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Journal of Chromatography A, 1216 (2009) 449469

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review

Analytical methods for the determination of bisphenol A in food


Ana Ballesteros-Gmez, Soledad Rubio , Dolores Prez-Bendito
Department of Analytical Chemistry, Facultad de Ciencias, Edicio Anexo Marie Curie, Campus de Rabanales, 14171 Crdoba, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Food constitutes the primary route for human exposure to bisphenol A (BPA), one of the highest vol-
Available online 3 July 2008 ume chemicals produced worldwide. The estrogenic properties of BPA, its wide dispersive use and the
recent extensive literature describing low-dose BPA effects in animals, have raised concerns about its
Keywords: possible adverse effects on human health. A reliable health risk assessment of BPA relies basically on its
Bisphenol A unambiguous identication and accurate quantication in food, and the aim of the present review is to
Review
give an overview of the analytical methods reported so far for the determination of BPA in these matri-
Endocrine disrupters
ces. Emphasis is placed on the main strategies developed for sample treatment, which usually consists
Food analysis
Sample treatment
of several laborious and time-consuming steps in order to achieve the required sensitivity and selectiv-
Mass Spectrometry ity. Separation, identication and quantitation of BPA is today reliably made with mass spectrometric
Chromatography methods, namely liquid chromatographymass spectrometry (LCMS) and gas chromatographymass
spectrometry (GCMS), and thus main attention is devoted to these techniques, but other methods using
LC coupled to uorescence or electrochemical detection, as well as immunochemical methods are also
covered. Recent and expected future developments are discussed.
2008 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
2. Sources and removal of background contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
3. Sample treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
3.1. Solvent-based extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
3.1.1. Solvent extraction (SE) and liquidliquid extraction (LLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
3.1.2. Microwave-assisted extraction (MAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
3.1.3. Pressurized liquid extraction (PLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3.2. Solid-phase extraction (SPE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3.2.1. Non-selective sorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3.2.2. Selective sorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
3.3. Less common extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
3.3.1. Solid-phase microextraction (SPME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
3.3.2. Stir bar sorptive extraction (SBSE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.3.3. Matrix solid-phase dispersion (MSPD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
4. Separation and detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.1. Liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.1.1. Fluorescence detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.1.2. Electrochemical detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.1.3. LCMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4.2. GCMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
4.3. Immunochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

Corresponding author. Tel.: +34 957 218643; fax: +34 957 218644.
E-mail address: qa1rubrs@uco.es (S. Rubio).
0021-9673/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.06.037
450 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

1. Introduction included in the list of substances subject to authorization in the


new policy on chemicals approved by the EU, REACH (Registration,
Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl) propane, is one of Evaluation, Authorisation and Restriction of Chemicals, Annex XIV)
the highest volume chemicals in the world [1,2]. Global demand for [26]. So, there are needs for research (new methods) and for revision
BPA is predicted to grow from 3.9 million tonnes in 2006 to about and optimization of existing methods in order to have reliable tools
5 million tonnes in 2010 [3]. Many countries throughout the world for risk assessment and control of human exposure to BPA.
have large production capacities for BPA, especially Germany, the Although different authors [18,20,27] and regulatory agencies
Netherlands, the USA and Japan. BPA capacity in West Europe was [6,7] have offered reviews concerning the toxicological properties
estimated at 830 million tonnes in 2000 [4], it grew by 4%/year from of BPA and the levels of this contaminant in human tissues and
2000 to 2006 and it has been predicted to grow by 2%/year in the uids, no review dealing with the determination of BPA in food
20062010 period [3]. The main market for BPA is the production has been reported so far. Here, we summarize the state-of-the-
of polycarbonate with the second largest outlet being epoxy resins. art of the analytical methodologies developed, including strategies
Other uses include ame retardants, unsaturated polyester resins for removal of background contamination, sample preparation and
and polyacrylate, polyetherimide and polysulphone resins [5,6]. A separation and detection of BPA. Conventional methods and recent
wide variety of food contact materials stand out among their uses, developments will be critically discussed in terms of simplicity,
mainly derived from polycarbonates (infant feeding bottles, table- robustness, sample size, cost, consumption of organic solvents, sen-
ware, microwave ovenware, storage containers, returnable water sitivity and selectivity, highlighting their main drawbacks and the
and milk bottles and water pipes) and epoxy resins (internal pro- need for future developments.
tective lining for food and beverage cans, coating on metal lids
for glass jars and bottles and surface-coating on drinking water
2. Sources and removal of background contamination
storage tanks and wine vats) [7]. The chemical structure and some
physico-chemical properties of BPA are shown in Table 1.
BPA is inherently ubiquitous in the environment. Background
The extensive use of BPA-based polymers, with ester bonds
contamination of BPA occurs at ng L1 levels and mainly arises from
subject to hydrolysis and non-polymerized monomer residues,
solvents, SPE columns, glassware, plastic ware and other reagents
has led to widespread environmental contamination. BPA con-
and laboratory tools. In general, heat-treated glassware (4 h at
centrations in the ranges 5320 ng L1 in river waters [810],
400 C) and solvent-washed materials are used as a precautionary
20700 ng L1 in sewage efuents [911], 2208 ng m3 in air
measure to prevent background contamination [28].
[1214], 0.2199 ng g1 in dust [1214] and 0.1384 ng g1 in food-
BPA concentrations around 0.02 g L1 have been found in
stuffs [1517] have been reported. Its presence in food is of special
Milli-Q water using highly sensitive methods (instrumental quan-
concern since it constitutes the primary route of human exposure
tication limit of 5 pg) [2931]. The contamination arose from the
[6,7,14]. The scientic panel on food additives, avourings, process-
plastics used in the purication system and was removed by l-
ing aids and materials in contact with food of the European Union
tering the water through a hydrophobic membrane (Empore disk).
(EU) has reported estimates of potential dietary exposure of 13,
Other authors, however, did not nd BPA contamination when they
5.3 and 1.5 g/kg body weight/day in 6- to 12-month-old breastfed
analysed different ultra-high quality waters, such as Water Pes-
infants, young children and adults, respectively [7]. The widespread
tanal from Riedel-de Han or those obtained from an Elgastast
human exposure to BPA has been highlighted by measurements
and a Millipore Milli-Q system, although in these experiments the
in human uids and tissues (reviewed in [18]). Concentrations
instrumental quantication limit was around 10 ng [32].
in blood and urine were on average in the 0.34.4 g L1 and
SPE cartridges (e.g. Oasis HLB from Waters and Bond Elut Cer-
0.479.5 g L1 ranges with a detection rate above 90% in most of
tify from Varian) have been known to cause BPA contamination at
the studies.
concentrations of around 0.04 g L1 [31]. The contamination was
The estrogenic activity of BPA was rst reported in 1993 [19].
effectively removed by pre-washing the cartridges which at least
The afnity of BPA for estrogen receptors is 10,000- to 100,000-
15 mL of methanol and was probably derived not from the sorbent
fold weaker than that of estradiol, so it has been considered a very
material, but from the manufacturing process. Sample loading in
weak environmental estrogen. However, a large number of recent
SPE can introduce strong BPA contamination when glass syringes
in vitro studies have shown that the effects of BPA are mediated by
are utilized, mainly caused by the adhesive used to x the needle
both genomic and non-genomic estrogen-response mechanisms,
[30,32]. Using a peristaltic pump with vinyl tubes, the BPA con-
with the disruption of the cell function occurring at doses as low
tamination became insignicant although the contamination was
as 1 pM (0.23 ng L1 ) (reviewed in [20]). Recent reports also indi-
important for two BPA derivatives (i.e. 4-tert-butylbenzoic acid,
cate the potential of BPA to disrupt thyroid hormone action [21], to
BBA, and 4-tert-butylphenol, t-BP). Contamination from SPE sam-
cause proliferation of human prostate cancer cells [22] and to block
ple loading can be almost completely eliminated by replacing the
testosterone synthesis [23] at very low part-per-trillion doses. This
vinyl tubes with Viton tubes from DuPont [32].
extensive new literature concerning low-dose effects of BPA has
given rise to controversy about the BPA limit values set by reg-
ulatory agencies for consumer health protection and a new risk 3. Sample treatment
assessment has been strongly recommended [20]. Currently, the
tolerable daily intake (TDI) set by the EU Commission [7] and the There are a wide variety of BPA-containing foods including fresh
reference dose (RfD) established by EPA [24] is 0.05 mg BPA/kg and canned solid and liquid samples (Fig. 1). Determination of
body weight/day. This value was derived by applying a 100-fold BPA in these matrices often requires extensive sample prepara-
uncertainty factor to the currently accepted overall Non-Observed- tion prior to instrumental analysis. The typical steps within food
Adverse-Effect Level (NOAEL) of 5 mg/kg. On the other hand, a sample preparation include pre-treatment, extraction, clean-up,
specic migration limit (SML) for BPA from food contact plastic concentration and sometimes derivatization, and constitute the
materials of 600 ng g1 was set by the EU Commission in 2004 bottleneck in current food analysis (Fig. 1). The solid samples
[25]. are usually rst homogenized while the liquid ones are ltered
Because of the high volume, wide dispersive use and endocrine and/or centrifuged. Special treatments can be required depend-
disrupting and toxic properties of BPA, it is a clear candidate to be ing on the matrix composition; e.g. carbonated beverages are
A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469 451

Table 1
Physico-chemical properties of bisphenol A

Chemical structure Molecular mass Water solubility (mg L1 ) log Kow b pKa H donor and acceptor suma

227 89 3.25 9.7 4

a
Calculated using Advanced Chemistry Development (ACD/Labs) Software V9.04 for Solaris.
b
Logarithm of the octanolwater partition coefcient.

degassed [16,33], samples with high protein content may require 3.1. Solvent-based extraction
their removal by precipitation and sh tissues are crushed and
freeze-dried before homogenization. Canned foods containing both 3.1.1. Solvent extraction (SE) and liquidliquid extraction (LLE)
liquid and solid portions are usually ltered and treated sepa- Solvent extraction (SE) is still the most common technique for
rately. the isolation of BPA from solid foodstuffs (cf. Table 3). Liquidliquid
Solvent extraction and SPE are the most widely used techniques extraction (LLE), despite being a reliable technique for the extrac-
for the isolation of BPA from solid and liquid samples, respectively, tion of BPA from liquid foods, has been used to a lesser extent than
mainly because of its simplicity and wide-range applicability. Other SPE. Most extraction methods have been developed for specic food
techniques (e.g. MAE, PLE, MSPDE, SPME, SBSE, cf. Fig. 1), although matrices such as sh [38,39], fruit and vegetables [40,41], yoghurt,
scarcely used so far, may improve the extraction of BPA in terms cream, butter, and pudding [42], infant formula powders [43], pet
of sample sizes, automation and solvent consumption. The extracts foods [44] and mineral water [45], and only few of them are applica-
containing BPA are commonly subject to extensive clean-up and in ble to a wide range of foods. In this respect, Goodson et al. proposed
this respect liquidliquid extraction and SPE are preferred. Con- a method for the extraction of BPA and isomers of bisphenol F from
centration of BPA by solvent evaporation of the nal extract is a variety of canned products, including sh, fruit, vegetables, bever-
an inevitable step because of its low concentration in food (cf. ages, soup, desserts, infant formula, meat and pasta [16], which was
Table 2). In general, the combination of different techniques is usu- subsequently applied by Thomson et al. to a wide array of foodstuffs
ally inevitable and methods become frequently matrix-dependent. [17].
Only those methods intended to study the migration of BPA from The amount of sample required for the extraction of BPA from
unused commercial cans by extraction with food-simulating liquids solid an liquid food is usually in the ranges 0.530 g (being 5 g
are simpler [3437]. Table 3 gives some representative examples the most frequent selected size [39,40,41,44,46]) and 1050 mL
of sample treatment procedures for the analysis of BPA-containing [1517,33], respectively, although higher sample sizes have also
foods. Below, the main techniques used for extraction and clean-up been proposed, e.g. 120 g for canned jalapeno peppers [47] and
are discussed in detail. 500 mL for mineral water [45]. Among solvents, acetonitrile is

Fig. 1. Analytical methodologies for the determination of bisphenol A in foodstuffs. Abbreviations: SE, solvent extraction; MAE, microwave-assisted extraction; PLE, pres-
surized liquid extraction; MSPS, matrix solid-phase dispersion; LLE, liquidliquid extraction; SPE, solid-phase extraction; SPME, solid-phase microextraction; SBSE, stir bar
sorptive extraction; FL, uorescence detection; ED, electrochemical detection.
452 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

Table 2
Levels o BPA in foods reported by several authors

Foodstuff Number of samples (number of BPA concentration in Reference


positive quantiable samples) positive quantiable
samples

Fruit and vegetables 14(8) 18.495 ng g1 a Yoshida et al. [40]


10(6) 29458 ng mL1 b Brotons et al. [48]
10(10) 948 ng g1 Goodson et al. [16]
33(11) 1224 ng g1 Thomson et al. [17]
10(10) 535 ng g1 Braunrath et al. [78]

Milk 8(2) 7.1115.2 ng g1 Maragou et al. [65]


10(3) 0.410 ng g1 Shao et al. [114]

Milk or soy-based infant formula 5(5) 44113 ng g1 Kuo and Ding [43]
14(14) 0.113.2 ng mL1 Biles et al. [15]

Fish 8(4) 24109 ng g1 Thomson et al. [17]


10(9) 1043 ng g1 Goodson et al. [16]
2(2) 2.143 ng g1 a Braunrath et al. [78]
7(5) 0.31.0 ng g1 Shao et al. [28]

Seafood 5(5) 13213 ng g1 Basheer et al. [51]

Meat 6(2) 2998 ng g1 Thomson et al. [17]


2(2) 9.622.0 ng g 1 Braunrath et al. [78]
20(8) 0.37.1 ng g1 Shao et al. [28]

Eggs 10(1) 0.5 ng g1 Shao et al. [114]

Soup and sauces 8(4) 1121 ng g1 Thomson et al. [17]


10(4) 821 ng g1 Goodson et al. [16]
4(4) 9.637.6 ng g1 Braunrath et al. [78]

Infant formula, other than milk or non-specied 13(11) 9384 ng g1 Goodson et al. [16]

Honey 107(17) 333 ng g1 Inoue et al. [62]


Pet foods 26(26) 13206 ng g1 Kang and Kondo [44]

Wine (vat) 10(8) 0.22.1 ng mL1 Brenn-Struckhofova and Cichna-Markl [79]


(Bottle) 48(37) 0.21.6 ng mL1 Brenn-Struckhofova and Cichna-Markl [79]

Soft-drinks 7(6) 0.13.4 ng mL1 Braunrath et al. [78]


Mineral water (PET bottles) 9(9) 310 ng L1 Toyooka and Oshige [45]
a
Concentrations referred to the solid portion of the container.
b
Concentrations referred to the liquid portion of the container.

usually preferred for extraction of solid foods [16,17,39,40,42,44,46] of sample treatment procedures [38,4044,46]. After that, the evap-
although others like acetone [41], methanol [39,47] and ethanol oration of elutes, reconstitution with organic solvent and ltration
[43] may also extract BPA efciently. Liquid foods are extracted with (0.200.5 m) completes the sample treatment. Although overall
ethyl acetate [45], chloroform [48] or dichloromethane [33]. As a recoveries of these procedures are usually well above 75%, low
general rule, repeated extractions are usually necessary to ensure recoveries (<50%) due to matrixanalyte interactions were reported
the complete isolation of BPA. Typical overall solvent consump- by Thomson et al. for a variety of foods [17]. Kuo et al. also obtained
tion, including repeated extractions and washing, is between 40 low recoveries (18%) and high relative standard deviation (RSD)
and 300 mL. Extraction times range from 10 min to 120 min using (24%) in a specic type of infant formula powders, namely a hypoal-
stirring or sonication to favour equilibrium partition. Sodium anhy- lergenic brand, this fact being related to the presence of hydrolyzed
drous sulphate is frequently added to the sample before [40,41] or enzymes inuencing the SPE [49]. Table 3 compares several solvent-
after extraction [16,17] in order to remove trace amounts of water based extraction methods in terms of overall solvent consumption,
in the organic layer. The importance of controlling the enzymatic concentration factors and recoveries, among other characteristics.
degradation of BPA during extraction in samples such as fresh fruit
and vegetables has been highlighted since it leads to poor recover- 3.1.2. Microwave-assisted extraction (MAE)
ies [41]. Degradation may be prevented by addition of 0.1N HCl to MAE is based on the application of microwave energy to the sam-
the extractant solvent [41]. ple during extraction, which is consequently agitated and heated
Because of the limited selectivity of solvent-based extractions quickly. It constitutes a good alternative to the solvent extraction of
there is a need for extensive clean-up prior to instrumental anal- solid and semi-solid food samples because good extraction efcien-
ysis. Removal of lipids from the extract is essential for samples of cies can be achieved using less solvent and shorter extraction times.
animal origin (e.g. sh, meat) since they can signicantly reduce The application of MAE to the extraction of BPA-containing foods
the analytical performance of LC and GC. Lipidic material affects has been a rather limited and restricted to sh [50,51]. Low sample
the active surface of the stationary phase in LC and degrades the sizes (typically between 0.2 and 1 g of thawed and crushed tissue)
resolution power of the column. In GC/MS, lipids accumulate in the were subject to MAE with 20 mL of dichloromethane:methanol (2:1,
injection port, column and ion source. Fat removal is mainly made v/v) [50] or 10 mL (20% water content) of tetramethylammonium
by liquidliquid extraction with n-heptane [16], trimethylpentane hydroxide (TMOH) and 1 mL n-nonane [51] for 1520 min. SPE (Sep-
[17] and n-hexane [39,40,46] or by freezing the lipids in the extract Pak NH2 ) [49] and Oasis-HLB [50] were used for clean-up of the
at 24 C for 40 min followed by ltration [38]. The use of SPE for extracts with the subsequent solvent evaporation and reconstitu-
clean-up of the BPA-containing extracts becomes inevitable in most tion prior to LC/MS [50] or GC/MS [51]. Average recoveries were
Table 3
Sample preparation methods for determination of BPA in foodstuffs and food simulants

Sample type (size) Sample preparation steps Extraction conditions Overall solvent Solvent BPA recovery (R) and Phase volume Separation/detection Reference
consumptiona (mL) evaporation b
RSD (%) ratio for liquid technique
samples

Infant formula - Diluted with 100 mL - SPE (PS-DVB) 34 Yes R(n = 3): 67106 2.57.5 LCFL (conrmation Biles et al.
concentrates (milk water by GCMS) [15]
and soy-based
products)
(1030 mL)
- SPE extraction R(n = 3): 227

Fish (10 g) - Solvent extraction - Methanol (70 mL) 115125 Yes R: 105120 GCMS Gyong et al.
[38]
- Delipidation (freezing - Sonication (30 min) RSD: 514 (n = 7)
lipid)
- SPE clean-up (Oasis HLB
and Florisil cartridges)
- Derivatization

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


Vegetables, desserts, - Solvent extraction - Acetonitrile (40 mL 4575 (5 for beverages) Yes R: 81103 10 for GCMS Goodson et
sh, soup (20 g) (except for beverages) except for infant beverages al. [16]
formula, 20 mL)
Infant foods (10 g) - Clean-up of crude RSD(mean in a can of
extracts of fatty samples salmon, n = 6): 4.6
by extraction with an
apolar solvent
Beverages (50 mL) - Derivatization

Vegetables, fruit, Similar to those used by Similar to those used by 75 (5 for beverages) Yes R: 42112 10 for GCMS Thomson et
sh, soup and Goodson et al. [16] Goodson et al. [16] beverages al. [17]
sauces, canned
meat, spaghetti,
baked beans,
infant foods (20 g)
Beverages (50 mL) RSD(mean in a can of
beetroot, n = 8)
Fatty-food simulant - Freeze-drying (for tuna - Acetonitrile (20 mL) for 38 (food simulants) Yes R(mean): 91 (food LCFL (conrmation Mungua-
(commercial sh). food simulant simulant), 77 (tuna by GCMS) Lpez et al.
sunower oil) (5 g) sh) [39]

Tuna sh (7 g). - Solvent extraction - Methanol (100 mL) for 205 (tuna sh) RSD(mean): 5 (food
tuna sh simulant), 9 (tuna
sh)
- Clean-up of crude
extracts by extraction
with an apolar solvent
Beverages (10 mL) - Solvent extraction Dichloromethane 60 Yes R: 99.5105.7 50 GCMS Varelis and
(60 mL) Balafas [33]
- Two times aqueous RSD(n = 5): 1.33.3
back-extraction for
clean-up
- Derivatization

Fruit, vegetables and Solid portion of the can: - Solid portion: 176 (solid portion) Yes R(mean in corn 5 for the liquid LCUV Yoshida et
corn (5 g solid solvent extraction, acetonitrile (130 mL) infusion, n = 3): 8790 portion al. [40]
portion, 10 mL clean-up of the crude
liquid portion) extract by extraction
with an apolar solvent
and subsequent SPE
(Florisil)

453
454
Table 3 (Continued )

Sample type (size) Sample preparation steps Extraction conditions Overall solvent Solvent BPA recovery (R) and Phase volume Separation/detection Reference
consumptiona (mL) evaporation b
RSD (%) ratio for liquid technique
samples

Liquid portion: dilution - Liquid portion: SPE 46 (liquid portion) RSD(mean in corn
1:1 with water, SPE (Oasis HLB) infusion, n = 3): 34
extraction and
subsequent SPE clean-up
(Florisil)
Fruit and vegetables - Solvent extraction - Acetone (70 mL) 142 Yes R(n = 5): 82100 LCFL Kang et al.
(5 g) [41]
- SPE clean-up (Oasis HLB - Mechanical shaker RSD(n = 5): 422
and Florisil) (10 min)
Pet foods for cat and Pet food: solvent - Pet food: acetonitrile 176 (pet food) Yes R(mean, n = 5 pet 5 for water LCFL Kang and
dogs (5 g) extraction, clean-up of (130 mL) food): 95.4 Kondo [44]
the crude extracts with

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


an apolar solvent and
subsequent SPE (Florisil)
Water food simulant Water (food simulant): - Water: SPE (Oasis HLB) 13.5 (water) RSD(mean, n = 5 pet
(5 mL) SPE extraction food): 10
Canned foods in oily, - Solvent extraction - Acetonitrile (100 mL) 192 Yes R(mean, n = 10): LCFL Sun et al.
aqueous or acidic 8614 [46]
media (5 g)
- Clean-up of the crude RSD(mean, n = 10):
extracts with an apolar 2.65.1
solvent and subsequent
SPE (Oasis HLB)
Powdered infant - Solvent extraction - Ethanol:water (50:50, 11 Yes R(n = 3): 1897 GCMS Kuo and
formula (0.5 g) v/v) (10 mL) Ding [43]
- Ultra-centrifugation Stirring with magnetic RSD(n = 3): 524
bar (10 min)
- Clean-up of crude - Ultra-sonication (2 h,
extract by SPE (C18 ) 4 C)
- Derivatization

Human colostrum - Protein precipitation SPE extraction (Oasis 4 Yes R(mean, n = 2): 102.6 5 ELISA Kuruto-
(1 mL) HLB) Niwa et al.
[61]
- SPE extraction RSD(mean, n = 2):19%

Honey (10 g) - Dilution with water SPE extraction (PS-DVB) 15 Yes R(n = 6): 99.9103 LCFL Inoue et al.
(50 mL) [62]
- SPE extraction RSD(n = 6): 5.36.6

Breast milk (100 L) - Enzymatic hydrolysis SPE (RAM, LiChrosphere 0.19 No R(n = 5): 97106 LCMS/MS Ye et al. [73]
- Protein precipitation RP-18 ADS) RSD(mean, n = 50):
8.211.4
- SPE extraction on-line
with LCMS/MS
Mineral water (2 L), - SPE extraction SPE (Oasis HLB) 13 Yes R(n = 5): 82.196.5 2000 for LCMS/MS Shao et al.
drinking water mineral and [60]
(2 L) and soda drinking water
beverages (50 mL) and 50 for
soda
beverages
RSD(n = 5): 2.97.1
A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469 455

in the ranges 4749% and 7879% for spiked liver or muscle tissue
Maragou et

Tokooya et

Lpez et al.
Mungua-
from rainbow trout (RSD 4.14.7% [50]) and 92111% (RSD 38%
al. [65]

al. [45]
[51]) for a variety of seafood including prawns, crabs, cockles, white

[47]
clams, squids and sh.

3.1.3. Pressurized liquid extraction (PLE)

LCFL (conrmation
PLE, also known as accelerated solvent extraction (ASE), involves
the use of liquid solvents at elevated temperatures (40200 C) and

by GCMS)
pressures (10002500 psi). Under these conditions, solvents have
enhanced solvation power and have increased the extraction rates.
LCMS

LCED

The application of PLE to food analysis has been recently reviewed


[52,53]. Although scarcely used for the extraction of BPA-containing
foods, the suitability of PLE for extracting samples of animal [28,54]
and vegetal [32] origin prior to LC/MS has been proved. The solvents
used have been dichloromethane for meat (pork, meat, rabbit, duck
and chicken) [28], acetonen-hexane (1:1, v/v) for sh liver [54] and
5000

methanol for cereals [32]. Sodium sulphate [54] and diatomaceous


earth [32] have been utilized as dispersing agents. The optimisa-
tion of the extraction conditions for PLE includes the study of the
solvent type, temperature, pressure, extraction time and number
RSD(n = 6): 2.112.5

(food simulant), 91
(jalapeno peppers)

(jalapeno peppers)
RSD(mean, n = 3):1
R(mean, n = 3): 63

R(mean, n = 3): 89

of cycles and this can be performed using statistical experimental


(food simulant),6
R(n = 6): 83106

design procedures to minimise the number of experiments [32].


Lipid removal from meat is carried out by mixing the samples with
celite and putting activated neutral alumina on the bottom of the
RSD

PLE cells [28], while its removal from sh liver is made by extraction
with n-hexane after PLE [54]. Further clean-up of PLE-extracts by
SPE (Oasis HLB [32], Florisil 60 [54] and aminopropyl sorbents [28])
is always required to achieve selectivity. The recoveries obtained in
the above applications were from 81% to 104% (RSD 49%) for cere-
als [32], 53% (RSD 20%) for sh liver [54] and from 91% to 100% for
Yes
Yes

Yes

meat [28].
Abbreviations: SPE, solid-phase extraction; PS-DVB, polystyrene divinylbenzene; RAM, restricted access material.

3.2. Solid-phase extraction (SPE)

SPE is by far the most used technique for both the extraction
of BPA-containing liquid foods and the clean-up of crude extracts
after solvent extraction. The basis of SPE [55] and its application
in food analysis [5658] have been comprehensively reviewed.
300 mL

275 mL

The selection of a suitable sorbent for BPA will mainly be gov-


Overall solvent consumption per sample during the different sample preparation steps.
12

erned by its properties (moderately polar character and presence


of hydrogen acceptor/donor groups, cf. Table 1) and the type of
food matrix. Both, non-selective and selective sorbents have shown
excellent efciency for the isolation and clean-up of BPA from food
- Methanol (270 mL)

- Stirring (1 h) and

(cf. Table 3).


allowed to stand
Ethyl acetate

3.2.1. Non-selective sorbents


overnight
SPE (C18 )

The divinylbenzene/N-vinylpyrrolidone copolymer (OASIS HLB


from Waters, 30200 mg) has been the most used sorbent to date.
The hydrophilic N-vinylpyrrolidone polymer affords good wet-
tability of the sorbent and acts as a hydrogen acceptor, while
(8:1, v/v) to desestabilize

the hydrophobic divinylbenzene polymer provides reversed-phase


1 mL water (condensed)

water:methanol 18 mL
- Reconstitution with

retention for BPA. It offers advantages over classical silica sorbents,


and 6.4 mL water

Solvent extraction
Solvent extraction

i.e. high specic area (800 m2 /g), possibility to dry out during the
- SPE extraction

Spiked samples, RSD as repeatability.

extraction procedure without reducing its ability to retain BPA and


the emulsion
(powdered).

like other polymeric resins, stability over the entire pH range [59].
- Addition

Among other liquid foods, OASIS HLB has been applied to the isola-
tion of BPA from the aqueous portion of canned fruit and vegetables
[40], the leachate originated from the treatment of empty pet food
cans with distilled water at 121 C for 30 min [44], drinking water
and soda beverages [60], human colostrum [61] and highly viscous
and powdered)

Jalapeno peppers
Milk (condensed

samples, such as honey [62]. Removal of proteins and lipids was


Mineral water

required for human colostrum samples prior to SPE. For this pur-
(500 mL)

pose, the samples were diluted with distilled water, treated with
(120 g)
(1 g)

acetonitrile or 2-propanol, centrifuged and ltered [61]. The size


a

of samples was in the ranges 150 mL [40,44,61] and 10 g [62]


456 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

although the breakthrough volume increased up to 2 L for drinking and expensive, as is the case for immunosorbents and molecularly
water [60]. imprinted polymers. Their application in food analysis, and specif-
On the other hand, as OASIS HLB efciently removes hydrophilic ically to the determination of BPA, is emerging but a rather limited
and lipophilic interferences [63] and it has been applied to the so far.
clean-up of a variety of foods after solvent extraction, namely sh
(to eliminate the co-extracted polar lipids) [38], fruit and veg- 3.2.2.1. Restricted access materials (RAMs). The term restricted
etables (total content or solid portion) [40,41] and a variety of access materials (RAMs) was introduced by Desilets et al. in 1991
canned foods [46]. A second clean-up step with a normal-phase [71]. These sorbents were particularly developed for the on-line
SPE sorbent (Florisil, a synthetic magnesium silicate) was neces- clean-up/extraction of biological samples (such as plasma and
sary in some applications, namely in the treatment of sh, fruit and serum) in order to perform a high throughput analysis. RAMs com-
vegetable samples [38,40,41]. Retention on Florisil mainly occurs bine size exclusion of proteins and other macromolecules with the
through adsorption, and clean-up has to be carried out from sam- simultaneous enrichment of low molecular mass analytes at the
ple extracts previously evaporated and redissolved in a non-polar inner pore surface, which are retained by conventional mechanisms
organic solvent such as n-hexane. Overall recoveries in all the cited (hydrophobic, ionic or afnity interactions) [72]. The size exclusion
applications were above 80%. is achieved by a physical diffusion barrier (pore diameter) or by a
Chemically bonded reversed-phase silica (C18 ) has been pro- chemical barrier created by a polymer at the outer surface of the
posed as a SPE sorbent for the isolation of BPA from mineral particles.
water and wines [64] and canned condensed and powdered milk A RAM (LiChrosphere RP-18 ADS from Merck) has been used
[65]. Milk samples were diluted with watermethanol (8:1, v/v) for the simultaneous on-line SPE-LCMS/MS analysis of BPA, other
in order to obtain extracts as clean as possible. The addition of phenolic compounds and triclocarban in breast milk [73]. The RAM
water reduced the viscosity of the sample, thus resulting in a bet- consists of a bonded reverse-phase covering the internal pore (C4 ,
ter ow rate during SPE, while the addition of methanol aimed at C8 or C18 ) and a surface modied with hydrophilic groups (glyceryl-
the destabilization of the milk emulsion. It was argued that if milk propyl, i.e. diol moieties), which acts as a physical barrier (pore size
fat globule membranes are not disrupted, BPA might not be effec- 6 nm) that excludes the macromolecules. The method involves the
tively and reproducibly extracted. Milk samples with a fat content addition of the internal standard (100 L, 13 C12 -BPA) to the sam-
above 3.5% were previously diluted with water. SPE recoveries for ple, the enzymatic hydrolysis of the conjugated BPA, protein and
BPA ranged between 57 and 85% and the internal standard [2 H16 ] lipid removal by the addition of 2-propanol, and centrifugation to
bisphenol A, BPA-d16 was used for quantitation [65]. The use of extract the clean supernatant (200 L), which is subject to on-line
polystyrene-divinylbenzene (PS-DVB, Isolute, and hydroxylated PS- SPE-LCMS/MS. It can also be used to measure the free BPA by omit-
DVB, Isolute ENV+) sorbents in this application did not improve ting the enzymatic treatment. Recoveries for BPA were between 94
recoveries despite they offer hydrophobic and  interactions for and 106% and the precision of the method was in the range 519%.
retention of BPA. Contrariwise, recoveries higher than 85% were
obtained using PS-DVB sorbents for the isolation of BPA from water 3.2.2.2. Immunosorbents (ISs). Immunoafnity columns (IAC) or
diluted and acidied honey [62], water diluted, centrifuged and l- the so-called immunosorbents (ISs) are made by covalently bond-
tered infant formula and canned vegetables and fruit juices [15,66] ing antibodies onto an appropriate support. They provide unique
and untreated canned drinks [67]. Recoveries were always higher selectivity on the basis of molecular recognition, which is par-
than 85%. ticularly suited to complex food matrices. ISs can be designed
Finally, multi-mode phases (Isolute multi-mode cartridges) for targeting a single analyte or by exploiting cross-reactivity to
have also been proposed for isolation of BPA from instant coffee extract a whole class of structurally related compounds. Although
[68]. Multi-mode phases were introduced to exploit multiple-mode most applications are for biological and environmental samples
interactions and were originally designed for the screening of drugs (reviewed in [74]), ISs have also been exploited for the extraction
and metabolites [69,70]. Besides exploiting the multiple inter- of foods and are commercially available for the analysis of natural
actions to retain mixtures of analytes with different properties, toxins [75,76]. The main drawbacks of this technique is the cost to
these sorbents have been lately used to obtain clean extracts for produce the antibodies and the short-life of columns.
a compound that can be retained by multiple interactions. Isolute Several methods have been reported that use solgel
multi-mode cartridges combine cationic, anionic and non-polar immunoafnity chromatography for the clean-up of liquid foods or
functionalities. Recoveries in coffee ranged from 85% to 89% (RSD: crude extracts prior to the determination of BPA by LCuorescence
47%). detection. Non-antibody-containing solgel precolumns were on-
In general, the use of SPE for isolation of BPA from liquid line coupled with the immunoafnity columns in order to prevent
foods offers two advantages as compared with LLE, namely higher them from the clogging caused by the small particles present in
selectivity and lower solvent consumption. On the other hand, con- the sample extracts. The approach was applicable to a variety of
centrations factors, enhanced by solvent evaporation, are similar for foods including canned beverages, fruits, vegetables [77,78], fat-
SPE and LLE. The SPE of liquid foods usually requires further clean- containing foodstuffs (e.g. tuna, cream, potato soup) [78] and wine
up and great care must be taken regarding the small particulates [79]. The ISs produced by immobilization of the antibodies into
present in the sample extracts that can produce low recoveries and the pores of a solgel generated silica matrix are simpler, cheaper
irreproducibility by adsorption of analytes or clogging. A detailed and more versatile than those prepared by the covalent coupling
comparison between different SPE and solvent extraction methods of polyclonal antibodies to CNBr-activated Sepharose 4B [80]. The
is given in Table 3. binding capacity of the IAC columns for BPA was 280 ng and good
batch to batch reproducibility was found, being reusable at least 15
3.2.2. Selective sorbents times. Substances showing high cross-reactivity (>1%) did not affect
A variety of highly selective SPE sorbent materials have been the determination of BPA since they were efciently separated by
developed that are especially suitable for the determination of trace the LC system.
contaminants in such complex samples as foods, thus perform- In general, food samples have to be treated to some extent
ing extraction and clean-up in one step. On the other hand, the in order to make them compatible with immunoafnity chro-
development of these smart materials is time-consuming, complex matography. Dilution with water is essential since organic solvents
A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469 457

to 81% in wines [79]. RSD values for these samples were between
1 and 21%. The variable and low recoveries obtained for IAC were
explained on the basis of the matrix-dependent nature of the previ-
ous solvent-based BPA extraction and the inuence of co-extracted
matrix components on the BPAantibody interactions.

3.2.2.3. Molecularly imprinted polymers (MIPs). Molecularly


imprinted polymers (MIPs) are synthetic polymers having molec-
ular recognition ability for a target analyte [81]. MIPs offer some
advantages over ISs such as stability against organic solvents,
strong acids and bases and heating. Furthermore, they permit
larger sample volumes and reusability, and their synthesis is easier
since they are not dependent on antibody production. Currently a
number of approaches have been used to prepare BPA imprinted
polymers, which have been applied to the determination [8285]
and removal [86] of BPA and other phenolic estrogen pollutants in
environmental waters. Novel architectures to develop BPA-MIPs,
such as hybrid molecularly imprinted membranes [86], molecu-
larly imprinted polyethersulphone (PES) microspheres [87] or a
hybrid RAM-MIP [88] have been described in the literature.
Food applications of MIPs have been proposed in a lesser extent
(reviewed in [89]) although it is an emerging area with promising
developments. To the best of our knowledge, only one applica-
tion has been developed for the MIP-based extraction of BPA from
food [90]. A combinatorial approach was used to simplify the opti-
misation procedure. Methacrylic acid (MAA) and 4-vinylpyridine
(4-VP) were assessed as functional monomers, ethylene glycol
dimethacrylate (EDMA) and trimethylolpropane trimethacrylate
(TRIM) as cross-linkers, and methanol, acetonitrile and toluene
as solvents. The initiator used was 2,2 -azobis methylbutyroni-
trile (AIMN) and the template was BPA. Stable isotope labelled
compounds like BPA-d16 [88] and structure analogues as p-tert-
butylphenol [84], have also been used as templates in other
approaches to avoid undesirable template leakage from MIP and the
consequent carry over effects. The optimal components selected
were 4-VP (4 mmol) and TRIM (12 mmol), which provided a higher
degree of cross-linking, AIMN (0.88 mmol) and toluene (150 mL),
which provided lower non-specic binding. The MIP-based clean-
up/extraction/concentration method permitted the determination
of BPA (recovery 78%; RSD 10%, n = 3), free of co-extractives, in the
ltered aqueous phase of canned foods.

3.3. Less common extraction techniques

Miniaturised sorptive extraction techniques such as solid-phase


microextraction (SPME) and stir bar sorptive extraction (SBSE) have
the capability of improving the isolation and clean-up of contam-
inants from food in terms of solvent consumption, automation
and sample handling reduction, however their application to the
Fig. 2. Chromatograms obtained for (a) BPA standard solution (10 ng mL1 ), (b) a extraction of BPA from food is still limited to date. Likewise, matrix
wine sample subject to clean-up by solid-phase extraction (C18 cartridge) and (c)
the same wine sample cleaned-up by immunoafnity chromatography [79].
solid-phase dispersion (MSPD), which has the potential of simpli-
fying the extraction of solid samples, has been also scarcely applied
to the extraction of BPA from foods. Below, these applications are
cause irreversible denaturation of antibodies. Liquid foods like commented highlighting their main benets and drawbacks.
beverages and wines are degassed and diluted 1:1 with phosphate-
buffered saline (PBS) solution (pH 7) to enable binding of BPA to 3.3.1. Solid-phase microextraction (SPME)
the immunosorbent. Solid foods such as fruits, vegetables, goulash, The SPME device consists of a fused-silica bre coated with an
soup and sh are extracted with acetonitrile, back extracted with appropriate stationary phase attached to a modied microsyringe.
n-hexane to remove lipids in fatty matrices, ltered and diluted The extraction can be performed by suspending the bre in the
(1:10) with water prior to clean-up with ISs. Compared to C18 -based vapour phase above the liquid sample (headspace (HS)-SPME) or by
SPE, IAC was superior in removing food matrix interferences as it direct immersion into the sample, (DI)-SPME, being the desorption
is shown in Fig. 2 for wine samples cleaned-up with C18 and IAC. carried out thermally, by exposing the bre in the injection port of
Recoveries for BPA using IAC strongly depend on the food matrix. a gas chromatograph, or chemically, when coupling with LC [91].
Thus, some of the values obtained were: 53% in peas, 75% in peaches HS-SPME followed by GCMS is ideally suited to the extraction
[77], 27% in goulash, 103% in a lemon soft drink [78], and from 74% of volatile compounds from foods (reviewed in [92]) since analyte
458 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

determinations are practically interference-free, but no applica- the bar is achieved by GC thermal desorption or elution with a
tions related to BPA in food have been developed so far. DI-SPME LC solvent. The technique was rst developed for the extraction of
followed by GCMS has been applied to the determination of BPA organic compounds from aqueous samples, but today a variety of
in aqueous food simulants [93] and water from plastic contain- applications in the environmental, food and biomedical areas have
ers and tableware [94]. DI-SPME has also been proposed prior to been reported (reviewed in [101]).
LCFL for the fast screening of BPA in canned foods, just analyz- Since PDMS is the unique commercially available coating, the
ing the aqueous phase of the can [95]. Different stationary phases application of SBSE to polar and semi-polar compounds requires
with the capability to withstand high injector temperatures (poly- derivatization. Thus, the extraction of BPA from water [102104]
dimethylsiloxane, PDMS; carboxen/PDMS; PDMS/divinylbenzene, has been carried out by BPA derivatization with acetic anhydride,
DVB; carbowax CW/DVB and polyacrylate, PA) were assessed for immersion of the bar in the solution and thermal desorption-
the extraction of BPA by DI-SPMEGC [93,94]. The coating PA gave GCMS analysis. In this way, the LODs for BPA were in the
the best recoveries on the basis of its similar polarity to BPA. Among range 0.62 ng L1 . Despite new SBSE coatings are necessary to
the coating materials tested for extraction of BPA by DI-SPMELC, extend the scope of SBSE, only a few ones have been reported
the most polar bres gave the best results. Recoveries were 6% for up to now. It is worth noting the dual-phase twisters (short
PDMS-DVB, 7% for PA, 58% for PDMS and 90% for CW, so the lat- PDMS tubes closed at both ends with magnets, with an inner
ter was selected as optimal. The effect of the ionic strength on cavity that is packed with activated carbon adsorbent), which
BPA recoveries was signicant and the three methods included the were proposed to improve recoveries of polar compounds [105],
addition of salt, namely 7.5% (w/w) [93,95] and 15.4% (w/w) [94]. and two selective materials, i.e. a molecularly imprinted polymer
Maximum sorption occurred at neutral pH [93,95] and the sorption (MIP) for the extraction of monocrotophos [106] and a restricted
process was carried out at room temperature [95] or at 5055 C access media (RAM) for the extraction of caffeine and related
[93,94]. metabolites [107]. In this respect, a novel SBSE coating based
The three methods were sensitive enough for the screening or on polydimethylsiloxane/-cyclodextrin (PDMS/-CD) has been
migration assessment of BPA (quantitation limits were 3.8 mg L1 recently developed and applied to the extraction of BPA (and other
[95] and 0.010.02 mg L1 [93,94]), but they also presented serious estrogenic compounds) from environmental water, drinking water
drawbacks. Thus, the automation of SPME with LCFL through the and leachate from one-off dishware, prior to determination by
interface supplied by Supelco was not successful, being the off-line LCFL [108]. The PDMS/-CD coating was prepared by a solgel
mode more efcient that the automated one in terms of sensitiv- technique, which provided direct chemical binding of the stationary
ity and peak resolution [95]. This fact was explained on the basis phase to the glass substrate and resulted in higher stability com-
of the higher concentration factor in the off-line mode (67) and pared to the physical deposition technique frequently employed for
the slower introduction of the analytes into the LC system in the coating. The introduction of the cyclodextrin permitted to increase
dynamic mode, which resulted in broader peaks [95]. In fact, Eis- the recovery of polar compounds due to the high number of func-
ert and Pawliszyn [96] later developed the in-tube SPME technique, tional hydroxyl groups in the -CD molecule and the selectivity
which uses an inner-wall coated capillary as the extraction medium provided by its molecular recognition ability. The extraction was
and it is more suitable for LC automation. In-tube SPE has been suc- carried out by immersion of the stir bar in the sample (5 mL) for
cessfully applied to the extraction of BPA and other estrogens from 40 min (non-equilibrium conditions) at room temperature. Desorp-
environmental waters [97,98], but not in food samples yet. Other tion of BPA was made with 100 L of methanol for 2 min under
weak point of the SPMELCFL method was the reproducibility ultrasonic agitation (ratio of sample volume over extract volume
(RSD was 22% for 100 mg L1 of BPA). With regard to the determi- of 50). The PDMS/-CD coated stir bar provided better selectivity
nation of BPA by SPMEGCMS, the variability was high between and sensitivity for polar compounds than a PDMS coated stir bar
series (mean RSD 1013%) and consequently a calibration for each and a PDMS-CD coated bre. The lower method performance
series of analysis was recommended [93,94]. On the other hand, for SPME was attributed to the smaller stationary phase volume
despite only aqueous simulants or drinking water were analyzed, and the introduction of a third competitive phase in the stirring
the low recoveries resulting from the effect of matrix components step, namely the Teon coated magnetic bar. The LOD for BPA was
(765%) [94,95], made advisable the use of the standard addition 8 ng L1 , with recoveries between 86 and 116% and RSDs in the
method for BPA quantitation. The low recoveries were related to range 0.710.7%.
competitiveness between BPA and the matrix components for bind-
ing to the limited volume of stationary phase, which led to analyte 3.3.3. Matrix solid-phase dispersion (MSPD)
displacement [94]. To our knowledge no study related to the use of MSPD was rst reported in 1989 [109] and it is well suited to the
SPME for extraction of BPA from more complex food matrices has extraction of solid, semi-solid and/or highly viscous food and bio-
been reported so far. In fact, the use of DI-SPME in food matrices logical matrices (reviewed in [110,111]). The sample is mixed with
should need exhaustive clean-up to ensure the reproducibility and a sorbent such as C18 bonded silica, sodium sulphate or diatoma-
the robustness of the bres, which can be affected by adsorption of ceous earth, followed by washing with a small volume of solvent
proteins or clogged by particles, similarly to that occurring in the or packing the dispersant sorbent material into a SPE mini-column
analysis of BPA in other complex matrices such as human plasma before elution. MSPD is simple and versatile and offers the possi-
[99]. bility of performing extraction and clean-up in one step [112,113],
this resulting in a drastic shortening of analysis times and solvent
3.3.2. Stir bar sorptive extraction (SBSE) consumption.
SBSE uses a stir bar into a sealed glass tube that is coated with MSDP has been recently reported for the simultaneous extrac-
polydimethylsiloxane (PDMS) to extract solutes [100]. In compar- tion of BPA, nonylphenol and octylphenol from eggs and milk [114]
ison to SPME, a larger amount of stationary phase (50250 times prior to LC/MS/MS determination. The samples (1 g) were blended
higher) is used and hence better recoveries, sensitivities and sample with 1 g of C18 powder just for 5 min, packed and eluted with 10 mL
capacity are achieved. Like in SPME, the stir bar can be immersed of ethanol. This dispersant agent gave better recoveries for BPA
into the liquid sample or can be held in the headspace above the than graphite carbon black (GCB) because of the strong adsorp-
liquid or the solid sample to determine volatiles and semi-volatiles tion of BPA to GCB. The eluent was evaporated to dryness and the
(headspace sorptive extraction, HSSE). Removal of analytes from residue was redissolved in dichloromethane/hexane and subject to
Table 4
LC-based analysis for the determination of BPA in food

Sample Other analytes than BPA I.S. Sample preparation LC column LC parameters Detector parameters Linear range, method Reference
(main steps) LOD or LOQ, recoveries
(R)

Cereals 4-n-Nonylphenol PLESPE XTerra MS C18 , Mobile phase: ESI(-)-MS: capillary LR: 0.080.8 g g1 Carabias-Martnez
100 mm 2.1 mm, 3.5 m 0.0025 M ammonium voltage: 3.0 kV, cone (standard addition), et al. [32]
(Waters System). formate buffer (pH 3.1, voltage: 40 V, source LOD: 43 ng g1 ; R:
adjusted with formic temperature: 120 C, 81104% (PLE
acid)/methanol with desolvation recoveries)
gradient elution, temperature: 250 C,
0.2 mL min1 , Inj: 50 L desolvation gas (N2 ):
410 L h1 , cone gas
(N2 ): 50 L h1
4-tert-Butylphenol Post-column addition
of a strong base
[1,8-diazabicyclo-
(5,4,0)undec-7-en],

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


10 L min1
2,4-Dichlorophenol
2,4,5-Trichlorophenol
Pentachlorophenol
4-tert-Butylbenzoic acid

Fruit and vegetables SESPE Symmetry Shield RP18 , Mobile phase: Fl: 275/300 nm LR: 101000 ng mL1 ; Kang et al. [41]
150 mm 4.6 mm I.D., water/acetonitrile LOD: 1.3 ng g1 ; R:
3.5 m with a guard (60:40, v/v); 82101%
column, 10 mm 2.1 mm 1.2 mL min1 ; Inj:
I.D., 3.5 m (Millipore). 50 L.
Canned pet foods SESPE (total Symmetry Shield RP18 Mobile phase: Fl: 275/300 nm LR: 501000 ng mL1 ; Kang and Kondo
content) 150 mm 4.6 mm I.D., water/acetonitrile LOD: 2 ng mL1 [44]
3.5 m (Millipore (60:40, v/v), 40 C, (water), 3 ng g1 (pet
Corporation, Milford, MA, 1 mL min1 , Inj: 50 L. foods); R: 95%
USA).
SPE (leaching from
empty cans)
Canned foods of oily, BADGE SESPE Nucleosil-100 C18 , Mobile phase: Fl: 235/317 nm LR: 2002000 ng mL1 ; Sun et al. [46]
aqueous of acidic media. 250 mm 4 mm I.D., 5 m water/acetonitrile with LOD: 4.57.9 ng g1 ; R:
(Hichrom Limited, gradient elution, 25 C, 87105% (food
Berkshire, UK). 0.4 mL min1 , Inj: simulant)
10 L.
BADGE-H2 O
BADGE-2H2 O
BADGE-H2 O-HCl
BADGE-HCl
BADGE-2HCl

Mineral water p-Butylphenol LLE Inertsil ODS-2, Mobile phase: ED, coulometric LOD: 5.7 g L1 ; Tokooya et al. [45]
150 mm 4.6 mm I.D., water/acetonitrile detection with a dual R(mean): 63%
5 m (GL Sciences, Tokyo). (40:60) containing 0.2% electrode analytical cell
H3 PO4 ; 40 C;
1 mL min1 ; Inj: 10 L.
p-tert-Butylphenol E1 : 400 mV (vs Pd)
p-Hexylphenol E2 : 650 mV (vs Pd)
p-tert-Octylphenol Guard cell: 700 mV (vs
Pd)
p-Nonylphenol
p-Heptylphenol
Nonylphenol

459
460
Table 4 (Continued )

Sample Other analytes than BPA I.S. Sample preparation LC column LC parameters Detector parameters Linear range, method Reference
(main steps) LOD or LOQ, recoveries
(R)

p-Octylphenol

Fish 4-tert-Octylphenol BPA-d16 MAESPE Zorbax Eclipse XDB-C18 , Mobile phase: APCI(-)-MS: capillary LR: Pedersen and
150 mm 2.1 mm I.D. water/methanol with voltage 3 kV, corona 20.710044 ng mL1 , Lindholst [50]
gradient elution, current: 40 A, LOD: 0.1 ng mL1
0.4 mL min1 , Inj: not fragmentor voltage: (water), 50 ng g1
specied. 100 V, drying gas ow (sh); R: 4979%
5 L min1 , nebulising (absolute recovery,
gas pressure 60 psi, sh)
vapourising
temperature 280 C
Meat Octylphenol 4-n-Nonylphenol PLE-SPE Symmetry C18 , Mobile phase: water ESI(-)-MS/MS: capillary LR: 1500 ng mL1 ; Shao et al. [28]
150 mm 2.1 mm I.D., (0.1% voltage: 3.5 kV, cone LOD: 0.3 ng g1 ; R:

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


3.5 m. ammonia)/methanol voltage: 70 V, source 9297%
with gradient elution, temperature: 100 C
0.2 mL min1 , Inj:
10 L.
Nonylphenol

Water and soda beverages Octylphenol 4-n-Nonylphenol SPE Symmetry C18 , Mobile phase: water ESI(-)-MS/MS Capillary LR: 1500 ng mL1 ; Shao et al. [60]
150 mm 2.1 mm I.D., (0.1% voltage: 3.5 kV; LOD: 0.01 ng L1
3.5 m. ammonia)/methanol desolvation (drinking water),
with gradient elution, temperature: 350 C 0.6 ng L1 (soda
40 C, 0.2 mL min1 , Inj: beverage); R: 8297%
10 L.
Nonylphenol

Honey Bisphenol F SPE Quantitation: CAPCELL PAK Mobile phase: Fl: 275/300 nm LR: 25500; LOD: Inoue et al. [62]
C18 , 150 mm 4.6 I.D., water/acetonitrile 2 ng g1 ; R:
5 m) (Shiseido Co. Tokyo, (70:30, v/v); 93.9116.4%
Japan). 1.2 mL min1 ; Inj:
10 L.
Conrmation: CAPCELL Mobile phase: water ESI(-)-MS: Capillary
PAK MG C18 (0.01 acetic voltage: 3.5 kV;
(250 mm 2 mm, 5 m acid)/acetonitrile with fragmentor voltage:
(Shiseido Co. Tokyo, Japan) gradient elution; 140 V, 220 V;
0.3 mL min1 ; Inj: 5 L. desolvation
temperature: 350 C
Milk BPA-d16 SPE LiChrospher 100 RP-18, Mobile phase: ESI(-)-MS: capillary LR: 5700 ng mL1 ; Maragou et al. [65]
250 mm 4 mm I.D., 5 m water/methanol voltage: 3.5 kV, cone LOD: 0.7 ng mL1 ; R:
(30:70, v/v), voltage: 70 V, source 52% (absolute
0.9 mL min1 , Inj: 10 L temperature: 500 C recovery), 101%
(relative recovery)
Aqueous portion of canned BADGE SPME XTerra MS C18 , Mobile phase: Fl: 275/305 nm LR: 10500 ng mL1 ; Nern et al. [95]
foods 100 mm 4.6 mm, 5 m water/acetonitrile LOD: 1.1 ng mL1 ; R:
(Waters, Milford, MA, USA). (50:50, v/v); 765%
1.2 mL min1 ; Inj:
20 L.
BADGE-diol
BADGE-2HCl
BFDGE
BFDGE-diol
Breast milk 4-tert-Octylphenol 13
C12 -BPA On-line SPE Two ChromolithTM Mobile phase: APCI(-)-MS: needle LR: 0.1100 ng mL1 ; Ye et al. [73]
(RAM) Performance RP-18, water/methanol with voltage: 3 V, curtain LOD: 0.28 ng mL1 ; R:
100 mm 4.6 mm I.D. gradient elution, gas ow: 20 au, 94% (SPE recoveries)
(Merck KGaA, Germany). 0.75 mL min1 , Inj: collision gas ow: 9 au,
100 L. nebulizer gas ow:
50 au, nebuliser gas
temperature: 500 C
ortho-Phenylphenol
2,4-Dichlorophenol
2,5-Dichlorophenol
2,4,5-Trichlorophenol
2,4,6-Trichlorophenol
2-Hydroxy-4-
metoxybenzophenone
Beverages, fruits and IAC Spherisorb S ODS1, Mobile phase: 50 mM Fl: 275/305 nm LR: 0.5100 ng mL1 ; Braunrath and
vegetables 250 mm 4.6 mm I.D., sodium acetate buffer LOD: 0.1 ng mL1 , Cichna [77]
5 m (Knauer, Berlin, (adjusted to pH of 4.8 1.11.6 ng g1 ; R:

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


Germany). with acetic 5375%
acid)/acetonitrile
(60:40, v/v);
1 mL min1 ; Inj:
100 L.
Wine IAC Quantitation: LiChrospher Mobile phase: Fl: 275/305 nm LR: 0.5100 ng mL1 ; Brenn-
60 RP-Select B, water:acetonitrile LOD: 0.1 ng mL1 ; R: Struckhofova and
250 mm 4.0 mm I.D., (70:30, v/v); 7481% Cichna-Markl [79]
5 m (Merck). 1 mL min1 ; Inj:
100 L.
Conrmation: HP-Hypersil Water (0.01 M sodium ED (coulometric
PDS, 250 mm 4 mm I.D., acetate buffer, pH 4.8 electrode array
5 m adjusted with acetic detection). Potentials
acid):acetonitrile with set at 300, 400, 500,
gradient elution; 550, 600, 650 and
0.6 mL min1 ; Inj.: 750 mV against Pd
100 L. reference electrodes
Eggs and milk Octylphenol 4-n-Nonylphenol MSPDSPE Symmetry C18 , Mobile phase: water ESI(-)-MS/MS: capillary LR: 1500 ng mL1 , Shao et al. [114]
150 mm 2.1 mm I.D., (0.1% voltage: 3.5 kV, cone LOD: 0.1 ng g1 (eggs
3.5 m ammonia)/methanol voltage: 70 V, and milk); R: 7983%
with gradient elution, Multiplier voltage: (eggs), 8694% (milk)
40 C, 0.2 mL min1 , Inj: 650 V, desolvation gas
10 L ow (N2 ): 550 L h1
source temperature:
100 C, desolvation
temperature: 300 C
Nonylphenol

Abbreviations: PLE, pressurized liquid extraction; SPE, solid-phase extraction; Inj., injection volume, ESI, electrospray ionization, SE, solvent extraction; Fl, uorescence detection; BADGE, bisphenol A diglycidyl ether; LLE,
liquidliquid extraction; ED, electrochemical detection; MAE, microwave-assisted extraction; APCI, atmospheric pressure chemical ionization; BFDGE, bisphenol F diglycidyl ether; SPME, solid-phase microextraction, RAM,
restricted access material.

461
462 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

SPE with aminopropyl to remove the lipidic fraction of the samples.


Mean recoveries for BPA ranged from 82% to 91% and the mean RSD
values for eggs and milk were 6% and 4%, respectively.

4. Separation and detection

The determination of BPA in foodstuffs requires the use of highly


sensitive and selective techniques due to the trace levels at which
it is frequently found (cf. Table 2) and the complexity of food matri-
ces. Although the SML set by the EU commission is relatively high
(600 ng g1 ), the reported low-dose effects of BPA has given rise
to the development of analytical methods with LODs low enough
to asses the human exposure at these levels. The determination
of BPA in food is mainly carried out by LC/FL, LC/MS and GC/MS.
Liquid chromatography offers the advantage of simplicity over GC
for which derivatization step is necessary, while the latter provides
higher peak resolution. Other techniques like LCelectrochemical
detection (LCED) and immunoassays have been used in a lesser
extent. Detailed information about chromatographic and detection Fig. 3. Fluorescence spectra (excitation wavelength 275 nm, slit spectral band-pass
conditions for BPA determination is summarized in Tables 4 and 5. 20 nm) for bisphenol A in (1) water, (2) acetonitrile and (3) methanol. BPA concen-
trations: (1) 25 mg L1 and (2 and 3) (1 mg L1 ).

4.1. Liquid chromatography


4.1.2. Electrochemical detection
Electrochemical detection (ED) of BPA is based on the well
LC of BPA is usually carried out in reversed-phase C18 columns.
known electroactivity of the phenolic groups present in the
Mobile phases vary according to the detector coupled to LC. Water
molecule (cf. Table 1). LCED has been used for the determination
and acetonitrile are the commonest binary solvents when uores-
of BPA in biological uids [31,116119] and water [2931,45]. Inoue
cence detection is used while water and methanol are preferred for
et al. [31] compared the instrumental detection limits obtained
ESI-MS and APCI-MS. Elution conditions highly depend on the ana-
for BPA with LC coupled to electrochemical, FL and UV detectors
lytes to be determined along with BPA and food matrices. Thus, it is
[31]. The electrochemical detector (Coul Array Model 6210, ESA,
frequent to determine BPA with other phenols, endocrine disrup-
USA) consisted of four cells, working at the following potentials;
tors and migrants from food packaging (cf. Table 4) and in this case
350, 430, 570 and 650 mV and provided a method LOD of 0.5 pg,
gradient elution is always performed. Run times range between
which was 3000- and 200-times lower than those obtained with UV
15 (e.g. [32]) and 40 min (e.g. [60]) depending on the number of
and FL detectors using the same injection volume (50 L). Similar
contaminants determined and matrix composition. LC is usually
mass sensitivity (e.g. LOD of 57 g for an injected sample volume of
performed at room temperature but temperatures up to 40 C are
10 L [45]) has been obtained by different authors [29,30]. The pH
sometimes recommended [e.g. 32 and 60] to reduce analysis time
and electrolyte content of the mobile phase inuence the electron-
and increase the reproducibility.
transfer rate constants, so they have to be optimised in order to
get maximum sensitivity. Isocratic elution is recommended [31,45],
4.1.1. Fluorescence detection otherwise rather large equilibrium times will be required for mea-
BPA shows native uorescence with excitation and emission surements, this being a main drawback of ED.
wavelengths at 275 and 305 nm, respectively, which keep con- LCED has also been used for the analysis of BPA in food
stant in the solvents more frequently used in LC mobile phases, simulants (water, acidied water and water:ethanol) using a
namely water, acetonitrile and methanol. The uorescence inten- home-made electrochemical detector [36]. A chemically modied
sity of BPA is, however, much higher in organic media (cf. Fig. 3) and electrode (CME) was prepared by coating a glassy carbon electrode
thus the sensitivity in LC will be dependent on the mobile phase with a Ni-Protoporphyrin IX dimethyl ester lm according to the
composition. Typical instrumental quantitation limits for BPA by procedure previously described by the same authors [120]. The CME
LCuorescence detection are in the range 550 ng mL1 . was especially designed to prevent the increase in the background
The technique is well suited to the determination of BPA in current caused by the high potential required for the oxidation
very different food matrices such as beverages, wines, pet foods, of phenolic compounds on glassy carbon, which in the case of
honey, fruit, vegetables, goulash and sh. Sample preparation nor- BPA leads to a decreased sensitivity. The mobile phase consisted
mally includes clean-up using non-selective sorbent-based SPE of methanol:water (60:40) containing 10 mM KNO3 and 0.25 mM
[41,44,46,62], immunoafnity columns [7779] or SPME extrac- H2 SO4 as supporting electrolytes and the method LOD was 4 pg
tion [95] followed by solvent evaporation. Typical detection limits after a concentration step. Despite the proved suitability of ED for
for BPA in foods using uorescence detection are in the range the determination of BPA in drinking water and water-based food
0.12 ng mL1 and 15 ng g1 . simulants, LCED has not been applied to common complex food
The identication of BPA in the sample is only based on reten- matrices yet.
tion times, so the possibility of interference from other uorescent
food migrants from can coatings, e.g. bisphenol A diglycidyl ether 4.1.3. LCMS
(BADGE), bisphenol F diglycidyl ether (BFDGE) or novolacs glycidyl The use of mass spectrometry can reduce sample treatment and
ethers (NOGE) [115,116], should be always considered since they even may enable the extraction of an analyte at the detection
may produce false-positives. Indeed, conrmation by LC/MS after stage of a method by selection of specic ions or transitions. How-
quantication by LCuorescence detection has sometimes been ever, good sample preparation is still necessary in LCMS methods
employed [62]. (cf. Table 4) since it determines the presence of matrix components
Table 5
GC-based analysis for the determination of BPA in food

Sample Other analytes than I.S. Sample preparation Derivatization GC column Temperature MS parameters Linear range, LOD or Reference
BPA (main steps) program and LOQ, recoveries (R)
injection

Vegetables fruit, sh BPA-d14 SE or LLE Acetic anhydride DB-5MS, 120 C held for BPA diacetyl: m/z LOQ: 10 ng g1 Thomson et
soup and sauces, 30 m 0.25 mm 2 min, ramped at 228, m/z 213. (samples <1% fat), al. [17]
canned meat, 10 C min1 to 20 ng g1 (samples
spaghetti and baked 280 C. Inj: splitless >1% fat); R: 42112%.
beans, infant foods,
beverages
I.D., 0.25 m (J&W, I.S.: m/z 224
Folsom, CA, USA).

Fish 4-tert-Butylphenol BPA-d16 SESPE MTBSTFA, 100 L to DB-5MS, 100 C held for TBDMS derivate LR: 0.550 ng g1 ; Gyong et al.
the dried SPE eluent 30 m 0.25 mm 1 min, ramped at BPA: m/z 441, m/z LOD: 0.41 ng g1 ; R: [38]
(75 C, 30 min) 3 C min1 to 200 C, 470 105120%.
held for 1 min,
ramped at
20 C min1 to

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


280 C, held for
5 min. Inj: splitting
ratio 1:10
4-n-Butylphenol I.D., 0.25 m (J&W, I.S.: m/z 452, m/z 470
Folsom, CA, USA)
4-Pentylphenol
4-n-Hexylphenol
4-tert-Octylphenol
4-n-Heptylphenol
Nonylphenol
4-Octylphenol

Beverages BPA-d14 LLE Triuoroacetic HP-5 Trace Analysis, 100 C held for O- LR: Varelis and
anhydride, 200 L 25 m 0.2 mm I.D., 1 min, ramped at bis(triuoroacetyl) 0.0150 g mL1 ; Balafas [33]
added to the SPE 0.33 m 25 C min1 to derivate of BPA: m/z LOD: 1 ng g1 ; R:
eluent (30 min, 150 C, ramped at 405, m/z 420 1897%
room temperature) 5 C min1 to 210 C,
(60 C, 5 min to ramped at
remove 25 C min1 to
derivatization 280 C, held for
reagent in excess) 2.5 min. Inj: splitless
I.S.: m/z 416, m/z 434

Infant formula Daidzein Chrysene-d12 SESPE BSTFA:TMCS: DTE DB-5MS, 100 C held for O-bis(trimethylsilyl) LR: 0.0150 g Kuo and
(1000:10:2, v/v/w), 30 m 0.25 mm 2 min, ramped at derivate of BPA: m/z mL1 ; LOD: 1 ng g1 ; Ding [43]
100 L added to the 10 C min1 to 372 and m/z 357 R: 1897%
dried SPE eluent 250 C, ramped at
(80 C, 30 min) 5 C min1 to 300 C,
hold for 5 min. Inj:
1 L, splitless
Genistein I.D., 0.25 m (J&W, I.S.: m/z 284 and m/z
Folsom, CA, USA) 269
Seawater and seafood 4-tert-Butylphenol BPA-d14 LLESPE (seawater) BSTFA, 100 L, to DB-5MS, Inj: 1 L O-bis(trimethylsilyl) R: 7497% (sweater), Basheer et
evaporated SPE 30 m 0.25 mm derivate of BPA: m/z 92111% (seafood) al. [51]
eluate (60 C, 357
30 min)
4-n-Butylphenol MAESPE (seafood) I.D., 0.5 m (J&W, I.S.: m/z 416 LOD: 6.30 ng L1
Folsom, CA, USA) (water), 1.35 ng g1
(seafood)
4-Pentylphenol
4-n-Hexylphenol

463
464
Table 5 (Continued )

Sample Other analytes than I.S. Sample preparation Derivatization GC column Temperature MS parameters Linear range, LOD or Reference
BPA (main steps) program and LOQ, recoveries (R)
injection

4-tert-Octylphenol
4-n-Heptylphenol
4-Nonylphenol

A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469


2,4-Dichlorophenol
Pentachlorophenol

Food simulant BADGE SPME HP-5 MS, 200 C held for BPA: m/z 213, m/z LR: 0.018 g g1 Salafranca
30 m 0.25 mm, 2 min, ramped at 228 (distilled water), et al. [93]
0.25 m 10 C min1 to 0.0210 g g1
270 C, hold 15 min. (water with 3%
acetic acid),
0.023 g g1 (water
with 10% ethanol)
LOD: 0.1 ng g1
(distilled water),
1 ng g1 (water with
3% acetic acid),
2 ng g1 (water with
10% ethanol)
R

Food simulant SPME BSTFA:TMCS (99:1, DB-5MS, 80 C held for 2 min, O-bis(trimethylsilyl) LR: Chang et al.
v/v), 7 L (using the 30 m 0.25 mm ramped at derivate of BPA: m/z 0.001100 g L1 [94]
vapour of the 20 C min1 to 372, m/z 357
derivatization 280 C, hold 1 min
reagents in the
headspace on the
bre) (65 C, 30 s)
I.D., 0.5 m (J&W, LOD:0.4 ng L1
Folsom, CA, USA)
R: 13.8%

Abbreviations: SE, solvent extraction; LLE, liquidliquid extraction; MTBSTFA, N ,N -methyl-(tert-butyldimethylsilyl) triuoroacetamide; TBDMS, tert-butyldimethylsilyl; BSTFA, N-O-bis(trimethylsilyl) triuoroacetamide; TMCS,
trimethylchlorosilane, DTE, 1,4-dithioerythritol; MAE, microwave-assisted extraction; SPME; solid-phase microextraction; BADGE, bisphenol A diglycidyl ether.
A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469 465

affecting ionisation efciency and background noise, and conse- abundance were reported in the MS2 ion-trap spectrum, namely the
quently detection and quantication limits. Furthermore, clean ion [MHC6 H5 OH] m/z 133, resulting from the cleavage of the
extracts are preferred to extend the column life and spend less time hydroxybenzyl group, and the ion [MHC9 H10 O] m/z 93, formed
on the instrument maintenance. Anyway, LCMS based BPA meth- by the loss of hydroxyphenyl propyl.
ods offer higher condence in identication than LCFL and LCEC. On the other hand, the most abundant ion in the mass spectrum
Compared to GCMS, the time-consuming derivatization step of of the commonest internal standard, BPA-d16 (molecular mass 244),
BPA is not required. was [(MD2 +H2 )H] m/z 241 instead of [MD] m/z 242, owing
The LCMS analysis of BPA is exclusively carried out using to the deuterium/hydrogen exchange in the hydroxyl groups when
atmospheric pressure ionization interfaces, namely electrospray BPA-d16 was dissolved in a protic medium, which led to its imme-
ionization (ESI) and atmospheric pressure chemical ionization diate transformation in BPA-d14 (molecular weight 242) [124]. The
(APCI), both in negative mode. ESI is more frequently used than MS/MS spectrum of BPA-d16 showed as base peak the fragment ion
APCI because it generally provides better sensitivity (cf. Table 4). at m/z 223, which was attributed to the loss of the CD3 radical, and a
Thus, instrumental quantitation limits for BPA of 5 [65] and second fragment at m/z 142 related to the loss of a phenol-d4 group
20.7 ng mL1 [53] have been reported using ESI(-) and APCI(-), together with a deuterium atom derived from a CD3 group [125].
respectively. In both studies, a quadrupolar mass analyzer was used Applications of LCESI(-)-MS to the determination of BPA
and the injected sample volume was 10 L. Using ESI(-) and a include cereals [32] and milk [65], while LCMS/MS has been
triple quadrupole mass analyzer the instrumental LOQ decreased used for the analysis of water and soda beverages [60], meat [28]
up to 1 ng mL1 (injected sample volume: 10 L) [28,60,73]. To our and milk and eggs [114], after extraction with PLE [32,28], SPE
knowledge, ion-trap instruments have not been used for the anal- [60] and MSDP [114]. Method detection limits were in the ranges
ysis of BPA in food samples yet, although the analysis of BPA in 0.743 ng g1 and 0.0010.3 ng g1 for MS and MS/MS detection,
environmental samples using ESI(-) ion-trap MS was at least six respectively. On the other hand, two methods using APCI(-) have
times more sensitive than APCI [121], since the fragmentation of the been developed for the determination of BPA. The rst one uses
deprotonated molecule (quantitation ion) by loss of CH3 occurred MAE for extraction of BPA from sh followed by LCMS analysis
in APCI even under mild temperature conditions. [50]. The second one determines BPA in breast milk based on an
The response in ESI-MS for BPA is strongly dependent on the on-line SPE(RAM)tandem MS system [73]. LODs were in the range
mobile phase composition [60,65]. Thus, mobile phases made up 0.150 ng g1 .
of methanolwater gave higher response for BPA standard solu-
tions than those consisting of acetonitrilewater owing to the lower 4.2. GCMS
boiling point of the former, which favoured the desolvation of
the electrospray droplets [65]. The response in acetonitrilewater GCMS provides higher resolution and lower detection limits
increased by 3-, 4-fold under the addition of modiers such as than LCMS for the determination of BPA in food, although the
0.5% [122] and 0.01% of ammonia or 0.01% acetic acid [65]. Con- need for a derivatization step makes the GC-based methods labour
trary to these studies, the presence of additives in methanolwater intensive and introduces new sources of errors, mainly due to con-
mobile phases has been known to decrease the response for BPA. tamination. On the other hand, since the presence of lipids can
Thus, basic additives, which were added to promote deprotona- signicantly reduce the analytical performance of GC [126,127],
tion of BPA, resulted in strong ionization suppression [121,123], and extensive clean-up is required for fatty foods, such as sh [39]. Like
the addition of ammonia (0.01% in water) or acetic acid (0.01% in LC/MS methods, the use of an internal standard is common, being
water) decreased the signal by 1.5- and 3-fold for BPA standard solu- deuterated BPA-d16 and BPA-d14 the most used surrogates.
tions and spiked milk, respectively [65]. However, in some cases the GCMS with electron impact (EI) ionization has widely been
opposite effect has been reported, e.g. the addition of 0.1% ammonia used for the conrmation of BPA in food analysis [15,36,39,66,128].
to methanol:water increased the response for BPA [60]. No derivatization of BPA is required for this application. The EI
Most MS methods for BPA include the addition of an internal mass spectrum and the normal fragmentation pathway for BPA are
standard (I.S.) to overcome sample preparation losses and matrix depicted in Figs. 4a and 5. The base peak in this spectrum corre-
effects (suppression or enhancement of the signal), which lead to sponds to the loss of a methyl group ([C14 H13 O2 ]+ , m/z 213) from
low method absolute recoveries. The most used internal standards the molecular ion ([C15 H16 O2 ]+ , m/z 228). This can be rationalised
have been 4-nonylphenol (when alkylphenols were also deter- in terms of resonance stabilization of the resulting tert-benzylic
mined), deuterated BPA-d16 and isotope labelled 13 C12 -BPA. The carbocation (cf. fragment 3, Fig. 5). An alternative minor fragmen-
importance of using an I.S. was highlighted by Maragou et al. [65], tation pathway involves the loss of one of the aryl groups from the
who overcame the losses caused by signal suppression (around molecular ion to give a tert-benzylic carbocation ([C9 H8 O]+ , m/z
20%) and those generated during sample preparation (estimated 135, fragment 4, Fig. 5) and the subsequent loss of methane to give
to be around 28%), by using the internal standard BPA-d16 . In this a fragment ion at m/z 119 (fragment 5, Fig. 5).
way, the mean relative recovery of the method was 101% (mean Quantitation of BPA by GCMS requires the derivatization of
absolute recovery of 52%) [65]. the analyte in order to improve its separation and detection. The
Independently of the type of analyzer and ionization source, the inclusion of a derivatization step leads to sharper peaks for BPA
most abundant ion in the BPA mass spectrum, and therefore used and consequently, better separation from other analytes and co-
for quantitation purposes, was [MH] m/z 227. The mass spec- extracted matrix components, in addition to higher sensitivity (sub
trum obtained with quadrupole instruments also contained the ng g1 level) [37]. Silylation and acetylation have been by far the
fragment ions m/z 211 and 212, formed by the additional loss of oxy- most used derivatization procedures, which are usually carried out
gen, [MHO] , and a methyl radical, [MHCH3 ] , respectively. by adding 100200 L of the corresponding reagent to the dried
Using mobile phases of acetonitrile0.01% NH3 in water, an ion with extract and allowing the mixture to stand for 3060 min under
m/z 113 was detected in the BPA mass spectrum, which was related room temperature or at 6580 C.
to the loss of both acidic protons [M2H]2 . The [MHCH3 ] m/z Silylation of the active hydrogens of BPA is mainly made using
212 was the most prominent product ion obtained by LCMS/MS, N-O-bis(trimethylsilyl) triuoroacetamide (BSTFA) containing 1% of
so it was used for conrmation and/or quantitation of BPA in all trimethylchlorosilane (TMCS) [37,43,51,94]. The addition of TMCS
the quoted methods (cf. Table 4). Other fragments of lower relative favours the formation of a single derivative, since the reaction
466 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

Fig. 4. Electron impact mass spectra of (a) bisphenol A, (b) BPA O-bis(triuoroacetyl) derivative and (c) BPA O-bis(trimethylsilyl) derivative [33] and [47].

of BSTFA with analytes having different hydroxyl groups, such as bis(trimethylsilyl)] was similar to those of BPA (cf. Fig. 4c). The base
BPA, can generate several derivatives, thus reducing the sensitivity peak corresponded to the loss of a methyl group ([C20 H29 Si2 O2 ]+ ,
and selectivity of the analysis [43,128,129]. The EI mass spectrum m/z 357) from the molecular ion ([C21 H32 Si2 O2 ]+ , m/z 372), which
and the fragmentation pattern of the BPA derivative formed [O- was also present in the spectrum [43,94]. Quantitation of BPA is

Fig. 5. Electron impact fragmentation pathways for bisphenol A and its O-bis(trimethylsilyl) and O-bis(triuoroacetyl) derivatives [33].
A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469 467

routinely carried out at one (m/z 357) [94] or two (m/z 357 and [134]). They have also been used for the determination of food con-
372) [43] masses of this derivative. taminants such as microorganisms, bacterial toxins, mycotoxins,
Silylation of BPA has also been carried out using N ,N -methyl- pesticides, anabolic hormones and veterinary drugs (reviewed in
(trimethylsilyl) triuoroacetamide (MSTFA) and N ,N -methyl- [134,135]). Immunoassays are easy to perform, give good sensitiv-
(tert-butyldimethylsilyl) triuoroacetamide (MTBSTFA) [38]. Both ity and specicity and they require neither qualied personnel nor
reagents provided BPA derivatives with diagnostic ions for the expensive equipment.
characterization of their identities in the mass spectra. The charac- The application of immunochemical techniques to the determi-
teristic ions for the trimethylsilyl (TMS) and tert-butyldimethylsilyl nation of BPA in foods is rather recent [136140]. Up to now, the
(BDMS) BPA derivatives were [M15]+ , at m/z 357 and 441, respec- determinations have focused on the analysis of liquid foods, mainly
tively. These ions, which appeared as base peaks in the mass spectra, milk, water and food simulants, using polyclonal mammalian [136]
can be used as quantication ions for BPA in the GCMS SIM mode. and chicken [137] antibodies in enzyme linked immunosorbent
The molecular ions for TMS (m/z 372) and BDMS (m/z 456) deriva- assays (ELISA). The detection limits ranged from 0.05 ng mL1 to
tives were used for BPA conrmation. No difference in reaction 500 ng mL1 , mainly depending on the immunogen and the type
yields and responses was found between them, but TBDMS gave of antibody produced. As a small molecule, BPA is not able to initi-
higher storage stability (it was stable after 8 days while the TMS ate an immune response itself and needs to be conjugated with a
derivative decreased to 60% due to hydrolysis at this time). In addi- protein to form a complete antigen. Consequently, the production
tion, the retention time for TBDMS derivative was longer than that of antibodies for BPA has involved the attachment of the pro-
for TMS (41 and 37 min, respectively, under the experimental con- tein bovine serum albumin (BSA) to the derivatized analyte or to
ditions referred in [38]), which resulted in better separation from an structural analogue (e.g. [4,4-bis(4-hydroxyphenyl)valeric acid,
the earlier eluted interferences. BHPVA]) in order to avoid the loss of part of the structural char-
Acetylation of the hydroxyl groups of BPA with acetic anhydride acteristics of BPA [138,139]. Kuruto et al. proposed the use of a
[16,17] or triuoroacetic anhydride [33] is other frequent procedure commercially available ELISA kit (EcoAssay BPA kit supplied by
to obtain BPA derivatives for GCMS. Fig. 4b includes the mass spec- Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) for the determina-
trum for the O-bis(triuoroacetyl) derivative and its fragmentation tion of BPA in human colostrum [140]. This kit was able to detect
pattern. As usual, the base peak in the spectrum corresponded to glucoronide-conjugated BPA as well as free BPA. The preparation of
the fragment ion [M15]+ (m/z 405) formed from the ion molecu- the sample (1 mL) involved the addition of acetonitrile (3 mL) and
lar (m/z 420) by the loss of a methyl group. It was proved that the the subsequent centrifugation for protein removal. The supernatant
O-bis(triuoroacetyl) derivative of BPA was more sensitive than the was evaporated to dryness, reconstituted with 1 mL of phosphate
corresponding trimethylsilyl derivative, which was a consequence and subject to SPE (Oasis HLB). The mean recovery of spiked sam-
of the higher molecular mass of the former and the monoisotopic ples was 102 19.0% and the detection limit of the method was
nature of uorine [33]. The higher molecular mass led to more 0.3 ng mL1 . The same methodology was applied to determination
favourable signal-to-noise ratios and the monoisotopic nature of of BPA in maternal serum and amniotic uid [141].
uorine prevented the reduction in ion intensity that results from
the distribution of the current between isotopomeric ions, like it
occurs for silicon. 5. Conclusions
Derivatization of the internal standard (e.g. BPA-d14 ) has been
found essential to maintain its isotopic purity during chromato- The determination of BPA in food is a requirement to support
graphic separation on a fused-silica capillary column [33], since it the enforcement of legislation and assess the risk of human expo-
degrades owing to the deuteronproton (2 HH) exchange in the sure to BPA low-doses. At present, LCuorescence detection is
aromatic portion of the molecule. This phenomenon also occurs still frequently used and it gives satisfactory quantitative results,
in other monodeuterated phenols [130] and has been attributed but LCMS and GCMS are becoming more attractive because
to the aromatic electrophilic exchange of deuterium atoms with they provide more selective and, therefore, more reliable methods.
active hydrogen atoms located on the internal surface of the Proper identication of BPA is usually made by GCMS or LC-triple
column [131]. In this respect, although the TMS derivative of BPA- quadrupole instruments.
deuterated derivatives reduces the loss of isotopic purity, the use Sample preparation still constitutes the key-step for the deter-
of O-bis(triuoroacetyl) derivative is preferred to prevent 2 HH mination of BPA in food and it is the origin of the main drawbacks
exchange in the column, since its stability is higher [33] and in in the available methodologies, independently of their applicabil-
addition, as previously quoted for BPA, its higher molecular mass ity to BPA as the only analyte or to a large number of contaminants
and the monoisotopic nature of uorine makes this derivative more (e.g. BPA along with other phenols, endocrine disruptors and/or
sensitive. migrants from food packaging). Solvent extraction and SPE are by
GCMS is the most sensitive technique for the determination of far the most used extraction techniques for both isolation of BPA
BPA in foods. Stuart et al. [132] compared the instrumental LODs for and clean-up of matrix components. Techniques such as SPME and
BPA obtained by different techniques, the values being 0.8 ng for SBSE, which offers advantages in terms of solvent consumption,
GCMS without derivatization, 0.004 ng for GCMS using methyl sample size and ease of automation have to overcome some seri-
derivate (0.5 M methanolic solution of phenyltrimethylammonium ous drawbacks before becoming suitable for the determination of
hydroxide), 1.6 ng for LCUV (275 nm) and 1.0 ng for LC/ESI(-)-MS. BPA in food. Other alternatives such as MSPD, excellent in specic
A lower instrumental LOD (0.51 pg) has been reported by using an aspects, have not reached widespread utilization yet.
acetyl derivate [133]. On the other hand, method detection limits There are two critical aspects in the sample preparation pro-
were usually in the ranges 0.42 ng g1 and 0.46.3 ng L1 . cedures developed, which are rather general in food contaminant
analysis, namely the low selectivity of solvents and the exhaustive
4.3. Immunochemical methods purication required to isolate the analyte from matrix compo-
nents. As a general rule, repeated solvent extractions are required to
Immunochemical methods have widely been utilized in food completely isolate BPA from food matrices which involves the use of
analysis for the detection and quantication of proteins, enzymes, large volumes of organic solvent that subsequently has to be evap-
vitamins and other naturally occurring substances (reviewed in orated for further treatment or analyte concentration. This makes
468 A. Ballesteros-Gmez et al. / J. Chromatogr. A 1216 (2009) 449469

the respective sample treatments laborious and time-consuming. [22] Y.B. Wetherill, C.E. Petre, K.R. Monk, A. Puga, K.E. Knudsen, Mol. Cancer Ther.
In this respect, alternative strategies that provide high extraction 7 (2002) 515.
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Purication of samples becomes essential because matrix com- [24] Integrated Risk Information System (IRIS), Bisphenol A (CASRN 80-05-7), U.S.
ponents constitute the main source of errors in the determination Environmental Protection Agency, Washington, DC, 1988. Available on line at
http://www.epa.gov/iris/subst/0356.htm.
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considerably reduce quantication accuracy in all the techniques. als and articles intended to come into contact with foodstuffs (Ofcial Journal
On the other hand, purication of samples is also required for satis- of the European Communities L71 8).
[26] EC, Regulation (EC) No. 1907/2006 of the European Parliament and of the
factory long-term chromatographic system performance during the Council of 18 December 2006, concerning the Registration, Evaluation, Autho-
analysis of a range of samples. SPE is by far the most used technique risation and Restriction of Chemicals (REACH), establishing a European
for sample purication, although proteins and lipids must be previ- Agency, amending Directive 1999/45/EC and Repealing Council Regulation
(EEC) No. 793/93 and Commission Regulation (EC) No. 1488/94 as well
ously removed to avoid clogging of the sorbent. The use of selective
as Council Directive 76/769/EEC and Commission Directives 91/155/EEC,
sorbents such as RAMs, immunosorbents and MIPs should increase 93.67/EEC, 93/105/EC and 2000/21/EC, 2006.
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[39] E.M. Mungua-Lpez, S. Gerardo-Lugo, E. Peralta, S. Bolumen, H. Soto-Valdez,
The authors gratefully acknowledge nancial support from Food Addit. Contam. 22 (2005) 892.
Spanish MEC (Project CTQ2005-00643). A. Ballesteros-Gmez [40] T. Yoshida, M. Horie, Y. Hoshino, H. Nakazawa, Food Addit. Contam. 18 (2001)
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acknowledges to the Spanish MEC the doctoral fellowship awarded
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