A Review of Separation Methods For The Determination of Estrogens and Plastics-Derived Estrogen Mimics From Aqueous Systems

Analytica Chimica Acta 696 (2011) 626
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Review
A review of separation methods for the determination of estrogens and plastics-derived estrogen mimics from aqueous systems
Alesha D. LaFleur, Kevin A. Schug
Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, USA
a r t i c l e
i n f o
a b s t r a c t
Recent methods of separation and detection for the quantication of trace-level concentrations of selected endocrine disrupting compounds (EDCs) from aqueous systems are reviewed. A brief introduction of the selected EDCs (natural and synthetic estrogens and plastics-derived xenoestrogens), including their characteristics and importance, is presented. Sample preparation and extraction trends are discussed. Various types of separation techniques are presented, with the express goal of emphasizing time and costeffective methods that isolate and quantify trace-levels of multiple endocrine disruptors from aqueous systems. 2011 Elsevier B.V. All rights reserved.
Article history: Received 7 December 2010 Received in revised form 11 March 2011 Accepted 28 March 2011 Available online 9 April 2011 Keywords: Endocrine disruptor Estrogen Phthalate Phenol Chromatography Review
Abbreviations: 4-CP, 4-cumylphenol; 4-OP, 4-(tert-octyl)phenol; -E2 or -E2, 17 -or 17 -estradiol; AP, alkyl phenols (AP); APCI, atmospheric pressure chemical ionization; APEO, alkylphenolic ethoxylate; APPI, atmospheric pressure ionization; BBP, butylbenzyl-phthalate; BPA, bisphenol A (2,2-bis(4-hydroxyphenyl)propane); BPAF, bisphenol AF; CAD, charged aerosol detection; CE, capillary electrophoresis; CI, chemical ionization; CL, chemiluminescence; CPE, cloud point extraction; CZE, capillary zone electrophoresis; DAD, diode array detection; DBP, di-butyl-phthalate; DCHP, di-cyclohexyl-phthalate; DEHP, di-(2-ethylhexyl)-phthalate; DEHP, di-(2-ethylhexyl)-phthalate; DEP, di-ethyl-phthalate; DES, Diethylstilbestrol; DHP, di-n-hexyl-phthalate; DIBP, di-iso-butyl-pthalate; DIDP, di-decyl-phthalate; DINP, di-iso-nonyl-phthalate; DOP, dioctyl phthalate; DVB, divinylbenzene; E1, estrone; E1-3G, estrone-3-glucuronide; E1-3S, estrone-3-sulfate; E2-3G, estradiol-3-glucuronide; E2-3S, estradiol-3-sulfate; E3, estriol; E3-16G, estriol-16-glucuronide; E3-3G, estriol-3-glucuronide; E3-3S, estriol-3-sulfate; EC, electrochemical; ECD, electron capture detection; EDCs, endocrine disrupting compounds; EE2, 17 -ethynyl-estradiol; EI, electron impact; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; EU, European Union; FID, ame ionization detection; FL, uorescence; FMPTS, 2-uoro-1-methylpyridinium p-toluenesulfonate; GC, gas chromatography; GCE, glassy carbon electrode; HILIC, hydrophilic interaction liquid chromatography; HPLC, high performance liquid chromatography; IC, ion chromatography; ISO, International Organization for Standardization; IT, ion trap; LLE, liquidliquid extraction; LOD, limit of detection; LPME, liquid-phase microextraction; LVSEP, large-volume sample stacking in-line concentration; MISPE, molecularly imprinted solid-phase extraction; MRM, multiple reaction monitoring; MS, mass spectrometry; MSTFA, N-methyl-N-trimethylsilyl-triuoracetamide; MTBSTFA, n-methylN-(tert-butyldimethyltriuoroacetamide); PAE, phthalate acid esters; PAH, polycyclic aromatic hydrocarbon; PDMS, polydimethylsiloxane; PET, poly(ethylterephthalate); PFBBr, pentauorobenzyl bromide; PFBOCl, pentauorobenzoyl chloride; PTA-OH, phenyltrimethylammonium hydroxide; PVC, poly(vinyl chloride); QqQ, triple quadrupole; RI, refractive index; SBSE, stir bar sorptive extraction; SIM, single ion monitoring; SPE, solid phase extraction; SPME, solid phase microextraction; SRM, single-reaction monitoring; TMCS, trimethylchlorosilane; TOF, time-of-ight; UHPLC, ultra-high-performance liquid chromatography; US EPA, United States Environmental Protection Agency; UV, ultraviolet; WHO, World Health Organization. Corresponding author at: 700 Planetarium Pl., Mailbox 19065, Arlington, TX 76019-0065, USA. Tel.: +1 817 272 3541; fax: +1 817 272 3808. E-mail address: kschug@uta.edu (K.A. Schug). 0003-2670/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.03.054
A.D. LaFleur, K.A. Schug / Analytica Chimica Acta 696 (2011) 626
Alesha LaFleur received her M.S. from the Department of Chemistry and Biochemistry at the University of Texas at Arlington (UTA) in 2010. She received her B.S. degree in Biochemistry in 1997 from Texas Wesleyan University. She has been employed in the pharmaceutical industry for 13 years with a focus on surgical devices and pharmaceutical packaging.
Kevin Schug is assistant professor in the Department of Chemistry and Biochemistry at the University of Texas at Arlington (UTA). Kevin received his B.S. degree from the College of William and Mary and his Ph.D. degree in Chemistry from Virginia Tech under Prof. Harold McNair. He performed post-doctoral research in the laboratory of Prof. Wolfgang Lindner at the Institute for Analytical Chemistry and Food Chemistry at the University of Vienna in Austria. He joined UTA in 2005. His research has been focused on the theory and application of separation science and mass spectrometry for solving a variety of analytical and physical chemistry problems.
1. Introduction As environmental and social concerns about water quality increase and industries move toward an awareness of the life cycle of their products and packaging, the study of the environmental impact of endocrine disrupting compounds derived from manufactured materials will become more prevalent. Understanding which and how much biologically active compounds are in a body of water or products for human consumption is important not just to scientists and environmentalists, but also to governments, pediatricians, geneticists, and the general public. It is critical that the scientic community be responsible for generating reliable methods and data that support clinical and environmental studies being conducted in this area. Analytical chemists must provide the most accurate, sensitive, analytically robust methods for the isolation, identication, and quantication of these compounds, which can be present in trace amounts in aqueous systems. The goal of this review is to detail recent methods (from 2005 to 2010) developed for the quantication of select endocrinedisrupting compounds (EDCs) from aqueous systems. Focus is placed on various natural and synthetic estrogens, estrogen conjugates, and chemical additives used in the plastics industry that can act as estrogen mimics (Tables 1 and 2). The latter group is limited mainly to plasticizers and anti-oxidants used in the plastics manufacturing process. Extensive studies have been conducted for other known or suspected synthetic endocrine disruptors (pesticides, dioxins, ame retardants, parabens), as well as many naturally occurring compounds (isoavones and other phytoestrogens in plants) and their potential effects. They will not be presented here and the reader is instead referred to a number of excellent papers on these topics [13]. Instead of reviewing all of the available methods, this paper will focus on modern methods with recovery values greater than 80% and limits of detection (LOD) in the nanogram per milliliter range or better (ng g1 ). In this way, the methods presented here are not comprehensive, but will be those demonstrated to be better-suited for laboratory testing of trace-level EDCs from aqueous systems. We will rst present the selected EDCs, including their characteristics and importance for investigation. Next, we briey cover common sample preparation methods for isolating the identied EDCs from aqueous systems. Finally, methods of separation and detection for the quantication of trace levels of the selected endocrine disrupting compounds are discussed. These methods were selected with the express goal of highlighting time and cost-efcient ways to effectively isolate and quantify trace levels of multiple endocrine disruptors from aqueous systems. A list of acronyms used throughout this review and their meanings has been included for the readers convenience.
Table 1 Select estrogen structures. Compound Abbreviation Chemical structure
Estrone
E1
HO
OH
17 -Estradiol
E2
HO
OH
17 -Estradiol
E2
HO
OH OH
Estriol
E3
HO
OH
17 -Ethynylestradiol
EE2
HO
OH
Mestranol
MES
O Me
OH
Dienestrol
DIE
HO
OH
Diethylstilbestrol
DES
HO
8 Table 2 Select plastics-derived xenoestrogen structures. Phthalate compound
Abbreviation
Chemical structure
O O O
Di-methyl-phthalate DMP
O O O O
Di-ethyl-phthalate
DEP
O O O O
Di-n-propyl-phthalate
DPP
O O O O
Di-butyl-phthalate
DBP
O O O O
Di-amyl-phthalate
DAP
O O O O
Di-n-hexyl-phthalate
DHP
O O O O
Di-n-octyl-phthalate
DOP
O O O O
Di-nonyl-pthalate
DNP
O O
O O
Di-iso-nonyl-phthalate DINP
O
O O O
Di-(2-ethylhexyl)-phthalate
DEHP
A.D. LaFleur, K.A. Schug / Analytica Chimica Acta 696 (2011) 626 Table 2 (Continued) Phthalate compound Abbreviation Chemical structure
O O O
Di-decyl-phthalate DIDP
O O O O
Di-iso-butyl-phthalate
DIBP
O
O O O
Butylbenzyl-phthalate
BBP
O O O
Di-cyclohexyl-pthalate DCHP
Bisphenol A
BPA
HO
F F F F F F HO
OH
Bisphenol AF
BPAF
OH
4-tert-Octylphenol
4-OP
HO
nC8H17
n-Octylphenol
OP
HO
nC 9H19
4-Nonylphenol NP
HO
Ph HO
HO
4-Cumylphenol
4-CP
m-Methyl phenol
MP
4-tert-Butylphenol
4-BP
HO
The endocrine system is an integrative system that controls the cell function and activities of mammals, amphibians, birds, sh, and various invertebrates by communicating through chemical messengers called hormones. The endocrine system uses hormones to act as messengers that regulate reproduction, metabolism, growth and development, natural defenses to stress, as well as water, electrolyte, and nutritional balance of the blood [6]. The rst and most crucial step of healthy endocrine system functioning is
1.1. Signicance and importance Endocrine disruptors are any externally originating chemical compound, either natural or synthetic, that interferes with normal endocrine function. These compounds are thought to affect the binding, synthesis, signaling, or decomposition of essential hormones [2,4,5]. An example of how EDCs can interfere with receptor sites is outlined in Fig. 1.
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Fig. 1. (a) Normal hormone activates the receptor at the appropriate level and time. (b) Hormone blocker interferes with the signal from the body hormones. (c) Hormone disrupters give a weaker or stronger-than-normal signal at inappropriate times compared to the bodys hormones. Adapted from [2].
hormone-receptor binding. The receptor increases or decreases the rate of a target cells activity depending upon: (a) the concentration of a specic hormone in the blood, (b) the number of receptors available in the target cells, and (c) the binding strength between the hormone and the receptor [7]. The scientic and medical communities have discovered a number of compounds that disrupt normal estrogen signaling processes [4,8]. This research has provided a link between the presence of estrogens and estrogen mimics in our environment and the increasing reports of gender mutations and reproductive dysfunction in a number of species of wildlife, including sh, amphibians, birds, and mammals [9,10]. In the past, the effects of single compounds have been studied independently, but now mixtures of low-concentration EDCs are suspected of acting together to impact endocrine functions [10]. Currently, there are few conclusive studies or regulations regarding a safe dose of some of these biologically active compounds when acting independently, much less as mixtures. In fact, there is evidence to suggest that the current linear dose response of the standard toxicological model does not accurately predict the toxicity of some EDCs [8]. Even so, the implications of the research on EDCs, through environmental risk assessments and chemical monitoring, are leading to the discussion and recommendation of regulations by various governmental and scientic agencies [11]. The demand for environmental analysis is driven mostly by regulatory agencies with the major motivator being the potential impact of synthetic chemical contaminants to public and environmental safety. In the United States, the US Environmental Protection Agency (US EPA) has been authorized to screen all manufacturing or processing chemicals and formulations for potential endocrine activity when drinking water and/or food supply lines are at risk for contamination [12]. In Europe, the European Community on Risk Assessment and Directive on the Classication of Dangerous Substances proposes limits and strategies to deal with EDC contamination in waterways. In March 2000, Europe adopted a document titled, Community strategy for endocrine disrupters: A range of substances suspected of interfering with the hormone systems of humans and wildlife. This spurred the development of analytical and environmental monitoring tools and programs for EDCs [13]. The European Union (EU), US EPA, and the World Health Organization (WHO) have also characterized and proposed acceptable limits of certain EDCs in drinking water [12]. For example, the EPA maximum limit for di-(2-ethylhexyl)-phthalate (DEHP), a phthalate commonly found in drinking water, is 6 ng mL1 [14,15]. Various medical industries comply with the International Organization for Standardization (ISO), which has proposed testing strategies to assess chemical contaminants and leachable compounds from medical devices in its guidance, ISO 10993. A leachate is a chemical or component from either the device itself, or the product packaging (plastics, inks, paper, or adhesives) that can migrate from or through the plastic and into the product or biological sys-
tem. The phasing out of certain plasticizers in poly(vinyl chloride) (PVC) is a recent example of industry regulation in Europe. The EU opted to ban the use of certain phthalates in toys and infant teething products in 2005 and to completely eliminate the use of the material in medical devices in 2009 [16]. Legislation involving other synthetic materials and their additives (poly(carbonate), poly(ethylterephthalate) (PET), bisphenol A (BPA)) are currently being considered in a number of states in the US as well as by other nations [8]. These were mainly prompted by study ndings that potential endocrine disruptors were leaching from these materials into products, foods, medical devices, water, and milk or infant formula [14,17]. 1.2. Compound classes of interest Known and potential endocrine disruptors in the environment originate from many sources including pharmaceuticals, personal care products, synthetic compounds from polymers (xenoestrogens), pesticides, naturally occurring compounds in plants and animals, and inorganic and organometallic compounds. Estrogenic steroid hormones were selected for the focus of this paper because of their propensity for strong bio-activity at lower concentrations (Table 1). The scope of xenoestrogens discussed here has been narrowed specically to certain additives and plasticizers used in the plastics industry that have demonstrated estrogenic effects (Table 2). 1.2.1. Estrogens and estrogen conjugates Estrogens are steroids that play important roles as sex hormones in animals. These hormones are potent and can cause profound effects at very low concentrations. The free estrogens selected for review in this publication promote target organ responses nearly instantaneously, whereas other steroid hormones can take hours to register any effect. Reproductive interferences and effects have been observed from estrogens and estrogen conjugates in water from concentrations as low as 1 ng L1 [18]. They are natural estrogens, such as estrone (E1), 17 - or 17 -estradiol ( -E2 or -E2), estriol (E3), and 17 -ethynyl-estradiol (EE2). These are produced by animals or can be found in certain pharmaceuticals [10,19]. E2 is biodegraded to E1, which degrades to E3 in the aquatic environment. The -form of estradiol is the synthetic hormone used in both pharmaceuticals and for livestock. These compounds persist in water due to incomplete removal from wastewater treatment facilities. Free estrogens are rarely detected in urine, however, they metabolize in the body to sulfate and glucuronide conjugates which are then eliminated in urine. The most prevalent conjugates found in adult female urine by researchers were g mL1 levels of conjugated E3, -E2, and E1 [20]. Conjugated estrogens can also be found in prescription medications as sulfated estrogen salts to treat hormonal imbalances, post-menopausal symptoms, and osteoporosis,
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among other conditions [21]. These conjugates are less likely to exhibit signicant biological activity; however, they are converted back into their free forms during water treatment techniques and regain their potency [22,23]. Table 1 depicts select structures of the estrogens covered in this review. 1.2.2. Plastics-derived xenoestrogens Non-steroidal synthetic estrogens are known as xenoestrogens. Table 2 displays a number of compounds linked to this classication. Xenoestrogens are suspected of disrupting reproduction in humans and wildlife, increasing the incidences of cancers of the breast and testes, and impacting the neural development of fetuses and young children. Studies linking these health effects to xenoestrogens are currently being conducted; this increases the importance placed upon the quality of analytical data used to estimate the concentrations of these compounds in the environment [2426]. Some antioxidants and plasticizers in the manufacture of plastics have been shown to be estrogenically active. Many industrial compounds that have demonstrated estrogenic activity contain phenol groups and can be found in water. Generally, plasticizer additives have low molecular weights and can migrate from packaging material into consumer products or an aqueous system and become indirect food additives or contaminants in the environment. Recent studies conducted on bottled water in both glass and plastics found EDCs in 60% of the water samples tested. The highest concentrations were found in plastic bottles, suggesting that the plastics contribute to higher levels of contamination in water [27]. This review will focus predominately on analytical methods to determine phthalates and phthalic acid esters (PAEs), phenolic antioxidants (specically the para-OH containing compounds), and bisphenol A. 1.2.2.1. Phthalates (phthalic acid esters). Today, phthalate acid esters (PAEs) are one of the top offenders in the growing list of suspected EDCs used in common household products, cosmetics, detergents, ame retardants, plastics, inks, adhesives, metal food can liners, and medical devices. Specic phthalates (DBP, DEHP, BBP, DEP and DHP) (Table 2) are thought to disrupt the endocrine system by competing with 17 -estradiol for binding to the estrogen receptor [14,28]. Their entry into the environment occurs directly from the worldwide production of tons of plastic materials every year and indirectly via volatile emissions (incinerations) and leaching from their parent polymeric material [13,14,29]. Phthalates are not chemically bound in plastics and as such, can leach into the environment over time [14]. Often found in fatty foods from packaging or leaching from medical devices, di-(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) are a few of the additives generating growing interest. Toxicological evaluations have indicated that the lower molecular weight phthalates (DEP) are irritating to the eyes, nose, and throat. Other larger molecular weight phthalates (DEHP, BBP, DINP, and DIDP) are suspected carcinogens, as well as toxic to the liver, kidneys, and reproductive organs [14]. These lipophilic compounds have low solubility in water and have been found to bio-accumulate in fats. They are susceptible to photo-degradation through free radical attack, biodegradation, and to a lesser extent, hydrolysis. 1.2.2.2. Phenolic additives. The phenolic additives of interest for this review are polymer additives that have an unhindered phenolic OH in the para-position. Phenolic antioxidants are used to reduce free radical growth and deactivate the formation of hydroperoxides. These antioxidants maintain certain physical qualities desired in the plastics; however, not all are without health or environmental risks. Alkyl phenols, used as antioxidants and plasticizers in the plastics industry, have been shown to stimulate the growth of breast cancer cells [3]. Recent studies demonstrated
that branching at the -carbon in alkyl phenols with 89 carbon chain lengths show the greatest estrogenic effects [22]. Alkyl phenols and other phenolic compounds have been found to contain weak estrogenic activity at g L1 concentration levels [22,30]. The compound 2,2-bis(4-hydroxyphenyl)propane, commonly known as bisphenol A (BPA), synthesized by condensation of phenol with acetone, is a building block of polycarbonates. A related compound that is potentially more harmful to human health is the chemical bisphenol AF (BPAF). BPAF is used as a cross-linker in uoroelastomer gaskets and hoses used in food processing equipment and as a monomer in a multitude of polymers used for electronic devices and optical bers. Both BPA and BPAF are thought to mimic estrogens and react with the estrogen receptors in the body; however, a recent study by a group of biochemists in Japan has found that the uorine atoms of BPAF bind to beta estrogen receptors 50 times more effectively than BPA [31,32]. Suggestions of estrogenic and anti-androgenic activity in vitro prompted the National Toxicology Program of National Institute of Environmental Health Sciences and the National Institutes of Health (U.S. Department of Health and Human Services) to suggest comprehensive toxicological characterization of BPAF in 2008. A comprehensive summary of the endocrine activity of 36 bisphenol A analogs and derivatives, including BPAF, is published on the National Institutes of Health website as a part of a chemical information prole of BPAF [33]. 1.3. Technical challenges for trace level EDC determination The goal of many scientists is to nd a method for routine use that is fast, sensitive, reproducible, inexpensive, relatively green, and can simultaneously detect trace levels of estrogens and multiple EDCs in aqueous systems. Analytical determination of EDCs has been rened in the last decade, but is not without remaining challenges and difculties. Specically, the diverse chemical characteristics of the analytes require a variety of analytical approaches. It is rare to nd one method that is capable of determining trace levels of different classes of compounds in a single run. Also, the efcient and complete isolation and extraction of the target compounds from the sample matrix presents another set of challenges as they can be chemically reactive or unstable in nature, necessitating unique handling techniques. In spite of sample treatment (and sometimes because of it) the ability to obtain a reliable background level as a starting point to track contamination can be extremely difcult. Nearly every body of water tested contains one or more forms of naturally occurring estrogenic compounds. In a related issue, incidences of sample contamination have been discovered to originate from common laboratory or sample processing practices from sources such as storage containers, contaminated solvents, lters, tubing, pipette tips, septa, and processing equipment [13,14]. For example, it was reported that rinsing or pre-washing SPE cartridges with methanol and replacing vinyl tubes with VitonTM tubing from the SPE sample loading apparatus minimized potential BPA contamination in a trace-level study [34]. Not only is contamination a potential issue, but the loss of component due to sample processing has also been observed. Specic extraction techniques have been shown to cause low recoveries of alkyl phenols, for example, due to a loss in the tubing used to carry an extract to a sample vial [35]. The practice of precipitating proteins from liquid samples has also been problematic for similar reasons. Losses due to the solvents used for precipitation combined with the hydrophobic behavior of the analytes are also possible [34]. Another challenge that remains a stumbling block in EDC determination is overcoming matrix effects on the analysis. Most samples are complex matrices of sludge, pharmaceutical drugs, fats, proteins, salts, sugars, or other unwanted material, as far as
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analytical determination is concerned. Matrix issues can impact the quality of the analysis, therefore, proper sample treatment is essential in order to isolate and quantitate trace levels of EDCs. Ultra-trace and trace-level quantitation limits also present challenges, particularly when the quantity of sample available for testing is limited. Large quantities of sample are desirable in order to lower detection limits, but the higher the volume of sample that is pre-concentrated, the more likely the matrix will interfere [36]. Effective isolation of a particular compound can be complicated by a sample matrix that contains many similar interfering components. In addition, sample matrices can also complicate detection by causing an increase in the background signal, lowering recoveries, or introducing chromatographic interferences which can affect reproducibility and accuracy. Signal suppression or enhancement is readily observed in the literature when environmental sample matrices are analyzed by electrospray or atmospheric pressure ionization interfaces with mass spectrometry techniques [37,38]. This issue was the focus of a study by Benijts et al. [37] that evaluated the matrix effects of environmental samples (surface, rain, ground, channel, wastewater, and industrial efuents) on a mass spectrometry method (HPLCESI-MS/MS) for the quantitation of EDCs. They minimized the impact of sample matrix due to sample origin through extensive sample cleanup (removing many co-eluting matrix components by solid phase extraction) and using isotopically labeled internal standards for quantitation. Even after taking extra steps for sample cleanup, the researchers were unable to overcome the suppressive effects (exceeding 50% in some cases) on the less polar, later-eluting alkyl phenolss (4t-octylphenol, 4-octylphenol, and nonylphenol) in environmental water matrices. This was attributed to the lack of efciency in the removal of hydrophobic matrix components by solid phase extraction. In trace analysis, it may become desirable to improve the signal intensity by concentrating the sample. The relationship between the sample volume to be concentrated and matrix effects is complicated and must be considered when selecting an analytical method. The volume of sample available is a key consideration if multiple assays are required to identify and quantify various EDCs [35]. Some general recommendations for trace analysis of steroid hormones and EDCs are to avoid sample contact with plastic materials, storage, and transfer equipment. It is essential to collect a blank sample and controls to run with every analysis. Treated, cleaned, deactivated glassware, lab supplies, solvents, and personal protective equipment must be used to avoid contamination [39,40]. It is not recommended to make more than one injection from a sample vial [14]. Trace analysis requires additional attention to potential sources of contamination on the part of the analyst, and special efforts and expense must be made in order to obtain quality data.
Common examples of isolation techniques observed in the literature to achieve separation of EDCs from aqueous matrices are: liquidliquid extraction (LLE), solid phase extraction (SPE), cloud point extraction (CPE), solid phase microextraction (SPME), and stir bar sorptive extraction (SBSE). The topic of sample preparation techniques has been addressed extensively in a number of exceptional books and reviews; therefore, they will not be discussed in great detail in this review [4144]. Once the component of interest has been isolated from the aqueous sample matrix, it may be necessary to improve the sensitivity of the method. Chemical derivatization is a form of sample treatment that is commonly used to improve sensitivity and selectivity in both HPLC and GC methods. There are a number of disadvantages to using chemical derivatization. The chemicals required are typically toxic, and while the technique greatly increases the sensitivity of the methods used, they can be labor intensive. In addition, derivatization agents can quickly degrade column performance if not properly or completely quenched after the designated reaction period. In some methods, the derivatization agent cannot distinguish between structurally related compounds, in effect, producing the same derivatives for two different compounds [45]. The sample treatment may also interfere with the separation and shape of the analyte peaks. Sample pre-treatment is essential in many cases, however, and allows the analytical chemist to effectively isolate and analyze specic compounds of interest from complex aqueous matrices. The most commonly observed derivatization reactions seen in the literature for this review were esterication or silyzation of the phenolic OH on the compound of interest. These derivatization techniques were used to dramatically improve the volatility of the compound of interest (for GC analysis), or to add functional groups that increased the specicity and sensitivity for particular types of detection. For example, a sensitive uorescence method was developed to analyze estrogens in urine using p-nitrobenzoyl chloride [46]. In a separate study, a number of derivatization agents were evaluated for optimal detection of steroid estrogens in water. The analysts observed that derivatization increased the mass spectral signal intensity of the component of interest 212 times compared to an untreated sample [47]. Unfortunately, derivatization is targeted for a specic group of analytes, often excluding related compounds and metabolites that are also present in the samples [40]. This selectivity, although sometimes desirable, requires the use a variety of methods in order to accomplish the analysis of a single sample; a situation that increases time and cost of sample analysis. 3. Analytical applications In this section we will briey introduce instrumentation used to separate and detect natural and synthetic estrogens and plasticsderived xenoestrogens from aqueous systems. Each application will then be discussed as it applies to the particular analytical methods including any observations regarding the strengths and or weaknesses of the technique. Due to the range in polarities of the classes of EDCs discussed, the techniques considered for this review will also cover a rather wide range. Summaries of select GC and HPLC methods for trace EDC determination are displayed in Tables 3 and 4, respectively. 3.1. Gas chromatography (GC) Gas chromatography (GC) is an indispensible chromatographic technique for the detection of volatile or semi-volatile compounds of interest. There are a number of different injection modes available, a variety of columns useful for separation, and several
2. Sample treatment Generally the rst goal of any assay is isolating the components of interest from the sample matrix into an injectable solution at concentrations detectable by the selected analytical separation system. In many cases, the matrix is complex and interferes with detection of low-level compounds. The most common and simplistic techniques used to minimize matrix effects are to isolate the components of interest through various extraction methods. The proper sample treatment can considerably improve the limit of quantitation and detection for the compounds of interest. The most effective sample treatment uses minimal sample, alters it as little as possible, and has a limited number of steps. Analytical laboratories aim to develop sample preparation techniques that are accurate, reproducible, robust, simple, cost-effective, time-efcient, and safe.
Table 3 GC methods for trace-level determination of endocrine disrupting compounds from aqueous systems. Separation technique GCMS/MS GCMS Stationary phase 5% phenyl, 95% fused silica (BPX-5 25 m 0.22 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (HP-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness) Cross-linked 5% methyl silicone (HP-5MS of 30 m 0.25 mm I.D., 0.25 m lm thickness) DB5-MS (60 m 0.32 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (HP-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (DB-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness) TRB-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness (5% diphenyl, 95% dimethylpolysiloxane) 5% diphenyl, 95% dimethylpolysiloxane (HP-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness) DB-1MS (30 m 0.25 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (DB-5HT 15 m) with a 1 m polysiloxane guard column 5% diphenyl, 95% dimethylpolysiloxane (DB-5MS 30 m 0.25 mm I.D., 0.5 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (DB-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (DB-5MS 30 m 0.25 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (HP-5 15 m 0.25 mm I.D., 0.25 m lm thickness) 35% phenyl, 65% methyl polysiloxane (DB35-MS 30 m 0.25 mm I.D., 0.25 m lm thickness) 5% diphenyl, 95% dimethylpolysiloxane (DB-5 60 m 0.25 mm I.D., 0.25 m lm thickness) 5% phenyl, 1% vinyl-methylpolysiloxane (SE-54 25 m 0.32 mm I.D.) 8% phenyl polycarborane-siloxane, 1 (HT8 50 m 0.22 mm I.D., 0.25 m lm thickness) with guard column Detection EI-multiple ion trap-MS EI-MS Sample matrix Ultrapure water Surface water Sample preparation technique SPE, derivatization SPE & LLE with derivatization SPME thermal desorption SPE, derivatization SPE, derivatization Compound of interest E2, E2, E1, EE2 EE2, E2, 4-NP, BPA Limit of detection LOQ 0.25 ng L1 Phenols 56 ng L ; EE2 50 ng L1 ; E2 300 ng L1 330 ng L1 0.020.10 ng L1 0.33.0 ng L
1 1
Reference [23] [49]
GCMS GCMS GCMS
EI-MS Magnetic sector EI-MS
River and sea water Wastewater, river water, drinking water Wastewater
DMP, DEP, DBP, DEHP BPA, OP, NP, E1, E2, E2, EE2 E1, E2, E3, MES, and estrogen conjugates BPA
[40,50,91] [50] [92] A.D. LaFleur, K.A. Schug / Analytica Chimica Acta 696 (2011) 626
GCMS
EI-MS
Water and aqueous extracts of plastics Drinking water
SPME with Headspace derivatization Stir bar sorptive extraction SPE with derivatization
0.4 ng L1
[52]
Large volume injection (LVI)/GCMS GCMS
EI-quadrupole
DBP, BBP
340 ng L1
[56]
EI-quadrupole
Wastewater
GCMS GCMS
EI-IT EI-magnetic sector
Groundwater Wastewater
SPE, derivatization SPE and derivatization
DMP, DEP, 4OP, 4-t-OP, 4-NP, DBP, BPA, BBP, BEHP, DOP Steroid estrogens and NPs NP, phthalate esters, E1, E2, E2, E3, EE2, Phthalates, alkylphenols, E1, E2 DEP, DBP, BBP
LOQ 20400 ng L1
[53]
Estrogens: 24 ng L1 ; NP: 500 ng L1 1.5172 ng L1
[93] [94]
GCMS
EI-MS
Wastewater
Stir bar sorptive extraction Modied SPE with ATD
2 ng L1
[18]
GCMS
EI-quadrupole
Industrial ultrapure water Tap and drinking water
3695 ng L1
[57]
GCMS
EI-quadrupole
LPME
DMP, DEP, DAP, DnBP, BBP, DCHP, DEHP Phthalates, alkylphenols, BPA 4-n-NP, 4-t-OP, 4-NP, BPA, E1, E2 DMP, DEP, DBP, BBP, DEHP, DOP DMP, DEP, DBP, DAP, DEHP, DnOP, DNP, DIDP DMP, DnBP, DnOP
0.020.05 g L1
[58]
GCMS GCMS
EI-quadrupole CI-quadrupole
Wastewater Surface water
SPME SPE and derivatization
354 ng L1 0.22.0 ng L
1
[59] [54]
GCMS GC-FID
EI-MS FID
GC-ECD
ECD
Wastewater and stormwater Puried water and 180 g L1 NaCl aqueous extract Wastewater
LLE SPME
0.050.1 g L1 630 ng L1
[95] [60]
LLE
2030 ng L1
[62]
Limit of quantitation (LOQ).
13
14
different detectors qualied for the detection of EDCs. A downside of using GC as a chromatography system is that direct injection of an aqueous matrix should be avoided due to the likely degradation of system and column performance. These criteria require reliable and efcient sample preparation steps that risk introducing errors and technical issues associated with treatment techniques such as derivatization or extraction. Even so, GC is a very well-established technique that offers extremely robust instrumentation that is widely available in nearly any laboratory setting. 3.1.1. MS detection The most common analytical approach reported for the trace determination of estrogen mimics, xenoestrogens, and estrogens is GC with mass spectrometry (GCMS). MS is the most suitable method for the simultaneous detection and quantication of mixtures of trace EDCs in aqueous systems because they provide the selectivity and sensitivity to analyze complex samples. Gentili et al. have written a comprehensive review of mass spectrometry methods available to analyze alkyl phenols, chlorinated phenols, and steroidal estrogens [22]. Another useful review of MS analysis of EDCs (alkyl phenols, polychlorinated compounds such as dioxins, furans and biphenyls, polybrominated diphenyl ethers, phthalates and steroid sex hormones) in aquatic environmental samples was written by Petrovic et al. in 2002 [40]. The determination of estrogens and progestogens by GCMS, HPLCMS, and HPLCMS/MS has also been reviewed [48]. The consensus of most of these reviews is that mass spectrometry is undeniably the best technique to examine trace levels and provide essential identication and quantication of EDCs in aqueous systems. Two distinct advantages of MS are: (a) it has mass-selective detection that is compound specic, and (b) it is subject to less interference when compared to other types of detectors [45]. These benets do come at a price; typically, the instrumentation is more complex and costly than standard HPLC or GC equipment, and it requires highly skilled technicians to maintain a properly functioning system. MS also involves intense data analysis and a thorough understanding of matrix effects and fragmentation patterns. The future of applying MS detection to EDC trace analysis is becoming more promising as these techniques increase in number and become more commonplace in typical analytical laboratories. The GCMS methods will be organized and discussed by analyte type: estrogens and estrogen conjugates and then, plastics-derived xenoestrogens (including phenols and phthalates). 3.1.1.1. Estrogens and estrogen conjugates. GC separations of estrogens and progestogens are performed using various columns and typical temperature programs (45300 C) with helium carrier gas [40]. The stability, sensitivity, and precision of the methods have been improved by derivatization of the hydroxyl groups of the steroid ring. Derivatization of estrogens improves volatility and helps alleviate thermal decomposition [48]. These improvements come at the cost of time, as well as accuracy. In addition, poor detection of EE2 and MES, compounds which contain an ethynyl group, was observed because derivatization techniques failed to substitute the adjacent OH group (Table 1). In spite of these observed drawbacks, an advantage of using GCMS with electron impact over HPLCMS methods for synthetic and natural steroids is the availability of mass spectral libraries useful for characterization and identication [40]. A sensitive GCMS/MS procedure using SPE with derivatization agent MSTFA mixture (N-methyl-N-trimethylsilyl-triuoracetamide, ammonium iodide, and ethanethiol) was capable of detecting a group of natural and synthetic estrogens in water at trace levels (0.255 ng L1 ) with rather good recoveries in water (105 20%) and acceptable R2 values (0.94) [23]. This study exhibits how efcient and targeted
derivatization of EDCs can lower the detection limits by GCMS considerably. 3.1.1.2. Plastics-derived xenoestrogens. Phenolic EDCs. Combined determinations of xenoestrogens are challenging due to the various factors related to sample preparation and storage, extraction, and derivatization of different types of compounds (alcohols, phenols, and acids). Mol et al. recommended SPE if hormones are a part of the EDC group to be extracted from aqueous samples [49]. They preferred this approach over LLE due to improved response and peak shapes of estrogens and a lessening of matrix effects; however, LLE was the preferred method if hormone levels were not a part of the analysis. Unfortunately, if SPE is used, alkyl phenol recoveries can be affected due to loss during ltration of the aqueous sample or adsorption to an SPE lter. Only the most volatile degradation products of alkylphenolic compounds, such as alkyl phenols (AP) and alkylphenolic ethoxylates (APEOs) with fewer than 4 ethoxy groups, can be analyzed without derivatization [40,49]. Often, silylation is used because it can react with all these analytes. Specically, much success has been achieved using the reagent n-methyl-N-(tert-butyldimethyltriuoroacetamide), or MTBSTFA [49]. However, there are a number of methods capable of detecting EDCs at a reasonably sensitive concentration without derivatization. Specically, a GCMS method was able to separate and identify different isomers of the alkyl chain in underivatized 4-nonylphenol (Fig. 2), whereas it appears as a single broad peak in HPLCMS. Bisphenol A, a polar compound, was able to be detected in water by GCMS at a detection limit of 0.11 g L1 underivatized, but the sensitivity was improved using pre-concentration, LLE, derivatization to silyl BPA or pentauorobenzoylate ester. BPA-pentauorobenzoylate was detected using negative chemical ionization MS with methane as the reagent gas at a LOD of 20 pg L1 [34,40,50]. This clearly demonstrates the advantages of using derivatization to improve MS detection of phenolic EDCs. In another interesting study evaluating advantages and disadvantages of derivatization, three different techniques were used for the detection of phenolic endocrine disruptors in marine samples [51]. GCMS with and without derivatization was compared to HPLCESI-MS without derivatization. In addition, an HPLC method with UV detection was discussed. The compounds analyzed were octylphenol, nonylphenol, cumylphenol, and bisphenol A. The authors found that derivatization of the phenolic compounds with 0.5 M methanolic solution of phenyltrimethylammonium hydroxide (PTA-OH) greatly improved sensitivity and peak shape by GCMS. This reagent was selected due to its ability to methylate alcohols, phenols and carboxylic acids in wet/aqueous media. The GCMS method was linear (R2 0.99) and sensitivity was increased two orders of magnitude, to the ng L1 range when derivatization was used. In this case, the GCMS method was found to be more sensitive than either HPLC with ultraviolet (UV) detection or HPLCMS. The LOD of BPA was improved shortly thereafter when Chang et al. achieved a detection limit of 0.4 ng L1 in using SPME in conjunction with GCMS and headspace derivatization (bis(trimethylsilyl)triuoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) vapor) [52]. The sole focus of this paper was BPA and no other potential EDCs were analyzed. One year later, a method featuring SPE-GCMS with silylation was able to successfully separate and detect phthalate esters, APs, BPA, and their chlorinated derivatives in wastewater with recoveries greater than 95% in the g L1 range [53]. In a separate experiment, seven estrogenic phenolic compounds (4-tert-octylphenol, 4-nonylphenol, bisphenol-A, triclosan, estrone, estradiol, and diethylstilbestrol) were simultaneously extracted and analyzed from surface water using GCMS with negative chemical ionization. The samples were extracted by SPE and later derivatized using pentauorobenzoyl chloride (PFBOCl) [54]. With the exception
Table 4 HPLC methods for the determination of trace-level endocrine disrupting compounds from aqueous systems. Separation technique HPLCMS/MS HPLCMS/MS HPLCMS/MS Stationary phase Synergi Max-RP C12 (250 mm 4.6 mm I.D., 4 m) Purospher STAR-RP-18e (55 mm 2 mm I.D., 3 m) Zorbax Extend-C18 column (150 mm 1 mm I.D., 3.5 m) Luna C-18 (100 mm 2 mm I.D., 3 m) with C-18 guard column (4 mm 2 mm, 3 m) LC-18 (250 mm 4 mm I.D., 5 m) with precolumn Synergi Max-RP C12 (250 mm 4.6 mm I.D., 4 m) SunFire C18 column (150 mm 2.1 mm I.D., 5 m) Purospher STAR-RP-18e (125 mm 2 mm I.D., 5 m) with guard (4 mm 4 mm, 5 m of same packing) BDS Hypersil C18 (150 mm 4.6 mm I.D., 5 m) Zorbax Eclipse XDB C18 (100 mm 2.1 mm I.D., 3.5 m) HILIC TSKgel Amid-80 (150 mm 2.0 mm I.D., 5 m) with guard cartridge Hypersil GOLD C18 (50 mm 2.1 mm I.D., 3 m) with guard (2 mm 2 mm, 5 m of same packing) Purospher STAR-RP-18e (125 mm 2 mm I.D., 5 m) with guard (4 mm 4 mm, 5 m of same packing) Waters Acquity C18 (50 mm 2.1 mm I.D., 1.7 m) Betasil C18 (150 mm 2.1 mm I.D., 3 m) LiChrospher 100 RP-18 (250 mm 4 mm I.D., 5 m) LiChrospher 100 RP-18 (250 mm 4 mm I.D., 5 m) with guard column (4 mm 4 mm, 5 m of same packing) Detection ESI-APCI-QqQ ESI-QqQ ESI-QqQ Sample matrix Deionized water Deionized water Lake water Sample preparation technique SPE SPE SPE Compound of interest E2, EE2 NP E1, E2, E2, EE2, E3, and estrogen conjugates E1, E2, EE2, E3, DES Limit of detection 1.0 ng L1 1 ng L1 0.13.1 ng L
1
Reference [96] [72] [38]
HPLCMS/MS
ESI-IT
Deionized water and environmental water Surface water Deionized water and wastewater Water and wastewater Wastewater
SPE
0.52.0 ng L1
[67] A.D. LaFleur, K.A. Schug / Analytica Chimica Acta 696 (2011) 626
HPLCMS/MS HPLCMS/MS HPLCMS/MS HPLCMS/MS
ESI-QqQ ESI-QqQ and APCI QqQ ESI-QqQ ESI-QqQ
SPE SPE SPE, derivatization Immunosorbent extraction
BPA, E1, E2, EE2, E3 E1, E2, E3, EE2 E1, E2, EE2 E1, E2, E3, EE2
0.10.4 ng L1 0.30.6 ng L1 0.40.7 ng L1 0.42 ng L1
[36] [97] [98] [69]
HPLCMS/MS HPLCMS/MS HPLCMS/MS
APCI-QqQ ESI-QqQ Ion S pray-Q-IT
Cell culture medium Surface and ground water Urine
SPE SPE SPE
E1 and six metabolites E1, E2, E2, EE2, E3 Estrogen conjugates (sulfate and glucuronide) E3, E2, EE2, E1
0.91.4 ng mL1 0.010.2 ng L1 21000 ng L

1
[64] [99] [21]
HPLCMS/MS
APPI-QqQ
Surface and wastewater
SPE
350 ng L1
[71]
HPLCMS/MS
ESI-QqQ
Milli-Q water and river water
SPE
E2-17G, E1-3S, E2, EE2, E1
0.030.85 ng L1
[100]
UPLCMS/MS HPLCMS HPLCMS HPLCMS
Q-TOF ESI-quadrupole ESI-MS ESI and APCI-MS
Wastewater Wastewater Water Groundwater
SPE Immunosorbent extraction On-line SPE SPE
E1, E2, E3, EE2 E2, E1 E3, E2, E1, DES BPA, E2-17G, E1-3S, E3, E2, EE2, E1
56 ng L1 0.070.18 ng L1 <1 ng L
1
[69] [66] [67] [68]
0.536.30 ng L1
15
16
Table 4 (Continued) Separation technique HPLCMS HPLCMS HPLC-DAD Stationary phase BetaBasic C18 (150 mm 2.1 mm I.D., 3 m) Zorbax Eclipse XDB-C8 (50 mm 2.1 mm I.D., 3.5 m) LiChrospher 100 RP-18 (250 mm 4 mm I.D., 5 m) LiChrospher 100 RP-18 (250 mm 4.6 mm I.D., 5 m) Hypersil ODS-C18 (150 mm 4.0 mm I.D., 5 m) Tracer excel 120 OctaDecilSilica-A column (150 mm 4.0 mm I.D., 5 m) Eurospher-100 ODS (250 mm 4.0 mm I.D., 5 m) ODS-C18 (250 mm 4.6 mm I.D., 5 m) Diamonsil-C18 (250 mm 4.6 mm I.D., 4 m) Supelcosil LC-18 (100 and 250 mm 4.6 mm I.D., 5 m) Detection ESI and APCI-QqQ ESI-quadrupole DAD (200, 225, 240 nm) UV (280 nm) UV, 225 nm (190400 nm) DAD Sample matrix Milli-Q, drinking, river, and wastewater Physiological saline solutions LC-grade water, drinking, ground, and surface water Milli-Q and river water Intravenous injection solutions Water and urine Sample preparation technique SPE, derivatization Not applicable SPE Compound of interest E1, E2, E3, EE2 DMP, DEP, BBP, DBP E3, E2, EE2, E1, DES, MES MES, 4-NP, 4-t-OP BPA, DEP, DPP, DBP, OP, NP, DAP, DHP, DEHP, DOP E1, E2, E2, EE2, MES, DES DMP, DEP, DBP DEP, DEHP, DCHP DEP, DPP, DBP, DCHP, DEHP E3, E2, E2, EE2, E1, DES, MES, BPA, DMP,DBP, DOP, 4-BP, DES BPA 4-NP, BPA, EE2, E2, E2, E3 BPA EE2 BPA BPA, E2, E2, EE2, E1, DES, 4-t-BP BPA, DMP, EE2 Limit of detection <22 ng L1 0.9924.07 ng mL 1020 ng L1
1
Reference [47] [63] [65]
HPLC-UV HPLC-DAD
SPME SPE in tube microextraction Stir bar sorptive extraction with liquid desorption Micro dialysis enrichment Cloud point extraction SPE with ionic liquid mixed hemimicelles SPE
0.31.1 g L1 LOQ 110 ng mL

1
[55] A.D. LaFleur, K.A. Schug / Analytica Chimica Acta 696 (2011) 626 [73]
HPLC-DAD
25100 g L1
[103]
HPLC-UV HPLC-UV HPLC-UV HPLC-DAD
UV (225 nm) UV (226 nm) UV (226 nm) DAD
Water Environmental water Environmental water Environmental water
0.34 g L1 1.03.8 ng mL1 0.120.17 g L1 0.21.6 ng L1
[101] [74] [75] [78]
HPLC-DAD HPLC-FL
HPLC-FL HPLC-CL HPLC-CL HPLC-ED HPLC with amperomeric detection
Kromasil-C18 (250 mm 4.6 mm I.D., 5 m) Luna C18 (250 mm 4.0 mm, 5 m) with guard column (4 mm 3 mm) Hypersil ODS-C18 (150 mm 4.6 mm I.D., 5 m) CapcelPak ODS (250 mm 4.6 mm I.D.) Daisopak-SP-120-5-ODS-BP (250 mm 4.6 mm I.D., 5 m) LiChrospher 100 RP-18 (250 mm 4.6 mm I.D., 5 m) Luna C18 (150 mm 4.6 mm I.D., 5 m)
DAD (271 nm) FL
Tap water, sea water Urine
SPME SPE, derivatization
2.4 g L1 2.78.3 g L1
[77] [46]
FL (
em
ex = 276 nm, = 306 nm)
Urine Plasma Water River and wastewater Ground and tap water
SPE, derivatization Not applicable Not applicable SPME SPE
0.2 g L1 12 pg 0.38 g L1 0.060.08 g L

1
[80] [82,102] [82,84] [55] [81]
CL CL ED Carbon nanotube modied glassy carbon electrode 1.0 V
0.0980.340 M
Limit of quantitation (LOQ).
17
Fig. 2. GCMS chromatogram of 4-t-octylphenol and isomers of 4-nonylphenol. Reprinted from [40], Copyright 2002, with permission from Elsevier.
of high recoveries for estradiol (E2 135163%) and low recoveries for diethylstilbestrol (DES 7173%), the phenolic compounds had recoveries between 86% and 118% for 5 ng L1 spiked concentrations with detection limits between 0.2 and 2.0 ng L1 . These ndings demonstrate the equivalent sensitivity of chemical ionization when compared to electron impact for the quantication of phenolic EDCs in environmental water using GCMS. Phthalates. Phthalate esters have been detected using various MS methods including electron impact (EI-MS) and chemical ionization (CI-MS), in both the positive or negative modes. EI-MS was reported to have the most sensitive detection limits and is the recommended method for quantication of phthalate esters; although, it cannot give comprehensive qualitative information, such as molecular mass or the nature of the alcohol moiety in the molecule [40]. SPME using various polyacrylate bers, coupled to GCMS, have been compared for the concentration of six phthalates found in aqueous samples. The optimal ber for this purpose was indicated to be a 65- m polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated ber [14,55]. A low LOD (0.006 ng g1 (approximately 6 ng L1 )) of DEHP in aqueous samples using a SPME-GCMS was reported [14]. Researchers successfully used SBSE with a large volume injection GC method (LVI-GCMS) for ultra-trace phthalates in drinking water with instrumental LOD values of 0.150.60 g L1 for the simultaneous analysis of six phthalate acid esters and one adipate ester [56]. A drawback of this method is the total run time: slightly less than 25 min for chromatographic separation plus the extensive stir-bar extraction and concentration performed on the aqueous samples prior to analysis. Method improvements might be made in the future to make this a more time-efcient, high throughput method. It is also important to note that the recoveries of spiked water samples (0.40 g L1 ) were rather low for all of the analytes except DBP and BBP. These lower recoveries were explained by the higher hydrophobic nature of the phthalate esters; this was speculated to be the cause of adsorption of the analytes onto the sampling ask glass walls. The researchers were unsuccessful in attempts to use addition of methanol to reduce the adsorption phenomena [56]. The authors did not indicate if any other solvents were investigated for this purpose. Using SPE and automated thermal desorption GCMS, Liu et al. was able to achieve 3695 ng L1 detection limits for ve phtha-
late esters with recovery rates in ultrapure water between 15% and 101% [57]. Again, low recoveries were seen for some of the analytes of interest. This time, the explanation was that dioctyl phthalate (DOP) was not adequately adsorbed onto or desorbed from the SPE sorbent (Tenax TA). The authors also investigated different sample storage containers and concluded that storage in borosilicate glass bottles results in surface sorption of certain phthalates with longer alkyl chains. The loss of PAEs over storage time was not observed when samples were stored in PFA bottles (peruoralkoxy copolymer). This method shows promise in that it attempts to use an automated thermal desorption directly into a capillary GC column to analyze trace PAEs in ultrapure water for high-volume analyses. This would eliminate the solvent elution procedure needed for conventional SPE methods [57]. A separate method using liquidphase microextraction (LPME) GCMS demonstrated a number of advantages for qualitative and quantitative determination of select phthalate esters in water samples. The technique required minimal sample volume (10 mL), as well as a limited volume of organic solvent (7 mL of 1-dodecanol). It was reported to be simple and cost effective with no sample carryover and little, if any, matrix issues from complex samples [58]. The detection limits and extraction times were comparable with other microextraction methods (2050 ng L1 ; 25 min). Optimally, a selected method would be able to analyze a number of EDCs in a single run with minimal time and sample volume required. One multi-residue SPE-GCMS method was found to be sensitive enough to detect 42 priority contaminants in wastewater, including alkyl phenols, phthalates, and PAHs, in the ng L1 to g L1 concentration ranges [59]. In addition, this method only required a 50 mL sample for analysis. In another study, a different sample preparation technique, SPME using assorted polyacrylate bers, coupled to GCMS, was compared for the concentration of six phthalates found in aqueous samples. The optimal ber for this purpose was found to be a 65- m polydimethylsiloxane/divinylbenzene (PDMS/DVB), yielding a method detection limit for DEHP of approximately 6 ng L1 in aqueous sample [14,55]. 3.1.2. Other detection techniques Flame ionization detectors (FID) are commonplace and preferred because they are versatile, simple, and readily accessible to most typical analytical laboratories and gas chromatographs. The
18
sensitivity required for trace determination of EDCs may be suboptimal, however. Electron capture detection (ECD) is useful due to its extremely powerful selectivity and sensitivity for halogenated compounds, peroxides, quinones, and nitro groups, but it does not exhibit the sensitivity or linear response range required for trace level analysis for the EDCs presented in this paper. ECD requires special radioactivity disposal and safety training because it contains a -emitting source, usually nickel-63 or tritium, to ionize the carrier gas [60]. Most of the GC work being conducted currently makes use of MS detection; however, several studies were able to separate and quantify EDC mixtures using GC-FID. In their study of phthalate acid esters (PAE) in plastics, Li et al. reported that sodium chloride is often added to the sample to increase the ionic strength and enhance the amount of neutral analytes extracted by and SPME ber [61]. They examined different concentrations and revealed that the responses for most compounds that were part of their study for the PAEs were the greatest when there was 180 g L1 NaCl. This group also evaluated extraction times and desorption temperatures to optimize SPME with a GC-FID method. Their reported detection limits ranged from 6 to 84 ng L1 for the simultaneous extraction and quantication of eight PAEs using a calix[4]arene ber [61] from 180 g L1 sodium chloride. The separation of the eight compounds with GC-FID was successful in less than 12 min, demonstrating the potential separation efciency of GC-FID for EDC analysis. In a separate study, however, researchers addressed the negative impact of salt concentrations on phthalate compounds with lower water solubility, causing a decrease in response. They opted to avoid using NaCl altogether due to degraded peak shape, poor resolution, and the deterioration of the coating of the stir bars used in their sample extractions [56]. A study of the removal of six phthalates from a French wastewater treatment plant used an EPA method for organic chemical analysis of municipal and industrial wastewater. The analysis used GC with ECD and a very involved efuent extraction method that included a number of highly toxic solvents and compounds (hexane, dichloromethane, and mercury) [62]. The detection limits and recovery values for three of the six phthalates studied met the criteria of this paper: DMP (20 ng L1 ), DnBP (30 ng L1 ), and DnOP (20 ng L1 ). While ECD detection has shown good sensitivity for phthalates, they have weaker specicity than other detection methods [34]. In this case, the detection ranges reported for GC-ECD offer no advantage when compared to previously reported methods for the direct analysis (no extraction steps using toxic solvents) of aqueous samples with HPLCMS [63].
3.2.1. MS detection Mass spectrometry is widely considered to be the most sensitive and discerning detector for HPLC analysis. The main advantage of HPLCMS over GCMS is that the determination of trace levels of EDCs can be made without the tedious, and sometimes toxic, sample preparation process of derivatization [40]. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), in combination with single ion monitoring (SIM), single-reaction monitoring (SRM), or multiple reaction monitoring (MRM) techniques, are the most commonly found MS techniques in the recent literature. These approaches offer versatility, sensitivity, and selectivity for trace analysis in general [45]. The drawbacks of using ESI-MS and APCI-MS detection include matrix-induced signal suppression and isobaric spectral interferences from the sample that affect the sensitivity of some methods used to detect estrogens and other EDCs in aqueous environments [66]. The literature demonstrates that the triple quadrupole (QqQ) MS is preferred over a single quadrupole or ion trap analyzers for quantitative determination of phenols, due to its superior selectivity, higher signal-to-noise ratios and capability for using high speed when using MRM acquisition modes. QqQ reduces the possibility of false positives observed using single quadrupole due to poor selectivity caused by the presence of isobaric compounds in the matrix. Timeof-ight MS (TOF-MS) increases the selectivity further through improved resolution and mass accuracy of detected analytes. Modern TOF detectors are especially useful in trace qualitative and quantitative analysis when presented in a hybrid format, after an ion trap (IT-TOF) or quadrupole (Q-TOF) mass analyzer. The Gentili et al. review outlines and examines the advantages and disadvantages of many recent MS methods relevant to the analysis of phenols and steroidal estrogens [22]. LCMS techniques which use APCI and ESI in the negative ion mode have been published for the detection of phenolic compounds, which are weakly acidic. The steroidal estrogens, however, can be detected by both negative and positive-ion modes. Automated online methods have also increased in importance. The on-line SPE of aqueous samples with subsequent analysis by HPLC with diode array detection (DAD) or HPLCESI-MS is described by Petrovic et al. as a fully automated process that is capable of analyzing target compounds in the ng L1 concentration range [40]. This kind of development ensures mass spectrometrys place in standard analytical laboratories involved in high throughput, fully automated techniques. The HPLCMS methods are grouped by applications for the quantication of estrogens, and then those suited for quantication of plastics-derived xenoestrogens (phenols and phthalates). 3.2.1.1. Estrogens and estrogen conjugates. A good review detailing mass spectrometric applications for the determination of EDCs in aquatic environmental samples was published in 2002 [40]. The methods included in the review were published from about 1994 to 2002, so may not apply to the scope of this paper; however, they do serve as bench-marks for progress that has been made in the past decade in the detection of EDCs using mass spectrometric techniques. Previously, the major obstacles of HPLCMS were unreliable interfaces [40]. The structural information derived from the data and the sensitivity was limited when thermospray or particle beam techniques were used. In the Petrovic review, it is clear that the advent of atmospheric pressure ionization and interfaces such as electrospray (ESI) and APCI overcame many of these limitations. We found these techniques well represented in the publications from the past ve years. Generally, the ESI interface was reported to provide detection limits one order of magnitude better that the APCI interface for a number of estrogens; however this trend was refuted by Hsu et al. when they concluded that APCI-MS/MS with switching between positive and negative modes was the optimal
3.2. High performance liquid chromatography (HPLC) The need to analyze complex aqueous samples directly can be lled by liquid phase separation methods, including HPLC and capillary electrophoresis (CE). HPLC is a valuable analytical technique that is conventional to most laboratories conducting trace analysis. The hardware has a history of robust laboratory performance and there are a number of suitable methods available for determination of EDCs. The combination of HPLC with mass spectrometry (HPLCMS) has been used more frequently in recent research due to the sensitivity, specicity, and speed MS can offer. Detectors of all kinds, from refractive index (RI), ultraviolet (UV), diode array detection (DAD), uorescence (FL), chemiluminescence (CL), and electrochemical (EC) have been investigated for trace analysis of EDCs. Generally, these techniques were less sensitive than GC or MS methods and many did not meet the criteria of this review; however, these detectors are commonplace in analytical laboratories, affordable, and versatile, so will be mentioned as potential areas for future research.
19
Fig. 3. Summed ion chromatograms (SIC) of raw (A) and immunosorbent-treated (B) extracts of sewage efuent analyzed by negative polarity HPLCESI-MS. Note the absence of signal for EE2, which should elute between E2 and equilin-d4, but was not retained on the immunosorbent. Reprinted with permission from [66]. Copyright 2001 American Chemical Society.
method for simultaneously detecting E1 and six of its metabolites at concentration levels of 0.91.4 ng mL1 [64]. In the mid to late 1990s and early 2000s, the majority of the HPLC methods used C18 stationary phases with a wateracetonitrile gradient mobile phase. This trend does not appear to have changed much in the past ve years. While acetonitrile is preferred as a strong eluent for HPLC with UV detection, it has been reported to leave carbon deposits on an ESI capillary. In the study that made mention of this, methanol did not appear to affect the negatively charged stainless steel tip in the same manner as acetonitrile [51]. This information may be helpful when selecting a mobile phase for this type of analysis in the future. The majority of the MS work was conducted in negative ionization mode to obtain the desired sensitivity. An on-line SPE method using HPLCESIMS in negative ionization mode (SIM) was reported in 2001. This method set the standard because it was capable of detecting E1, E2, E3, and EE in water at concentrations less than 1 ng L1 . This was an exciting development as full automation of the method was also achieved [65]. Also reported in 2001, an immunoafnity extraction method, in combination with HPLCESI-MS was able to minimize isobaric noise in the chromatograms (SIM) and achieve less than 1 ng L1 detection limits for E1 and E2 and recoveries of 88107% in aqueous systems [66]. Fig. 3 demonstrates the improvement in sensitivity for immunosorbent-treated efuent compared to raw sample. The method was unable to detect the analytes prior to sample treatment (Panel A); however, the response of E1 and E2 were excellent post treatment (Panel B). E3 was not retained due to the extreme specicity of the sorbent. This trait can be benecial for quantitation of a single analyte, however, limits the use of this technique for more sweeping types of analysis. While tandem MS can be used to solve issues with isobaric noise, such methods do still experience suppression effects, which occur in the MS source during ion formation. Other studies that did not report these suppression effects used graphitized carbon black SPE isolation (instead of C18 ) to increase the selectivity of estrogen extraction from aqueous samples [66]. In addition, Ferguson et al. [66] found that the acidication of aquatic samples resulted in an increased co-extraction of some interfering sample matrix components and may not be necessary during sample preparation and storage. They also reported that E2 remains stable with no detectable degradation for up to six days when stored at 4 C without acidication or the addition of formaldehyde to reduce
bacterial oxidation. In order to achieve the low detection limits, the samples were quantied using stable-isotope deuterium-labeled internal standards. In the 2002 review, Petrovic et al. [40] mentioned the comparable quantitation limits for synthetic and natural steroids between ESI in the negative ionization mode and APCI interface operating in the positive ionization mode (LOQ between 0.08 and 0.6 ng L1 and LOQ between 0.5 and 1 ng L1 , respectively). Nearly eight years ago, the article challenged the scientic community to improve the limits of detection for ethynylestradiol and other steroid estrogens as they were suspected of affecting organisms at 1 ng L1 . Future detection goals were set between 0.1 and 1 ng L1 . With the continued development of immunoafnity cartridges and other advanced extraction and MS techniques, we will examine if this goal has been achieved in a manner that is robust and cost-effective for analytical screening of aqueous matrices. Only one year after the Petrovic et al. review, another review from the University of Barcelona detailed HPLCMS/MS methods which improved the limits of quantitation and analyte identication [48]. They indicated that 0.110 g L1 detection limits for estrogens were achieved using SIM mode with the QqQ, but a single-quadrupole was better for detecting progestogens (0.4 g L1 ). As of 2003, progestogens were best detected by MS with an ESI interface [67]. Again, hope was placed in emerging HPLCMS capabilities (TOF and ion-trap) for routine applications involving low ng L1 levels of EDCs in aquatic samples. Not only new MS technology, but also new ideas for minimizing the matrix effects in environmental samples were needed to achieve the trace levels of EDCs required. The matrix effect problems often observed in HPLCMS has not been completely resolved, nor was the mechanism of interference exhibited for different matrix compounds fully understood. Ion suppression of analytes is thought to be caused by precipitation or co-precipitation with other matrix components in APCI. A competition for access to the droplet surface in ESI is believed to occur between the analytes and some matrix constituents [37]. The ultimate goal for EDC analysis is to develop a robust method that can detect different classes of EDCs in a single automated analysis. This goal was partially achieved when an HPLCAPCI/MS technique in the negative ionization mode for the quantication of estrogens and bisphenol A was developed in 2004 [68]. While the recovery values for most of the estrogens and estrogen conjugates were rather good (91119%), bisphenol A had a slightly lower recovery of 81% in spiked groundwater. The detection limits were fairly good for all of the analytes, however, and were below 15 ng L1 . In a separate study by Lin et al. in 2007 [47], an evaluation of three derivatization agents with HPLCAPCI/MS and HPLCESI/MS methods for the detection of trace-level estrogens (E1, E2, E3, and EE2) in different aqueous matrices was made. They found that the sensitivity of phenolic estrogens in complex matrices such as river water was improved by derivatization using pentauorobenzyl bromide (PFBBr). When the sample matrix was less complex, like drinking water, the derivatization agent that was found to be the most effective was dansyl chloride [47]. The derivatization reagents improved the sensitivity of phenolic EDCs by reacting with the phenol group to form ethers (Fig. 4). Lin et al. noted that the improvement in sensitivity was not equivalent among all of the estrogens in their experiment, specically estriol (E3). The researchers determined that they could demonstrate little control over which of estriols three hydroxyl groups reacted with the derivatization reagent (2-uoro1-methylpyridinium p-toluenesulfonate (FMPTS), in this case) to give the characteristic fragmentation patterns. Unfortunately, the detection limits for this study were estimated using an assumption of linearity without any verication through representative
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Fig. 4. Scheme of selected derivatization reactions: (a) dansyl chloride derivatization, (b) 2-uoro-1-methylpyridinium p-toluenesulfonate (FMPTS) derivatization (c) pentauorobenzyl bromide (PFBBr) derivatization. Adapted from [47].
curves. The estimated LODs were in the ng L1 range, which are not unreasonable based upon the data of similar APCI methods that used untreated (non-derivatized) samples. A recent paper by an environmental chemistry group in Barcelona, Spain used an immunosorbent kit (enzyme-linked immunosorbent assay or ELISA) initial screening method with ultra-performance liquid chromatography-quadrupole time of ight mass spectrometry (UPLC-Q-TOF-MS). This is the rst example we were able to nd that used modern ultra-high pressure liquid chromatography systems with TOF for the determination of 17- -estradiol (E2) in water samples [69]. The ELISA kit was used to perform an inexpensive initial screen for E2 to determine samples that needed further analysis using the more complex, expensive analytical technique. In this manner, the authors were hoping to eliminate the expense of unnecessary testing. They veried the ELISA results with HPLCMS/MS (QqQ) instrument. The authors found that ELISA often overestimates the concentrations of E2 in the water samples due to the positive interference of estrogen conjugates, and residual estrogens, progestogens, and phytoestrogens present in the sample [69]. Treatment with SPE procedure outlined in Fig. 5 was suggested in order to help eliminate these potential interferences. The lowest concentration for ELISA detection was reported at 2.5 ng L1 . The detection limits of the ELISA happened to be below those of the HPLCMSMS method used to verify the ELISA approach. While it appeared that some samples displayed a false positive due to the MS detection discrepancy, no false negatives were generated using the ELISA screen. These ndings indicate that immunoassay screening methods work well for rapid initial estimation of the presence of ng L1 levels of E2 in drinking water. Of further interest, the TOF-MS method which was evaluated used a shorter (50 mm) C18 chromatographic column with sub-2 m particles. This reduced the run time to about 12 min and increased the separation efciency.
While the TOF did demonstrate its ability to detect and quantitate estrogens and phytoestrogens at ng L1 concentrations, it did not improve the detection limits for estradiol in aqueous samples, as hoped. While the great majority of liquid chromatographic separations have featured C18 stationary phases, one related study was conducted using a hydrophilic interaction liquid chromatography (HILIC) separation in conjunction with ESI-MS detection [21]. The 2 mm diameter HILIC column improved the sensitivity in ESI-MS for estrogen metabolites (estrogen sulfate and glucuronide conjugates) due to the increased linear velocity of the mobile phase when compared to a typical reversed phase column. HILIC is well known for providing improved ESI-MS detection limits in comparison with reversed phase separation strategies for a wide variety of analyses [70]. The method did not use hydrolysis or derivatization and was able to achieve detection limits in the ng L1 range for many of the estrogen metabolite analytes in urine (estrone-3sulfate (E1-3S), estrone-3-glucuronide (E1-3G), estradiol-3-sulfate (E2-3S), estradiol-3-glucuronide (E2-3G), estriol-3-sulfate (E3-3S), estriol-3-glucuronide (E3-3G), estriol-16-glucuronide (E3-16G)) [21]. Another benet of this particular method was the low sample volume required to achieve the detection limits (1 mL) with a simple SPE pre-treatment. Another example of a method designed to be automated and use only 1 mL of sample was reported by Viglino et al. in 2008 [71]. This method featured online SPE and atmospheric pressure ionization (APPI). APPI was used to avoid a derivatization step, but still achieved ng L1 detection limits for natural and synthetic estrogens, progesterone, and synthetic progestogens in surface and wastewater. The complexity and cost of this system may be a deterrent for most typical analytical laboratories (Fig. 6), but the conclusions indicate that the authors succeeded in developing an automated method capable of using only 13 mL of
21
Fig. 5. General SPE procedure to eliminate sample interference for E2 screening by ELISA. Adapted from [69].
Fig. 6. Schematic diagram of the automated SPE tandem LCMS/MS system. Reprinted from [71], Copyright 2008, with permission from Elsevier.
22
sample and rapidly analyzing eight EDCs with a total cycle time of 15 min [71]. 3.2.1.2. Plastics-derived xenoestrogens. Phenolic EDCs. The alkyl phenols, octylphenol and nonylphenol, are readily detected using negative ionization mode in both ESI and APCI interfaces. The ESI interface is favored over APCI, however, because of its improved sensitivity (nearly 4050 times more sensitive) [40]. Sample cleanup to minimize matrix effects is important for phenolic compounds. Specically, SPE has been shown to be useful, but is not always successful at removing all of the hydrophobic matrix components, which then can suppress the ionization of late-eluting alkyl phenols [37]. To assist with this problem and to improve precision and reliability, costly stable isotopically labeled internal standards can be used. They have been effective for this purpose, except for the compounds 4-octylphenol and 4-tert-octylphenol, which continued to exhibit nearly 50% matrix suppression. Ionization enhancement was demonstrated for most of the EDCs that were a part of Benijts et al.s study after changes were made to acidify the mobile phase. Bisphenol A, a polar compound was detected primarily by GCMS in the mid-1990s, but HPLCMS work was starting to be conducted as well [40]. At that time, the detection limit for BPA using an HPLCAPCI/MS method was reported to be 100 ng L1 . By 2004, the BPA levels detected using APCI with QqQ were much improved. A method described by Lagana et al. was able to simultaneously detect various natural and synthetic estrogens (E1, E2, E3, and EE2) and BPA in the low ng L1 range. The method was also able to detect NP, but the detection limits were considerably higher (36182 ng L1 depending on the sample matrix) [36]. NP was detected along with related halogenated derivatives one year earlier using HPLCMSMS at detection limits of about 12 ng L1 in water samples [72]. Stuart et al. was able to study select xenoestrogens in marine samples comparing GCMS, HPLCMS, and HPLC-UV methods [51]. While this article does not specically focus on aqueous sample matrices, the side-by-side comparisons of the performance of each analytical technique are of great interest. The xenoestrogens studied that met the criteria of greater than 80% recovery values were BPA, 4-cumylphenol (4-CP is used in the rubber, plastic, and adhesive industry as an anti-oxidant), and 4-(tert-octyl)phenol (4-OP is a by-product of alkyl phenol polyethoxylates that are surfactants added to soap, paint, herbicide, and pesticide formulations). They found that the GCMS method was able to improve sensitivity two orders of magnitude when the phenolic compounds were derivatized with phenyltrimethylammonium hydroxide (PTA-OH). The different method detection limits were compared and the conclusion was drawn that the most sensitive method for the determination of phenolic EDCs was GCMS (0.004 ng to 0.010 ng). The HPLCESI-MS() method had detection limits ranging from 1.0 to 2.4 ng while the HPLC-UV method detection limits were not too far removed at 1.6 to 4.7 ng. Phthalates. Earlier in the decade, phthalate esters (PAEs) were mostly analyzed by GCMS or HPLCAPCIMS with minimal work using HPLCESI-MS. The detection limits reported were in the 100 ng L1 range [40]. A compilation of analytical methods for the determination of phthalic acid esters in various types of samples (air, water, sewage sludge, foods, plastics, and so forth) was presented by Gomez-Hens and Aguilar-Caballos in 2003 [14]. They outlined the environmental and economic interest in PAEs due to their signicant presence as plasticizers and additives in industry and their impact on human health and the environment [14]. The EPA maximum limit for DEHP in drinking water was set at 6 ng mL1 ; therefore, the limit of quantitation must be equal to or less than this regulation. Phthalates have limited solubility in water, and sample preparation typically involves LLE, SPE, or SPME.
The reported preference for use with HPLC analysis is SPME using a 65- m PDMS/DVB-coated ber [14]. Fewer HPLCMS methods are available than GCMS or GC-FID methods for the separation of phthalates due to the lower detection limits that can be achieved by GC. For example, GCMS using SPME had a reported LOD for DEHP of 0.006 ng g1 (ng mL1 ) in tap water and SPE-HPLC-UV had an LOD of 0.1 ng g1 in natural water. In 2008, Perez Feas et al. reported LOD values for the phthalates DMP, DEP, BBP, and DBP in the low ng mL1 range using HPLCESI-MS in positive ionization mode in saline solutions [63]. Many kinds of MS analyzers have been used for the analysis of phthalates (quadrupole, triple quadrupole, ion traps, and magnetic sector instruments); however, due to the high propensity for contamination, many false positives or erroneously high recoveries can be determined on instrumentation that is so uniquely selective and sensitive. Special precautions must be taken to minimize contamination during sample preparation, such as: (a) avoiding all contact with plastics, (b) vigorously cleaning and rinsing all glassware with certied phthalate-free solvents, and (c) understanding potential sources of contamination in the laboratory, such as particulate matter in air, contaminated water or solvents, plastic, and compounds adsorbed on glass. Smaller column dimensions are beginning to appear more frequently in the literature. In the method presented by Perez Feas et al. [63], a 3.5 m 2.1 mm internal diameter and 50 mm length C8 stationary phase was used together with an HPLCESI-MS (single quadrupole). A gradient method with a ten minute run time and a ten minute equilibration time was used. The separation of four phthalates was accomplished at room temperature in less than ten minutes. The detection limits for the four phthalates, without any derivatization or other sample pretreatment, ranged from 0.99 to 24 ng mL1 in saline. The opportunities for improvement in this area might include using a thermostated column compartment at an increased temperature to further reduce the separation time of the analytes, as well as subjecting the samples to some form of pre-concentration or derivatization to improve sensitivity. 3.2.2. UV detection DAD and UV detectors are the most commonly used detectors for HPLC methods due to their ease of use and applicability to compounds with UV chromophores. UV detectors are simple and inexpensive to use and maintain and are the preferred detector for most analytical laboratories. UV detectors have demonstrated the capability to provide detection limits at or below g L1 concentrations for select EDCs [45]. A good example of a fully automated SPE-LC method with diode array detection was described in a study of estrogens and progestogens in water in 2001 [65]. Full automation is desirable for improving sample throughput, economic efciency, simplicity, sensitivity, and reproducibility. In this case, the method detection limits ranged from 10 to 20 ng L1 with 96112% recoveries and relative standard deviations less than 3% (n = 6) in water. The method consisted of gradient elution over a 40 min run time while monitoring UV detection at 200, 225, and 240 nm. One of the ndings in this study was that the SPE cartridge (polymeric PLRP-S 1525 m Polymer Laboratories, Church Stretton), while advertised by the manufacturers specications as reusable, was not recommended by the authors to be used more than one time per sample. They found that the more polar compounds, such as estriol, would no longer be retained after a single use. The apolar compounds were able to be analyzed from a cartridge reused up to 12 times; however, the peak shape suffered after the sixth use, when increased tailing was observed. Another automated analysis featured SPME-HPLC with UV detection at 225 nm in which liquid medicines and intravenous injection solutions were analyzed without any pre-treatment for BPA, alkyl phenols, and phthalates [73]. The LOD for these compounds, how-
23
ever, were much higher than the estrogen and progestogen analysis described previously: 0.14.0 ng mL1 . Less efcient recoveries were also observed for this method. This may be due to the challenging behavior of alkyl phenols and phthalates in general. The method showed promise in that the separation of BPA, alkyl phenols and phthalates was accomplished in less than 12 min with the total extraction and desorption of the analytes from the samples taking only 35 min. An interesting nding of this particular study was that DEHP, which can be leached from medical polyvinylchloride administration set tubing or containers, is more susceptible to migrate into product formulations that contain the solublizing agent polysorbate 80. This illustrates the potential uses of fully automated methods that are simple, rapid, selective, and sensitive for the screening of liquid medicines and intravenous injection solutions for BPA, alkyl phenols, and phthalates without extensive sample pre-treatment. A remarkable development occurred in 2007 when cloud point extraction HPLC-UV was used to detect and quantitate di-ethyl-phthalate, di-(2-ethylhexyl)-phthalate and di-cyclohexylphthalate with detection limits between 1.0 and 3.8 ng mL1 in only 10-mL of environmental water samples [74]. This method allowed trace amounts of phthalate to be analyzed while minimizing the environmental impact of the sample extraction considerably by using the non-ionic surfactant Triton X-114 as an extraction solvent. Approximately 10-mL of sample was concentrated with Triton X-114 and Na2 SO4 for an hour in a thermostatic bath held at 45 C. The Na2 SO4 was added in order to increase the density of the aqueous phase, thereby facilitating the phase separation. The phases of the samples were separated using centrifugation for about 5 min at 3500 rpm. From the surfactant-rich phase, 20 L was injected directly for HPLC analysis at 226 nm. The column dimensions in this method were large and the gradient required a minimum of 22 min. An opportunity for analytical development would be to make use of smaller bore columns with smaller particle sizes or fused core technology, or newer stationary phases, maximizing efciency by minimizing time and solvent usage required to separate the analytes of interest. Other sample preparation applications for the isolation and detection of phthalates from water have recently been explored. For example, ve different phthalates in environmental water samples were separated and detected with a method that applied ionic liquid mixed hemimicelle solid-phase extraction with HPLC-UV. The reported detection limits for this method were 0.120.17 g L1 [75]. Concentration factors of 600 were achieved using this SPE method based on Br-coated silica mixed hemimicelles. The hydrophobic interactions of the PAEs enabled the retention and then easy release/desorption without carryover from the SPE. Again, the HPLC analysis was conducted at 226 nm and a run time in excess of 25 min was required. Other innovative developments include the use of unique materials for SPME or molecularly imprinted solid-phase extraction (MISPE) techniques with HPLCUV. The MISPE method incorporated a molecularly imprinted bulk polymer with a selectivity for a given analyte or group of structurally related species (in this case, chlorophenols) [76]. The advantages of these molecularly imprinted polymers are: (a) their selectivity, (b) cost, (c) ability to be used in a wide range of operating conditions, and (d) their chemical and mechanical stability. Also, smaller sample volumes (10 mL) were able to be processed with recoveries in the 90% range. The calibration curves of spiked water samples were run from 0.05 to 2.0 mg L1 , but the authors did not attempt to extend the curve down into the trace level range, even though the reported detection limits for the phenolic analytes were between 0.6 and 1.1 g L1 . In another study, the determination of phenols in aqueous samples by direct immersion of a SPME platinum ber coated with carbon nanotubes was accomplished [77]. A carbon nanotube coat-
ing on a Pt ber was explored due to the inherent issues known for most commercial and some laboratory-made SPME bers, such as temperature and chemical instability, swelling, lower extraction efciencies for polar compounds, and high likelihood of breakage. When compared to a commercial polyacrylate SPME ber, the SWCNT ber was found to be similar or superior for extraction of phenol, m-methylphenol, bisphenol A, a nitrophenol, and two chlorophenols. In addition, the detection range of the method for the various phenols ranged from 0.9 to 3.8 g L1 with recoveries (n = 3; 200 g L1 ) greater than 87% in seawater and greater than 92% in tap water. The authors observed an increased RSD when using separate bers for replicate analyses. When the same ber was used for replicate analyses, the RSD remained below 10%. This is indicative of a potential need to improve the reproducibility between different bers (ber-to-ber variation). In other work, Zarzycki et al. conducted a study that successfully separated a group of 27 suspected EDCs in environmental samples using temperature-dependent inclusion chromatography, where eluents were modied with -cyclodextrin or hydroxypropyl- cyclodextrin and the system was operated at a temperature of 47 C [78]. The authors focused on the effect of temperature on the simultaneous separation and detection of various natural and articial steroids, as well as a number of non-steroidal EDCs including BPA, a few additional phenols, and a number of phthalates by HPLC-UV. From the regression analysis of the calibration curves, the best correlation coefcients were demonstrated when the mobile phase did not contain modiers. They concluded that temperature is a critical parameter for HPLC selectivity of these compounds when using macrocyclic mobile phase additives. Explorations of different derivatization reagents, extraction techniques, column technology, and operating conditions continue to be evaluated in order to identify applications that best separate and quantify mixtures of trace-level EDCs from complex aqueous matrices. 3.2.3. Other detection techniques 3.2.3.1. Fluorescence detection (FL). Fluorescence detectors are specic and sensitive for uorescing species. The sensitivity is typically more than one order of magnitude better than absorbance detectors; however in the area of EDCs, they appear to perform no better than UV detection, as a whole. Species that do not ordinarily uoresce can be treated with reagents which complex with the analytes to form uorescent derivatives. Phenolic analytes can be derivatized in this manner using reagents such as dansyl chloride. BPA can be detected with excitation and emission wavelengths of 275 and 305 nm, respectively, but because the intensity of BPA is higher in organic media, response is dependent upon the composition of the mobile phase [34]. Markham et al. applied HPLC with uorescence detection for the analysis of bisphenol A in river water samples. The quantitation limit of the method was about 3 g L1 [49,79]. By 2008, typical quantitation limits for BPA by uorescence detection were still being reported in the 550 g L1 range, indicating that no signicant improvement in detection limits had been achieved in nearly a decade. A need to test large numbers of samples in a fast, economic manner exists and has encouraged the development of faster, more efcient extraction methods for removing BPA from aqueous matrices. The majority of methods use SPE or LLE, but researchers have developed a short, 20-min, sample treatment using reverse micelle microextraction of BPA from urine that achieved a detection limit for BPA of 0.197 g L1 [80]. One weakness of uorescence detection is the potential for interference from various other migrants from packaging which are also actively uorescing compounds. This can cause erroneously high recoveries and false-positives for BPA. For this reason, conrmation of the identity of the detected compound using HPLCMS or an in-line DAD is encouraged [34,80]. An example of natural estrogens and phenolic EDC detection by
24
uorescence after derivatization with p-nitrobenzoyl chloride has been reported [46]. The method was applied to the determination of three endogenous estrogens, -E2, -E2, and E3, as well as -EE2, NP, and BPA, in urine. The detection limits for the analytes ranged from 2.7 to 8.3 g L1 , which was more than adequate for this purpose as the compounds that were detected in the urine samples are typically at g L1 to mg L1 levels. Interestingly, the detection limits were dened as a signal-to-noise ratio of 2 (the LOD is traditionally dened as a signal-to-noise ratio of 3). The recoveries of the six analytes using this method were all greater than 85%, and the relative standard deviations and correlation coefcients were also satisfactory (%RSD < 5, r > 0.999, n = 5). 3.2.3.2. Electrochemical detection (ED). Electrochemical detectors (ED) offer high sensitivity and widespread applicability based upon the oxidation or reduction of organic functional groups. Phenolic groups are electroactive and easily detected by HPLC-ED. Electrochemical detection of BPA was found to be much more sensitive (0.5 pg detection limit) than HPLC with FL or UV detection [34]. ED sensitivity is inuenced by both the pH and electrolyte concentration of the mobile phase. In addition, due to the long equilibration times needed for ED, isocratic elution is recommended over gradient methods. Even with those weaknesses, a SPME-HPLC method was reported using a gradient with UV and ED. The detection limits reported were between 60 and 80 ng L1 for various phenols including BPA, as well as various estrogens [55]. The UV detector had to be used instead of the ED for the later-eluting compounds, which were affected by the baseline drift of the increased acetonitrile composition of the gradient. In a study conducted in 2007, Vega et al. used a modied glassy carbon electrode (GCE) at +1.0 V to quantify estrogenic phenols in river water by amperometry [81]. The limits of detection using signal to noise ratio of 3 were reported to be ng mL1 range for BPA and EE2. Good correlation coefcients (>0.999) with low relative standard deviations were reported (<4% for n = 10 samples). ED was not optimal for all of the EDCs in this study, however. Of the phenols examined, the compound 4nonylphenol behaved differently by voltammetric detection than BPA. The oxidation of NP nearly completely transforms it to one of its oxidation products, a polymerized lm. This lm collects on the surface of the electrode, inhibiting detection. This nding illustrates that understanding both the characteristics of the analytes and their oxidation products are important when selecting ED as a detection technique for EDC analyses. 3.2.3.3. Chemiluminescence (CL). A novel approach using chemiluminescence (CL) detection with HPLC has been published as a simple, low cost, selective and sensitive method of detection for various EDCs. While delivering these advantages, CL also delivers the drawback of requiring additional pumps and rapid mixing needs for post-column CL reagent addition. In addition, the rapid and intense CL reactions require the adjustment of a reaction coil used between the mixing device and the detector. The length and diameter are variables that must be adjusted and optimized in order to allow the maximum CL emission to be observed. There are a multitude of variables that must be carefully optimized in a system utilizing CL, such as the composition of mobile phase, reaction temperature, and pH, among others [82]. In one example, the phenols had to be extracted from the aqueous matrix (in this case, urine) using SPE, as well as derivatized with dansyl chloride prior to analysis. The LOD for this method was about 0.33.1 g L1 . Compounds containing phenolic moieties exhibit CL response from direct oxidation with acidic potassium permanganate, however, there were no studies cited for this technique for the determination of phenolic compounds derived from polymeric materials. One study, cited by Gamiz-Gracia et al., simultaneously detected twenty phenolic compounds in wine samples. This study was conducted using
a reaction of the phenolic compounds with cerium (Ce(IV)) and rhodamine 6G and had limits of detection in the ng mL1 range (1.582.1 ng mL1 ) [83]. The study conducted by Zhang et al. does not include any of the EDCs mentioned here. It appears that this is an area of potential development for endocrine-affecting phenols. Another chemiluminescence detection method included in GamizGracia et al.s review was able to detect bisphenol A leached into hot water from a baby bottle at a detection limit of 0.38 g L1 [84]. The sample was minimally processed but, in order to improve the detection and baseline, was treated with SPE. Another chemiluminescence application using HPLC was used for the determination of estradiol in very small volumes of rat plasma. In standard, the detection limit of this method for estradiol was 8 pg and in plasma it was determined to be 24 pg [85]. Chemiluminescence might offer better detection limits than UV as well as cheaper, simpler ways to analyze EDCs from aqueous samples, however there has been little recent activity using this detection technique. 3.3. Capillary electrophoresis (CE) CE or capillary zone electrophoresis (CZE) has been explored for polymer analysis and monitoring of endocrine disruptors recently by a variety of research groups [8690]. CE enables separation of various analytes in one run according to their molecular size, charge/mass ratio, and isoelectric points (differences in electrical eld-induced migration properties of the analytes and the run buffer) [60]. CE typically has low sample volume requirements, fairly rapid analysis (average of 320 min), and the ability to analyze a large number of samples quickly. CE also exhibits higher column efciencies than HPLC and freedom to alter or improve separation efciencies by changing buffer composition. The limitations of CE applied to trace determination of EDCs involve sensitivity, sample matrix interferences, and sample ionic strength. The CE methods explored by Reagan et al. were CZE and micellar electrokinetic chromatography (MEKC) using photo-diode array detection from 190 to 300 nm [87]. Micellar electrokinetic capillary methods involve a modication of the buffer using a surfactant at concentrations (termed critical concentration) at which micelles form. This technique permits the separation of lower molecular weight aromatic phenols. The authors were able to separate and detect various estrogens and phenolic EDCs. They explored the potential of three different methods for the detection and quantication of a variety of EDCs in aqueous samples, and found the limitations of the CZE method to be poor reproducibility and variable migration times of the target compounds dependent upon the presence of other analytes in the sample mixture. Using MEKC, they were able to separate 19 target analytes with some variation in migration time. The study included calibration curves for each analyte with acceptable linear correlations and RSDs for each; however, they did not publish any projected detection limits of their optimized method. Derivatization of phenolic compounds in surface water using peroxyoxalate together with dansyl chloride for CE with chemiluminescence detection (CE-CL) has also been studied [86]. In this particular study, a CE-CL method was able to separate fteen kinds of phenolic compounds using a running buffer solution of SDS and acetonitrile. Six were determined to be linear over three orders of magnitude with detection limits approaching 107 M. The analyte recoveries were rather low (6792% with chlorophenols having the highest recoveries) for the six phenolic compound curves. A separate research group studied aqueous and non-aqueous CE to separate halogenated phenolic and bisphenolic compounds in aqueous samples. These compounds are mostly used as ame retardants and are, therefore, not the focus of this review, however the study bears mention because it reported g L1 limits of quantitation for the analytes using large-volume sample stacking in-line concentration with photo-diode array UV detection
25
(LVSEP-CE-DAD). The analysis of wastewater samples by this technique began with SPE, and used methanol as an eluent, which could be directly injected into the CE system [89]. In light of this study, the CE does show some promise for the determination of phenolic EDCs in aqueous systems when levels are expected to be in the g L1 range. Also in 2005, aqueous free solution capillary electrophoresis (FSCE) was used to separate derivatized bisphenol A ethoxylate dimethacrylates (Bis-EMA). The different average number of ethoxy groups per Bis-EMA in dental composite material was determined by FSCE due to the high resolution power of the technique [88]. This technique has been used to monitor the release of growth hormone from various co-polymeric devices made of vinylpyrrolidone-hydroxyethyl methacrylate. The ability to monitor the release of a drug and the resorption rate of the polymeric delivery systems on drug release is another interesting and useful property of this FSCE method. A potential exists for this type of analytical method to be explored as a monitoring technique for the release of EDCs from polymeric medical devices or other plastics into aqueous systems over time. Implantable drug delivery devices that deliver active compounds, such as hormones, are the current targets of such research [88]. More recently, CE with amperomeric detection was used by Ha et al. to demonstrate a wide detection range for BPA (nM to mM range) [90]. This would be useful for monitoring samples that were expected to contain varying amounts of this compound. The research tested a modication using a twisted serpentine microchannel conguration with a CE-AD device that enabled the separation and detection of a group of phenolic compounds from aqueous extractions of Styrofoam food packaging in about 3 min [90]. While this novel approach is not yet commercially available, the sensitivity and speed of detection reported in this preliminary research inspires additional attention to the CE with amperometric detection for fast screening for EDCs in aqueous systems.
4. Conclusion Much emphasis has been placed on the detection of endocrine disrupting compounds in aqueous sample matrices, such as, environmental waters, beverages, foods or medicines for human consumption, as well as plastics used for packaging or medical devices that can leach into aqueous environments. The scientic community is learning more about how these compounds affect the endocrine systems of a variety of animals, from sh to mammals. The concentrations of a single EDC, or mixtures of EDCs, that have been found to impact the endocrine system appear surprisingly low, in some instances less than 1 ng L1 . These ndings are driving the detection limits of the analytical methods routinely used to monitor these endocrine disruptors lower than they have ever been before. Varieties of sample preparations and analysis techniques are being explored in search of an inexpensive method that offers ultra-trace-level detection limits and high sample throughput for the simultaneous detection of multiple EDCs in aqueous systems. When comparing the methods reported in Tables 3 and 4, we found the majority of them involve mass spectrometry detection. MS offers a wide range of selectivity as well as the sensitivity to detect many of the estrogens, estrogen conjugates, and xenoestrogens mentioned in this review. GCMS remains the most useful and sensitive method for the detection of trace levels of volatile EDCs and their volatile derivatives; however, HPLCMS using ESI or APCI appears to be approaching GCMS sensitivities without the requirement of derivatization. These methods may offer appealing options to analytical laboratories wishing to avoid complex and lengthy sample work-ups. Comparisons of sample preparation revealed that much use was made of SPE and related extractions,
such as SPME, over the more common and accepted LLE. This trend reduces the amounts of toxic solvents that are required for sample treatment, minimizing overall environmental impact and cost. In addition, smaller volumes of sample were required in many recent methods, which are appealing when sample availability or storage space is limited, or costly. Opportunities to improve chromatography (separation, efciency, detection limits) exist through the new column dimensions and chemistries available in the market today, as well as the higher pressure rapid resolution HPLC and UHPLC systems that exist. Other areas available for exploration and improvements involve the use of the newer types of detectors, such as charged aerosol detection (CAD) or TOF-. Automation is also highly desirable and, in at least one instance, has demonstrated promise using online SPE coupled with HPLCMS/MS [68]. This type of progress offers benets to the research, as well as the nancial bottom line. A vast amount of research has been conducted in this eld and a number of elegant methods have been proposed for analysis of EDCs. In a modern industrial setting, however, the focus is to make the complicated simple by nding the most efcient path to the end goal. Methods that meet these performance criteria are desirable: such as those using simple equipment that is easy and inexpensive to obtain and maintain; systems that offer the exibility and sensitivity to be used for other types of analyses; and methods that have the capability of being fully automated with high sample throughput. Only a few of the published research methods met these more selective criteria, and we would like to highlight two of them. Tan et al. [18] developed a GCMS method using stir-bar sorptive extraction with simultaneous derivitization that was capable of detecting select estrogens, alkyl phenolss, and phthalates at the ng L1 level. It may be possible to fully automate the sample preparation and analysis for this method. The reproducibility for this method was slightly elevated (RSD 10%) and might offer an area for future improvement. The second method, an HPLC method, was able to separate and detect select estrogens, phenols, and phthalates from 0.2 to 1.6 ng L1 using UV detection [78]. This method is optimal for a typical analytical laboratory because it is sensitive to trace levels and uses cheaper UV/DAD detection instead of MS or MS/MS. This system is simple, with very little sample workup and the method has the capability for high sample throughput. The identication and quantication of compounds in the environment that are found to disrupt the endocrine system will continue to be a relevant research topic into the next decade. The attention EDCs are receiving in the media will certainly spur more research, and potentially more regulation of water quality, acceptable levels of leachable EDCs from plastics, and other related legislation. The need for selective, accurate, sensitive, and robust methods will continue to grow. The identication of analytical techniques for trace-level EDC analysis will help to inform policy and support other scientic elds of investigation, such as biochemistry, toxicology, food science, pharmaceutical science, package engineering, and polymer science. Acknowledgements The authors would like to thank Kayunta Johnson-Winters and Deborah Rowan for proof-reading, professional opinions, and thoughtful suggestions during the preparation of this manuscript. References
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A Review of Separation Methods For The Determination of Estrogens and Plastics-Derived Estrogen Mimics From Aqueous Systems

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Analytica Chimica Acta 696 (2011) 626

Contents lists available at ScienceDirect

Analytica Chimica Acta

Table 1 Select estrogen structures. Compound Abbreviation Chemical structure

8 Table 2 Select plastics-derived xenoestrogen structures. Phthalate compound

Reference [23] [49]

GCMS GCMS GCMS

EI-MS Magnetic sector EI-MS

Water and aqueous extracts of plastics Drinking water

Large volume injection (LVI)/GCMS GCMS

EI-IT EI-magnetic sector

SPE, derivatization SPE and derivatization

Estrogens: 24 ng L1 ; NP: 500 ng L1 1.5172 ng L1

Stir bar sorptive extraction Modied SPE with ATD

Industrial ultrapure water Tap and drinking water

Wastewater Surface water

SPME SPE and derivatization

Limit of quantitation (LOQ).

Reference [96] [72] [38]

HPLCMS/MS HPLCMS/MS HPLCMS/MS HPLCMS/MS

ESI-QqQ ESI-QqQ and APCI QqQ ESI-QqQ ESI-QqQ

SPE SPE SPE, derivatization Immunosorbent extraction

0.10.4 ng L1 0.30.6 ng L1 0.40.7 ng L1 0.42 ng L1

[36] [97] [98] [69]

HPLCMS/MS HPLCMS/MS HPLCMS/MS

APCI-QqQ ESI-QqQ Ion S pray-Q-IT

Cell culture medium Surface and ground water Urine

SPE SPE SPE

0.91.4 ng mL1 0.010.2 ng L1 21000 ng L

[64] [99] [21]

Surface and wastewater

Milli-Q water and river water

E2-17G, E1-3S, E2, EE2, E1

UPLCMS/MS HPLCMS HPLCMS HPLCMS

Q-TOF ESI-quadrupole ESI-MS ESI and APCI-MS

Wastewater Wastewater Water Groundwater

SPE Immunosorbent extraction On-line SPE SPE

[69] [66] [67] [68]

Reference [47] [63] [65]

0.31.1 g L1 LOQ 110 ng mL

HPLC-UV HPLC-UV HPLC-UV HPLC-DAD

UV (225 nm) UV (226 nm) UV (226 nm) DAD

Water Environmental water Environmental water Environmental water

0.34 g L1 1.03.8 ng mL1 0.120.17 g L1 0.21.6 ng L1

[101] [74] [75] [78]

HPLC-FL HPLC-CL HPLC-CL HPLC-ED HPLC with amperomeric detection

DAD (271 nm) FL

Tap water, sea water Urine

SPME SPE, derivatization

ex = 276 nm, = 306 nm)

SPE, derivatization Not applicable Not applicable SPME SPE

0.2 g L1 12 pg 0.38 g L1 0.060.08 g L

[80] [82,102] [82,84] [55] [81]

CL CL ED Carbon nanotube modied glassy carbon electrode 1.0 V

Limit of quantitation (LOQ).

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