bisphenol a (bpa) and its source in foods in japanese markets

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Bisphenol A (BPA) and its source in foods in JapanesemarketsJ. Sajiki a , F. Miyamoto a , H. Fukata b c , C. Mori b c , J. Yonekubo d e & K. Hayakawa ea Chiba Prefectural Institute of Public Health , 666-2 Nitona-cho, Chuo-ku, Chiba City, Chiba260-8715, Japanb Department of Bioenvironmental Medicine, Graduate School of Medicine , ChibaUniversity , Chiba 260-8670, Japanc Environmental Health Science Project for Future Generations , Graduate School ofMedicine , Chiba University, Chiba 260-8670, Japand Nihon Waters K.K., Katokichi Shin-Osaka Building , Osaka 532-0011, Japane Graduate school of Natural Science and Engineering , Kanazawa University , Kanazawa920-1192, JapanPublished online: 20 Feb 2007.

To cite this article: J. Sajiki , F. Miyamoto , H. Fukata , C. Mori , J. Yonekubo & K. Hayakawa (2007) Bisphenol A (BPA) and itssource in foods in Japanese markets, Food Additives & Contaminants, 24:1, 103-112, DOI: 10.1080/02652030600936383

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Food Additives and Contaminants, January 2007; 24(1): 103–112

Bisphenol A (BPA) and its source in foods in Japanese markets

J. SAJIKI1, F. MIYAMOTO1, H. FUKATA2,3, C. MORI2,3, J. YONEKUBO4,5,& K. HAYAKAWA5

1Chiba Prefectural Institute of Public Health, 666-2 Nitona-cho, Chuo-ku, Chiba City, Chiba 260-8715, Japan,2Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan,3Environmental Health Science Project for Future Generations, Graduate School of Medicine, Chiba University,

Chiba 260-8670, Japan, 4Nihon Waters K.K., Katokichi Shin-Osaka Building, Osaka 532-0011, Japan, and5Graduate school of Natural Science and Engineering, Kanazawa University,

Kanazawa 920-1192, Japan

(Received 11 May 2006; revised 24 July 2006; accepted 31 July 2006)

AbstractThe determination of bisphenol A (BPA) and/or bisphenol A diglycidyl ether (BADGE) in foods sold in Japanese marketsand in water leached from six epoxy resin cans with similar diameters was carried out using high-performanceliquid chromatography (HPLC) with electrochemical detection (LC/ECD), LC-mass spectrometric detection (LC/MS)and LC-tandem mass spectrometric detection (LC/MS/MS). BPA concentrations were 0–842 ng g�1 for 48 canned foods,0–14 ng g�1 for 23 foods in plastic containers, and 0–1 ng g�1 for 16 foods in paper containers. No BADGE was detected inthree canned foods. There was no difference in leaching concentrations of BPA into glycine buffers at pHs 8 and 11, andwater. The amounts of BPA leached into water from six epoxy resin cans held at 121�C for 20 min were almost the sameas the cans’ contents and were much higher than the amounts leached from cans held at or below 80�C for 60 min.The amount leached depended on the type of can, but not on the amount of BADGE leached from the cans. Considerablymore BPA than BADGE leached to water from six cans. Two cans whose contents had high concentrations of BPA showedno BADGE leaching even at 121�C, suggesting the different kinds of epoxy resin can linings from others. The results implythat the main source of human exposure to BPA is food from cans with linings that contain high percentages of BPAas an additive or an unforeseen contaminant.

Keywords: Bisphenol A (BPA), foods in Japanese markets, source of pollution, can linings

Introduction

Endocrine-disrupting chemicals have been acontroversial issue for more than a decade. Themanufacture of some chemicals such as dichloro-diphenyl-trichloroethane (DDT) and polychlori-nated biphenyls (PCBs) has been prohibited, andofficial anti-pollution measures have been taken todecrease environmental dioxins in Japan due to theiradverse effects. However, some chemicals that maybe potential endocrine disruptors are still beingmanufactured and are in widespread use. One ofthese is bisphenol A (BPA). Many researchers havereported that the endocrine-disrupting effect of BPA

on people can be ignored, based on the followingdata:

. Exposure of humans to BPA from foods andwine as estimated by European UnionScientific Bodies (0.0005–0.009 mg kg�1

day�1) is far lower than the reference dose(RfD¼ 0.05 mg kg�1 day�1) calculated by theUS Environmental Protection Agency(USEPA) as being a safe daily dose forhumans over a lifetime of exposure (Gray andCohen 2004).

. BPA is converted into a harmlessmetabolite, BPA-glucuronide, which has less

Correspondence: J. Sajiki. E-mail: [email protected]

ISSN 0265–203X print/ISSN 1464–5122 online � 2007 Taylor & FrancisDOI: 10.1080/02652030600936383

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oestrogenicity in the mammalian liver (Volkelet al. 2002).

However, it has been reported that BPA transfersfrom a maternal body to its foetus (Miyakoda et al.1999; Zalko et al. 2003), causes abnormality ofreproductive organs (Vom Saal et al. 1998; Gupta2000), advances female puberty (Howdeshell et al.1999), and changes behaviour (Adriani et al. 2003;Kubo et al. 2003) in experiments using mammals.Thus, there is concern that BPA may influencehumans throughout the foetal period.

BPA is used for the manufacture of polycarbonateplastics and epoxy resins, which are used in babybottles, protective linings of food cans, and compo-sites and sealants for dentistry. Oral intakes fromthese sources are considered to be a possible route ofBPA exposure for humans. BPA determination infoods was carried out in laboratories in Japan around10 years ago, and migration from canned foods, suchas 213 ng ml�1 for drinks and 602 ng g�1 for cornedbeef, was shown (Kawamura et al. 1999; Imanakaet al. 2001). An abnormally high BPA value(26 000 ng g�1) was also reported in food packagingmade from recycled paper (Ozaki et al. 2004). Sincethen many companies supplying not only babybottles and food containers for children, but alsofood cans and food packages have been careful withthe use and management of BPA. BPA is, however,still being detected in human biological samples suchas serum and urine (Matsumoto et al. 2003; Calafatet al. 2005; Fukata et al. 2006), indicating thecontinued presence of a source of BPA exposure.

Due to its high boiling point (398�C at760 mm Hg) and low vapour pressure (4E�8 mmHg), BPA is seldom evaporated (Staples et al. 1998),therefore it is considered that exposure routes ofBPA via air do not need to be considered. BPA hasnot been detected in tap water in spite of the use ofpolyvinyl chloride (PVC) for water pipes, becausethe processing of drinking water supplies, such aschlorination, ultraviolet light radiation and ozona-tion, is effective in destroying oestrogenic com-pounds (Lee et al. 2004). As BPA is easilydegraded by various kinds of active oxygen (Sajikiand Yonekubo 2002), it is speculated that BPA intap water could be degraded by the action of activeoxygen produced by the addition of chlorine forsterilization, even if it is leached from PVC pipes.Although dental materials such as composites andsealants were proposed as an oral exposure route ofBPA, nowadays the risk is considered to be low dueto its disappearance soon after dental treatment(Sasaki et al. 2005).

The major source of human BPA exposure seemsto be food packed in various kinds of containerssuch as can linings. BPA levels in recycled paper

products are reported to be ten or moretimes higher than those in virgin products (Ozakiet al. 2004). The aim of the present study was todetermine daily BPA intakes from foods packed invarious kinds of containers and to identify BPAmigration pathways from containers. To investigatethe possibility of degradation of BPA from epoxyresin used in food cans, BPA and bisphenol Adiglycidyl ether (BADGE), a monomer of epoxyresin, were determined using LC/ECD, LC/MS, orLC/MS/MS.

Materials and methods

Materials

Eighty-seven regularly consumed foods in threedifferent kinds of containers (cans, plastics andpapers) were purchased from supermarkets inChiba Prefecture and the Tokyo metropolitan area.There were 48 cans (seven fish, five meats, 16 fruitsand vegetables, 18 soups and sauces and two others),23 plastic containers (three beverages, five choco-lates and cookies, four soups and sauces, seven fastfoods and four others), and 16 paper containers(nine drinks, five dairy foods, and two fast foods),and these are listed in Table I. Food in plastic andpaper containers were stored in a refrigeratorand BPA extraction was performed within two daysof purchase. BPA extraction from canned foods wasperformed immediately after removal of the lid.

Reagents and instruments

Authentic BPA and BADGE were purchased fromWako Pure Chemicals (Tokyo, Japan) and KantoKagaku (Tokyo, Japan), respectively. LC-gradeethanol (EtOH) and acetonitrile (Wako PureChemicals, Tokyo, Japan) were used in this study.Other chemicals were of special grade (Wako PureChemicals, Tokyo, Japan). An Oasis HLB (Waters,MA, USA) used for the solid-phase extraction ofBPA and BADGE was washed with 3.5 ml EtOHfollowed by 3.5 ml water before use. BPA-free waterprepared using ODS-silica Sep-pak cartridges(Waters, MA, USA) was used throughout theexperiments after deionization of tap water usingMilli-RX12� (which uses a combination of reverseosmosis and electric deionization) (Nihon Millipore,Tokyo Japan). Glass tubes for BPA analysis werepre-washed with 99% ethanol. A multi-blender mill(with stainless steel cups, Nihonseiki Kaisha Ltd,Tokyo, Japan) was used for homogenizing foods.

Analytical instruments and analytical conditions

Determination of BPA in foods and water samplesleached from cans was carried out using LC/ECD

104 J. Sajiki et al.

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Tab

leI.

BP

Aco

nce

ntr

atio

ns

inva

riou

sfo

od

s.

Can

ned

food

sn

gg�

1C

ann

edfo

od

sF

ood

sin

pla

stic

con

tain

ers

Food

sin

pap

erco

nta

iner

s

Con

ten

tsB

PA

con

cen

trat

ion

(ng

g�1)

Con

ten

tsB

PA

con

cen

trat

ion

(ng

g�1)

Con

ten

tsB

PA

con

cen

trat

ion

(ng

g�1)

Con

ten

tsB

PA

con

cen

trat

ion

(ng

g�1)

Fish

Soupsandsauces

Drinks

Beverages

Tu

na

(IP

)2

Pott

age

sou

p2

Coff

eed

rin

k0.3

Veg

etab

leju

ice

n.d

.T

un

a23

Corn

crea

mso

up

1C

off

eed

rin

k1

Yogu

rt0.3

Tu

na

8M

ush

room

sso

up

11

Nat

ura

lw

ater

n.d

.A

lcoh

ol

(Sak

e)n

.d.

Tu

na

6C

hic

ken

sou

p(I

P)

77

Confectionaries

Alc

oh

ol

(Sak

e)n

.d.

Sal

mon

13

Cre

amso

up

(IP

)156

Cooki

es4

Alc

oh

ol

(Sak

e)n

.d.

Sal

mon

and

vege

tab

les

1C

on

som

me

sou

p(I

P)

6C

ooki

es3

Lac

tic

acid

dri

nk

n.d

.M

acke

rel

3T

om

ato

sou

p(I

P)

48

Cooki

es(I

P)

1L

acti

cac

idd

rin

kn

.d.

Meats

Corn

crea

mso

up

(IP

)74

Cooki

es(I

P)

14

Ora

nge

juic

en

.d.

Bee

f9

Bro

ccoli

Cre

amso

up

(IP

)19

Wh

ite

choco

late

1V

eget

able

juic

en

.d.

Ch

icke

n4

Fis

hm

ixso

up

(IP

)27

Soupsandsauces

Cream

andmilk

Bee

f10

Mea

tsa

uce

n.d

.S

auce

n.d

.C

ream

0.2

Pork

(IP

)20

Mea

tsa

uce

(IP

)11

Sau

cen

.d.

Cre

am0.2

Pork

(IP

)10

Mea

tsa

uce

(IP

)13

Veg

etab

leso

up

3C

ream

1Fruitsandvegetables

Lob

ster

crea

mso

up

(IP

)5

Corn

pott

age

n.d

.C

ow

’sm

ilk

n.d

.G

rap

es(I

P)

n.d

.B

row

nsa

uce

(IP

)428

Fastfoods

Cow

’sm

ilk

n.d

.P

each

(IP

)n

.d.

Bro

wn

sau

ce(I

P)

547

Mix

edsa

nd

wic

hes

3Fastfoods

Pin

eap

ple

(IP

)n

.d.

Bro

wn

sau

ce(I

P)

842

Tu

na

san

dw

ich

es3

Gra

tin

Tr

Bea

ns

(IP

)17

Ch

icke

nB

roth

(IP

)9

Tu

na

san

dw

ich

es3

Gra

tin

n.d

.C

orn

5Others

Sp

agh

etti

and

mea

tsa

uce

n.d

.C

orn

(IP

)3

Coco

nu

tm

ilk

(IP

)247

Tu

na

bre

ad8

Corn

(IP

)20

Coco

nu

tm

ilk

(IP

)56

Tu

na

bre

ad6

Asp

arag

us

(IP

)78

Tu

na

bre

adn

.d.

Mu

shro

om

s(I

P)

4Others

Tom

ato

5F

resh

stra

wb

erri

es2

Tom

ato

(IP

)12

Fre

shb

eef

0.2

Tom

ato

(IP

)26

Fre

shtu

na

n.d

.Y

ou

ng

corn

(IP

)44

Pu

mp

kin

seed

s(I

P)

n.d

.M

ush

room

s(I

P)

36

Hea

rtof

Pal

m(I

P)

15

Stu

ffed

olive

s(I

P)

8

IP,

imp

ort

ed;

n.d

.,n

ot

det

ecte

d.

Dat

aar

em

ean

valu

esof

du

plica

tes

mea

sure

db

yL

C/E

CD

.

BPA and its source in foods in Japanese markets 105

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or LC/MS. To confirm BPA in food, a few sampleswere analysed by LC/MS/MS. BADGE wasmeasured by LC/MS/MS. Analytical instrumentsand conditions were as follows.

Determination of BPA

The LC/ECD system consisted of an HPLC, ModelLC-10 AD (Shimadzu, Kyoto, Japan) equipped withan electrochemical detector (Coulochem II 5200A,ESA, MA, USA). Detector conditions were asfollows: guard cell potential, E: 600 mV; analyticalcell potentials: E1: 300 mV; E2: 550 mV; sensitivity:1 A. Separation was carried out using a Shim-PackVP-ODS column (150� 4.6 mm i.d., Shimadzu) at40�C under isocratic conditions using an acetoni-trile–water–phosphoric acid (40:60:0.2) mobilephase. The flow rate and injection volume were1.0 ml min�1 and 50 ml, respectively.

The LC/MS system was Waters Alliance 2690equipped with Waters ZQ2000 mass detector(Milford, MA, USA). Ionization conditions were asfollows; ESI negative, capillary voltage: 3.5 kV, conevoltage: 39 V; source block temperature: 130�C,desolvation temperature: 390�C. Separation wascarried out using a Symmetry C18 column(150�2.1 mm i.d., Waters, MA, USA) at 40�Cunder isocratic conditions with an acetonitrile–water(40:60) mobile phase. The flow rate and injectionvolume were 0.25 ml min�1 and 10ml, respectively.Selected ion monitoring mode (m/z¼ 227, M–H�)was used for BPA quantitative analysis.

The LC/MS/MS system consists on WatersAcquity UPLC system and Waters QuattroPremier API MS/MS analyser (Milford). Ionizationconditions were as follows: ESI negative conevoltage, 37 V; collision energy, 20 eV; collision gaspressure, 0.05 Mpa (Ar); source block temperature,130�C; dissolution temperature, 390�C. Separationwas carried out using an Acquity BEH C18 column(1.7 mm, 50� 2.1 mm i.d. Waters) at 40�C, using alinear gradient of acetonitrile in water as the mobilephase, 0–1 min 25% acetonitrile, 1–7 min 25–75%acetonitrile, 7–9 min 75% acetonitrile. The flow rateand injection volume were 0.35 ml min�1 and 5ml,respectively. Selected reaction mode (SRM), aprecursor ion of 227 as (M–H)� and a product ionof 212, were used for BPA quantitative analysis.

Determination of BADGE

The LC/MS/MS system consists of Waters Alliance2690 and Waters Quattro Premier API MS/MSanalyser (Milford). Ionization conditions were asfollows: ESI positive, cone voltage, 25 V; collisionenergy, 20 eV; collision gas pressure, 0.05 MPa (Ar);source block temperature, 130�C; dissolution tem-perature, 390�C. Separation was carried out using

a Symmetry C18 column (3.5 mm, 150�2.1 mmi.d., Waters) at 40�C under isocratic conditions witha 5 mM ammonium acetate/methanol (30:70)mobile phase. The flow rate and injection volumewere 0.25 ml min�1 and 5ml, respectively. The SRMions were a precursor ion of 358 as (MþNH4)þ,and a product ion of 191 for BADGE analysis.

Methods

Determination of BPA and BADGE in foods. Afterhomogenizing all contents of canned foods and 50 gof other foods with a multi-blender mill, 2–5 gsamples were taken for the BPA extraction. Sampleswere prepared according to the procedure shown inFigure 1 and were analysed for BPA using LC/ECDand/or LC/MS. Identification of BPA was carried outusing LC/MS/MS for three samples with high BPAconcentrations. BADGE contents in three samplesprepared for BPA determination were analysed usingLC/MS/MS. Can contents showing the highest BPAvalue were divided into seven glass tubes (50 ml) toinvestigate the change in BPA concentration as afunction of time at room temperature.

Determination of BPA and BADGE in water leached

from cans. After removal of the contents, six emptycans with similar diameters (66–70 mm) were

50 g food (entire contents in the case of canned food)

homogenize

5 g (2 g of fatty foods)

extract BPA with 15 ml acetonitrile

extract BPA with 10 ml acetonitrile

supernatantadd 120 ml water

Apply to solid extraction column (OASIS)

wash with 5 ml 15% ethanolwash with 5 ml water

wash with 10 ml petroleum ether

elute BPA with 5 ml ethyl acetate

dry under N2

dissolve in 10 ml acetonitrile/water (40:60)

Samples for determination by LC/ECD, LC/MS and/or LC/MS/MS

Figure 1. Extraction of BPA and BADGE from foods.


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washed with water and dried at room temperature.A total of 50 ml water or 50 mM glycine buffer (pH 8and 11) were put into the cans and allowed to standat 20�C (room temperature), 60�C, and 80�Cfor 60 min, and 121�C (autoclave) for 20 min.The experiments were performed from the lowesttemperature to the highest using the same cans.After cooling, 20 ml samples were applied directly toan Oasis-HLB column and BPA and BADGEwere extracted in the manner shown in Figure 1.Finally, each extract was dissolved in 1 mlacetonitrile/water (40:60) for LC/ECD or LC/MS/MS analysis.

Identification of epoxy resin in can linings. To identifymaterials of six can linings, attenuated total reflec-tion-infrared spectroscopy (ATR-IR, FT/IR-4200type A, JASCO Co., Tokyo, Japan) was used. Afterfinishing BPA or BADGE leaching experimentsfrom cans to various solvents, the bottom of canswere cut by can opener. ATR-IR spectra of thelinings were directly obtained using an attachmentATR PRO 410-S (JASCO).

Results

Determination of BPA and BADGE in foods

The limits of detection (LODs) of BPA and BADGEwere calculated using the standard deviation of thefive to seven replicates measurements of standardsolutions. LODs of BPA by LC/ECD, LC/MS andLC/MS/MS were 0.2 ng ml�1 (%RSD¼ 2.9, n¼ 5),0.1 ng ml�1 (%RSD¼ 3.2, n¼ 7), and 0.1 ng ml�1

(%RSD¼1.2, n¼7), respectively. Table II showsthe recoveries of BPA added to various food samplesusing LC/ECD or LC/MS. Although recoveries ofBPA from strawberries were high (137.6%) for LC/ECD and low (58.2%) for LC/MS, values for otherfoods (66–109% for LC/ECD and 82–129% for LC/MS) were satisfactory. BPA concentrations in foodsas determined by LC/ECD are shown in Table I.BPA concentrations were 0–842 ng g�1 in cannedfood (0.8–23 ng g�1 in fish, 4–20 ng g�1 in meats,0–78 ng g�1 in fruits and vegetables, 0–842 ng g�1 insoups and sauces, and 56–247 ng g�1 in others),0–14 ng g�1 for foods in plastic containers (0–0.6in beverages, 0.7–14 ng g�1 in confectionaries,0–3 ng g�1 in soups and sauces, 0–8 ng g�1 in fastfood, and 0–2 ng g�1 in others), and 0–0.5 ng g�1 infood in containers made of paper (0–0.3 ng g�1

in drinks, 0–0.5 ng g�1 in dairy foods, not detectedin fast foods). Of the food tested, soups andsauces in cans showed significantly high BPAvalues. Vegetables in cans (78 ng g�1 in asparagus,44 ng g�1 in young corn, and 36 ng g�1 in

mushrooms) contained significantly high BPAlevels, although no BPA was detected in cannedfruits.

The correlation between BPA values measured byLC/MS (A) and those measured by LC/ECD (B) for48 foods which were below 100 ng g�1 as determinedby LC/ECD was:

Y ðBÞ ¼ 0:888XðAÞ þ 2:868, r ¼ 0:7702 ðp < 0:01Þ:

BPA values of three kinds of food as measured byLC/ECD (74, 34 and 29 ng g�1) were higher thanthose measured for the same samples by LC/MS/MS(69, 29 and 12 ng g�1). Changes in BPA concentra-tions in a sauce which showed high BPA migrationfrom the can at room temperature are shown inFigure 2. The BPA content on day 7 was signifi-cantly lower than at the start. The correlationbetween BPA values (Y) and preserving days (X)was:

Y ¼ �20:30X þ 318:43:

No BADGE was detected in the three kinds ofcanned food.

Table II. Recoveries of BPA from various food.

Recoveries (%)

Food LC/ECD LC/MS

Water 79.3 81.7Consomme soup 97.3 129.4Tuna can food 109.0 97.3Tomato can food 65.5 92.7Creamy gratin 80.7 82.7Fresh strawberry 137.6 58.2Fresh beef 71.6 –Fresh tuna 83.9 –

Data are means of duplicates.BPA (5 ng g�1) was spiked to each 5 g food except spiking2 ng ml�1 to fresh beef and to fresh tuna.

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6 7

Days

BP

A c

once

ntra

tion

(ng/

g)

a,b

a

b b

c

Figure 2. Changes in BPA concentrations in the contents of cannumber 6 as a function of the number of days kept at roomtemperature (20�C). Data are mean values of duplicates.Significant differences exist between different superscript lettersand results with identical superscript letters did not show asignificant difference.


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Determination of BPA and BADGE in water

The LOD of BADGE by LC/MS/MS was0.05 ng ml�1 (%RSD¼ 3.55, n¼ 8). BADGE recov-ery from water was 97.4%. BPA and BADGEconcentrations of the contents of six cans used inthis experiment are shown in Table III. No BADGEwas detected in the contents of cans which containedsignificantly high BPA levels (numbers 4 and 6).Table IV shows the BPA concentrations leached towater or glycine buffers (pH 8 and 11) from cans as afunction of ambient temperature. BPA concentra-tions leached from cans to those water samplesincreased with a rise in temperature, but were verysmall at room temperature. In particular, the BPAleaching velocity (26.4 ng ml�1 min�1) from cannumber 6 to water at 121�C was 338 times higherthan that at 80�C (0.08 ng ml�1 min�1). BPA con-centrations leached from cans by the glycine buffers(pH 8 and 11) were similar to those for water.BADGE concentrations leached from cans to waterat 80�C for 60 min and at 121�C for 20 min areshown in Table V. There was no difference inBADGE concentrations between these tempera-tures. There was no significant relationship betweenBADGE concentrations and BPA concentrations in

the can contents. BADGE was not leached from cannumbers 4 and 6 to water at either temperature.

Identification of epoxy resin in can linings

Materials from six can linings were identified asepoxy resin, and a typical spectra of can number 6is shown in Figure 3. Eight significant peaks, C-Hbend vibration of hydroxyl group at 826 cm�1, epoxyanti-symmetric vibration at 944 cm�1, ether sym-metric vibration of aromatic compound at1038 cm�1, ether anti-symmetric vibration of aro-matic compound epoxy anti-symmetric vibration at1231 cm�1, C¼C stretching vibration of benzenering at 1507 and 1606 cm�1, C-H stretching vibra-tion at 2964 cm�1 and O-H stretching vibration ofbenzene ring at 3393 cm�1 were detected in thelinings of six cans.

Discussion

The first finding of suspected oestrogenicity of BPAwas reported by Krishnan et al. (1993), whereMCF-7 cells grown in media prepared with waterautoclaved in polycarbonate exhibited higher pro-gesterone receptor levels than cells grown in media

Table III. BPA and BADGE concentrations in the contentsof cans used in the experiment.

Cannumber Contents

BPAconcentrations

in the contents1

BADGEconcentrations

in the contents2

1 Fish 3 –2 Vegetable 8 –3 Consomme soup 6 –4 Cream soup 48 n.d.5 Cream soup 5 n.d.6 Sauce 428 n.d.

1 Data are mean values of duplicates measured by LC/ECD.2 Data are mean values of duplicates measured by LC/MS/MS.–, Not measured.

Table IV. BPA concentrations leached to water or 50 mM glycine buffers in cans at various ambient temperatures.

Treatment conditions of cans1

Water Glycine buffer (pH 8) Glycine buffer (pH 11)

Can number 20�C 60�C 80�C 121�C 20�C 60�C 20�C 60�C

1 n.d. 0.1 0.3 9 n.d. 0.1 0.1 0.12 n.d. 0.2 0.6 8 n.d. 0.2 0.0 0.23 0.1 0.1 0.1 4 0.1 0.0 0.0 0.04 n.d. 0.1 2 39 n.d. 0.1 0.1 0.15 n.d. 0.1 0.2 4 0.1 0.1 0.0 0.06 n.d. 0.8 5 528 0.2 1 0.3 1

Data are mean values (ng ml�1) of duplicates measured by LC/MS.1 A total of 50 ml water or 50 mM glycine buffers in cans was allowed to stand for 60 min except for number 6 for 20 min at 121�C.

Table V. BADGE concentrations leached to water from cansallowed to stand at 80�C for 60 min and at 121�C for 20 min.

BADGE concentrations (ng ml�1)

Can number 80�C 121�C

1 0.4 0.42 0.6 0.53 0.3 0.34 Tr n.d.5 0.1 0.16 Tr n.d.

Data are mean values of triplicates measured by LC/MS/MS.Tr, trace; n.d., not detected.


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prepared with water autoclaved in glass. Despite theoutcomes confirming the oestrogenicity of BPA withmany kinds of biomaterials, output and use of BPAhas continued until now as it has been reported to beof low risk to humans (European Union 2003;Kamrin 2004). BPA supply and demand is increas-ing yearly in Japan. The supply of 576 213 t anddemand of 426 674 t in 2003 were 1.35 and 1.11times higher, respectively, than 5 years ago.

It has been reported that the risk from BPA is largeduring the foetal stage. Regarding this risk, it hasbeen reported that low dose exposure (2–20 ng g�1)in maternal animals induced a permanent increase inthe size of the preputial glands but a decrease in thesize of the epididymides and the daily spermproduction in the offspring of mice (Vom Saalet al. 1998) and aggressive behaviour in male mice(Kawai et al. 2003). Recently, reports on antithyr-oidal function and adverse effects on immune ornervous systems by BPA that cannot be explained bythe oestrogenicity of BPA have increased (Sekizawaand Itou 2005). These findings imply that it isnecessary to analyse carefully the large amounts ofresults obtained by researchers.

Fujimaki et al. (2004) reported that the dailyintake of BPA among Japanese pregnant women wasestimated to be in the range of 0.3–7.9 mg day�1

(median < 2 mg day�1), which is far below the accep-table daily intake (ADI) level. They also calculatedthat the maximum level reached was one-tenth thatof the lowest level at which adverse effects of BPA

(2 mg kg�1 day�1) were reported for pregnant mice(Vom Saal et al. 1998). The daily intake of BPAestimated from the BPA concentrations in hospitalmeals, in which it is considered that care has beentaken, for example, to use cooking implements thathad avoided BPA exposure or to use wrapping filmsthat did not contain BPA, was 0.42 mg day�1, whichcould have originated from tuna meat in cans(Higuchi et al. 2004).

BPA migration into food is generally considered tobe caused mainly by recycled paper containers,probably coming from additives of inks used invirgin paper, and can lining materials (Ozaki et al.2004). In the present analysis, BPA values in foodsfrom cans were relatively high, though the valuesin foods from plastic and paper containers were low.The three canned foods that showed the highestBPA values (canned brown sauce at 428, 547 and842 ng g�1) had the same brand name and manu-facturer but were bought from different places atdifferent dates. This indicates that these three canswould be made by the same manufacturer with thesame lining material. On the other hand, BPAconcentrations in meat sauce from other cans,which had similar ingredients but different manu-facturers, were low (0–13 ng g�1). The differentBPA values seem to be caused by differences in thecans. BPA concentrations leached from six cans towater at 121�C were similar to those of theircontents, which indicated that BPA migration fromcans could occur during the sterilization stage.

Figure 3. ATR-IR spectra of the linings in can number 6. Assignments of the major eight peaks show a typical spectrum of epoxy resin.


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Munguia-Lopez et al. (2005) reported that leachingof high levels of BPA (646.5 mg kg�1) to fatty foodstimulants from tuna fish cans coated with organosolresin after the commercial heating process. Thus,BPA coming from cans might have been intended foruse as an additive, and may be an unexpectedcontaminant in the lining materials of the cans. If anadult weighing 50 kg ingested such canned food(net weight¼290 g/can) with 842.3 ng g�1 BPA, thehighest BPA in this study every day, the BPAexposure is estimated to be 4.89 mg kg�1 day�1,which corresponds to around half the ADI(0.01 mg kg�1 day�1) proposed by the EuropeanScientific Committee of Food. Such high concentra-tions of BPA in canned foods are a health concern.Thus, it is necessary to continue the monitoring ofBPA concentrations in canned food.

Epoxy resins are the main material used for liningcans. Indeed, materials for linings of six cans used inthis study were identified as epoxy resin. BADGE,a monomer of epoxy resin, was leached to waterfrom four of the six cans at a very low level(<0.5 ng ml�1) when they were allowed to stand atsterilization temperature, although no BADGE wasdetected in the contents of three cans analysed in thepresent study. BADGE concentrations leached towater from cans held at 121�C for 20 min were muchlower than those of BPA. It has been reported thatthere is no problem in using epoxy resins for liningcans, due to the lower oestrogenicity of BADGEcompared to BPA (Poole et al. 2004; Strieber et al.2004). Pardo et al (2006) reported that no BADGEpeak was detected in three canned fish and five babyfoods. Commission Regulation (EC) No. 1895/2005(European Commission 2005) reported that specificmigration limit (SML) of certain epoxy derivatives isrestricted below 9 mg kg�1 in food. We can concludethat it is probably safe to use epoxy resins withoutBPA for can linings.

BPA and BADGE concentrations leached to waterfrom can number 2 were 35.9 and 1.6 nM,respectively, after 20 min at 121�C. Whereas noBADGE was detected in either the contents or watersamples after leaching at 121�C from can numbers 4and 6, whose contents showed high BPA concentra-tions, it indicates that BPA and BADGE concentra-tions depend on epoxy resins used for can linings.Relatively high contamination of BPA in resinmonomers such as bisphenol-A dimethacrylate (bis-DMA) and BADGE from a manufacturer has beenreported (Schmalz et al. 1999). These findingsindicate the possibility of BPA contamination as animpurity in epoxy resins for can linings. Althoughit is not clear whether BPA coming from cans havebeen intended for use as an additive or is anunexpected contaminant, the selling of canned

foods possessing a high concentration of BPA inmarkets is a critical problem for human health.

In this study, a low level of BPA was detectedin fresh strawberries. There is no data on thedetection of BPA in fresh vegetables and fruit inJapan. In Italy, however, detection of BPA at levelsof 0.25–1.0mg g�1 in eight out of 14 fresh vege-tables were reported (Vivacqua et al. 2003). As thestrawberries used in this study were packed in apolyethylene terephthalate (PET) case with a plasticfilm at room temperature, which is the normal waythis is sold in Japan, it is not likely that BPAcould have leached from the packaging materials.Nowadays in Japan strawberries are usually plantedin greenhouses where some kinds of plastic stretchseats (polyethylene, PVC, etc.) are covered with soilto keep a constant temperature in soil and to helpsatisfactory growth of plants, and PVC panels areused for the walls of greenhouses. If these plasticmaterials contain BPA, migration of BPA fromthese via the atmosphere (and also soil and water)cannot be ruled out. It is necessary to monitor BPAmigration to fresh vegetables and fruits planted ingreenhouses.

In the present study, two different instrumentalmethods (LC/ECD and LC/MS) for detection ofBPA and LC/MS/MS for a confirmation of BPA wereused. Each has its merits and limitations. LC/ECD iselectrically specific for phenol compounds but notselective for BPA. LC/MS is specific for molecularweight of BPA but not selective for BPA. LC/MS/MSis the most specific and selective for BPA but isexpensive and not commonly used for analysis. Thefact that a high correlation (r¼ 0.7702, p< 0.01)between BPA values measured by LC/MS (A) andLC/ECD (B) for 48 foods was observed and that BPAvalues of three kinds of foods measured by LC/ECDwere similar to those measured by LC/MS/MSindicates that two approaches could be used forBPA analysis in food. This agrees with the datareported for BPA by the two instrumental methods inserum (Sajiki et al. 1999). Isocratic solvent systemswere used for analyses of BPA using LC/ECD andLC/MS in food as the similar manner to serum.As food samples contain many kinds of compoundswith longer retention time, application of gradientsolvent program is recommended in point of timeconsuming and higher quality of data for BPAanalysis.

Conclusions

Results for the determination of BPA in 87 foodsindicated that can lining materials are suspected tobe the main source of BPA exposure for humans.BPA leaching from cans might be attributed to the


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high temperature used for the sterilization of cannedfoods. It is necessary to monitor BPA concentrationsin canned foods in the future. In comparison withBPA concentrations, BADGE concentrations inwater that leached from epoxy resin cans at 121�Cwere far below that level, suggesting that the use ofepoxy resin without BPA for can linings has a lowrisk from the viewpoint of oestrogenicity.

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bisphenol a (bpa) and its source in foods in japanese markets

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