Determination of Bisphenols and Related Compounds in Honey and

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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

ACS Paragon Plus Environment


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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,

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the detected bisphenols in honey probably derive from a source other than the final packaging.

12

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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

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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

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mammary gland in animals2. Recently, the safe level (the “tolerable daily intake” or TDI) was

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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

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BPA alternatives (analogues) that share similar structures and are currently under surveillance

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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

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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

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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

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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

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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-

65

d16), obtained from Sigma Aldrich (Steinheim, Germany), was used as a surrogate standard.

66

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

71

(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-

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European countries as well as directly from Slovenian honey producers in 2015 and 2016 (SI-

78

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

80

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

82

“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;

126

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

135

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,

179

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

201

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

203

under constant conditions (1 h at 60 °C). MTBSTFA with 1 % TBDMCS gave the highest

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abundances of the derivatized species. Various reaction times (1 h, 2 h, 4 h, 16 h and

205

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).

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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;

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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

221

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 %

226

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

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extracted matrix blank at two concentrations: 2.5 and 20 ng g-1 honey (n = 3). The results are

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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.

235

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

238

honey samples. Table 2

239

240

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.

257

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


Page 15 of 34


15 304

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).



Page 16 of 34

16 328

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


Page 17 of 34


17 352

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



Page 18 of 34

18 376

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


Page 19 of 34


19 400

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;


BPAP

=

bisphenol

AP;


Page 20 of 34

20 424

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;


Page 21 of 34


21 446

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447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496

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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).

559



Page 24 of 34

24

FIGURE CAPTIONS Figure 1: The detected compounds (positive hits) in honey samples.


Page 25 of 34


25

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



Page 26 of 34

26

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


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

Page 27 of 34


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



Page 28 of 34

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


Page 29 of 34


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


< LOD


Page 30 of 34

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


Page 31 of 34


31 S28b

(unknown material)

S28c S29a S29c


S29d S30a S30b S30c


S31a S31b S31c


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.



Page 32 of 34

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


Page 33 of 34


33

GRAPHIC FOR TABLE OF CONTENTS



84x47mm (96 x 96 DPI)


Page 34 of 34

Determination of Bisphenols and Related Compounds in Honey and

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