealed the presence of 6 out of the 11 investigated
381
compounds, while food simulant contained only BPA and BPAF in measurable
382
concentrations under simulating conditions (Tables 5 and 7). The concentrations of BPA,
383
BPAF or both varied in the analyzed honey and food simulant samples. Considerably higher
384
concentrations of BPA and/or BPAF were detected in honey samples from plastic or foil
385
sachets, i.e. the samples H8, H23, H28 and H33 (BPA: 0.909 – 18.6 ng g-1; BPAF: 12.0 –
386
35.4 ng g-1) compared to the food simulant in the corresponding containers (BPA: < LOD –
387
3.85 ng mL-1; BPAF: < LOD – 2.02 ng mL-1; Tables 5 and 7). The opposite was observed in
388
samples from jars with lids (H30 for both compounds and in H6 and H31 for BPA). Here,
389
higher concentrations of BPA and BPAF were detected in food simulant (BPA: 3.95 –
390
42.2 ng mL-1; BPAF: 0.714 – 19.8 ng mL-1) compared to honey, where all samples, except for
391
H6 (BPA: 0.475 ng g-1) contained concentrations < LOD. The higher concentrations in honey
392
samples can be explained by additional contamination from the sources other than final
393
packaging. Another option is also that the tested FCMs (empty ones and the ones filled up
394
with honey) were not from the same lot of FCM manufacturer or that the simulation testing
395
did not mimic the actual storage conditions. The last two reasons can also explain the opposite
396
effect, where only food simulant was contaminated. Other tested honey samples and their
397
corresponding empty containers (H5, H29 and H34 vs. S5a-d, S29a-d and S34a-b) contained
398
comparably low concentrations of BPA and BPAF (Tables 5 and 7).
399
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Exposure assessment
401
Dietary exposure from honey to BPA has been estimated for each sample by combining
402
results from our study with the median and high consumption (P95) levels for honey. In order
403
to evaluate the consumer risk to BPA, exposure estimates were compared with established
404
TDI. For BPA absorption an absorption factor 1 was used. The analytical data were not
405
grouped since the samples are not representative for Slovenian market and safety assessment
406
was done for each sample. Consumption data for honey was obtained from the EFSA
407
Comprehensive European Food Consumption Database41. Exposure to BPA for adults was
408
calculated with the consumption data from Slovenian dietary survey CRP 2008 (median
409
20 g day-1; large consumer P95 40 g day-1) and for children from the Austrian dietary survey
410
ASNS 2010 (median 16 g day-1; P95 30 g day-1). The average estimated dietary exposures to
411
BPA for adults (body weight; bw = 70 kg) are from 0.01 ng kg bw-1 day-1 to 30.5 ng kg bw-1
412
day-1, with the highest estimate 61.1 ng kg bw-1 day-1 at the 95th percentile of consumption.
413
Dietary exposure from honey is higher for children due to their lower body weight (bw
414
=12 kg), for average consumers it is 0.07 ng kg bw-1 day-1 to 143 ng kg bw-1 day-1. The
415
highest estimate at the 95th percentile for the sample H13 amounts to 267 ng kg bw-1 day-1,
416
which is 6.7 % of the tolerable daily intake (TDI). These estimations show that the established
417
TDI value for BPA (4 µg kg bw-1 day-1)41 would not be exceeded with any of the tested
418
honeys.
419
420
ABBREVIATIONS USED
421
BPA
422
BPB = bisphenol B; BPC = bisphenol C; BPE = bisphenol E; BPF = bisphenol F;
423
BPS = bisphenol S; BPZ = bisphenol Z; u´ = combined uncertainty; DHBP = 2,4-
=
bisphenol
A;
BPAF
=
bisphenol
AF;
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BPAP
=
bisphenol
AP;
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dihydroxybenzophenone; EDC = endocrine disrupting compound; U = expanded uncertainty;
425
FCM
426
butyldimethylsilyl)-N-methyltrifluoroacetamide; MTBSTFA with 1 % TBDMCS = N-(tert-
427
butyldimethylsilyl)-N-methyltrifluoroacetamide
428
MSTFA = N-methyl-N-(trimethylsilyl)trifluoroacetamide); PC = polycarbonate plastics; SML
429
= specific migration limit; SPE = solid-phase extraction, TDI = tolerable daily intake.
430
ACKNOWLEDGEMENT
431
The authors would like to thank local honey producers for donating representative FCMs and
432
their willingness for collaboration with us and to Andreja Zorič from National Laboratory of
433
Health, Environment and Food (Slovenia) for her advices within the study design.
434
SUPPORTING INFORMATION DESCRIPTION
435
Additional information regarding the present study, marked in the text as SI-1 (additional
436
information of the investigated compounds, SI-2 (honey and FCM samples), SI-3 (blanks and
437
contamination) and SI-4 (derivatization process), is available free of charge via the Internet at
438
http://pubs.acs.org.
439
FUNDING
440
This work was financially supported by the EU through the ISO-FOOD project (Era chair for
441
isotope techniques in food quality, safety and traceability, Grant agreement No: 621329) and
442
by the Slovenian Research Agency (Program groups P1-0143 and P3-0395 and Projects L1-
443
5457, L1-7544, N1-0047 and J1-6744).
444
NOTES
445
The authors declare no competing financial interest.
= food contact material; HPP = 4-cumylphenol; MTBSTFA =
with
1%
N-(tert-
tert-butyldimethylchlorosilane;
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18. Yang, Y.; Yu, J.; Yin, J.; Shao, B.; Zhang, J. Molecularly Imprinted Solid-Phase Extraction for Selective Extraction of Bisphenol Analogues in Beverages and Canned Food. J. Agric. Food Chem. 2014, 62 (46), 11130-11137. 19. da Silva, P. M.; Gauche, C.; Gonzaga, L. V.; Costa, A. C. O.; Fett, R. Honey: Chemical composition, stability and authenticity. Food Chem. 2016, 196, 309-323. 20. Wiest, L.; Buleté, A.; Giroud, B.; Fratta, C.; Amic, S.; Lambert, O.; Pouliquen, H.; Arnaudguilhem, C. Multi-residue analysis of 80 environmental contaminants in honeys, honeybees and pollens by one extraction procedure followed by liquid and gas chromatography coupled with mass spectrometric detection. J. Chromatogr. A 2011, 1218 (34), 5743-5756. 21. Lambert, O.; Veyrand, B.; Durand, S.; Marchand, P.; Bizec, B. L.; Piroux, M.; Puyo, S.; Thorin, C.; Delbac, F.; Pouliquen, H. Polycyclic aromatic hydrocarbons: Bees, honey and pollen as sentinels for environmental chemical contaminants. Chemosphere 2012, 86 (1), 98-104. 22. Juan-Borrás, M.; Domenech, E.; Escriche, I. Mixture-risk-assessment of pesticide residues in retail polyfloral honey. Food Control 2016, 67, 127-134. 23. Silva, A. S.; Cruz Freire, J. M.; Sendón, R.; Franz, R.; Paseiro Losada, P. Migration and Diffusion of Diphenylbutadiene from Packages into Foods. J. Agric. Food Chem. 2009, 57 (21), 10225-10230. 24. Inoue, K.; Murayama, S.; Takeba, K.; Yoshimura, Y.; Nakazawa, H. Contamination of xenoestrogens bisphenol A and F in honey: safety assessment and analytical method of these compounds in honey. J. Food Comp. Anal. 2003, 16 (4), 497-506. 25. Tomasik, P., Chemical and Functional Properties of Food Saccharides. Sikorski, Z. E., Ed. U.S., Florida, 2004. 26. European Committee for Standardization, European Standard EN 13130-1: Materials and articles in contact with foodstuffs. Plastics substances subject to limitation. Brussels, 2004. 27. Kujawski, M. W.; Namieśnik, J. Challenges in preparing honey samples for chromatographic determination of contaminants and trace residues. TrAC Trends in Anal. Chem. 2008, 27 (9), 785-793. 28. Gallart-Ayala, H.; Núñez, O.; Lucci, P. Recent advances in LC-MS analysis of foodpackaging contaminants. TrAC Trends in Anal. Chem. 2013, 42, 99-124. 29. Eurolab, Measurement Uncertainty Revised: alternative approaches to uncertainty evaluation, Technical Report 1/2007. 2007. 30. A. Williams; Ellison, S. L. R., Eurachem/CITAC guide: Quantifying Uncertainty in Analytical Measurement. 2012. 31. International Organisation for Standardisation, Guide to the Expression of Uncertainty in Measurement (GUM), Geneva. 2008. 32. Pivnenko, K.; Pedersen, G. A.; Eriksson, E.; Astrup, T. F. Bisphenol A and its structural analogues in household waste paper. Waste Management 2015, 44, 39-47. 33. Goldinger, D. M.; Demierre, A.-L.; Zoller, O.; Rupp, H.; Reinhard, H.; Magnin, R.; Becker, T. W.; Bourqui-Pittet, M. Endocrine activity of alternatives to BPA found in thermal paper in Switzerland. Regul. Toxicol. Pharmacol. 2015, 71 (3), 453-462. 34. Lee, S.; Liao, C.; Song, G.-J.; Ra, K.; Kannan, K.; Moon, H.-B. Emission of bisphenol analogues including bisphenol A and bisphenol F from wastewater treatment plants in Korea. Chemosphere 2015, 119, 1000-1006. 35. Caballero-Casero, N.; Lunar, L.; Rubio, S. Analytical methods for the determination of mixtures of bisphenols and derivatives in human and environmental exposure sources and biological fluids. A review. Anal. Chim. Acta 2016, 908, 22-53. 36. Vinas, P.; Campillo, N.; Martinez-Castillo, N.; Hernandez-Cordoba, M. Comparison of two derivatization-based methods for solid-phase microextraction-gas chromatography-mass spectrometric determination of bisphenol A, bisphenol S and biphenol migrated from food cans. Anal. Bioanal. Chem. 2011, 397 (1), 115-125. 37. Grumetto, L.; Montesano, D.; Seccia, S.; Albrizio, S.; Barbato, F. Determination of Bisphenol A and Bisphenol B Residues in Canned Peeled Tomatoes by Reversed-Phase Liquid Chromatography. J. Agric. Food Chem. 2008, 56 (22), 10633-10637. 38. Fasano, E.; Bono-Blay, F.; Cirillo, T.; Montuori, P.; Lacorte, S. Migration of phthalates, alkylphenols, bisphenol A and di(2-ethylhexyl)adipate from food packaging. Food Control 2012, 27 (1), 132-138.
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39. Cao, X.-L.; Corriveau, J. Migration of Bisphenol A from Polycarbonate Baby and Water Bottles into Water under Severe Conditions. J. Agric. Food Chem. 2008, 56 (15), 6378-6381. 40. Maragou, N. C.; Makri, A.; Lampi, E. N.; Thomaidis, N. S.; Koupparis, M. A. Migration of bisphenol A from polycarbonate baby bottles under real use conditions. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2008, 25 (3), 373-83. 41. European Food Safety Authority (2015) Bisphenol A immune system safety to be reviewed. https://www.efsa.europa.eu/en/press/news/160426a (accessed 26th May).
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FIGURE CAPTIONS Figure 1: The detected compounds (positive hits) in honey samples.
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TABLES Table 1: Retention times (Rt) and characteristic fragment ions of derivatized compounds. Compound Rt (min) Ions (m/z)a BPA-d16 16.33 452, 470 BPA 16.43 441, 456, 249 BPAF 14.09 315, 225, 357 BPAP 22.50 503, 269, 518 BPB 17.41 441, 221, 470 BPC 17.54 469, 484, 263 BPE 16.10 427, 442, 235 BPF 15.91 179, 428, 221 BPS 22.41 229, 478, 379 BPZ 21.23 247, 289, 496 HPP 11.23 311, 326, 269 DHBP 15.25 385, 427, 105 a: Quantification ions are in bold.
Table 2: SPE recoveries for honey and food simulant samples determined at low (L = 2.5 ng g-1; n = 3) and high (H = 30 ng g-1; n = 3) concentration.
BPA BPAF BPAP BPB BPC BPE BPF BPS BPZ HPP DHBP
Honey (% ± RSD) L H 92.2 ± 7.5 104 ± 4 95.0 ± 12.5 97.0 ± 5.1 90.9 ± 13.3 98.4 ± 4.3 93.2 ± 6.6 102 ± 4 98.1 ± 7.9 104 ± 4 98.3 ± 7.8 111 ± 3 99.0 ± 6.7 91.8 ± 15.3 24.6 ± 7.2 20.9 ± 9.0 78.6 ± 10.5 88.8 ± 2.0 105 ± 4 104 ± 5 101 ± 6 104 ± 4
Food simulant (% ± RSD) L H 98.5 ± 4.4 92.8 ± 5.6 71.6 ± 10.5 74.6 ± 11.0 88.6 ± 6.5 77.4 ± 5.7 96.3 ± 5.0 91.5 ± 6.1 98.6 ± 5.4 90.1 ± 6.0 97.3 ± 2.1 92.5 ± 7.0 92.9 ± 7.1 93.4 ± 7.7 19.5 ± 17.9 22.3 ± 25.5 91.9 ± 5.4 85.7 ± 4.0 123 ± 11 117 ± 2 70.2 ± 30.8 91.1 ± 14.2
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Table 3: Validation parameters for honey. Accuracyb LOD (%) Slope (ng g-1) 1 2 L H BPA 0.997 0.996 6.50 2.03 0.508 0.128 BPAF 0.996 0.994 27.5 2.72 3.861 0.0645 BPAP 0.998 0.996 26.7 0.897 0.209 0.0476 BPB 0.998 0.996 12.6 1.36 0.774 0.0209 BPC 0.976 0.996 31.0 3.02 0.097 0.0476 BPE 0.983 0.995 48.9 2.37 0.346 0.0747 BPF 0.993 0.998 29.2 0.390 7.38 0.000597 BPS 0.990 0.997 43.0 4.23 0.149 0.0649 BPZ 0.998 0.995 2.80 1.62 0.0603 0.147 HPP 0.999 0.998 25.6 1.01 1.00 0.0396 DHBP 0.994 0.999 27.6 3.33 4.00 0.00338 a: R2 values 1 and 2 refer to lower and higher calibration curves, respectively. b: L = lower concentration (2.5 ng g-1); H = higher concentration (30 ng g-1). R2 valuea
LOQ (ng g-1) 0.428 0.215 0.157 0.0697 0.159 0.249 0.00199 0.216 0.489 0.132 0.0112
Method repeatabilityb L H 10.2 4.90 2.36 17.0 10.4 12.1 11.2 4.05 4.11 11.8 7.42 10.8 6.60 14.1 4.26 16.0 8.58 6.82 7.23 17.0 9.87 15.6
Filtr. recovery (%) 103 104 103 100 80.8 102 100 99.4 102 99.1 87.5
Expanded Uncertainty (U; %) L H 37.40 10.03 29.69 20.19 30.92 20.17 34.15 6.99 49.88 46.90 17.37 13.60 18.74 20.55 44.28 41.43 44.87 22.78 18.38 20.21 31.95 18.45
Table 4: Validation parameters for food simulant (10 % EtOH). 2
R
Accuracya (%)
Slope
LOD (ng mL-1)
L H BPA 0.992 25.3 3.78 0.475 0.0506 BPAF 0.985 15.5 5.37 0.992 0.0494 BPAP 0.995 37.3 0.0213 0.170 0.0453 BPB 0.993 34.8 2.89 0.630 0.0101 BPC 0.991 44.2 2.59 0.257 0.0324 BPE 0.992 33.9 3.21 0.235 0.0144 BPF 0.995 26.6 1.86 1.803 0.0557 BPS 0.967 61.1 7.45 0.0397 0.0907 BPZ 0.995 35.9 1.14 0.125 0.0574 HPP 0.991 4.49 4.31 0.436 0.0104 DHBP 0.988 20.4 4.83 0.696 0.111 a: L = lower concentration (2.5 ng mL-1) and H = higher concentration (30 ng mL-1).
LOQ (ng mL-1) 0.169 0.165 0.151 0.0336 0.108 0.0470 0.183 0.302 0.191 0.0346 0.370
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Method repeatabilitya L 5.38 10.3 4.13 7.05 5.50 3.02 1.96 38.5 6.70 20.1 9.84
H 3.50 6.87 4.31 5.23 2.98 4.86 1.84 20.7 7.51 12.3 11.6
Expanded Uncertainty (U; %) L H 49.02 13.92 48.83 13.26 45.37 35.60 50.00 12.10 46.88 27.54 49.46 27.19 42.22 7.48 63.57 53.66 44.23 44.93 49.42 21.11 49.95 20.27
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Journal of Agricultural and Food Chemistry
27
Table 5: The average concentrations ± SD of detected bisphenols in honey (n = 3, except for samples H15 and H16: n = 1). Sample
Container
Frequency of detection (%) jar + coated lid H1 (unknown material) H2 PP5 plastic bottle plastic bottle H3 (unknown material) H4 PET1 plastic bottle H5 H6 H7 H8 H9 H10 H11
H12
H13
H14 H15 H16
LDPE4 plastic bottle jar + coated lid (unknown material) jar + coated lid (unknown material) plastic tube (unknown material) plastic pouch (unknown material) plastic pouch (unknown material) plastic container with coated foil (unknown material) plastic container with coated foil (unknown material) plastic container with coated foil (unknown material) plastic container with coated foil (unknown material) foil sachet (unknown material) plastic container with coated foil (unknown material)
Concentration (ng g-1) BPA
BPAF
BPE
BPF
BPS
BPZ
75
64
17
17
3
14
97.7 ± 5.8
< LOD
< LOD
< LOD
< LOD
< LOD
75.3 ± 2.4
< LOD
< LOD
6.89 ± 0.84
< LOD
< LOD
1.84 ± 0.40
< LOD
< LOD
< LOD
< LOD
< LOD
2.88 ± 0.08
< LOD
< LOD
< LOD
< LOD
< LOD
0.561 ± 0.028
< LOD
< LOD
< LOD
< LOD
< LOD
0.475 ± 0.059
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
5.96 ± 0.39
< LOD
< LOD
< LOD
28.8 ± 2.7
35.4 ± 2.5
< LOD
31.6 ± 3.9
< LOD
< LOD
1.47 ± 0.11
17.5 ± 2.7
5.58 ± 0.55
< LOD
< LOD
< LOD
1.15 ± 0.11
20.3 ± 3.0
6.66 ± 0.45
< LOD
< LOD
< LOD
17.5 ± 3.5
< LOD
< LOD
< LOD
< LOD
< LOD
14.5 ± 0.8
7.63 ± 1.15
< LOD
< LOD
< LOD
< LOD
107 ± 11
34.2 ± 1.0
< LOD
3.57 ± 0.46
< LOD
5.61 ± 1.12
3.45 ± 0.42
34.5 ± 2.1
< LOD
< LOD
< LOD
< LOD
< LOD
20.4
< LOD
< LOD
< LOD
< LOD
1.38
13.0
< LOD
< LOD
< LOD
< LOD
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Journal of Agricultural and Food Chemistry
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28 H17 H18 H19 H20
H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35
plastic bottle (unknown material) PET1 plastic bottle plastic bottle (unknown material) LDPE4 plastic container with coated foil plastic container with coated foil (unknown material) PET1 plastic bottle plastic bottle (unknown material) CPAP 81 paper/plastic composite container jar + coated lid (unknown material) jar + coated lid (unknown material) plastic container with coated foil (unknown material) foil sachet (unknown material) jar + coated lid (unknown material) jar + coated lid (unknown material) jar + coated lid (unknown material) jar + coated lid (unknown material) plastic container with coated foil (unknown material) jar + coated lid (unknown material) PET1 plastic bottle
8.44 ± 0.72
28.8 ± 9.8
< LOD
< LOD
< LOD
< LOD
1.19 ± 0.21
7.24 ± 3.46
< LOD
< LOD
< LOD
< LOD
< LOD
16.2 ± 2.1
< LOD
< LOD
< LOD
< LOD
43.0 ± 2.6
47.7 ± 8.0
< LOD
1.69 ± 0.14
< LOD
9.39 ± 1.61
1.84 ± 0.50
53.5 ± 11.0
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
302 ± 20
< LOD
0.909 ± 0.299
29.7 ± 7.1
8.18 ± 0.55
< LOD
< LOD
28.4 ± 3.0
4.72 ± 1.12
39.8 ± 6.3
< LOD
< LOD
< LOD
< LOD
1.03 ± 0.03
47.2 ± 9.9
< LOD
< LOD
< LOD
< LOD
77.7 ± 6.7
12.8 ± 1.6
< LOD
< LOD
< LOD
< LOD
36.6 ± 3.2
< LOD
< LOD
< LOD
< LOD
5.05 ± 1.17
18.6 ± 0.7
17.1 ± 3.6
< LOD
1.88 ± 0.10
< LOD
7.95 ± 0.59
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
< LOD
12.8 ± 0.7
< LOD
< LOD
< LOD
0.522 ± 0.120
9.49 ± 3.02
< LOD
< LOD
< LOD
< LOD
18.0 ± 1.9
12.0 ± 2.6
3.66 ± 0.34
2.15 ± 0.34
< LOD
< LOD
0.364 ± 0.089
3.89 ± 0.52
< LOD
< LOD
< LOD
< LOD
< LOD
13.4 ± 2.8
< LOD
< LOD
< LOD
< LOD
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Journal of Agricultural and Food Chemistry
29 H36
PET1 plastic bottle
< LOD
7.80 ± 1.58
< LOD
< LOD
< LOD
Table 6: The reported concentrations of bisphenols in various foodstuffs. Compounds BPA and BPF BPA and BPB
13 bisphenols
13 bisphenols
8 bisphenols
3 bisphenols and biphenyl 5 bisphenols
Detected concentration
Units
Sample
Reference
BPA: < LOD – 33.3 BPF: < LOD BPA: < LOD – 115.3 BPB: < LOD – 85.7 BPA: < LOD – 1.26 BPF: < LOD – 1.00; BPS, DMBPS, 2 BPF isomers, BPE, BPB, BPAP, BPZ, BPAF, TBBPA, BPP: < LOD BPS: 2.4 BPF: 0.39 BPF, DMBPS, 2 BPF isomers, BPE, BPB, BPAP, BPZ, BPAF, TBBPA, BPP: < LOD BPA: < LOD – 9.97 BPAF: < LOD – 0.028 BPAP: < LOD – 0.185 BPB: < LOD – 0.017 BPF: < LOD – 4.63 BPP: < LOD – 0.562 BPS: < LOD – 0.609 BPZ: < LOD – 0.076 biphenyl: < LOD – 0.58 BPA: < LOD – 4.44 BPF: < LOD – 13.98 BPZ: < LOD – 0.92 BPA: < LOD – 0.607 BPF: < LOD – 0.218 BPB, BPE, BPS: < LOD
ng g-1
Honey
24
ng g-1
Canned peeled tomatoes
37
ng mL-1
Alcoholic and nonalcoholic beverages
ng g-1
Beef ravioli (among other foodstuffs)
ng g-1
Various foodstuffs
ng mL
Alcoholic and nonalcoholic beverages; filling liquids of canned vegetables and fruits
ng mL-1
Alcoholic and nonalcoholic beverages
-1
7
15
17
14
16
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< LOD
Journal of Agricultural and Food Chemistry
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30
7 bisphenols out of 10 target compounds
2 bisphenols and biphenyl
BPA: < LOD – 9.7 BPAF: < LOD – 0.052 BPF: < LOD BPS: < LOD – 0.036 BPA: < LOD – 58 BPAF: < LOD – 0.070 BPF: < LOD – 35 BPS: < LOD – 0.22 biphenyl: < LOD – 34.1 BPA: < LOD – 65.6 BPS: < LOD – 34.1 biphenyl: < LOD – 358 BPA: < LOD – 317 BPS: < LOD – 170
ng mL-1
Beverages 18
ng g-1
Canned Foods
ng g-1
Canned food 36
ng mL-1
Supernatant of the canned food
Table 7: The average concentrations ± SD of BPA and BPAF in food simulant from 35 containers (n = 3). Three consecutive tests were performed only in case of glass jars with lids. BPA (ng mL-1)
Container S5a S5b S5c
LDPE4 plastic bottle
S5d S6a S6b
jar + coated lid (unknown material)
S8a S8b S8c
plastic tube (unknown material)
S8d S23a S23b S28a
plastic bottle (unknown material) foil sachet
BPAF (ng mL-1)
1st test
2nd test
3rd test
1st test
2nd test
3rd test
0.395 ± 0.181
/
/
< LOD
/
/
< LOD
/
/
0.462 ± 0.037
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
6.33 ± 0.91
1.36 ± 0.25
1.24 ± 0.12
< LOD
< LOD
< LOD
7.83 ± 1.33
2.44 ± 0.22
< LOD
< LOD
0.630 ± 0.140
< LOD
< LOD
x
/
/
< LOD
/
/
< LOD
x
/
/
< LOD
/
/
< LOD
x
/
/
< LOD
/
/
< LOD
x
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
/
/
x
3.85
/
/
x
2.02
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Journal of Agricultural and Food Chemistry
31 S28b
(unknown material)
S28c S29a S29c
jar + coated lid (unknown material)
S29d S30a S30b S30c
jar + coated lid (unknown material)
S31a S31b S31c
jar + coated lid (unknown material)
S31d S33a S33b S33c S33d S33e
/
/
1.09x
/
/
0.819x
/
/
1.90x
/
/
x
x
S28d S29b
1.16x
plastic container with coated foil (unknown material)
1.00
/
/
1.98
/
/
0.852 ± 0.209
0.561 ± 0.064
< LOD
< LOD
< LOD
< LOD
1.23 ± 0.22
< LOD
< LOD
< LOD
< LOD
< LOD
1.41 ± 0.17
0.485 ± 0.044
< LOD
< LOD
< LOD
< LOD
2.43 ± 1.04
0.650 ± 0.054
0.502 ± 0.074
< LOD
< LOD
< LOD
42.2 ± 4.8
26.9 ± 0.9
16.5 ± 1.5
2.15 ± 1.12
< LOD
< LOD
30.9 ± 2.6
18.4 ± 2.2
15.7 ± 0.9
0.714 ± 0.309
< LOD
< LOD
42.0 ± 1.4
29.7 ± 2.1
22.3 ± 0.4
19.8 ± 0.16
0.377 ± 0.065
1.50 ± 0.12
17.5 ± 1.9
10.1 ± 0.3
10.2 ± 0.4
< LOD
< LOD
< LOD
32.5 ± 5.0
15.8 ± 1.2
11.5 ± 1.3
< LOD
< LOD
< LOD
22.2 ± 2.1
13.0 ± 1.3
9.93 ± 0.43
< LOD
< LOD
< LOD
3.95 ± 0.90
2.07 ± 0.34
0.388 ± 0.234
< LOD
< LOD
< LOD
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
< LOD
/
/
S33f
< LOD / / < LOD / / jar + coated lid 1.50 ± 0.28 0.678 ± 0.150 0.616 ± 0.058 < LOD < LOD < LOD S34a (unknown S34b 1.67 ± 0.31 < LOD 0.658 ± 0.078 < LOD < LOD < LOD material) x: The analysis could not be performed in triplicate as there was not enough sample available. Only one sample was analysed.
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32
FIGURE GRAPHICS
Number of positive hits
Figure 1: 12 BPA 10 BPAF
8
BPE
6
BPF
4
BPS
2
BPZ
0
Declared origin of honey
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Journal of Agricultural and Food Chemistry
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GRAPHIC FOR TABLE OF CONTENTS
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Journal of Agricultural and Food Chemistry
84x47mm (96 x 96 DPI)
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