1 chemistry analytical_methods

Food and Agriculture
Organization of the
United Nations

WHO/HSE/FOS/11.1

Background Paper on

Chemistry and Analytical Methods for Determination of
Bisphenol A in Food and Biological Samples

FAO/WHO Expert Meeting on Bisphenol A (BPA)
Ottawa, Canada, 2–5 November 2010

Prepared by

Xu-Liang Cao
Food Research Division, Bureau of Chemical Safety,
Health Canada, Ottawa, Ontario, Canada

Note to readers:
The first draft of this paper was prepared by the named author, and the paper
was then revised following discussions at the November 2010 meeting.

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1

Toxicological and Health Aspects of Bisphenol A

CONTENTS
1. Chemistry ........................................................................................................................................................2
2. Analytical methods..........................................................................................................................................4
2.1 Sample preparation ...............................................................................................................................4
2.1.1 Deconjugation with enzymes .....................................................................................................4
2.1.2 Solvent extraction ......................................................................................................................4
2.1.3 Solid-phase extraction..............................................................................................................19
2.1.4 Derivatization...........................................................................................................................19
2.1.5 Solid-phase microextraction.....................................................................................................20
2.1.6 Stir bar sorptive extraction .......................................................................................................20
2.1.7 Coacervative microextraction ..................................................................................................20
2.2 Separation and detection .....................................................................................................................20
2.2.1 Liquid chromatography–based methods ..................................................................................20
2.2.2 Gas chromatography–mass spectrometry.................................................................................22
2.2.3 Enzyme-linked immunosorbent assay......................................................................................23
2.3 Method validation ...............................................................................................................................24
3. Conclusions and recommendations ...............................................................................................................10
References ............................................................................................................................................................25

Sensitive and reliable analytical methods are available for the determination of bisphenol A (BPA) in both food
and biological samples. Solvent extraction and solid-phase extraction are the most commonly used and most
effective methods for the extraction of BPA in food and biological samples. Although isotope dilution methods
based on mass spectrometry and tandem mass spectrometry are the most reliable for the detection of BPA, many
of the results of BPA determination in both food and biological samples have been generated by methods that
are not based on mass spectrometry.

The majority of methods used to measure free and total BPA in food and biological samples have been validated
for certain performance parameters, such as accuracy, precision, recovery and limit of detection. Most methods
fulfil the requirements of single-laboratory validation. For biological samples, however, validation of methods
for conjugated BPA is very limited. By the current standards of analytical science, findings of BPA in food
samples and most biological samples are reliable. Nevertheless, care needs to be taken to avoid cross-
contamination with trace levels of BPA during sample collection, storage and analysis.

1. CHEMISTRY
Bisphenol A (BPA) is the common name for 4,4′-dihydroxy-2,2-diphenylpropane
(International Union of Pure and Applied Chemistry [IUPAC] name). Its chemical structure
and physicochemical properties are shown in Table 1. BPA is a white solid (available in
crystals or flakes) with a mild phenolic odour under ambient conditions. Its melting point is
155 °C, and its specific gravity is 1.060–1.195 g/cm3. BPA is generally considered to be a
moderately hydrophobic compound (octanol–water partition coefficient [Kow] of 103.4), with a
slight polarity due to the two hydroxyl groups. It is soluble in acetic acid and very soluble in
ethanol, benzene and diethyl ether (Lide, 2004). Although it was classified as insoluble in
water in the 85th edition of the CRC Handbook of Chemistry and Physics (Lide, 2004), BPA
is generally considered to be fairly soluble in water, with a solubility of 300 g/m3 at 25 °C.
BPA has a relatively high boiling point (398 °C at 101.3 kPa) and low vapour pressure (5.3 ×
10−6 Pa at 25 °C), and its concentration in air will be very low. Its air–water partition
coefficient (Kaw) is very low (10−9); thus, BPA is unlikely to evaporate from aqueous
solution. The very high value of its octanol–air partition coefficient (Koa; 2.6 × 1012) also
suggests that BPA in gaseous form will sorb strongly to solid surfaces (Cousins et al., 2002).

2

Chemistry and Analytical Methods

The pKa value of BPA is between 9.59 and 11.30; thus, BPA will be present mainly in its
molecular form in liquid media with pH lower than 7.

Table 1. Physicochemical properties of bisphenol A

Property Value
Chemical Abstracts Service registry number 80-05-7
Chemical structure

Other names 4,4′-dihydroxy-2,2-diphenylpropane
2,2-bis(4-hydroxyphenyl)propane
4,4′-isopropylidenediphenol
Formula C15H16O2
Molecular weight 228.29 g/mol
Melting point 155 °Ca
Boiling point 398 °C at 101.3 kPab
3b
Specific gravity 1.060–1.195 g/cm
3 b
Water solubility 300 g/m at 25 °C
−6 a
Vapour pressure 5.3 × 10 Pa at 25 °C
a
Log Kow 3.40
a
Log Kaw −9.01
Log Koa 12.41a
a
pKa 9.59–11.30
a
Cousins et al. (2002).
b
Staples et al. (1998).

The BPA molecule has a fairly strong fluorophore due to the conjugated π-electrons in the
two benzene rings, and thus it can be detected by fluorescence detector. Its chromophore is
relatively weak, and the sensitivity of ultraviolet (UV) detection is much lower than that of
fluorescence detection.

Although BPA is fairly stable in its solid form, it does not persist in the environment. Aerobic
biodegradation is the dominant loss process for BPA in river water and soil, and its
degradation half-life is about 4.5 days (Cousins et al., 2002). Its loss process in the
atmosphere is due to the rapid reaction with hydroxyl radicals, and the photo-oxidation half-
life for BPA in air is about 4 hours (Cousins et al., 2002). Chlorinated BPA can be found in
both wastewater and drinking-water, as BPA can be easily chlorinated by sodium
hypochlorite, a bleaching agent in paper factories and a disinfection agent in sewage
treatment plants (Fukazawa et al., 2001; Yamamoto & Yasuhara, 2002), and chlorine, a
chemical used in the disinfection of drinking-water (Gallard, Leclercq & Croue, 2004).

Two major applications of BPA are in the production of polycarbonate plastics and epoxy
resins. Polycarbonate is synthesized from BPA and phosgene gas (carbonyl dichloride),
whereas epoxy resins are produced from the reaction of BPA with epichlorohydrin.

3


2. ANALYTICAL METHODS
Various methods have been developed and used to determine BPA in food and biological
samples (Tables 2 and 3). Although some of the methods could be used for qualitative
screening purposes, such as the enzyme-linked immunosorbent assay (ELISA), quantitative
results were reported in all publications. Owing to the complex matrices of the food and
biological samples and the low concentrations of BPA (parts per billion [ppb] or sub–parts
per billion levels), extensive sample preparations (extraction, cleanup, concentration,
derivatization, etc.) prior to analysis by instruments such as gas chromatography (GC) and
liquid chromatography (LC) coupled with various detectors (mass spectrometer [MS], UV,
fluorescence detector, electrochemical detector [ECD], etc.) are required, even for qualitative
screening analysis.

2.1 Sample preparation

2.1.1 Deconjugation with enzymes

BPA in biological samples exists as both free BPA and conjugated BPA. The majority of
conjugated BPA is in the form of BPA-glucuronide, whereas only a small portion is in the
form of BPA-sulfate. In order to determine the total BPA in biological samples, conjugated
BPA needs to be deconjugated by hydrolysis with enzymes at 37 °C for a period ranging
from a few hours up to overnight. Among the published results (Table 3), most people used
only β-glucuronidase for deconjugation, whereas only a few used both β-glucuronidase and
sulfatase enzymes for deconjugation. In some of the studies, enzymes were not used
(Pedersen & Lindholst, 1999; Sajiki, Takahashi & Yonekubo, 1999; Inoue et al., 2000;
Watanabe et al., 2001; Sun et al., 2002, 2004; Kuroda et al., 2003; Mao et al., 2004; Volkel et
al., 2005; Xiao et al., 2006; Fernandez et al., 2007; Dirtu et al., 2008; Cobellis et al., 2009;
Markham et al., 2010); thus, the results could be for the free BPA only. As the ELISA
method determines total BPA (free BPA plus conjugated BPA), this deconjugation step with
enzyme is not needed in sample preparation. It should be mentioned that, depending on the
types of β-glucuronidase enzymes (Escherichia coli or Helix pomatia) used, BPA-sulfate
could also be deconjugated (Ye et al., 2005a).

2.1.2 Solvent extraction

Solvent extraction is one of the most common and effective techniques for the extraction of
BPA from food and biological matrices, and acetonitrile is the most frequently used solvent
for this purpose. The other role of acetonitrile is to precipitate the proteins in protein-rich
samples, such as infant formula, milk, urine and blood. Acids have also been used for protein
precipitation (Yoshimura et al., 2002). Non-polar solvents, such as n-hexane, n-heptane and
trimethylpentane, have also been used together with acetonitrile for the extraction of BPA
from fatty samples (Goodson, Summerfield & Cooper, 2002; Kang & Kondo, 2003;
Braunrath et al., 2005; Thomson & Grounds, 2005; Sun, Leong & Barlow, 2006; Fernandez
et al., 2007; Podlipna & Cichna-Markl, 2007; Grumetto et al., 2008; Lim et al., 2009).
Solvents other than acetonitrile have also been used occasionally for the extraction of BPA
from biological samples; examples include chloroform (Sun et al., 2002; Kuroda et al., 2003),
dichloromethane (Arakawa et al., 2004), methyl tert-butyl ether (MTBE) (Lee et al., 2008),
diethyl ether (Ouchi & Watanabe, 2002), dichloromethane and methanol (Pedersen &
Lindholst, 1999) and 2-propanol (Ye et al., 2006; Yi, Kim & Yang, 2010). Microwave was

4


Table 2. Methods for determination of BPA in food samples

Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference
detection precision
Fish, meat, fruit, Sample extracted with acetonitrile. Extracts n/a LC-ECD 0.2 ng/ml — 65.5–137.6% Sajiki et al.
vegetable, soup, cleaned up with SPE, eluted with ethyl acetate, 2.9% RSD (n = 5) (2007)
sauce, dried under nitrogen, reconstituted with
LC-MS 0.1 ng/ml — 58.2–129.4%
beverage, milk acetonitrile/water (40:60).
3.2% RSD (n = 7)
LC-MS/MS 0.1 ng/ml — 1.2% RSD (n = 7)
Infant formula Sample spiked with d6-BPA, extracted with n/a LC-MS/MS 0.15 ng/g 0.5 47% Ackerman et
acetonitrile, centrifuged. Supernatant cleaned up ng/g 1.4–5.5% RSD for al. (2010)
with SPE, eluted with chloroform, reduced to 1.7–9.8 ng/g; 2.9–
dryness under nitrogen, reconstituted with 18% RSD for 2.3–
methanol/water (50:50). 10.6 ng/g
Milk Milk protein precipitated with acetonitrile. n/a LC-MS 0.20 ng/ml — 97.1% (0.6 ng/ml); Yan et al.
Supernatant cleaned up further with PSA and 92.4% (15 ng/ml) (2009)
online SPE (C30). 15.0% RSD (0.5
ng/ml); 13.2% RSD
(15 ng/ml)
Honey Honey sample dissolved in water, applied to SPE n/a LC-fluorescence 2.0 ng/g — 103.6% for 5 ng/g; Inoue et al.
cartridge (GL-Pak PLS-2, polystyrene divinyl (275/300 nm); 99.9% for 50 ng/g (2003b)
benzene), eluted with methanol. LC-MS for 6.6% RSD for 5
confirmation ng/g (n = 6); 5.3%
RSD for 50 ng/g
(n = 6)
Fruit, vegetable Sample extracted with acetonitrile. Extract n/a HPLC-UV (228 — 5–10 84.5–90.1% Yoshida et
applied to SPE cartridge, eluted with nm) ng/ml 3.4–4.2% RSD (n = al. (2001)
acetone/heptane, evaporated to dryness and 3)
reconstituted with mobile phase.
Infant formula Sample diluted with water was applied to SPE Chloroform HPLC- 0.9 ppb — 86–104% Biles,
cartridge, eluted with chloroform and extract not fluorescence 2–27% RSD (n = 3) McNeal &
concentrated. The concentrated extract was derivatized for (235/317 nm); Begley
diluted with mobile phase for HPLC analysis. GC-MS GC-MS (for (1997)
analysis confirmation)

5


Table 2 (continued)
detection precision
Coffee drink Sample applied to SPE cartridge, eluted with n/a HPLC- — 2–10 85.3–96.2% Kang &
acetonitrile/water (40:60 v/v). fluorescence ng/ml 1.9–6.7% RSD (n = Kondo
(275/300 nm) 3) (2002)
Milk, dairy Sample blended with acetonitrile and hexane. n/a HPLC- 1–3 ng/ml — 76.9–101.8% Kang &
products Hexane phase extracted again with acetonitrile. fluorescence 3.4–24.6% RSD Kondo
Acetonitrile phase combined, filtered and (275/300 nm) (n = 5) (2003)
evaporated to dryness. Residue dissolved in
acetone/n-heptane (3:97 v/v) and applied to Sep-
Pak Florisil cartridge for cleanup. BPA eluted
with acetone/n-heptane (20:80 v/v), evaporated
to dryness and dissolved with mobile phase for
HPLC analysis.
Beverage Sample loaded onto Oasis HLB SPE cartridge, n/a LC-MS/MS 0.6 ng/l 2.0 ng/l 82.1–96.5% Shao et al.
eluted with methanol/dichloromethane (20:80 2.9–7.1% RSD (n = (2005)
v/v). 5)
Beverage, Beverage sample applied to immunoaffinity n/a HPLC- 0.1–9.3 0.4–0.8 27–103% Braunrath &
vegetable, column, eluted with acetonitrile/water (40:60 v/v). fluorescence ng/g ng/g 1.0–31% RSD (n = Cichna
fruit, soup, fish Fruit and vegetable sample extracted with (275/305 nm) (fish) 3) (2005);
acetonitrile twice, supernatants filtered and Braunrath et
applied to immunoaffinity column, eluted with al. (2005);
acetonitrile/water (40:60). Podlipna &
Fat-containing food sample extracted with Cichna-Markl
acetonitrile/hexane (1:1). Acetonitrile extracts (2007)
filtered and applied to immunoaffinity column,
eluted with acetonitrile/water (40:60).
Fish, meat Coacervative microextraction. n/a HPLC- — 15–29 97–111% Bendito et al.
0.2 g decanoic acid dissolved in 2 ml THF in fluorescence ng/g 2.1–7.1% RSD (n = (2009)
centrifuge tube; 8 ml water and 140 µl (276/306 nm) 3)
hydrochloric acid (0.5 mol/l) added. Mixed with
food sample, stirred and centrifuged. Coacervate
phase analysed by LC.

6


Table 2 (continued)
detection precision
Food simulant, Food simulant evaporated to dryness, dry n/a (for HPLC- — 0.12– 89.2–90.6% Munguia-
pepper residue redissolved in 5 ml of acetonitrile and HPLC); extract fluorescence 0.2 1.2–5.8% RSD Lopez &
filtered. not derivatized (224/310 nm); ng/g Soto-Valdez
Pepper sample blended with methanol, filtered. for GC-MS GC-MS for (2001);
Liquids evaporated to dryness, residue confirmation Munguia-
redissolved with 5 ml of acetonitrile and filtered. Lopez et al.
(2002)
Vegetable, Coacervative microextraction. n/a HPLC- — 9 ng/g 81–96% Garcia-Prieto
fruit 0.2 g decanoic acid dissolved in 4 ml THF in fluorescence 3% RSD (n = 6) et al. (2008a)
centrifuge tube, 36 ml of hydrochloric acid added (276/306 nm)
(1.3 mmol/l). Mixed with food sample, stirred and
centrifuged. Coacervate phase analysed by LC.
Vegetable, Sample extracted with acetonitrile and hexane. n/a HPLC- 4.5–7.9 13.7– 87.3–105.2% Sun, Leong
fruit, fish, meat Acetonitrile extract evaporated, dissolved in fluorescence µg/kg 24.1 0.20–2.96% RSD & Barlow
methanol/water (5:95 v/v), loaded onto SPE (235/317 nm) µg/kg (inter-day, n = 5); (2006)
cartridge (Oasis HLB), eluted with methanol, 0.04–2.82% RSD
methanol:ethyl acetate (50:50) and ethyl acetate. (intra-day, n = 5)
Extract evaporated to dryness, reconstituted with
acetonitrile/water (90:10 v/v).
Fish, Sample spiked with BPA-d14 and extracted with Acetic GC-MS in EI 2 ng/g 7 ng/g 81–103% Goodson,
vegetable, acetonitrile (n-heptane also used for fat anhydride mode 4.5% RSD for 11 Summerfield
infant formula, samples). Extract derivatized with acetic ng/g (n = 6) & Cooper
pasta, dessert, anhydride. Derivatized BPA extracted with n- (2002)
soup, heptane. Beverage samples derivatized directly.
beverage
Fish, meat, Sample spiked with BPA-d14 and extracted with Acetic GC-MS in EI — 10–20 42–112% Thomson &
vegetable, acetonitrile (trimethylpentane also used for fat anhydride mode ng/g 8% RSD for 28.6 Grounds
fruit, soup, samples). Extract derivatized with acetic ng/g (n = 8) (2005)
dessert, anhydride. Beverage samples derivatized
beverage directly.
Milk Milk sample deproteined with trichloroacetic acid, n/a HPLC- 0.2 ng/ml 0.5 93–102% Liu et al.
diluted with water (20-fold), dissolved in fluorescence ng/ml 6.6% RSD (n = 3) (2008)
methanol, filtered. Extracted with SPME fibre by (275/315 nm)

7


Table 2 (continued)
detection precision
direct immersion.
Fish Sample extracted with acetonitrile. Extract n/a HPLC- 1 ng/g — 70.7–72.9% Tsuda et al.
filtered and evaporated to dryness. Residue fluorescence 1.8–4.8% RSD (n = (2000)
dissolved in hexane, extracted with acetonitrile (275/300 nm) 5)
saturated in hexane. Acetonitrile layer
evaporated to dryness, dissolved in hexane,
applied to column packed with Florisil PR for
cleanup. BPA eluted with acetone and hexane
(3:7 v/v). Eluate evaporated to dryness,
reconstituted with methanol.
Vegetable Canned food (solid or liquid) diluted with water. BPA GC-MS in EI 0.01–0.03 0.033– 84–112% Vinas et al.
For derivatization with acetic anhydride, acetic derivatized mode ng/m 0.1 1.96–2.09% RSD (2010)
anhydride also added to sample solution with acetic ng/ml (n = 10)
together with buffer solution. Derivatized BPA anhydride or
extracted with SPME polyacrylate fibre by direct BSTFA
immersion. For derivatization with BSTFA, BPA
extracted with SPME polyacrylate fibre first, then
derivatized in the headspace above BSTFA.
Wine Wine sample mixed with PBS, pH adjusted to n/a HPLC- 0.1 ng/ml 0.2 74–81% Brenn-
7.0, filtered, applied to immunoaffinity column fluorescence ng/ml 10–15% RSD (n = Struckhofova
and eluted with acetonitrile/water (40:60 v/v). (275/305 nm) 3) & Cichna-
Markl (2006)
Milk Milk sample diluted with water, applied to C18 No GC-MS in EI 0.15 — 81% Casajuana &
SPE cartridge, eluted with derivatization mode µg/kg 5% RSD (n = 3) Lacorte
dichloromethane/hexane and ethyl acetate, and for BPA (2004)
cleaned up on Florisil column.
Tomato Sample extracted with acetonitrile. Acetonitrile n/a HPLC-UV (228 20 µg/kg 66.9 0.14–2.2% RSD Grumetto et
phase partitioned with hexane for fat removal. nm) µg/kg (inter-day); 0.04– al. (2008)
Extract evaporated, residue dissolved in 1.84% RSD (intra-
water/acetonitrile, applied to C18 SPE cartridge, day)
eluted with acetonitrile. Eluate evaporated to HPLC- 1.1 µg/kg 3.7 0.2–2.96% RSD
dryness, dissolved in hexane/ethyl acetate (96:4 fluorescence µg/kg (inter-day, n = 5);
v/v), applied to Florisil cartridge, eluted with ethyl

8


Table 2 (continued)
detection precision
acetate. Eluate evaporated, residue reconstituted (273/300 nm) 0.04–2.82% RSD
with acetonitrile. (intra-day, n = 5)
Infant formula Sample dissolved in ethanol/water (50:50 v/v), BPA GC-MS in EI — 1.0 79% Kuo & Ding
centrifuged. Supernatant filtered, applied to C18 derivatized mode ng/g 9% RSD (n = 5) (2004)
SPE cartridge, eluted with methanol. Eluate with BSTFA +
derivatized with BSTFA + TMCS. TMCS
Coffee, tea, Coffee, tea, fruit, vegetable: sample mixed with n/a HPLC- 3 ng/g — 95.4% Lim et al.
fruit, acetonitrile, centrifuged. Supernatant filtered, fluorescence 9.1% RSD (n = 5) (2009)
vegetable, dried, dissolved in acetonitrile/water (60:40 v/v). (275/315 nm)
fish, meat Fish, meat: sample extracted with acetonitrile
and hexane, centrifuged. Solid sample and
hexane phase extracted several times with
acetonitrile.
Milk Sample spiked with BPA-d16, diluted with n/a LC-MS (ESI) 1.7 ng/g 5.1 83–106% Maragou et
water/methanol (8:1 v/v), applied to C18 SPE ng/g 2.1–12.5% RSD al. (2006)
cartridge and eluted with methanol/water (90:10 (intra-day, n = 6);
v/v). Eluate evaporated to dryness and 5.2–17.6% RSD
reconstituted with water. (inter-day, n = 6)
Water SPME n/a HPLC- 1.1 ng/ml 3.8 22% RSD (n = 4) Nerin et al.
fluorescence ng/ml (2002)
(275/305 nm)
Egg, milk Milk or egg sample mixed with C18 powder, n/a LC-MS/MS 0.1 ng/g — 79.2–86.8% (egg); Shao et al.
packed into a column. BPA eluted with methanol. 85.7–93.9% (milk) (2007)
Eluate evaporated to dryness, residue 2.86–7.42% RSD
redissolved in dichloromethane/hexane (50:50), (egg, n = 5); 3.15–
applied to aminopropyl SPE cartridge for 5.29% RSD (milk,
cleanup. Eluted with methanol/acetone (50:50 n = 5)
v/v). Eluate evaporated to dryness and
reconstituted with mobile phase.
Infant formula Sample spiked with BPA-d16, mixed with BPA GC-MS in EI — 0.5 85–94% Cao et al.
acetonitrile, centrifuged. Supernatant applied to derivatized mode ng/g 2.8–5.0% RSD (n = (2008)
C18 SPE cartridge, eluted with acetonitrile in with acetic 6)

9


Table 2 (continued)
detection precision
water (50:50 v/v), evaporated to 3 ml. anhydride
Concentrated aqueous extract derivatized with
acetic anhydride.
Soft drinks Sample spiked with BPA-d16, applied to C18 BPA GC-MS in EI — 0.05 99.9–101% Cao,
SPE cartridge, eluted with acetonitrile in water derivatized mode ng/ml 1.3–6.6% RSD (n = Corriveau &
(50:50 v/v). Eluate evaporated to 3 ml. with acetic 7) Popovic
Concentrated aqueous extract derivatized with anhydride (2009)
acetic anhydride.
BSTFA, N-O-bis(trimethylsilyl)trifluoroacetamide; ECD, electron capture detector; EI, electron ionization; ESI, electrospray ionization; GC, gas chromatography; HPLC, high-
performance liquid chromatography; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MS, mass spectrometry; MS/MS, tandem mass
spectrometry; n/a, not applicable; ppb, parts per billion; PBS, phosphate buffered saline; PSA, primary–secondary amine; RSD, relative standard deviation; SPE, solid-phase
extraction; SPME, solid-phase microextraction; THF, tetrahydrofuran; TMCS, trimethylchlorosilane; UV, ultraviolet; v/v, volume per volume

10


Table 3. Methods for determination of BPA in biological samples

detection precision
Urine Acetonitrile added to urine sample, centrifuged n/a LC-MS/MS — 1.3–5 — Volkel,
to precipitate protein. Supernatant applied to µg/l Kiranoglu
online SPE (Oasis HLB). β-Glucuronidase & Fromme
added to urine sample for total BPA (2008)
determination.
Human Sample mixed with hydrochloric acid (0.2 mol/l), BPA derivatized HPLC- 0.04 ppb — 78.6% (serum); 77.7% Kuroda et
blood extracted with chloroform, evaporated to with fluorescent fluorescence (ascitic fluid) al. (2003)
serum, dryness. Fluorescent reagent DIB-Cl in reagent DIB-Cl (350/475 nm) 4.2% RSD (intra-day,
ascitic fluid acetonitrile added to the residue to label BPA. n = 6); 8.0% RSD
(inter-day, n = 3)
Human Sample mixed with formic acid, diluted with Pentafluoro- GC-MS in ECNI — 280 81.3–83.1% Dirtu et al.
serum water. Mixture loaded onto SPE (Oasis HLB) propionic acid mode pg/ml 1.6–5.1% RSD (intra- (2008)
cartridge, eluted with methanol/dichloromethane anhydride day); 2.4–14% RSD
(1:1 v/v), concentrated to 0.5 ml. Extract (inter-day)
cleaned up further on Florisil cartridge, eluted
with methanol/dichloromethane (5:1 v/v),
derivatized with pentafluoropropionic acid
anhydride.
Urine Glucuronidase enzyme added to sample, n/a LC-ECD 0.5 µg/l — 115% Liu, Wolff
incubated overnight at 37 °C, applied to C18 6.1% RSD (n = 9) & Moline
SPE cartridge, eluted with methanol. (2005)
Urine Urine sample hydrolysed with hydrochloric acid, BPA derivatized LC-fluorescence 2.7 µg/l — 95.9% Mao et al.
eluted on C18 SPE cartridge with dichloro- with fluorescent (228/316 nm) 3.92% RSD (2004)
methane. Extracts derivatized with fluorescent reagent p-
reagent p-nitrobenzoyl chloride. nitrobenzoyl
chloride
Urine β-Glucuronidase and sulfatase added to urine MtBSTFA + 1% GC-MS in 3 ng/ml 7 ng/ml 90–119% Moors et
sample for hydrolysis at 37 ΕC overnight. tBDMCS electron impact 4–6% RSD (intra- al. (2007)
Hydrolysed urine sample loaded to SPE ionization mode assay, n = 4); 10%
cartridge, eluted with acetonitrile/ethyl acetate RSD (inter-assay, n =
(1:1 v/v), eluate evaporated to dryness. Residue 6)
derivatized with MtBSTFA + 1% tBDMCS.

11


Table 3 (continued)
detection precision
Blood Blood plasma mixed well with hydrochloric acid BPA derivatized HPLC- 4.6 ppb — 101% Sun et al.
(0.2 mol/l), extracted with chloroform. Organic with fluorescent fluorescence 1.0–2.2% RSD (intra- (2002)
phase evaporated to dryness, residue reagent DIB-Cl (350/475 nm) day, n = 4); 5.6–6.3%
derivatized with DIB-Cl. RSD (inter-day, n = 6)
Blood Blood serum mixed with mobile phase n/a HPLC- 0.15 0.50 85.6% Cobellis et
(acetonitrile/phosphate buffer at pH 6.0 [35:65 fluorescence ng/ml ng/ml 2 al. (2009)
Linearity (r ): 0.989
v/v]). Perchloric acid (25% w/v) added to (273/300 nm);
precipitate proteins, centrifuged. Supernatant LC-MS for
filtered. confirmation
Urine Coacervative microextraction. Urine sample n/a LC-fluorescence 0.197 — 88–95% Garcia-
hydrolysed with β-glucuronidase enzyme. 0.1 g (276/306 nm) µg/l 4.5% RSD (n = 3) Prieto et
decanoic acid dissolved in 1 ml THF in al. (2008b)
centrifuge tube, mixed with hydrolysed urine
sample, stirred and centrifuged. Coacervate
phase analysed by LC.
Blood, Blood sample fortified with BPA-d8, extracted n/a LC-MS/MS 0.05 — 67–109% (urine); 98– Markham
urine with acetonitrile, centrifuged. (NESI) ng/ml 130% (blood) et al.
Urine sample fortified with BPA-d8, diluted with 1.4–33.6% RSD (2010)
water, loaded onto Oasis HLB SPE cartridge, (urine, n = 5); 4.4–20%
eluted with MTBE. Extract evaporated to RSD (blood, n = 5)
dryness, reconstituted with acetonitrile/water
(50:50 v/v).
Urine Urine sample mixed with PBS and centrifuged. n/a HPLC- 0.2 — 78% Schoring-
Supernatant applied to enzyme column fluorescence ng/ml 3.4% RSD (n = 4) humer &
containing β-glucuronidase and arylsulfatase, (275/305 nm); Cichna-
eluted with PBS. Extracts applied to LC-MS (ESI-ion Markl
immunoaffinity column, eluted with trap) for (2007)
acetonitrile/water (40:60 v/v). confirmation
Blood Blood serum diluted with PBS and applied to n/a HPLC- — — 91.8% Zhao et al.
immunoaffinity column. BPA eluted with fluorescence 7.1% RSD (n = 6) (2003)
methanol/water (80:20 v/v). Extract evaporated (230/315 nm)

12


Table 3 (continued)
detection precision
to dryness, redissolved in acetonitrile/water
(60:40).
Human Milk sample extracted with acetonitrile, n/a ELISA 0.3 — 102.6% ± 19.0% Kuruto-
colostrum centrifuged. Supernatant evaporated, and ng/ml Niwa et al.
residue dissolved in phosphate buffer and (2007)
applied to SPE cartridge (Oasis HLB). BPA
eluted with methanol/acetonitrile (3:1 v/v),
evaporated to dryness, reconstituted with
phosphate buffer.
Urine β-Glucuronidase added to urine sample for BPA derivatized GC-MS in 0.1 — 95–116% Kuklenyik
deconjugation overnight. Acetonitrile added to with PFBBr negative ng/ml 6–7% RSD (n = 19) et al.
the deconjugated sample. Derivatizing agent chemical (2003)
PFBBr in hexane (1:2) loaded onto the SPE ionization mode
cartridge (Bond Elute PPL). Deconjugated urine
sample loaded onto the SPE cartridge, and
derivatized BPA eluted from the cartridge with
acetonitrile and ethyl acetate. Extract
evaporated to dryness and reconstituted with
isooctane.
Urine β-Glucuronidase added to urine sample for BPA derivatized GC-MS in EI 0.02 0.1 98.8–101% Kawaguchi
deconjugation. Acetic anhydride added for with acetic mode ng/ml ng/ml 1.8–6.7% RSD (n = 6) et al.
derivatization. Derivatized BPA extracted into anhydride (2008)
the solvent (toluene) contained in the hollow
fibre connected to a syringe.
Blood, β-Glucuronidase/sulfatase added to sample for BPA derivatized GC-MS in — 0.1–0.05 93-94% (serum); 100– Geens,
urine deconjugation. Deconjugated sample applied to with PFBCl electron ng/ml 102% (urine) Neels &
SPE cartridge (Oasis HLB), eluted with capture– 9–16% RSD (serum, Covaci
methanol/dichloromethane (1:1 v/v). Eluate negative n = 3); 4–10% RSD (2009)
evaporated to dryness and derivatized with ionization mode (urine, n = 3)
PFBCl.
Blood Acetonitrile and hydrochloric acid (1 mol/l) BPA derivatized HPLC- 0.05 — 94.8–95.2% Watanabe
added to plasma sample. Centrifuged. with DIB-Cl fluorescence ng/ml 5.8–8.2% RSD (n = 4) et al.
Supernatant diluted with water, applied to SPE fluorescent (340/470 nm) (2001)

13


Table 3 (continued)
detection precision
cartridge (Oasis HLB) and eluted with methanol. reagent
Eluate evaporated to dryness, reconstituted with
acetonitrile, derivatized with fluorescent reagent
DIB-Cl.
Human milk Milk sample diluted with water, extracted with BPA derivatized HPLC- 0.11 — 70% Sun et al.
hexane, centrifuged. Aqueous layer extracted with DIB-Cl fluorescence ng/ml 0.9–8.7% RSD (intra- (2004)
with chloroform, organic layer evaporated to fluorescent (350/475 nm) day, n = 5); 4.7–10.4%
dryness, residue derivatized with DIB-Cl reagent RSD (inter-day, n = 5)
fluorescent reagent.
Urine β-Glucuronidase added to sample for BPA derivatized GC-MS in 0.1 — 83% Tsukioka
deconjugation. Deconjugated sample applied to with PFBBr negative ion ng/ml 7.4% RSD (n = 5) et al.
C18 SPE cartridge, eluted with methanol. chemical (2003)
Eluate was concentrated and derivatized with ionization
PFBBr. Derivatized sample cleaned up using a
Florisil column.
Serum, Ammonium acetate buffer, hexane, diethyl ether n/a HPLC- 1.4–2.8 — 78.6–95% Xiao et al.
tissues added to serum, centrifuged. Organic layer fluorescence ng/ml 0.1–3.0% RSD (intra- (2006)
evaporated to dryness, residue reconstituted (227/313 nm) assay, n = 7); 5.0–
with acetonitrile. 11.4% RSD (inter-
Tissue sample homogenized with ammonium assay, n = 7)
acetate buffer. Methanol and perchloric acid (4
mol/l) added, vortexed and centrifuged.
Ammonium acetate buffer added to
supernatant, loaded onto C18 SPE cartridge,
eluted with methanol. Eluate evaporated to
dryness, reconstituted with acetonitrile.
Urine β-Glucuronidase added to sample for BPA derivatized GC-MS/MS 0.38 — 62–124% Arakawa et
deconjugation. Deconjugated sample spiked with BSTFA ng/ml 9% RSD (n = 5) al. (2004)
with BPA-d16, extracted with dichloromethane.
Dichloromethane layer evaporated to dryness,
residue dissolved in hexane and applied to SPE
cartridge. BPA eluted with acetone, eluate
evaporated and derivatized with BSTFA.

14


Table 3 (continued)
detection precision
Urine β-Glucuronidase added to sample for BPA derivatized GC-MS in 0.12 — 101.6% Brock et al.
deconjugation. Formic acid and ammonium with PFBBr negative ng/ml 1.1–16% RSD (n = 3) (2001)
acetate buffer added to deconjugated sample, chemical
applied to C18 SPE column, eluted with ionization mode
methanol. Eluate derivatized with PFBBr.
Adipose Sample homogenized with hexane and BPA derivatized GC-MS in 0.5 — 95–105% Fernandez
tissue acetonitrile. Aqueous phase diluted with water, with BSTFA/TMCS electron impact ng/ml et al.
applied to C18 SPE cartridge, eluted with mode (2007)
diethyl ether/methanol (9:1 v/v). Eluate
derivatized with BSTFA/TMCS (1:1 v/v).
Human ELISA n/a ELISA — — — Ikezuki et
biological al. (2002)
fluids
Human Serum sample mixed with hydrochloric acid n/a HPLC- 0.05 79–87.3% Inoue et al.
serum (1 mol/l), methanol, water, applied to SPE electrochemical ng/ml 5.1–13.5% RSD (n = (2000)
cartridge, eluted with methanol. Eluate detection 6)
evaporated to dryness, residue reconstituted
HPLC-UV 150
with acetonitrile/water (50:50 v/v).
ng/ml
HPLC- 10 ng/ml
fluorescence
Human Semen sample acidified with hydrochloric acid, n/a LC-MS — 0.5 100.5% (relative); Inoue et al.
semen spiked with BPA-d16 and mixed with water. ng/ml 71.2% (absolute) (2002)
Then applied to SPE cartridge, eluted with ELISA — 2.0 4.7% RSD (n = 6)
methanol. ng/ml
Human SBSE BPA derivatized GC-MS in 20–100 100–500 95.2–100.7% Kawaguchi
body fluids β-Glucuronidase added to urine, plasma or with acetic electron impact pg/ml pg/ml 6.3–9.6% RSD (n = 6) et al.
saliva sample buffered with ammonium acetate anhydride ionization mode (2004)
for deconjugation. Deconjugated sample diluted
with water, derivatized with acetic anhydride,
extracted with stir bar coated with PDMS, and
then thermally desorbed.

15


Table 3 (continued)
detection precision
Blood Blood serum hydrolysed with β- n/a HPLC- 0.625 — 91–95% Lee et al.
glucuronidase/sulfatase overnight, extracted fluorescence µg/l 3.61–14.83% RSD (2008)
with MTBE. MTBE extract evaporated to (227/313 nm) (n = 5)
dryness, residue reconstituted with 60%
acetonitrile.
Human ELISA n/a ELISA 0.3 — 81.9–97.4% Ohkuma et
serum ng/ml al. (2002)
Urine Urine sample extracted with diethyl ether twice. n/a HPLC-ECD 0.2 — 103% Ouchi &
Ether phase evaporated to dryness, residue ng/ml 3–12% RSD (n = 4) Watanabe
reconstituted with acetonitrile. (2002)
β-Glucuronidase and buffer solution added to
urine sample to determine total BPA.
Fish tissue Dichloromethane/methanol (2:1 v/v) added to n/a LC-MS (APCI) — 50 ng/g 49–79% Pedersen
tissue sample, extracted for 25 min in 2.7–10% RSD (intra- & Lindholst
microwave extraction apparatus. assay, n = 6); 3.7– (1999)
Dichloromethane phase evaporated to dryness, 14.7% RSD (inter-
redissolved in methanol/hexane (1:20), applied assay, n = 6)
to SPE cartridge (Sep-Pak NH2), eluted with
methanol. Eluate evaporated to dryness,
redissolved in methanol.
Blood Serum or plasma sample diluted with water, n/a HPLC-ECD 0.2 — 93% Sajiki,
applied to SPE cartridge, eluted with ethyl ng/ml 2.9% RSD (n = 5) Takahashi
acetate. Eluate evaporated to dryness, &
redissolved in acetonitrile/water (40:60 v/v). Yonekubo
HPLC-MS (ESI) 0.1 — 93%
(1999)
ng/ml 7.0% RSD (n = 5)
Urine, Sample diluted with methanol, centrifuged, n/a LC-MS/MS 1.14 3.42 Recovery: 92–121% Volkel,
blood acetonitrile added, centrifuged again. ng/ml ng/ml (BPA); 90–120% Bittner &
plasma (BPA in (BPA in (BPA-gluc) Dekant
urine); urine); (2005)
10.1 26.3
ng/ml ng/ml
(BPA- (BPA-

16


Table 3 (continued)
detection precision
gluc in gluc in
urine) urine)

Maternal Serum or fluid applied to SPE cartridge, eluted n/a ELISA 0.2 — 3.5–10.8% RSD (intra- Yamada et
serum, with methanol/acetonitrile (3:1). Eluate ng/ml assay); 5.3–8.4% RSD al. (2002)
amniotic evaporated to dryness, reconstituted with (inter-assay)
fluid phosphate buffer for ELISA analysis.

Urine Sample mixed with enzyme solution (β- n/a HPLC-MS/MS 0.4 — 100% Ye et al.
glucuronidase/sulfatase in ammonium acetate (negative ion ng/ml 8–17% RSD (n = 60) (2005a)
buffer; 1 mol/l; pH 5.0) for deconjugation APCI)
overnight. Deconjugated solution diluted with
formic acid (0.1 mol/l) and centrifuged, applied
to C18 SPE cartridge in the online SPE-HPLC-
MS/MS system, eluted with methanol/water
(50:50).
Urine Sample mixed with ammonium acetate buffer n/a HPLC-MS/MS 0.3 — 98–113% Ye et al.
(1 mol/l; pH 5.0), enzyme added for (negative ion ng/ml 8–13% RSD (n = 60) (2005b)
deconjugation overnight. Deconjugated solution APCI)
diluted with formic acid (0.1 mol/l) and
centrifuged, applied to SPE cartridge in the
online SPE-HPLC-MS/MS system.
Human milk Sample mixed with ammonium acetate buffer n/a HPLC-MS/MS 0.28 — 93.7% Ye et al.
(1 mol/l), enzyme added for deconjugation. (negative ion ng/ml 8.2–11.4% RSD (n = (2006)
2-Propanol added to deconjugated solution, APCI) 50)
centrifuged. Supernatant diluted with formic acid
(0.1 mol/l), applied to SPE cartridge in the
online SPE-HPLC-MS/MS system.
Human milk Sample mixed with ammonium acetate buffer n/a HPLC-MS/MS 0.3 — 105% Ye et al.
(1 mol/l), enzyme added for deconjugation (negative ion ng/ml 6.3–8.3% RSD (n = (2008)
overnight. Methanol added to deconjugated APCI) 40)
solution, centrifuged. Supernatant diluted with
formic acid (0.1 mol/l), applied to SPE cartridge

17


Table 3 (continued)
detection precision
in the online SPE-HPLC-MS/MS system.
Human milk β-Glucuronidase added to sample for n/a HPLC- 0.6 1.8 65–82% Yi, Kim &
deconjugation. Deconjugated sample extracted fluorescence ng/ml ng/ml <15% RSD Yang
with 2-propanol, centrifuged. Supernatant (225/305 nm) (2010)
evaporated to dryness, reconstituted in 60% LC-MS/MS 0.39 1.3
acetonitrile. ng/ml ng/ml
13
Human Serum sample spiked with [ C12]BPA and BPA derivatized GC-MS in 5 pg/ml 15 pg/ml 101–100.9% Yoshimura
serum mixed with formic acid (to prevent BPA with PFBBr negative 4.76–5.42% RSD (n = et al.
ionization and protein precipitation). Sample chemical 6) (2002)
applied to C18 SPE cartridge, eluted with ionization mode
methanol. BPA conjugated with GC-ECD 0.15 —
tetrabutylammonium hydrogen sulfate as the pg/ml
counter-ion in alkali solution. The ion-paired
BPA moved from the aqueous phase to the
organic phase as an ion-paired extraction and
derivatized with PFBBr.
Blood, Placental sample mixed with water and ethyl BPA derivatized GC-MS in — 0.1 — Schön-
placental acetate. Plasma sample mixed with ethyl with BSTFA electron impact ng/ml felder et al.
tissue acetate. Supernatant derivatized with BSTFA. ionization mode (2002)
APCI, atmospheric pressure chemical ionization; BSTFA, N-O-bis(trimethylsilyl)trifluoroacetamide; DIB-Cl, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride; ECD, electron
capture detector; ECNI, electron capture negative ionization; EI, electron ionization; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; GC, gas
chromatography; gluc, glucuronide; HPLC, high-performance liquid chromatography; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MS, mass
spectrometry; MS/MS, tandem mass spectrometry; MTBE, methyl tert-butyl ether; MtBSTFA, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide; n/a, not applicable; NESI,
negative electrospray ionization; PBS, phosphate buffered saline; PDMS, polydimethylsiloxane; PFBBr, pentafluorobenzylbromide; PFBCl, pentafluorobenzoylchloride; ppb,
parts per billion; SBSE, stir bar sorptive extraction; SPE, solid-phase extraction; tBDMCS, tert-butyldimethylchlorosilane; THF, tetrahydrofuran; TMCS, trimethylchlorosilane;
UV, ultraviolet; v/v, volume/volume; w/v, weight/volume

18


also used to assist the solvent extraction of BPA from fish tissues (Pedersen & Lindholst,
1999).

2.1.3 Solid-phase extraction

Further cleanup of the extracts from solvent extraction is almost always necessary to remove
the co-extracted interferences. Solid-phase extraction (SPE), either alone or in combination
with solvent extraction, is the technique used most often for the extraction of BPA from both
liquid and solid food and biological samples and further cleanup of the extracts from solvent
extraction. The C18 (chemically bonded silica) and the Oasis HLB (lipophilic divinylbenzene
with hydrophilic N-vinylpyrrolidone polymer) are the two SPE cartridges used most
frequently for both food and biological samples (Brock et al., 2001; Watanabe et al., 2001;
Yoshimura et al., 2002; Tsukioka et al., 2003; Kuo & Ding, 2004; Mao et al., 2004; Liu,
Wolff & Moline, 2005; Shao et al., 2005; Sun, Leong & Barlow, 2006; Xiao et al., 2006;
Fernandez et al., 2007; Kuruto-Niwa et al., 2007; Cao et al., 2008; Dirtu et al., 2008;
Grumetto et al., 2008; Volkel, Kiranoglu & Fromme, 2008; Cao, Corriveau & Popovic, 2009;
Geens, Neels & Covaci, 2009; Markham et al., 2010). Further cleanup with Florisil cartridge
is sometimes also required (Casajuana & Lacorte, 2004; Dirtu et al., 2008). Although solvent
extraction is always necessary for solid samples, it may not be essential for some liquid
samples. For example, honey (Yan et al., 2009), infant formula (Biles, McNeal & Begley,
1997), soft drinks (Shao et al., 2005; Cao, Corriveau & Popovic, 2009), milk (Casajuana &
Lacorte, 2004; Maragou et al., 2006), urine (Mao et al., 2004; Liu, Wolff & Moline, 2005;
Moors et al., 2007) and blood serum and plasma (Sajiki, Takahashi & Yonekubo, 1999) were
applied to SPE cartridges directly after dilution with water or deconjugation with enzyme.

Immunoaffinity columns were also used to extract BPA and clean up the extracts from
solvent extraction for food (Braunrath & Cichna, 2005; Braunrath et al., 2005; Brenn-
Struckhofova & Cichna-Markl, 2006; Podlipna & Cichna-Markl, 2007), urine
(Schoringhumer & Cichna-Markl, 2007) and blood samples (Zhao et al., 2003). Compared
with the extracts cleaned up by the non-selective C18 SPE cartridges, immunoaffinity
columns demonstrated better efficiencies in removing matrix interferences as a result of their
selectivity. As the extracts were analysed by an LC-based system, cross-reactivity of other
compounds is not really an issue compared with the ELISA method. However, application of
the immunoaffinity columns is still very limited. This may be due to 1) the sensitivity of this
method being similar to that of the conventional methods; 2) the current conventional
methods working well; and 3) the preparation process of the immunoaffinity column being
very tedious.

2.1.4 Derivatization

Extracts were rarely analysed directly by GC-MS without derivatization (Casajuana &
Lacorte, 2004). The additional step of derivatization in sample preparation is almost always
required for accurate and sensitive quantitative analysis using GC-based methods because of
the two hydroxyl groups in BPA. This is optional for qualitative GC analysis; extracts
without derivatization have been analysed by GC-MS for confirmation purposes (Biles,
McNeal & Begley, 1997; Munguia-Lopez & Soto-Valdez, 2001; Munguia-Lopez et al.,
2002). For analysis by GC-MS in electron impact ionization mode, the derivatization
chemicals used most frequently are acetic anhydride (Goodson, Summerfield & Cooper,
2002; Kawaguchi et al., 2004, 2008; Thomson & Grounds, 2005; Cao et al., 2008; Cao,
Corriveau & Popovic, 2009; Vinas et al., 2010) and N-O-bis(trimethylsilyl)trifluoroacetamide

19


(BSTFA) (Arakawa et al., 2004; Kuo & Ding, 2004; Fernandez et al., 2007; Vinas et al.,
2010), whereas pentafluoropropionic acid anhydride (Dirtu et al., 2008) and
pentafluorobenzylbromide (PFBBr) (Brock et al., 2001; Yoshimura et al., 2002; Kuklenyik et
al., 2003; Tsukioka et al., 2003) and pentafluorobenzoylchloride (Geens, Neels & Covaci,
2009) were used for the derivatization of BPA for GC-MS analysis in electron capture
negative ionization mode.

For LC analysis with fluorescence detection, a few publications also reported derivatizing
BPA with the fluorescent reagents 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-
Cl) (Watanabe et al., 2001; Sun et al., 2002, 2004; Kuroda et al., 2003) or p-nitrobenzyl
chloride (Mao et al., 2004) to improve sensitivity by adding a stronger fluorophore to BPA.

2.1.5 Solid-phase microextraction

Solid-phase microextraction (SPME) works well for volatile chemicals, but not for
semivolatile and non-volatile chemicals in general, especially in complicated matrices such as
food and biological samples. Most of the applications of SPME for BPA are for simple
matrices such as water. Limited applications of SPME were explored for the determination of
BPA in milk (Liu et al., 2008) and the liquids of canned vegetables (Vinas et al., 2010), but
major issues with this method for BPA, such as the high blank level of BPA in the SPME
fibre, carry-over and matrix effects, were not addressed. The SPME method coupled to GC or
LC analysis could be used as a qualitative screening method for BPA, but, again, in simple
matrices only, and it is unlikely to see wide application in food and biological samples for
quantitative determination of BPA.

2.1.6 Stir bar sorptive extraction

Similar to SPME, stir bar sorptive extraction (SBSE) could be used as a qualitative screening
method for BPA in simple matrices such as water. Its applications for BPA in food and
biological samples are very limited (Kawaguchi et al., 2004) owing to issues such as carry-
over and matrix effects.

2.1.7 Coacervative microextraction

Coacervative microextraction is almost the same as liquid-phase microextraction and has
been investigated for the determination of BPA in foods (Garcia-Prieto et al., 2008a; Bendito
et al., 2009) and urine (Garcia-Prieto et al., 2008b). However, the relatively high limits of
detection (LODs) make this method much less attractive.

2.2 Separation and detection

2.2.1 Liquid chromatography–based methods

As BPA can be analysed by LC directly without the derivatization step in sample preparation,
LC is the technique used most often for the determination of BPA in both food and biological
samples. Various detectors, including UV, fluorescence, ECD, MS and tandem mass
spectrometry (MS/MS), have been used for the detection of BPA.

20


(a) Liquid chromatography–ultraviolet

The chromophore in the BPA molecule is relatively weak, and the sensitivity of UV detection
is low; thus, UV is rarely used for the detection of BPA. The LOD of the UV method for
BPA is at least 15 times higher than that of fluorescence detection. The limit of quantification
(LOQ) of UV detection at an emission wavelength of 228 nm for BPA ranged from 5–10
ng/g to 67 ng/g (3.7 ng/g for fluorescence detection) for food (Yoshida et al., 2001; Grumetto
et al., 2008) to 150 ng/ml (10 ng/ml for fluorescence detection) for human serum (Inoue et
al., 2000).

(b) Liquid chromatography–fluorescence

Fluorescence detection is the most frequently used non-MS-based method for LC
determination of BPA in both food and biological samples. The fluorophore in the BPA
molecule is fairly strong. The most common excitation wavelength used is 275 nm, with
slight variation, although lower wavelengths ranging from 224 to 235 nm have also been used
(Biles, McNeal & Begley, 1997; Munguia-Lopez & Soto-Valdez, 2001; Zhao et al., 2003;
Mao et al., 2004; Sun, Leong & Barlow, 2006; Xiao et al., 2006; Lee et al., 2008; Yi, Kim &
Yang, 2010). The emission wavelength used, on the other hand, is more consistent, ranging
from 300 to 317 nm. Detection limits of the LC-fluorescence methods for BPA varied,
depending on the sample matrices and the extraction methods used, from as low as the sub–
parts per billion (i.e. sub–nanogram per gram) level for most methods to as high as 15–29
ng/g for some other methods (Sun, Leong & Barlow, 2006; Bendito et al., 2009).

Fluorescent reagents with stronger fluorophores were also used to derivatize BPA in
biological samples. With excitation and emission wavelengths for BPA derivatized with DIB-
Cl at 350 and 475 nm, respectively, LODs as low as 0.04–0.05 ppb were reported by Kuroda
et al. (2003) and Watanabe et al. (2001), but an LOD as high as 4.6 ppb was reported by Sun
et al. (2002), indicating that this method is still not mature enough for wide application. The
excitation and emission wavelengths (228 and 316 nm, the same as for non-derivatized BPA)
used by Mao et al. (2004) may not be optimized for BPA derivatized with the fluorescent
reagent p-nitrobenzoyl chloride, and the LOD (2.7 µg/l) is typical for non-derivatized BPA.

Owing to the complex matrices of food and biological samples, non-MS-based methods are
likely to generate false-positive results; thus, confirmation by MS is essential. However,
among all the results generated by LC-fluorescence methods, only a few investigators
confirmed the results by LC-MS (Inoue et al., 2003a; Schoringhumer & Cichna-Markl, 2007)
or GC-MS (Biles, McNeal & Begley, 1997).

(c) Liquid chromatography–electrochemical detector

Limited applications of ECD for LC determination of BPA in both food and biological
samples were reported (Sajiki, Takahashi & Yonekubo, 1999; Inoue et al., 2000; Ouchi &
Watanabe, 2002; Liu, Wolff & Moline, 2005; Sajiki et al., 2007). However, this method has
no more benefit in terms of LOD (sub-ppb levels) than the other non-MS-based methods and
thus will be unlikely to find wide application as MS-based instruments become more
affordable.

21


(d) Liquid chromatography–mass spectrometry or liquid chromatography–tandem mass
spectrometry

LC-MS or LC-MS/MS is the second most frequently used LC method after LC-fluorescence
for the determination of BPA in both food and biological samples, and it provides much more
confidence in peak identification based on the mass spectrum. The additional advantage of
MS-based methods is the use of isotope-labelled BPA, such as BPA-d16, BPA-d14 and
[13C]BPA. By spiking samples with isotope-labelled BPA at the beginning of the sample
extraction stage, matrix effect, loss of analyte, variations in extract volume, etc. can be
corrected; thus, the method will have better precision and accuracy. However, this advantage
has not been fully used in all LC-MS-based methods for BPA, and isotope-labelled BPA was
used in only some of the methods (Inoue et al., 2002; Volkel, Bittner & Dekant, 2005; Ye et
al., 2005a,b, 2006; Maragou et al., 2006; Ackerman et al., 2010; Markham et al., 2010).

Both negative ion electrospray ionization (ESI) and atmospheric pressure chemical ionization
(APCI) have been used to generate gas-phase ions in LC-MS. The most abundant ion in the
BPA mass spectrum is m/z 227 ([M-H]−), and it is used for the quantification of BPA in LC-
MS analysis in selected ion monitoring mode. In LC-MS/MS, one or more MS/MS
transitions of precursor ion m/z 227 to product ion m/z 133 or m/z 212 were monitored for the
quantification and confirmation of BPA.

Although LC-MS/MS provides more information on product ions and thus more confidence
in peak identification compared with LC-MS, the sensitivities of the two methods were
similar, around the sub-ppb level. The extremely low LOD (0.6 ng/l) of the LC-MS/MS
method reported by Shao et al. (2005) is questionable, as they failed to detect any BPA in
canned soft drink products (they should have been able to detect BPA with the claimed
LOD), and, in their later publication, the LOD of the same method for egg and milk was as
high as 0.1 ng/g (Shao et al., 2007).

The other advantage of LC methods, especially LC-MS-based methods, is that free BPA and
conjugated BPA in a sample extract could be separated by LC and detected simultaneously;
thus, deconjugation of the sample by enzymes is not needed. This was demonstrated by
Volkel, Bittner & Dekant (2005); LC-MS/MS was used to analyse BPA and BPA-
glucuronide in urine extracts simultaneously, with LOQs of 3.42 µg/l and 26.3 µg/l,
respectively. Two MS/MS transitions of precursor ion m/z 403 to product ions m/z 113 and
m/z 227 were monitored to quantify and confirm BPA-glucuronide.

2.2.2 GC-MS

GC-MS is also one of the methods frequently used for the determination of BPA in both food
and biological samples because of its higher resolution and lower LOD compared with LC-
MS methods, despite the tedious derivatization step required. Although derivatization of BPA
is not essential for confirmation purposes (Biles, McNeal & Begley, 1997), quantitative
determination of BPA using GC-MS without derivatization is rare (Casajuana & Lacorte,
2004). Symmetrical peaks could still be obtained for underivatized BPA with new GC
columns, especially with thick coating films, but the performance will start to deteriorate
after a few injections. Thus, derivatization of BPA is always recommended for quantitative
analysis by GC-MS. For analysis by GC-MS in electron impact ionization mode, the
derivatization chemicals used most frequently are the acetylation reagent acetic anhydride
(Goodson, Summerfield & Cooper, 2002; Kawaguchi et al., 2004, 2008; Thomson &

22


Grounds, 2005; Cao et al., 2008; Cao, Corriveau & Popovic, 2009; Vinas et al., 2010) and the
silylation reagents BSTFA with or without the stimulator trimethylchlorosilane (TMCS)
(Arakawa et al., 2004; Kuo & Ding, 2004; Fernandez et al., 2007; Vinas et al., 2010) and N-
(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MtBSTFA) with tert-butyldimethyl-
chlorosilane (tBDMCS) (Moors et al., 2007). Pentafluoropropionic acid anhydride (Dirtu et
al., 2008), PFBBr (Brock et al., 2001; Yoshimura et al., 2002; Kuklenyik et al., 2003;
Tsukioka et al., 2003) or pentafluorobenzoylchloride (Geens, Neels & Covaci, 2009) was
used for the derivatization of BPA for GC-MS analysis in electron capture negative
ionization or negative chemical ionization mode.

The electron ionization (EI) mass spectrum of BPA derivatized with acetic anhydride (BPA
diacetyl) is similar to that of underivatized BPA, with m/z 213 being the most abundant ion
(used for quantification) and other ions (m/z 228, 270, 312) used for confirmation. The most
abundant ion in the EI mass spectrum of BPA derivatized with BSTFA is m/z 357 (used for
quantification), and ion m/z 372 is used for confirmation. The molecular ion m/z 616 is the
most abundant for BPA derivatized with pentafluorobenzoylchloride in its electron capture
negative ionization mass spectrum, with m/z 406 [M-C6F5COCH3]− being the confirmation
ion. The most abundant ion for BPA derivatized with PFBBr is m/z 407, which is due to the
loss of a pentafluorobenzyl group from the pentafluorobenzyl diether of BPA during
chemical ionization (Brock et al., 2001; Kuklenyik et al., 2003; Tsukioka et al., 2003).
Although Yoshimura et al. (2002) claimed that only one of the two hydroxyl groups in BPA
was derivatized by PFBBr, and thus m/z 407 is the molecular ion, Brock et al. (2001)
confirmed the identity of the pentafluorobenzyl diether of BPA by its EI mass spectrum in
which both the molecular ion m/z 588 [M]+ and another ion m/z 573 [M-CH3]+ (the most
abundant ion) were observed.

Isotope-labelled BPA has been used in almost all GC-MS analyses of food and biological
samples for BPA. Although some of the early GC-MS methods showed relatively high LODs
(Goodson, Summerfield & Cooper, 2002; Thomson & Grounds, 2005), the majority of the
GC-MS methods for BPA showed good sensitivity, with LODs at sub-ppb levels.

A GC-MS/MS method is also reported for the determination of BPA in urine (Arakawa et al.,
2004). MS/MS transitions of precursor ion m/z 357 to product ions m/z 191, 267, 341 were
monitored for BPA derivatized with BSTFA. However, this method had no obvious benefit in
terms of LOD (0.38 ng/ml) compared with the GC-MS methods.

2.2.3 Enzyme-linked immunosorbent assay

Efforts were made in the early 2000s to develop ELISA methods for BPA (Ohkuma et al.,
2002). Commercial ELISA kits for BPA are now available (IBL International; Japan
EnviroChemicals Ltd) and have been used for the determination of BPA in biological
samples (Ikezuki et al., 2002; Yamada et al., 2002; Fukata et al., 2006; Kuruto-Niwa et al.,
2007). Although the ELISA method for BPA is convenient and popular among non-analytical
chemists, it should be used with care.

Cross-reactivity is one of the issues with the ELISA method. The ELISA method cannot
distinguish between free BPA and conjugated BPA, as both can generate responses with the
kit. Cross-reactivity of the ELISA kit for BPA from IBL International is as high as 85% for
BPA-glucuronide and 68% for BPA-sulfate. Cross-reactivities of chemicals with structures
similar to BPA are also relatively high: 15.6% for bisphenol B and 6.0% for bisphenol E for

23


the ELISA kit for BPA from Japan EnviroChemicals Ltd. Thus, ELISA results must be
confirmed by GC-MS or LC-MS for peak identity.

The ELISA method should be validated for the matrices to be applied, and results should be
compared with those obtained with well-established methods at different levels for accuracy.
As the ELISA method for BPA is not accurate at levels around its LODs (sub-ppb), it is not
suitable for the determination of BPA at low levels in any matrices.

Direct analysis for BPA without sample preparation using the ELISA method is possible only
for a simple matrix such as water. For food and biological samples, sample preparation and
treatment (solvent extraction followed by SPE, etc.) are still required to generate clean
extracts for analysis by ELISA (Kuruto-Niwa et al., 2007). It is thus logical to predict that
ELISA methods are unlikely to be applied widely for the determination of BPA in food and
biological samples, even for qualitative screening purposes. ELISA can be a good fast
screening method for BPA, but, again, only for samples with a simple matrix such as water.

2.3 Method validation

The published methods used for the determination of BPA in food and biological samples
have been validated for free BPA to a certain extent. Certified reference materials for BPA
are not available; thus, in-house reference materials have been used to check accuracy in
single-laboratory validations. Method performance parameters, summarized in Tables 2 and
3, were acceptable in general. For biological samples, however, there is almost no evidence
of the methods being validated for conjugated BPA. The only study in which the method was
validated for conjugated BPA does not involve the deconjugation step with enzymes to
convert conjugated BPA to free BPA, as the conjugated BPA was analysed directly with LC-
MS/MS together with free BPA (Volkel, Bittner & Dekant, 2005). This could be partly due to
the unavailability of conjugated BPA standards from reliable sources. Considering the fact
that results from biomonitoring have been used for BPA exposure assessments and the
majority of the BPA in biological samples is in the conjugated form, validation of methods
for conjugated BPA will be essential to ensure the validity of the results. Information on
validation of ELISA methods is very limited. No method performance parameters were
provided at all for the method used to determine BPA levels in human placenta samples
(Schönfelder et al., 2002); thus, the validity of those results is uncertain.

Proficiency test programmes for BPA, such as the Food Analysis Performance Assessment
Scheme (FAPAS) programme, are available, and some laboratories have participated in these
tests regularly or occasionally. Although most laboratories performed well with the analysis,
there are still some (about 10%) that reported unacceptable results, with z-scores greater than
2.0 in the 2009 and 2010 FAPAS proficiency tests for BPA. The samples used in proficiency
tests are usually simple matrices, such as alcohol or oil commonly used as food simulants in
migration studies. Thus, proficiency tests are limited in testing the method robustness, and
interlaboratory studies should be conducted using real food or biological samples.

3. CONCLUSIONS AND RECOMMENDATIONS
Sensitive and reliable analytical methods are available for the determination of BPA in both
food and biological samples. Solvent extraction and SPE are the most commonly used and
most effective methods for the extraction of BPA in food and biological samples. Although

24


isotope dilution methods based on MS and MS/MS are the most reliable for the detection of
BPA, many of the results of BPA determination in both food and biological samples have
been generated by non-MS-based methods.

The majority of methods used to measure free and total BPA in food and biological samples
have been validated for certain performance parameters, such as accuracy, precision,
recovery and LOD. Most methods fulfil the requirements of single-laboratory validation. For
biological samples, however, validation of methods for conjugated BPA is very limited; only
one study validated its method for conjugated BPA for some parameters. Proficiency testing
programmes for measuring BPA are available, and some laboratories have participated
regularly or occasionally, but validation of methods for BPA through interlaboratory
collaborative studies has not yet been conducted. It is difficult to rule out cross-contamination
with trace levels of free BPA during sample collection, storage and analysis because of the
ubiquitous presence of BPA in the environment.

The Expert Meeting recommends that:

• Analytical methods should be validated according to published guidelines for single-
laboratory validation, such as the IUPAC guidelines (Thompson, Ellison & Wood, 2002),
to include at least the following method performance parameters: LOD, LOQ,
repeatability, recovery, linearity and range of calibration curve.
• MS- or MS/MS-based isotope dilution methods should be used for the determination of
BPA whenever possible. Results from non-MS-based methods should be confirmed by
MS methods, especially for food and biological samples.
• The ELISA method could be used for screening purposes, but it is not adequate for the
quantitative determination of BPA in food and biological samples.
• Efforts should be made to produce commercially available, high-purity conjugated BPA
standards for method validation purposes for biological samples.
• Efforts should be made to avoid cross-contamination during sample preparation and
analysis, particularly when measuring unconjugated BPA concentrations, and method
blanks and certified reference materials (if available) should be included in the analysis.
• Laboratories are encouraged to participate in current proficiency testing programmes to
assess the reliability of the data they are producing.
• Interlaboratory studies should be conducted to validate methods for different types of
food and biological samples.

REFERENCES
Ackerman LK et al. (2010). Determination of bisphenol A in U.S. infant formulas: updated methods and
concentrations. Journal of Agricultural and Food Chemistry, 58:2307–2313.
Arakawa C et al. (2004). Daily urinary excretion of bisphenol A. Environmental Health and Preventive
Medicine, 9:22–26.
Bendito MD et al. (2009). Determination of bisphenol A in canned fatty foods by coacervative microextraction,
liquid chromatography and fluorimetry. Food Additives & Contaminants. Part A, Chemistry, Analysis,
Control, Exposure & Risk Assessment, 26(2):265–274.
Biles JE, McNeal TP, Begley TH (1997). Determination of bisphenol A migrating from epoxy can coatings to
infant formula liquid concentrates. Journal of Agricultural and Food Chemistry, 45:4697–4700.
Braunrath R, Cichna M (2005). Sample preparation including sol-gel immunoaffinity chromatography for
determination of bisphenol A in canned beverages, fruits and vegetables. Journal of Chromatography A,
1062(2):189–198.

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