Analytica Chimica Acta 696 (2011) 6–26

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Analytica Chimica Acta
journal homepage: www.elsevier.com/locate/aca

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 quantification 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, flame ionization detection; FL, fluorescence; FMPTS, 2-fluoro-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, liquid–liquid 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-trifluoracetamide; MTBSTFA, n-methylN-(tert-butyldimethyltrifluoroacetamide); PAE, phthalate acid esters; PAH, polycyclic aromatic hydrocarbon; PDMS, polydimethylsiloxane; PET, poly(ethylterephthalate); PFBBr, pentafluorobenzyl bromide; PFBOCl, pentafluorobenzoyl 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-flight; 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) 6–26

7

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 scientific 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, identification, and quantification 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 quantification 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, flame retardants, parabens), as well as many naturally occurring compounds (isoflavones 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 [1–3]. 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 g−1 ). 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 first present the selected EDCs, including their characteristics and importance for investigation. Next, we briefly cover common sample preparation methods for isolating the identified EDCs from aqueous systems. Finally, methods of separation and detection for the quantification of trace levels of the selected endocrine disrupting compounds are discussed. These methods were selected with the express goal of highlighting time and cost-efficient 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 reader’s convenience.

Table 1 Select estrogen structures. Compound Abbreviation Chemical structure

O

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

D. K. Phthalate compound A.8 Table 2 Select plastics-derived xenoestrogen structures.A. Schug / Analytica Chimica Acta 696 (2011) 6–26 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 O . LaFleur.

Significance and importance Endocrine disruptors are any externally originating chemical compound. The first and most crucial step of healthy endocrine system functioning is 1.A. metabolism. An example of how EDCs can interfere with receptor sites is outlined in Fig. synthesis. or decomposition of essential hormones [2. electrolyte. These compounds are thought to affect the binding. 1. The endocrine system uses hormones to act as messengers that regulate reproduction. amphibians.A. either natural or synthetic. fish. as well as water. signaling. that interferes with normal endocrine function. natural defenses to stress. LaFleur.D. .1.4. Schug / Analytica Chimica Acta 696 (2011) 6–26 Table 2 (Continued) Phthalate compound Abbreviation Chemical structure 9 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 O Di-cyclohexyl-pthalate DCHP O 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. growth and development. birds. K.5]. and nutritional balance of the blood [6]. and various invertebrates by communicating through chemical messengers called hormones.

foods.2.10]. Conjugated estrogens can also be found in prescription medications as sulfated estrogen salts to treat hormonal imbalances. (b) the number of receptors available in the target cells. For example. the European Community on Risk Assessment and Directive on the Classification of Dangerous Substances proposes limits and strategies to deal with EDC contamination in waterways. These compounds persist in water due to incomplete removal from wastewater treatment facilities. ISO 10993. hormone-receptor binding. In Europe. These hormones are potent and can cause profound effects at very low concentrations. Currently. inks. and osteoporosis. 1. In the United States.8]. such as estrone (E1). and mammals [9. the effects of single compounds have been studied independently. Even so. Reproductive interferences and effects have been observed from estrogens and estrogen conjugates in water from concentrations as low as 1 ng L−1 [18]. In the past. but now mixtures of low-concentration EDCs are suspected of acting together to impact endocrine functions [10]. .10 A. 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. The receptor increases or decreases the rate of a target cell’s activity depending upon: (a) the concentration of a specific hormone in the blood. a phthalate commonly found in drinking water. US EPA. These were mainly prompted by study findings that potential endocrine disruptors were leaching from these materials into products. synthetic compounds from polymers (xenoestrogens). 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]. personal care products. or the product packaging (plastics. Free estrogens are rarely detected in urine. In fact. Compound classes of interest Known and potential endocrine disruptors in the environment originate from many sources including pharmaceuticals. and E1 [20].” This spurred the development of analytical and environmental monitoring tools and programs for EDCs [13]. “Community strategy for endocrine disrupters: A range of substances suspected of interfering with the hormone systems of humans and wildlife. are leading to the discussion and recommendation of regulations by various governmental and scientific agencies [11]. medical devices. These are produced by animals or can be found in certain pharmaceuticals [10. The scientific and medical communities have discovered a number of compounds that disrupt normal estrogen signaling processes [4. 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. and (c) the binding strength between the hormone and the receptor [7]. naturally occurring compounds in plants and animals. 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]. Europe adopted a document titled. the implications of the research on EDCs. and 17 -ethynyl-estradiol (EE2). post-menopausal symptoms. 1. (b) Hormone blocker interferes with the signal from the body hormones. and inorganic and organometallic compounds. birds. (c) Hormone disrupters give a weaker or stronger-than-normal signal at inappropriate times compared to the body’s hormones. Estrogens and estrogen conjugates Estrogens are steroids that play important roles as sex hormones in animals. they metabolize in the body to sulfate and glucuronide conjugates which are then eliminated in urine. through environmental risk assessments and chemical monitoring. Various medical industries comply with the International Organization for Standardization (ISO).19]. which has proposed testing strategies to assess chemical contaminants and leachable compounds from medical devices in its guidance. Schug / Analytica Chimica Acta 696 (2011) 6–26 Fig. however.D.15]. -E2. The phasing out of certain plasticizers in poly(vinyl chloride) (PVC) is a recent example of industry regulation in Europe. The European Union (EU). LaFleur. K. They are natural estrogens. pesticides. The most prevalent conjugates found in adult female urine by researchers were g mL−1 levels of conjugated E3. 1. the EPA maximum limit for di-(2-ethylhexyl)-phthalate (DEHP).1. In March 2000. is 6 ng mL−1 [14. including fish. estriol (E3).or 17 -estradiol ( -E2 or -E2). or adhesives) that can migrate from or through the plastic and into the product or biological sys- tem. The scope of xenoestrogens discussed here has been narrowed specifically to certain additives and plasticizers used in the plastics industry that have demonstrated estrogenic effects (Table 2). (a) Normal hormone activates the receptor at the appropriate level and time. amphibians.A. water.2. whereas other steroid hormones can take hours to register any effect. 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]. Legislation involving other synthetic materials and their additives (poly(carbonate). The -form of estradiol is the synthetic hormone used in both pharmaceuticals and for livestock. much less as mixtures. paper. and the World Health Organization (WHO) have also characterized and proposed acceptable limits of certain EDCs in drinking water [12]. 17 . there are few conclusive studies or regulations regarding a safe dose of some of these biologically active compounds when acting independently.17]. A leachate is a chemical or component from either the device itself. 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 free estrogens selected for review in this publication promote target organ responses nearly instantaneously. Adapted from [2]. which degrades to E3 in the aquatic environment. E2 is biodegraded to E1. poly(ethylterephthalate) (PET). and milk or infant formula [14. bisphenol A (BPA)) are currently being considered in a number of states in the US as well as by other nations [8].

however. and processing equipment [13. filters. and throat. DINP. 1. contaminated solvents. Plastics-derived xenoestrogens Non-steroidal synthetic estrogens are known as xenoestrogens. Specific phthalates (DBP. Phthalates are not chemically bound in plastics and as such. Another challenge that remains a stumbling block in EDC determination is overcoming matrix effects on the analysis. Alkyl phenols and other phenolic compounds have been found to contain weak estrogenic activity at g L−1 concentration levels [22. necessitating unique handling techniques. adhesives. K. DEP and DHP) (Table 2) are thought to disrupt the endocrine system by competing with 17 -estradiol for binding to the estrogen receptor [14. incidences of sample contamination have been discovered to originate from common laboratory or sample processing practices from sources such as storage containers. 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. phthalate acid esters (PAEs) are one of the top offenders in the growing list of suspected EDCs used in common household products. or other unwanted material. These conjugates are less likely to exhibit significant biological activity. Technical challenges for trace level EDC determination The goal of many scientists is to find a method for routine use that is fast. used as antioxidants and plasticizers in the plastics industry.23].A. a recent study by a group of biochemists in Japan has found that the fluorine atoms of BPAF bind to beta estrogen receptors 50 times more effectively than BPA [31. Phenolic additives. LaFleur. For example. but is not without remaining challenges and difficulties. 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. 1. These antioxidants maintain certain physical qualities desired in the plastics. tubing. and bisphenol A. proteins. 1. the diverse chemical characteristics of the analytes require a variety of analytical approaches.A. metal food can liners. The practice of precipitating proteins from liquid samples has also been problematic for similar reasons.29]. BPAF is used as a cross-linker in fluoroelastomer gaskets and hoses used in food processing equipment and as a monomer in a multitude of polymers used for electronic devices and optical fibers.30].2. hydrolysis. Losses due to the solvents used for precipitation combined with the hydrophobic behavior of the analytes are also possible [34]. reproducible. and to a lesser extent. they are converted back into their free forms during water treatment techniques and regain their potency [22. Table 2 displays a number of compounds linked to this classification.D. Analytical determination of EDCs has been refined in the last decade. but the loss of component due to sample processing has also been observed. increasing the incidences of cancers of the breast and testes. not all are without health or environmental risks. Department of Health and Human Services) to suggest comprehensive toxicological characterization of BPAF in 2008. The highest concentrations were found in plastic bottles. synthesized by condensation of phenol with acetone. is published on the National Institutes of Health website as a part of a chemical information profile of BPAF [33]. sugars. fats. Nearly every body of water tested contains one or more forms of naturally occurring estrogenic compounds. and butyl benzyl phthalate (BBP) are a few of the additives generating growing interest. The compound 2. Also. and impacting the neural development of fetuses and young children.28].2-bis(4-hydroxyphenyl)propane. kidneys. BBP. 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. In a related issue. Many industrial compounds that have demonstrated estrogenic activity contain phenol groups and can be found in water. however. Often found in fatty foods from packaging or leaching from medical devices. Both BPA and BPAF are thought to mimic estrogens and react with the estrogen receptors in the body.S. inexpensive. this increases the importance placed upon the quality of analytical data used to estimate the concentrations of these compounds in the environment [24–26]. and reproductive organs [14]. Studies linking these health effects to xenoestrogens are currently being conducted. have been shown to stimulate the growth of breast cancer cells [3]. pipette tips.1. relatively “green”. di-n-butyl phthalate (DBP). commonly known as bisphenol A (BPA). cosmetics.2. and medical devices. A related compound that is potentially more harmful to human health is the chemical bisphenol AF (BPAF). Xenoestrogens are suspected of disrupting reproduction in humans and wildlife.2. Recent studies demonstrated that branching at the -carbon in alkyl phenols with 8–9 carbon chain lengths show the greatest estrogenic effects [22]. di-(2-ethylhexyl) phthalate (DEHP). nose. Other larger molecular weight phthalates (DEHP. Recent studies conducted on bottled water in both glass and plastics found EDCs in 60% of the water samples tested. including BPAF. however.2. pharmaceutical drugs. Not only is contamination a potential issue. flame retardants.14]. plastics. Today. They are susceptible to photo-degradation through free radical attack. Phthalates (phthalic acid esters).2. It is rare to find one method that is capable of determining trace levels of different classes of compounds in a single run.32].3.2. Specifically. Some antioxidants and plasticizers in the manufacture of plastics have been shown to be estrogenically active. suggesting that the plastics contribute to higher levels of contamination in water [27]. sensitive. as well as toxic to the liver. Most samples are complex matrices of sludge. salts.2. phenolic antioxidants (specifically the para-OH containing compounds). septa. 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 difficult. as far as . biodegradation. is a building block of polycarbonates. due to a loss in the tubing used to carry an extract to a sample vial [35]. for example. A comprehensive summary of the endocrine activity of 36 bisphenol A analogs and derivatives. Specific extraction techniques have been shown to cause low recoveries of alkyl phenols. Toxicological evaluations have indicated that the lower molecular weight phthalates (DEP) are irritating to the eyes. Schug / Analytica Chimica Acta 696 (2011) 6–26 11 among other conditions [21]. 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]. These lipophilic compounds have low solubility in water and have been found to bio-accumulate in fats. Generally. This review will focus predominately on analytical methods to determine phthalates and phthalic acid esters (PAEs). inks. Table 1 depicts select structures of the estrogens covered in this review. BBP. detergents. DEHP. 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. the efficient 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. can leach into the environment over time [14].14. and DIDP) are suspected carcinogens. and can simultaneously detect trace levels of estrogens and multiple EDCs in aqueous systems. Alkyl phenols. 1.

time-efficient. solvents. the techniques considered for this review will also cover a rather wide range. lab supplies. simple. the more likely the matrix will interfere [36]. later-eluting alkyl phenolss (4t-octylphenol. it may become desirable to improve the signal intensity by concentrating the sample. solid phase extraction (SPE). K. This selectivity. Effective isolation of a particular compound can be complicated by a sample matrix that contains many similar interfering components. 3. often excluding related compounds and metabolites that are also present in the samples [40]. a situation that increases time and cost of sample analysis. they can be labor intensive. Analytical applications In this section we will briefly introduce instrumentation used to separate and detect natural and synthetic estrogens and plasticsderived xenoestrogens from aqueous systems. Treated. and while the technique greatly increases the sensitivity of the methods used. For example. however. 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. although sometimes desirable. and nonylphenol) in environmental water matrices. a number of derivatization agents were evaluated for optimal detection of steroid estrogens in water. The relationship between the sample volume to be concentrated and matrix effects is complicated and must be considered when selecting an analytical method. In trace analysis. storage. In some methods. derivatization agents can quickly degrade column performance if not properly or completely quenched after the designated reaction period. Once the component of interest has been isolated from the aqueous sample matrix. deactivated glassware. This issue was the focus of a study by Benijts et al. In many cases. The analysts observed that derivatization increased the mass spectral signal intensity of the component of interest 2–12 times compared to an untreated sample [47]. Chemical derivatization is a form of sample treatment that is commonly used to improve sensitivity and selectivity in both HPLC and GC methods. the derivatization agent cannot distinguish between structurally related compounds. Gas chromatography (GC) Gas chromatography (GC) is an indispensible chromatographic technique for the detection of volatile or semi-volatile compounds of interest. proper sample treatment is essential in order to isolate and quantitate trace levels of EDCs. Even after taking extra steps for sample cleanup. Trace analysis requires additional attention to potential sources of contamination on the part of the analyst. therefore. The most common and simplistic techniques used to minimize matrix effects are to isolate the components of interest through various extraction methods. requires the use a variety of methods in order to accomplish the analysis of a single sample. 4-octylphenol. therefore. and personal protective equipment must be used to avoid contamination [39. . wastewater. Due to the range in polarities of the classes of EDCs discussed. The proper sample treatment can considerably improve the limit of quantitation and detection for the compounds of interest. Some general recommendations for trace analysis of steroid hormones and EDCs are to avoid sample contact with plastic materials. lowering recoveries. Sample treatment Generally the first 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.40]. In addition.38]. Sample pre-treatment is essential in many cases.D. Analytical laboratories aim to develop sample preparation techniques that are accurate.1. The volume of sample available is a key consideration if multiple assays are required to identify and quantify various EDCs [35]. 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. sample matrices can also complicate detection by causing an increase in the background signal. and industrial effluents) on a mass spectrometry method (HPLC–ESI-MS/MS) for the quantitation of EDCs. and allows the analytical chemist to effectively isolate and analyze specific compounds of interest from complex aqueous matrices. LaFleur. In a separate study. and has a limited number of steps. Ultra-trace and trace-level quantitation limits also present challenges. In addition. Schug / Analytica Chimica Acta 696 (2011) 6–26 analytical determination is concerned. producing the same derivatives for two different compounds [45].A. cleaned. rain. cloud point extraction (CPE). These derivatization techniques were used to dramatically improve the volatility of the compound of interest (for GC analysis). the researchers were unable to overcome the suppressive effects (exceeding 50% in some cases) on the less polar. it may be necessary to improve the sensitivity of the method. or to add functional groups that increased the specificity and sensitivity for particular types of detection. 3. and transfer equipment. solid phase microextraction (SPME). and several 2. reproducible. The sample treatment may also interfere with the separation and shape of the analyte peaks. Unfortunately. Common examples of isolation techniques observed in the literature to achieve separation of EDCs from aqueous matrices are: liquid–liquid extraction (LLE). The topic of sample preparation techniques has been addressed extensively in a number of exceptional books and reviews. There are a number of different injection modes available. robust. but the higher the volume of sample that is pre-concentrated. Summaries of select GC and HPLC methods for trace EDC determination are displayed in Tables 3 and 4. This was attributed to the lack of efficiency in the removal of hydrophobic matrix components by solid phase extraction. The most commonly observed derivatization reactions seen in the literature for this review were esterification or silyzation of the phenolic –OH on the compound of interest. a variety of columns useful for separation. 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. It is not recommended to make more than one injection from a sample vial [14]. particularly when the quantity of sample available for testing is limited. ground. a sensitive fluorescence method was developed to analyze estrogens in urine using p-nitrobenzoyl chloride [46]. Matrix issues can impact the quality of the analysis. and safe. cost-effective. It is essential to collect a blank sample and controls to run with every analysis. derivatization is targeted for a specific group of analytes. Large quantities of sample are desirable in order to lower detection limits. respectively. alters it as little as possible. and special efforts and expense must be made in order to obtain quality data. The most effective sample treatment uses minimal sample. The chemicals required are typically toxic. [37] that evaluated the matrix effects of environmental samples (surface. in effect. the matrix is complex and interferes with detection of low-level compounds. channel. they will not be discussed in great detail in this review [41–44]. and stir bar sorptive extraction (SBSE). There are a number of disadvantages to using chemical derivatization.12 A.

A. 95% dimethylpolysiloxane (HP-5 15 m × 0.02–0.2–2. EE2.D.1 g L−1 6–30 ng L−1 [95] [60] LLE 20–30 ng L−1 [62] Limit of quantitation (LOQ).25 m film thickness (5% diphenyl.. E3.D.25 m film thickness) 35% phenyl. DEP.0 ng L −1 [59] [54] GC–MS GC-FID EI-MS FID GC-ECD ECD Wastewater and stormwater Purified water and 180 g L−1 NaCl aqueous extract Wastewater LLE SPME 0. 95% dimethylpolysiloxane (DB-5MS 30 m × 0..91] [50] [92] A. DnBP. 95% dimethylpolysiloxane (HP-5MS 30 m × 0.10 ng L−1 0.25 m film thickness) 5% diphenyl. 95% dimethylpolysiloxane (HP-5MS 30 m × 0.. 0. DBP. BPA.25 m film thickness) DB-1MS (30 m × 0.D. Phthalates. DCHP. DnOP. DBP. BBP.25 mm I. E2. 0. 4-NP. DnBP.. BBP. 4-NP.25 m film thickness) TRB-5MS 30 m × 0.25 m film thickness) 5% diphenyl. DBP. DnOP 0. and estrogen conjugates BPA [40.25 m film thickness) 5% diphenyl.22 mm I.25 m film thickness) 5% phenyl.25 mm I.D.50. DAP. drinking water Wastewater DMP.25 mm I.25 mm I. derivatization SPE and derivatization DMP. E2. OP. 95% dimethylpolysiloxane (HP-5MS 30 m × 0. 95% dimethylpolysiloxane (DB-5HT 15 m) with a 1 m polysiloxane guard column 5% diphenyl. BBP 3–40 ng L−1 [56] EI-quadrupole Wastewater GC–MS GC–MS EI-IT EI-magnetic sector Groundwater Wastewater SPE..D.Table 3 GC methods for trace-level determination of endocrine disrupting compounds from aqueous systems. river water.D.25 m film thickness) with guard column Detection EI-multiple ion trap-MS EI-MS Sample matrix Ultrapure water Surface water Sample preparation technique SPE.25 m film thickness) DB5-MS (60 m × 0. 95% dimethylpolysiloxane) 5% diphenyl. 95% dimethylpolysiloxane (DB-5 60 m × 0. 0. MES. derivatization SPE & LLE with derivatization SPME thermal desorption SPE. DEHP. E2. DEP. 0. BPA 4-n-NP. BBP LOQ 20–400 ng L−1 [53] Estrogens: 2–4 ng L−1 . DEHP. 1% vinyl-methylpolysiloxane (SE-54 25 m × 0.25 m film thickness) 5% diphenyl. E1..05–0.D. 0. DIDP DMP. DBP.D. DOP Steroid estrogens and NPs NP.. E2. DEP.D. E1. alkylphenols. BEHP. 4OP. 95% dimethylpolysiloxane (DB-5MS 30 m × 0.D. 0.) 8% phenyl polycarborane-siloxane.3–3.25 mm I. alkylphenols.. DAP. DEP. derivatization SPE. 0. phthalate esters.. BPA. E2.32 mm I. NP: 500 ng L−1 1. E2 DMP. E2.. 0. DOP DMP. K. EE2 50 ng L−1 . 0. 13 . derivatization Compound of interest E2. EE2 E1. 0. Schug / Analytica Chimica Acta 696 (2011) 6–26 GC–MS EI-MS Water and aqueous extracts of plastics Drinking water SPME with Headspace derivatization Stir bar sorptive extraction SPE with derivatization 0.0 ng L −1 −1 Reference [23] [49] GC–MS GC–MS GC–MS EI-MS Magnetic sector EI-MS River and sea water Wastewater. Separation technique GC–MS/MS GC–MS Stationary phase 5% phenyl.D.25 ng L−1 Phenols 5–6 ng L . 95% dimethylpolysiloxane (DB-5MS 30 m × 0.25 m film thickness) 5% diphenyl.25 m film thickness) 5% diphenyl. LaFleur.25 m film thickness) 5% diphenyl. 65% methyl polysiloxane (DB35-MS 30 m × 0.32 mm I.4 ng L−1 [52] Large volume injection (LVI)/GC–MS GC–MS EI-quadrupole DBP. 4-t-OP.25 mm I. E1.5–172 ng L−1 [93] [94] GC–MS EI-MS Wastewater Stir bar sorptive extraction Modified SPE with ATD 2 ng L−1 [18] GC–MS EI-quadrupole Industrial ultrapure water Tap and drinking water 36–95 ng L−1 [57] GC–MS EI-quadrupole LPME DMP. E1. DEP. DNP.. 0.D. NP.. 0.25 mm I. BBP.22 mm I. DEHP Phthalates. 4-NP. 0.. E2 DEP.. 4-t-OP. EE2 EE2. E2.D. 0. 95% dimethylpolysiloxane (DB-5MS 30 m × 0.25 mm I.25 mm I.25 mm I. 95% fused silica (BPX-5 25 m × 0.25 mm I. DBP.02–0.25 mm I.D. BPA Limit of detection LOQ 0.D.D. 0..25 mm I.05 g L−1 [58] GC–MS GC–MS EI-quadrupole CI-quadrupole Wastewater Surface water SPME SPE and derivatization 3–54 ng L−1 0. 0. E2 300 ng L−1 3–30 ng L−1 0.5 m film thickness) 5% diphenyl. E3.. DEHP BPA.D. E1. 1 (HT8 50 m × 0.25 m film thickness) Cross-linked 5% methyl silicone (HP-5MS of 30 m × 0.D.

However. ammonium iodide. and steroidal estrogens [22]. and precision of the methods have been improved by derivatization of the hydroxyl groups of the steroid ring. Two distinct advantages of MS are: (a) it has mass-selective detection that is compound specific. 2). The LOD of BPA was improved shortly thereafter when Chang et al. 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. In spite of these observed drawbacks. 3. triclosan. These criteria require reliable and efficient sample preparation steps that risk introducing errors and technical issues associated with treatment techniques such as derivatization or extraction. In addition. A sensitive GC–MS/MS procedure using SPE with derivatization agent MSTFA mixture (N-methyl-N-trimethylsilyl-trifluoracetamide. MS is the most suitable method for the simultaneous detection and quantification of mixtures of trace EDCs in aqueous systems because they provide the selectivity and sensitivity to analyze complex samples. The samples were extracted by SPE and later derivatized using pentafluorobenzoyl chloride (PFBOCl) [54]. GC is a very well-established technique that offers extremely robust instrumentation that is widely available in nearly any laboratory setting. and diethylstilbestrol) were simultaneously extracted and analyzed from surface water using GC–MS with negative chemical ionization. With the exception . was observed because derivatization techniques failed to substitute the adjacent –OH group (Table 1).94) [23]. The consensus of most of these reviews is that mass spectrometry is undeniably the best technique to examine trace levels and provide essential identification and quantification of EDCs in aqueous systems. a method featuring SPE-GC–MS with silylation was able to successfully separate and detect phthalate esters. phthalates and steroid sex hormones) in aquatic environmental samples was written by Petrovic et al. In this case. an advantage of using GC–MS with electron impact over HPLC–MS methods for synthetic and natural steroids is the availability of mass spectral libraries useful for characterization and identification [40]. extraction. phenols and carboxylic acids in wet/aqueous media. 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. 4-nonylphenol. an HPLC method with UV detection was discussed.14 A. sensitivity. polychlorinated compounds such as dioxins. compounds which contain an ethynyl group. and HPLC–MS/MS has also been reviewed [48]. there are a number of methods capable of detecting EDCs at a reasonably sensitive concentration without derivatization. One year later. This clearly demonstrates the advantages of using derivatization to improve MS detection of phenolic EDCs. LLE was the preferred method if hormone levels were not a part of the analysis. They preferred this approach over LLE due to improved response and peak shapes of estrogens and a lessening of matrix effects. APs. The compounds analyzed were octylphenol. These improvements come at the cost of time. and their chlorinated derivatives in wastewater with recoveries greater than 95% in the g L−1 range [53]. and ethanethiol) was capable of detecting a group of natural and synthetic estrogens in water at trace levels (0. the instrumentation is more complex and costly than standard HPLC or GC equipment. The GC–MS method was linear (R2 ≥ 0. Specifically. LLE. however. In a separate experiment. and it requires highly skilled technicians to maintain a properly functioning system. Unfortunately. as well as accuracy. Often. poor detection of EE2 and MES.49]. Estrogens and estrogen conjugates.4 ng L−1 in using SPME in conjunction with GC–MS and headspace derivatization (bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) vapor) [52]. estrone. the GC–MS method was found to be more sensitive than either HPLC with ultraviolet (UV) detection or HPLC–MS. GC separations of estrogens and progestogens are performed using various columns and typical temperature programs (45–300 ◦ C) with helium carrier gas [40]. was able to be detected in water by GC–MS at a detection limit of 0. This study exhibits how efficient and targeted derivatization of EDCs can lower the detection limits by GC–MS considerably. much success has been achieved using the reagent n-methyl-N-(tert-butyldimethyltrifluoroacetamide). if SPE is used. to the ng L−1 range when derivatization was used. a polar compound.50]. have written a comprehensive review of mass spectrometry methods available to analyze alkyl phenols. cumylphenol. HPLC–MS. polybrominated diphenyl ethers.1–1 g L−1 underivatized. in 2002 [40].1. Even so.1. Schug / Analytica Chimica Acta 696 (2011) 6–26 different detectors qualified for the detection of EDCs. Another useful review of MS analysis of EDCs (alkyl phenols. and estrogens is GC with mass spectrometry (GC–MS). alkyl phenol recoveries can be affected due to loss during filtration of the aqueous sample or adsorption to an SPE filter.5 M methanolic solution of phenyltrimethylammonium hydroxide (PTA-OH) greatly improved sensitivity and peak shape by GC–MS. such as alkyl phenols (AP) and alkylphenolic ethoxylates (APEOs) with fewer than 4 ethoxy groups. phenols. The determination of estrogens and progestogens by GC–MS.40. Gentili et al. K. Only the most volatile degradation products of alkylphenolic compounds. and derivatization of different types of compounds (alcohols. Plastics-derived xenoestrogens. silylation is used because it can react with all these analytes.99) and sensitivity was increased two orders of magnitude. achieved a detection limit of 0. bisphenol-A.1. The stability. LaFleur. plastics-derived xenoestrogens (including phenols and phthalates). and acids). BPA. estradiol. nonylphenol. BPA-pentafluorobenzoylate was detected using negative chemical ionization MS with methane as the reagent gas at a LOD of 20 pg L−1 [34. a GC–MS method was able to separate and identify different isomers of the alkyl chain in underivatized 4-nonylphenol (Fig. three different techniques were used for the detection of phenolic endocrine disruptors in marine samples [51]. MS also involves intense data analysis and a thorough understanding of matrix effects and fragmentation patterns. derivatization to silyl BPA or pentafluorobenzoylate ester. and bisphenol A. whereas it appears as a single broad peak in HPLC–MS. can be analyzed without derivatization [40. typically. and (b) it is subject to less interference when compared to other types of detectors [45].25–5 ng L−1 ) with rather good recoveries in water (105 ± 20%) and acceptable R2 values (≥0. or MTBSTFA [49].1. but the sensitivity was improved using pre-concentration. Specifically. In another interesting study evaluating advantages and disadvantages of derivatization. The sole focus of this paper was BPA and no other potential EDCs were analyzed. furans and biphenyls.1. Combined determinations of xenoestrogens are challenging due to the various factors related to sample preparation and storage. chlorinated phenols. Derivatization of estrogens improves volatility and helps alleviate thermal decomposition [48]. In addition. This reagent was selected due to its ability to methylate alcohols. The authors found that derivatization of the phenolic compounds with 0. Mol et al. These benefits do come at a price. Phenolic EDCs. GC–MS with and without derivatization was compared to HPLC–ESI-MS without derivatization.A. The GC–MS methods will be organized and discussed by analyte type: estrogens and estrogen conjugates and then. seven estrogenic phenolic compounds (4-tert-octylphenol. recommended SPE if hormones are a part of the EDC group to be extracted from aqueous samples [49]. Bisphenol A.1. MS detection The most common analytical approach reported for the trace determination of estrogen mimics.2.1. 3.D. 3. xenoestrogens.

E1 E3. EE2.18 ng L−1 <1 ng L −1 [69] [66] [67] [68] 0.D.1 ng L −1 Reference [96] [72] [38] HPLC–MS/MS ESI-IT Deionized water and environmental water Surface water Deionized water and wastewater Water and wastewater Wastewater SPE 0. E3.7 ng L−1 0.5–2. E2. E1 5–6 ng L−1 0.. E2.9–1. E2-17G. 5 m) with guard column (4 mm × 4 mm. EE2 E1.4–0.53–6..D. 5 m of same packing) Waters Acquity C18 (50 mm × 2. 3 m) LC-18 (250 mm × 4 mm I.6 ng L−1 0.1 mm I.4 ng L−1 0. DES BPA. 4 m) SunFire C18 column (150 mm × 2. 3 m) with C-18 guard column (4 mm × 2 mm.1 mm I.. E2. E2.. EE2.6 mm I. E2. E3. EE2 0. EE2.D. E3 Estrogen conjugates (sulfate and glucuronide) E3.Table 4 HPLC methods for the determination of trace-level endocrine disrupting compounds from aqueous systems. E3.0 mm I. EE2.1 mm I.07–0.1 mm I. 5 m) LiChrospher 100 RP-18 (250 mm × 4 mm I.1–3. 3 m) Zorbax Extend-C18 column (150 mm × 1 mm I.D.7 m) Betasil C18 (150 mm × 2. EE2. E2. EE2 E1. 5 m of same packing) BDS Hypersil C18 (150 mm × 4.. E1-3S.6 mm I...1–0.. E3.A. 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.. E3. E1 0. 5 m) with guard cartridge Hypersil GOLD C18 (50 mm × 2. 5 m) with precolumn Synergi Max-RP C12 (250 mm × 4..5 m) HILIC TSKgel Amid-80 (150 mm × 2. Schug / Analytica Chimica Acta 696 (2011) 6–26 HPLC–MS/MS HPLC–MS/MS HPLC–MS/MS HPLC–MS/MS ESI-QqQ ESI-QqQ and APCI QqQ ESI-QqQ ESI-QqQ SPE SPE SPE.1 mm I. EE2 E2.3–0.D. derivatization Immunosorbent extraction BPA. 5 m) Zorbax Eclipse XDB C18 (100 mm × 2. E2.2 ng L−1 2–1000 ng L −1 [64] [99] [21] HPLC–MS/MS APPI-QqQ Surface and wastewater SPE 3–50 ng L−1 [71] HPLC–MS/MS ESI-QqQ Milli-Q water and river water SPE E2-17G. 4 m) Purospher STAR-RP-18e (55 mm × 2 mm I. 5 m) with guard (4 mm × 4 mm.0 ng L−1 1 ng L−1 0.. E3. E1. E3 E1.D. 1.D. 5 m) Purospher STAR-RP-18e (125 mm × 2 mm I. and estrogen conjugates E1. DES Limit of detection 1.D. E1 0.. 5 m) with guard (4 mm × 4 mm.D. Separation technique HPLC–MS/MS HPLC–MS/MS HPLC–MS/MS Stationary phase Synergi Max-RP C12 (250 mm × 4.D. EE2 NP E1..5 m) Luna C-18 (100 mm × 2 mm I.D.0 ng L−1 [67] A. 5 m of same packing) Purospher STAR-RP-18e (125 mm × 2 mm I.D.4 ng mL−1 0.01–0.D. E2.D. E1. 3 m) with guard (2 mm × 2 mm. K.D. E2. E2. E2.. E2. 3. LaFleur.. EE2. EE2.. E1-3S.85 ng L−1 [100] UPLC–MS/MS HPLC–MS HPLC–MS HPLC–MS Q-TOF ESI-quadrupole ESI-MS ESI and APCI-MS Wastewater Wastewater Water Groundwater SPE Immunosorbent extraction On-line SPE SPE E1.D.30 ng L−1 15 . E2. E2.4–2 ng L−1 [36] [97] [98] [69] HPLC–MS/MS HPLC–MS/MS HPLC–MS/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 E1.6 mm I. 3 m) LiChrospher 100 RP-18 (250 mm × 4 mm I.D.03–0. 3.D..

5 m) LiChrospher 100 RP-18 (250 mm × 4. MES.. E2. derivatization 2..4 g L−1 2. DPP.098–0..D. 4-BP. E1.D.DBP. E2.D. DES DMP. EE2.6 mm I. DES.1 mm I. DHP. drinking. DES.D. EE2.7–8. 5 m) Tracer excel 120 OctaDecilSilica-A column (150 mm × 4. E2. DEP. 3.3 g L−1 [77] [46] FL ( em ex = 276 nm. DEHP.07 ng mL 10–20 ng L−1 −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.D. 225 nm (190–400 nm) DAD Sample matrix Milli-Q.D. E1. 5 m) with guard column (4 mm × 3 mm) Hypersil ODS-C18 (150 mm × 4. 225.2 g L−1 12 pg 0. NP. 4-NP. DES BPA 4-NP. E2.6 mm I. ground.6 mm I.D. EE2 Limit of detection <22 ng L−1 0. MES. 5 m) DAD (271 nm) FL Tap water.3–4 g L−1 1. DEHP E3. BPA.0 mm I. 5 m) CapcelPak ODS (250 mm × 4.. DBP DEP.0 mm. DBP. E2. DES. EE2. EE2 DMP. DMP.0–3..6 mm I. 4 m) Supelcosil LC-18 (100 and 250 mm × 4.102] [82.D..84] [55] [81] CL CL ED Carbon nanotube – modified glassy carbon electrode 1.6 ng L−1 [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. 4-t-OP BPA. . derivatization Not applicable Not applicable SPME SPE 0. 5 m) Hypersil ODS-C18 (150 mm × 4. DBP. DCHP DEP. 4-t-BP BPA. E3. 5 m) Diamonsil-C18 (250 mm × 4.. BBP. E2. OP. LaFleur.6 mm I.3–1..D.17 g L−1 0. EE2.D. E2. E2. derivatization Not applicable SPE Compound of interest E1.. 3 m) Zorbax Eclipse XDB-C8 (50 mm × 2.D. and surface water Milli-Q and river water Intravenous injection solutions Water and urine Sample preparation technique SPE.08 g L −1 [80] [82..6 mm I. DBP E3.5 m) LiChrospher 100 RP-18 (250 mm × 4 mm I. E3 BPA EE2 BPA BPA..D.6 mm I. 5 m) Eurospher-100 ODS (250 mm × 4. E1. 5 m) Luna C18 (250 mm × 4. DMP.12–0.340 M Limit of quantitation (LOQ).99–24. Schug / Analytica Chimica Acta 696 (2011) 6–26 [73] HPLC-DAD 25–100 g L−1 [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 0.6 mm I. 240 nm) UV (280 nm) UV. DPP.D.A.6 mm I. 5 m) ODS-C18 (250 mm × 4.D. 5 m) Luna C18 (150 mm × 4. sea water Urine SPME SPE. DCHP. river.1 mm I..0 V 0.D.6 mm I. K.0 mm I. 5 m) Detection ESI and APCI-QqQ ESI-quadrupole DAD (200.16 Table 4 (Continued) Separation technique HPLC–MS HPLC–MS HPLC-DAD Stationary phase BetaBasic C18 (150 mm × 2. DEHP. E2.. and wastewater Physiological saline solutions LC-grade water. DOP E1.D. DOP.) Daisopak-SP-120-5-ODS-BP (250 mm × 4. DEP. EE2.. DAP. = 306 nm) Urine Plasma Water River and wastewater Ground and tap water SPE.D.2–1.1 g L−1 LOQ 1–10 ng mL −1 [55] A. E2. BPA.. MES MES.38 g L−1 0.8 ng mL−1 0. 5 m) LiChrospher 100 RP-18 (250 mm × 4. DEP.06–0.0 mm I. drinking.

have been compared for the concentration of six phthalates found in aqueous samples.40 g L−1 ) were rather low for all of the analytes except DBP and BBP. The . 2. and readily accessible to most typical analytical laboratories and gas chromatographs. was able to achieve 36–95 ng L−1 detection limits for five phtha- late esters with recovery rates in ultrapure water between 15% and 101% [57]. the explanation was that dioctyl phthalate (DOP) was not adequately adsorbed onto or desorbed from the SPE sorbent (Tenax TA). The researchers were unsuccessful in attempts to use addition of methanol to reduce the adsorption phenomena [56]. as well as a limited volume of organic solvent (7 mL of 1-dodecanol). Phthalate esters have been detected using various MS methods including electron impact (EI-MS) and chemical ionization (CI-MS). although. Phthalates. of high recoveries for estradiol (E2 135–163%) and low recoveries for diethylstilbestrol (DES 71–73%). Researchers successfully used SBSE with a large volume injection GC method (LVI-GC–MS) for ultra-trace phthalates in drinking water with instrumental LOD values of 0. Schug / Analytica Chimica Acta 696 (2011) 6–26 17 Fig. low recoveries were seen for some of the analytes of interest.m polydimethylsiloxane/divinylbenzene (PDMS/DVB). and PAHs. in both the positive or negative modes. Optimally. high throughput method. yielding a method detection limit for DEHP of approximately 6 ng L−1 in aqueous sample [14.55]. including alkyl phenols. SPME using assorted polyacrylate fibers.60 g L−1 for the simultaneous analysis of six phthalate acid esters and one adipate ester [56]. The optimal fiber for this purpose was found to be a 65.A. phthalates. The optimal fiber for this purpose was indicated to be a 65. The technique required minimal sample volume (∼10 mL). 3. Reprinted from [40]. This would eliminate the solvent elution procedure needed for conventional SPE methods [57].006 ng g−1 (approximately 6 ng L−1 )) of DEHP in aqueous samples using a SPME-GC–MS was reported [14].55]. These lower recoveries were explained by the higher hydrophobic nature of the phthalate esters. The detection limits and extraction times were comparable with other microextraction methods (20–50 ng L−1 . Other detection techniques Flame ionization detectors (FID) are commonplace and preferred because they are versatile.m polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber [14. 25 min). the phenolic compounds had recoveries between 86% and 118% for 5 ng L−1 spiked concentrations with detection limits between 0. The authors did not indicate if any other solvents were investigated for this purpose. GC–MS chromatogram of 4-t-octylphenol and isomers of 4-nonylphenol.15–0. In addition. Copyright 2002. was compared for the concentration of six phthalates found in aqueous samples. in the ng L−1 to g L−1 concentration ranges [59]. It was reported to be simple and cost effective with no sample carryover and little. this was speculated to be the cause of adsorption of the analytes onto the sampling flask glass walls. K. This time. matrix issues from complex samples [58]. In another study. A low LOD (0. this method only required a 50 mL sample for analysis. Method improvements might be made in the future to make this a more time-efficient. 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.1.2. a different sample preparation technique. One multi-residue SPE-GC–MS method was found to be sensitive enough to detect 42 priority contaminants in wastewater. 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. Again. if any. coupled to GC–MS.D. such as molecular mass or the nature of the alcohol moiety in the molecule [40]. It is also important to note that the recoveries of spiked water samples (0. Liu et al. coupled to GC–MS. The loss of PAEs over storage time was not observed when samples were stored in PFA bottles (perfluoralkoxy copolymer). LaFleur.0 ng L−1 . simple.A. with permission from Elsevier. EI-MS was reported to have the most sensitive detection limits and is the recommended method for quantification of phthalate esters. a selected method would be able to analyze a number of EDCs in a single run with minimal time and sample volume required. These findings demonstrate the equivalent sensitivity of chemical ionization when compared to electron impact for the quantification of phenolic EDCs in environmental water using GC–MS. it cannot give comprehensive qualitative information. A separate method using liquidphase microextraction (LPME) GC–MS demonstrated a number of advantages for qualitative and quantitative determination of select phthalate esters in water samples. SPME using various polyacrylate fibers. Using SPE and automated thermal desorption GC–MS. 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.2 and 2.

2. The HPLC–MS methods are grouped by applications for the quantification of estrogens. are the most commonly found MS techniques in the recent literature. so may not apply to the scope of this paper. LC–MS techniques which use APCI and ESI in the negative ion mode have been published for the detection of phenolic compounds. usually nickel-63 or tritium.1. LaFleur. HPLC is a valuable analytical technique that is conventional to most laboratories conducting trace analysis. The main advantage of HPLC–MS over GC–MS is that the determination of trace levels of EDCs can be made without the tedious. however. 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. including HPLC and capillary electrophoresis (CE). K. Generally. In their study of phthalate acid esters (PAE) in plastics. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Most of the GC work being conducted currently makes use of MS detection. causing a decrease in response. The combination of HPLC with mass spectrometry (HPLC–MS) has been used more frequently in recent research due to the sensitivity.2. due to its superior selectivity. and nitro groups. Modern TOF detectors are especially useful in trace qualitative and quantitative analysis when presented in a hybrid format. Electron capture detection (ECD) is useful due to its extremely powerful selectivity and sensitivity for halogenated compounds. Li et al. in combination with single ion monitoring (SIM). these techniques were less sensitive than GC or MS methods and many did not meet the criteria of this review. The steroidal estrogens. In a separate study. High performance liquid chromatography (HPLC) The need to analyze complex aqueous samples directly can be filled by liquid phase separation methods. however.1. single-reaction monitoring (SRM). to ionize the carrier gas [60]. it is clear that the advent of atmospheric pressure ionization and interfaces such as electrospray (ESI) and APCI overcame many of these limitations. from refractive index (RI). While ECD detection has shown good sensitivity for phthalates. These approaches offer versatility. and speed MS can offer. 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 L−1 NaCl. 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]. and versatile. and electrochemical (EC) have been investigated for trace analysis of EDCs. so will be mentioned as potential areas for future research. after an ion trap (IT-TOF) or quadrupole (Q-TOF) mass analyzer. MS detection Mass spectrometry is widely considered to be the most sensitive and discerning detector for HPLC analysis. They opted to avoid using NaCl altogether due to degraded peak shape. when they concluded that APCI-MS/MS with switching between positive and negative modes was the optimal 3. however. Previously. poor resolution. Timeof-flight MS (TOF-MS) increases the selectivity further through improved resolution and mass accuracy of detected analytes. affordable. In the Petrovic review. The methods included in the review were published from about 1994 to 2002. The structural information derived from the data and the sensitivity was limited when thermospray or particle beam techniques were used. The analysis used GC with ECD and a very involved effluent extraction method that included a number of highly toxic solvents and compounds (hexane. sample preparation process of derivatization [40]. and then those suited for quantification of plastics-derived xenoestrogens (phenols and phthalates). The hardware has a history of robust laboratory performance and there are a number of suitable methods available for determination of EDCs. The on-line SPE of aqueous samples with subsequent analysis by HPLC with diode array detection (DAD) or HPLC–ESI-MS is described by Petrovic et al. peroxides. however this trend was refuted by Hsu et al. chemiluminescence (CL). fully automated techniques. 3. 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 fiber [61]. . A good review detailing mass spectrometric applications for the determination of EDCs in aquatic environmental samples was published in 2002 [40]. ultraviolet (UV). quinones. Automated online methods have also increased in importance. 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 HPLC–MS [63]. ECD requires special radioactivity disposal and safety training because it contains a -emitting source. The separation of the eight compounds with GC-FID was successful in less than 12 min. but it does not exhibit the sensitivity or linear response range required for trace level analysis for the EDCs presented in this paper. the ESI interface was reported to provide detection limits one order of magnitude better that the APCI interface for a number of estrogens. however. Schug / Analytica Chimica Acta 696 (2011) 6–26 sensitivity required for trace determination of EDCs may be suboptimal. This kind of development ensures mass spectrometry’s place in standard analytical laboratories involved in high throughput. sensitivity. diode array detection (DAD).18 A. We found these techniques well represented in the publications from the past five years. can be detected by both negative and positive-ion modes. The literature demonstrates that the triple quadrupole (QqQ) MS is preferred over a single quadrupole or ion trap analyzers for quantitative determination of phenols. Generally. specificity. Estrogens and estrogen conjugates. The detection limits and recovery values for three of the six phthalates studied met the criteria of this paper: DMP (20 ng L−1 ). or multiple reaction monitoring (MRM) techniques. 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]. however.D. demonstrating the potential separation efficiency of GC-FID for EDC analysis. as a fully automated process that is capable of analyzing target compounds in the ng L−1 concentration range [40]. dichloromethane. and selectivity for trace analysis in general [45]. In this case. and mercury) [62]. 3. and DnOP (20 ng L−1 ). fluorescence (FL). and the deterioration of the coating of the stir bars used in their sample extractions [56]. the major obstacles of HPLC–MS were unreliable interfaces [40]. researchers addressed the negative impact of salt concentrations on phthalate compounds with lower water solubility. Detectors of all kinds. DnBP (30 ng L−1 ).A. 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. This group also evaluated extraction times and desorption temperatures to optimize SPME with a GC-FID method.1. Their reported detection limits ranged from 6 to 84 ng L−1 for the simultaneous extraction and quantification of eight PAEs using a calix[4]arene fiber [61] from 180 g L−1 sodium chloride. however.2. they have weaker specificity than other detection methods [34]. and sometimes toxic. 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. several studies were able to separate and quantify EDC mixtures using GC-FID. which are weakly acidic. these detectors are commonplace in analytical laboratories.

The method was unable to detect the analytes prior to sample treatment (Panel A).1 and 1 ng L−1 . Petrovic et al. The researchers determined that they could demonstrate little control over which of estriol’s three hydroxyl groups reacted with the derivatization reagent (2-fluoro1-methylpyridinium p-toluenesulfonate (FMPTS). This goal was partially achieved when an HPLC–APCI/MS technique in the negative ionization mode for the quantification of estrogens and bisphenol A was developed in 2004 [68]. progestogens were best detected by MS with an ESI interface [67]. methanol did not appear to affect the negatively charged stainless steel tip in the same manner as acetonitrile [51].5 and 1 ng L−1 . but a single-quadrupole was better for detecting progestogens (0. Lin et al. The detection limits were fairly good for all of the analytes. in this case) to give the characteristic fragmentation patterns. LaFleur. E2. While the recovery values for most of the estrogens and estrogen conjugates were rather good (91–119%). Reprinted with permission from [66]. An on-line SPE method using HPLC–ESIMS in negative ionization mode (SIM) was reported in 2001. In addition. and EE in water at concentrations less than 1 ng L−1 . nor was the mechanism of interference exhibited for different matrix compounds fully understood. E2.4 g L−1 ). it has been reported to leave carbon deposits on an ESI capillary. 3 demonstrates the improvement in sensitivity for immunosorbent-treated effluent compared to raw sample. the response of E1 and E2 were excellent post treatment (Panel B). Future detection goals were set between 0. In a separate study by Lin et al. A competition for access to the droplet surface in ESI is believed to occur between the analytes and some matrix constituents [37]. In the 2002 review. 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]. bisphenol A had a slightly lower recovery of 81% in spiked groundwater. With the continued development of immunoaffinity cartridges and other advanced extraction and MS techniques. This method set the standard because it was capable of detecting E1. an immunoaffinity extraction method. When the sample matrix was less complex. Note the absence of signal for EE2. As of 2003. In the mid to late 1990s and early 2000s. specifically estriol (E3). In order to achieve the low detection limits. review. the article challenged the scientific community to improve the limits of detection for ethynylestradiol and other steroid estrogens as they were suspected of affecting organisms at 1 ng L−1 . Only one year after the Petrovic et al. While acetonitrile is preferred as a strong eluent for HPLC with UV detection. method for simultaneously detecting E1 and six of its metabolites at concentration levels of 0. Ion suppression of analytes is thought to be caused by precipitation or co-precipitation with other matrix components in APCI. E3.9–1.08 and 0. Not only new MS technology. and EE2) in different aqueous matrices was made. like drinking water. K. E3 was not retained due to the extreme specificity of the sorbent. While tandem MS can be used to solve issues with isobaric noise. but also new ideas for minimizing the matrix effects in environmental samples were needed to achieve the trace levels of EDCs required. Summed ion chromatograms (SIC) of raw (A) and immunosorbent-treated (B) extracts of sewage effluent analyzed by negative polarity HPLC–ESI-MS. This was an exciting development as full automation of the method was also achieved [65]. This information may be helpful when selecting a mobile phase for this type of analysis in the future. Schug / Analytica Chimica Acta 696 (2011) 6–26 19 Fig. Unfortunately. noted that the improvement in sensitivity was not equivalent among all of the estrogens in their experiment. and were below 15 ng L−1 . which occur in the MS source during ion formation. however. Also reported in 2001. 4). The derivatization reagents improved the sensitivity of phenolic EDCs by reacting with the phenol group to form ethers (Fig. in 2007 [47]. [66] found that the acidification 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. in combination with HPLC–ESI-MS was able to minimize isobaric noise in the chromatograms (SIM) and achieve less than 1 ng L−1 detection limits for E1 and E2 and recoveries of 88–107% in aqueous systems [66]. such methods do still experience suppression effects.A. They found that the sensitivity of phenolic estrogens in complex matrices such as river water was improved by derivatization using pentafluorobenzyl bromide (PFBBr). however. Fig. hope was placed in emerging HPLC–MS capabilities (TOF and ion-trap) for routine applications involving low ng L−1 levels of EDCs in aquatic samples. Copyright 2001 American Chemical Society. The matrix effect problems often observed in HPLC–MS has not been completely resolved.4 ng mL−1 [64].A. Ferguson et al. Again.6 ng L−1 and LOQ between 0. the derivatization agent that was found to be the most effective was dansyl chloride [47]. They indicated that 0. which should elute between E2 and equilin-d4. The ultimate goal for EDC analysis is to develop a robust method that can detect different classes of EDCs in a single automated analysis. we will examine if this goal has been achieved in a manner that is robust and cost-effective for analytical screening of aqueous matrices. but was not retained on the immunosorbent. [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. respectively). In the study that made mention of this. This trend does not appear to have changed much in the past five years. The majority of the MS work was conducted in negative ionization mode to obtain the desired sensitivity. This trait can be beneficial for quantitation of a single analyte. the samples were quantified using stable-isotope deuterium-labeled internal standards. the detection limits for this study were estimated using an assumption of linearity without any verification through representative . 3. an evaluation of three derivatization agents with HPLC–APCI/MS and HPLC–ESI/MS methods for the detection of trace-level estrogens (E1. Nearly eight years ago. another review from the University of Barcelona detailed HPLC–MS/MS methods which improved the limits of quantitation and analyte identification [48]. the majority of the HPLC methods used C18 stationary phases with a water–acetonitrile gradient mobile phase. however.D. limits the use of this technique for more sweeping types of analysis.1–10 g L−1 detection limits for estrogens were achieved using SIM mode with the QqQ. E3. They also reported that E2 remains stable with no detectable degradation for up to six days when stored at 4 ◦ C without acidification or the addition of formaldehyde to reduce bacterial oxidation.

The method did not use hydrolysis or derivatization and was able to achieve detection limits in the ng L−1 range for many of the estrogen metabolite analytes in urine (estrone-3sulfate (E1-3S). estradiol-3-sulfate (E2-3S). as hoped. 6). and synthetic progestogens in surface and wastewater. Another benefit of this particular method was the low sample volume required to achieve the detection limits (1 mL) with a simple SPE pre-treatment.5 ng L−1 . 5 was suggested in order to help eliminate these potential interferences. They verified the ELISA results with HPLC–MS/MS (QqQ) instrument. Adapted from [47]. in 2008 [71]. 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]. progestogens. This method featured online SPE and atmospheric pressure ionization (APPI). progesterone. and phytoestrogens present in the sample [69]. This reduced the run time to about 12 min and increased the separation efficiency. Another example of a method designed to be automated and use only 1 mL of sample was reported by Viglino et al. Of further interest. APPI was used to avoid a derivatization step. the authors were hoping to eliminate the expense of unnecessary testing. While it appeared that some samples displayed a false positive due to the MS detection discrepancy.A. it did not improve the detection limits for estradiol in aqueous samples. In this manner. K. While the TOF did demonstrate its ability to detect and quantitate estrogens and phytoestrogens at ng L−1 concentrations. but still achieved ng L−1 detection limits for natural and synthetic estrogens. expensive analytical technique. and residual estrogens. 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. but the conclusions indicate that the authors succeeded in developing an automated method capable of using only 1–3 mL of . 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 lowest concentration for ELISA detection was reported at 2. (b) 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS) derivatization (c) pentafluorobenzyl bromide (PFBBr) derivatization. This is the first example we were able to find that used modern ultra-high pressure liquid chromatography systems with TOF for the determination of 17. estrone-3-glucuronide (E1-3G). The detection limits of the ELISA happened to be below those of the HPLC–MS–MS method used to verify the ELISA approach. These findings indicate that immunoassay screening methods work well for rapid initial estimation of the presence of ng L−1 levels of E2 in drinking water.20 A. The ELISA kit was used to perform an inexpensive initial screen for E2 to determine samples that needed further analysis using the more complex. The estimated LODs were in the ng L−1 range. Scheme of selected derivatization reactions: (a) dansyl chloride derivatization. The complexity and cost of this system may be a deterrent for most typical analytical laboratories (Fig.-estradiol (E2) in water samples [69]. 4. curves.D. estradiol-3-glucuronide (E2-3G). Spain used an immunosorbent kit (enzyme-linked immunosorbent assay or ELISA) initial screening method with ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). estriol-3-glucuronide (E3-3G). estriol-16-glucuronide (E3-16G)) [21]. Treatment with SPE procedure outlined in Fig. no false negatives were generated using the ELISA screen. estriol-3-sulfate (E3-3S). A recent paper by an environmental chemistry group in Barcelona. the TOF-MS method which was evaluated used a shorter (50 mm) C18 chromatographic column with sub-2 m particles. which are not unreasonable based upon the data of similar APCI methods that used untreated (non-derivatized) samples. LaFleur. The authors found that ELISA often overestimates the concentrations of E2 in the water samples due to the positive interference of estrogen conjugates. Schug / Analytica Chimica Acta 696 (2011) 6–26 Fig.

with permission from Elsevier. . Copyright 2008. K.A. General SPE procedure to eliminate sample interference for E2 screening by ELISA. Fig.D. LaFleur. Schematic diagram of the automated SPE tandem LC–MS/MS system. 6.A. 5. Adapted from [69]. Schug / Analytica Chimica Acta 696 (2011) 6–26 21 Fig. Reprinted from [71].

costly stable isotopically labeled internal standards can be used. The reported preference for use with HPLC analysis is SPME using a 65.1. While this article does not specifically focus on aqueous sample matrices. such as estriol. The LOD for these compounds. They outlined the environmental and economic interest in PAEs due to their significant presence as plasticizers and additives in industry and their impact on human health and the environment [14]. are readily detected using negative ionization mode in both ESI and APCI interfaces. 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]. without any derivatization or other sample pretreatment. Perez Feas et al.D. therefore. was able to simultaneously detect various natural and synthetic estrogens (E1. when increased tailing was observed.1 ng g−1 in natural water. The HPLC–ESI-MS(−) method had detection limits ranging from 1. while advertised by the manufacturer’s specifications as reusable. The alkyl phenols. the detection limit for BPA using an HPLC–APCI/MS method was reported to be 100 ng L−1 . plastic. but is not always successful at removing all of the hydrophobic matrix components. how- .’s study after changes were made to acidify the mobile phase. and so forth) was presented by Gomez-Hens and Aguilar-Caballos in 2003 [14]. and 240 nm. HPLC–MS. A method described by Lagana et al.7 ng. such as: (a) avoiding all contact with plastics.2. Smaller column dimensions are beginning to appear more frequently in the literature. and 4-(tert-octyl)phenol (4-OP is a by-product of alkyl phenol polyethoxylates that are surfactants added to soap. phthalate esters (PAEs) were mostly analyzed by GC–MS or HPLC–APCI–MS with minimal work using HPLC–ESI-MS. Full automation is desirable for improving sample throughput. which then can suppress the ionization of late-eluting alkyl phenols [37]. and phthalates [73]. octylphenol and nonylphenol. a polar compound was detected primarily by GC–MS in the mid-1990s. would no longer be retained after a single use. 3. Phthalates. ranged from 0. and HPLC-UV methods [51]. 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. A compilation of analytical methods for the determination of phthalic acid esters in various types of samples (air. The detection limits reported were in the 100 ng L−1 range [40]. SPE has been shown to be useful. The xenoestrogens studied that met the criteria of greater than 80% recovery values were BPA. water.A. In 2008. Bisphenol A. For example. They found that the GC–MS method was able to improve sensitivity two orders of magnitude when the phenolic compounds were derivatized with phenyltrimethylammonium hydroxide (PTA-OH). and reproducibility. the side-by-side comparisons of the performance of each analytical technique are of great interest. or SPME. and (c) understanding potential sources of contamination in the laboratory. sewage sludge. the method detection limits ranged from 10 to 20 ng L−1 with 96–112% recoveries and relative standard deviations less than 3% (n = 6) in water. LaFleur.004 ng to 0. the BPA levels detected using APCI with QqQ were much improved. 4-cumylphenol (4-CP is used in the rubber. and sample preparation typically involves LLE. because of its improved sensitivity (nearly 40–50 times more sensitive) [40]. 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. and pesticide formulations). sensitivity. Fewer HPLC–MS methods are available than GC–MS or GC-FID methods for the separation of phthalates due to the lower detection limits that can be achieved by GC. and magnetic sector instruments). was able to study select xenoestrogens in marine samples comparing GC–MS. reported LOD values for the phthalates DMP. In the method presented by Perez Feas et al.5 m 2. UV detectors are simple and inexpensive to use and maintain and are the preferred detector for most analytical laboratories.22 A. plastics. economic efficiency.2. and DBP in the low ng mL−1 range using HPLC–ESI-MS in positive ionization mode in saline solutions [63]. K.6 to 4.4 ng while the HPLC-UV method detection limits were not too far removed at 1. (b) vigorously cleaning and rinsing all glassware with certified phthalate-free solvents.2. UV detectors have demonstrated the capability to provide detection limits at or below g L−1 concentrations for select EDCs [45]. In this case. Many kinds of MS analyzers have been used for the analysis of phthalates (quadrupole.99 to 24 ng mL−1 in saline. Special precautions must be taken to minimize contamination during sample preparation. the limit of quantitation must be equal to or less than this regulation. except for the compounds 4-octylphenol and 4-tert-octylphenol. 3. Sample cleanup to minimize matrix effects is important for phenolic compounds. plastic. The detection limits for the four phthalates. SPE. They found that the more polar compounds. alkyl phenols. E3. Phenolic EDCs. however. GC–MS using SPME had a reported LOD for DEHP of 0. Schug / Analytica Chimica Acta 696 (2011) 6–26 sample and rapidly analyzing eight EDCs with a total cycle time of 15 min [71]. and adhesive industry as an anti-oxidant).m PDMS/DVB-coated fiber [14]. Plastics-derived xenoestrogens. simplicity. The apolar compounds were able to be analyzed from a cartridge reused up to 12 times. The different method detection limits were compared and the conclusion was drawn that the most sensitive method for the determination of phenolic EDCs was GC–MS (0.1 mm internal diameter and 50 mm length C8 stationary phase was used together with an HPLC–ESI-MS (single quadrupole). and compounds adsorbed on glass. At that time. herbicide. foods. The ESI interface is favored over APCI. A gradient method with a ten minute run time and a ten minute equilibration time was used. By 2004. many false positives or erroneously high recoveries can be determined on instrumentation that is so uniquely selective and sensitive. [63]. To assist with this problem and to improve precision and reliability. but HPLC–MS work was starting to be conducted as well [40]. but the detection limits were considerably higher (36–182 ng L−1 depending on the sample matrix) [36]. a 3.0 to 2.010 ng). BBP. Ionization enhancement was demonstrated for most of the EDCs that were a part of Benijts et al. NP was detected along with related halogenated derivatives one year earlier using HPLC–MS–MS at detection limits of about 1–2 ng L−1 in water samples [72]. was not recommended by the authors to be used more than one time per sample. DEP. The method was also able to detect NP. Earlier in the decade. contaminated water or solvents. the peak shape suffered after the sixth use. triple quadrupole. due to the high propensity for contamination. 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. The separation of four phthalates was accomplished at room temperature in less than ten minutes. 225. The method consisted of gradient elution over a 40 min run time while monitoring UV detection at 200.2. The EPA maximum limit for DEHP in drinking water was set at 6 ng mL−1 . however. E2. One of the findings in this study was that the SPE cartridge (polymeric PLRP-S 15–25 m – Polymer Laboratories. They have been effective for this purpose. such as particulate matter in air.006 ng g−1 (ng mL−1 ) in tap water and SPE-HPLC-UV had an LOD of 0. Phthalates have limited solubility in water. ion traps. Stuart et al. Specifically. Church Stretton). which continued to exhibit nearly 50% matrix suppression. paint. and EE2) and BPA in the low ng L−1 range. as well as subjecting the samples to some form of pre-concentration or derivatization to improve sensitivity. however.

Concentration factors of 600 were achieved using this SPE method based on Br-coated silica mixed hemimicelles.A. This may be due to the challenging behavior of alkyl phenols and phthalates in general. An interesting finding of this particular study was that DEHP. 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. and phthalates without extensive sample pre-treatment. smaller sample volumes (10 mL) were able to be processed with recoveries in the 90% range. 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. The column dimensions in this method were large and the gradient required a minimum of 22 min. lower extraction efficiencies for polar compounds. (c) ability to be used in a wide range of operating conditions. Other detection techniques 3. sample treatment using reverse micelle microextraction of BPA from urine that achieved a detection limit for BPA of 0. This is indicative of a potential need to improve the reproducibility between different fibers (fiber-to-fiber variation).cyclodextrin and the system was operated at a temperature of 47 ◦ C [78].1 g L−1 . such as temperature and chemical instability. The majority of methods use SPE or LLE. conducted a study that successfully separated a group of 27 suspected EDCs in environmental samples using temperature-dependent inclusion chromatography. When compared to a commercial polyacrylate SPME fiber. Zarzycki et al. A remarkable development occurred in 2007 when cloud point extraction HPLC-UV was used to detect and quantitate di-ethyl-phthalate. m-methylphenol.05 to 2. thereby facilitating the phase separation. Also. indicating that no significant improvement in detection limits had been achieved in nearly a decade. they appear to perform no better than UV detection. extraction techniques. (b) cost.197 g L−1 [80]. but the authors did not attempt to extend the curve down into the trace level range. rapid. as well as a number of non-steroidal EDCs including BPA. column technology. five 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. Other sample preparation applications for the isolation and detection of phthalates from water have recently been explored. but because the intensity of BPA is higher in organic media. The Na2 SO4 was added in order to increase the density of the aqueous phase. maximizing efficiency by minimizing time and solvent usage required to separate the analytes of interest.D. bisphenol A. Less efficient recoveries were also observed for this method. the HPLC analysis was conducted at 226 nm and a run time in excess of 25 min was required. typical quantitation limits for BPA by fluorescence detection were still being reported in the 5–50 g L−1 range. This illustrates the potential uses of fully automated methods that are simple. di-(2-ethylhexyl)-phthalate and di-cyclohexylphthalate with detection limits between 1. and (d) their chemical and mechanical stability. Species that do not ordinarily fluoresce can be treated with reagents which complex with the analytes to form fluorescent derivatives. BPA can be detected with excitation and emission wavelengths of 275 and 305 nm.A. Phenolic analytes can be derivatized in this manner using reagents such as dansyl chloride. 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. In other work.8 g L−1 with recoveries (n = 3. or newer stationary phases. which can be leached from medical polyvinylchloride administration set tubing or containers. Other innovative developments include the use of unique materials for SPME or molecularly imprinted solid-phase extraction (MISPE) techniques with HPLCUV. alkyl phenols. From the regression analysis of the calibration curves. When the same fiber was used for replicate analyses. the best correlation coefficients were demonstrated when the mobile phase did not contain modifiers.3. a few additional phenols. For example. An example of natural estrogens and phenolic EDC detection by . K. They concluded that temperature is a critical parameter for HPLC selectivity of these compounds when using macrocyclic mobile phase additives. the detection range of the method for the various phenols ranged from 0. LaFleur. were much higher than the estrogen and progestogen analysis described previously: 0. Fluorescence detection (FL). In addition. more efficient extraction methods for removing BPA from aqueous matrices. economic manner exists and has encouraged the development of faster.9 to 3. 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. response is dependent upon the composition of the mobile phase [34]. a nitrophenol. and sensitive for the screening of liquid medicines and intravenous injection solutions for BPA. This can cause erroneously high recoveries and false-positives for BPA. swelling. In another study. One weakness of fluorescence detection is the potential for interference from various other migrants from packaging which are also actively fluorescing compounds. 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.3.17 g L−1 [75].0 mg L−1 . where eluents were modified with -cyclodextrin or hydroxypropyl. and two chlorophenols. An opportunity for analytical development would be to make use of smaller bore columns with smaller particle sizes or fused core technology. A carbon nanotube coat- ing on a Pt fiber was explored due to the inherent issues known for most commercial and some laboratory-made SPME fibers. respectively. Again.8 ng mL−1 in only 10-mL of environmental water samples [74].1. From the surfactant-rich phase. The authors observed an increased RSD when using separate fibers for replicate analyses. the SWCNT fiber was found to be similar or superior for extraction of phenol. the RSD remained below 10%.2. The advantages of these molecularly imprinted polymers are: (a) their selectivity. The authors focused on the effect of temperature on the simultaneous separation and detection of various natural and artificial steroids.80]. is more susceptible to migrate into product formulations that contain the solublizing agent polysorbate 80. The reported detection limits for this method were 0.2. The phases of the samples were separated using centrifugation for about 5 min at 3500 rpm.1–4. The hydrophobic interactions of the PAEs enabled the retention and then easy release/desorption without carryover from the SPE. 3. 20 L was injected directly for HPLC analysis at 226 nm.0 ng mL−1 . the determination of phenols in aqueous samples by direct immersion of a SPME platinum fiber coated with carbon nanotubes was accomplished [77].0 and 3. and high likelihood of breakage. Fluorescence detectors are specific and sensitive for fluorescing species. Explorations of different derivatization reagents. confirmation of the identity of the detected compound using HPLC–MS or an in-line DAD is encouraged [34.6 and 1. 200 g L−1 ) greater than 87% in seawater and greater than 92% in tap water. For this reason. The quantitation limit of the method was about 3 g L−1 [49. and a number of phthalates by HPLC-UV.12–0. applied HPLC with fluorescence detection for the analysis of bisphenol A in river water samples. The calibration curves of spiked water samples were run from 0. By 2008. Schug / Analytica Chimica Acta 696 (2011) 6–26 23 ever. even though the reported detection limits for the phenolic analytes were between 0. selective.79]. chlorophenols) [76]. The sensitivity is typically more than one order of magnitude better than absorbance detectors. as a whole. but researchers have developed a short. Markham et al. however in the area of EDCs. The method showed promise in that the separation of BPA. 20-min. A need to test large numbers of samples in a fast.

sample matrix interferences. charge/mass ratio. however. It appears that this is an area of potential development for endocrine-affecting phenols. The authors were able to separate and detect various estrogens and phenolic EDCs. The detection limits reported were between 60 and 80 ng L−1 for various phenols including BPA. In standard. This finding 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. A separate research group studied aqueous and non-aqueous CE to separate halogenated phenolic and bisphenolic compounds in aqueous samples.38 g L−1 [84]. there were no studies cited for this technique for the determination of phenolic compounds derived from polymeric materials. CE also exhibits higher column efficiencies than HPLC and freedom to alter or improve separation efficiencies by changing buffer composition. simultaneously detected twenty phenolic compounds in wine samples. not the focus of this review. which was more than adequate for this purpose as the compounds that were detected in the urine samples are typically at g L−1 to mg L−1 levels. Vega et al. The recoveries of the six analytes using this method were all greater than 85%. They explored the potential of three different methods for the detection and quantification of a variety of EDCs in aqueous samples. in urine. In one example. 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. the rapid and intense CL reactions require the adjustment of a reaction coil used between the mixing device and the detector.D. Using MEKC. and pH. r > 0. The study included calibration curves for each analyte with acceptable linear correlations and RSDs for each. 3. -E2.3 g L−1 . due to the long equilibration times needed for ED. -E2.A.1 g L−1 . The CE methods explored by Reagan et al. inhibiting detection. as well as various estrogens [55]. The length and diameter are variables that must be adjusted and optimized in order to allow the maximum CL emission to be observed. however the study bears mention because it reported g L−1 limits of quantitation for the analytes using large-volume sample stacking in-line concentration with photo-diode array UV detection .3.2. CE enables separation of various analytes in one run according to their molecular size. Electrochemical detection of BPA was found to be much more sensitive (0. In addition. and BPA. Compounds containing phenolic moieties exhibit CL response from direct oxidation with acidic potassium permanganate. a polymerized film.5 pg detection limit) than HPLC with FL or UV detection [34]. The detection limits for the analytes ranged from 2. 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 mL−1 range (1. used a modified glassy carbon electrode (GCE) at +1.24 A. The LOD for this method was about 0. The UV detector had to be used instead of the ED for the later-eluting compounds. however there has been little recent activity using this detection technique. While delivering these advantages. which were affected by the baseline drift of the increased acetonitrile composition of the gradient. however. in order to improve the detection and baseline. The limits of detection using signal to noise ratio of 3 were reported to be ng mL−1 range for BPA and EE2. therefore. as well as derivatized with dansyl chloride prior to analysis. Micellar electrokinetic capillary methods involve a modification of the buffer using a surfactant at concentrations (termed “critical concentration”) at which micelles form.5–82. Even with those weaknesses. does not include any of the EDCs mentioned here. This film collects on the surface of the electrode. In addition. the phenols had to be extracted from the aqueous matrix (in this case. Electrochemical detectors (ED) offer high sensitivity and widespread applicability based upon the oxidation or reduction of organic functional groups. Chemiluminescence (CL). K. ED was not optimal for all of the EDCs in this study. A novel approach using chemiluminescence (CL) detection with HPLC has been published as a simple. simpler ways to analyze EDCs from aqueous samples.1 ng mL−1 ) [83]. fairly rapid analysis (average of 3–20 min).0 V to quantify estrogenic phenols in river water by amperometry [81].3. Phenolic groups are electroactive and easily detected by HPLC-ED. Another chemiluminescence detection method included in GamizGracia et al.. was treated with SPE. urine) using SPE. 3. The oxidation of NP nearly completely transforms it to one of its oxidation products.2. reaction temperature. among others [82]. NP. isocratic elution is recommended over gradient methods. Electrochemical detection (ED). a CE-CL method was able to separate fifteen kinds of phenolic compounds using a running buffer solution of SDS and acetonitrile. Schug / Analytica Chimica Acta 696 (2011) 6–26 fluorescence after derivatization with p-nitrobenzoyl chloride has been reported [46]. LaFleur. and the relative standard deviations and correlation coefficients were also satisfactory (%RSD < 5. 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]. The study conducted by Zhang et al. a SPME-HPLC method was reported using a gradient with UV and ED. 3. and E3. ED sensitivity is influenced by both the pH and electrolyte concentration of the mobile phase. were CZE and micellar electrokinetic chromatography (MEKC) using photo-diode array detection from 190 to 300 nm [87].3. the detection limit of this method for estradiol was 8 pg and in plasma it was determined to be 24 pg [85]. such as the composition of mobile phase.2. These compounds are mostly used as flame retardants and are. they did not publish any projected detection limits of their optimized method. they were able to separate 19 target analytes with some variation in migration time. The method was applied to the determination of three endogenous estrogens. 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 [86–90]. This technique permits the separation of lower molecular weight aromatic phenols. There are a multitude of variables that must be carefully optimized in a system utilizing CL. and isoelectric points (differences in electrical field-induced migration properties of the analytes and the run buffer) [60]. low cost. One study. In a study conducted in 2007.999) with low relative standard deviations were reported (<4% for n = 10 samples). Good correlation coefficients (>0. as well as -EE2. Of the phenols examined. Interestingly. CL also delivers the drawback of requiring additional pumps and rapid mixing needs for post-column CL reagent addition. The limitations of CE applied to trace determination of EDCs involve sensitivity.’s review was able to detect bisphenol A leached into hot water from a baby bottle at a detection limit of 0. cited by Gamiz-Gracia et al. CE typically has low sample volume requirements. and the ability to analyze a large number of samples quickly.7 to 8. The analyte recoveries were rather low (67–92% with chlorophenols having the highest recoveries) for the six phenolic compound curves. In this particular study. Six were determined to be linear over three orders of magnitude with detection limits approaching 10−7 M. the detection limits were defined as a signal-to-noise ratio of 2 (the LOD is traditionally defined as a signal-to-noise ratio of 3). selective and sensitive method of detection for various EDCs. however.3. n = 5). and sample ionic strength. The sample was minimally processed but.999. Chemiluminescence might offer better detection limits than UV as well as cheaper.3–3. the compound 4nonylphenol behaved differently by voltammetric detection than BPA. Another chemiluminescence application using HPLC was used for the determination of estradiol in very small volumes of rat plasma.

Conclusion Much emphasis has been placed on the detection of endocrine disrupting compounds in aqueous sample matrices. Schug / Analytica Chimica Acta 696 (2011) 6–26 25 (LVSEP-CE-DAD). and xenoestrogens mentioned in this review. These findings are driving the detection limits of the analytical methods routinely used to monitor these endocrine disruptors lower than they have ever been before. Toxicol. aqueous free solution capillary electrophoresis (FSCE) was used to separate derivatized bisphenol A ethoxylate dimethacrylates (Bis-EMA). 4.2 to 1. minimizing overall environmental impact and cost. Acknowledgements The authors would like to thank Kayunta Johnson-Winters and Deborah Rowan for proof-reading. The reproducibility for this method was slightly elevated (RSD 10%) and might offer an area for future improvement. or costly. which could be directly injected into the CE system [89]. [4] S Choi. as well as plastics used for packaging or medical devices that can leach into aqueous environments. Streets. HPLC–MS using ESI or APCI appears to be approaching GC–MS sensitivities without the requirement of derivatization. the CE does show some promise for the determination of phenolic EDCs in aqueous systems when levels are expected to be in the g L−1 range. Comhaire. The attention EDCs are receiving in the media will certainly spur more research. This would be useful for monitoring samples that were expected to contain varying amounts of this compound. B. Opportunities to improve chromatography (separation. Comparisons of sample preparation revealed that much use was made of SPE and related extractions. in some instances less than 1 ng L−1 . Minnesota Pollution Control Agency. [18] developed a GC–MS method using stir-bar sorptive extraction with simultaneous derivitization that was capable of detecting select estrogens. This type of progress offers benefits to the research. F. Dhooge. GC–MS remains the most useful and sensitive method for the detection of trace levels of volatile EDCs and their volatile derivatives. such as. and other related legislation. such as hormones. was able to separate and detect select estrogens. toxicology. pharmaceutical science. with very little sample workup and the method has the capability for high sample throughput. smaller volumes of sample were required in many recent methods. Toxicol. The identification of analytical techniques for trace-level EDC analysis will help to inform policy and support other scientific fields of investigation. Tan et al. and thoughtful suggestions during the preparation of this manuscript. 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]. or mixtures of EDCs. however. Methods that meet these performance criteria are desirable: such as those using simple equipment that is easy and inexpensive to obtain and maintain. Only a few of the published research methods met these more selective criteria. The identification and quantification 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 analysis of wastewater samples by this technique began with SPE. accurate. and potentially more regulation of water quality. In Vitro 17 (2003) 515–524. 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. [3] F. Implantable drug delivery devices that deliver active compounds. References [1] R. [2] S. acceptable levels of leachable EDCs from plastics. The need for selective. S. package engineering. MS offers a wide range of selectivity as well as the sensitivity to detect many of the estrogens. detection limits) exist through the new column dimensions and chemistries available in the market today. More recently. LaFleur. and used methanol as an eluent. to demonstrate a wide detection range for BPA (nM to mM range) [90]. The research tested a modification using a twisted serpentine microchannel configuration 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]. over the more common and accepted LLE. in at least one instance. and robust methods will continue to grow. In a modern industrial setting. This trend reduces the amounts of toxic solvents that are required for sample treatment. 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. The scientific community is learning more about how these compounds affect the endocrine systems of a variety of animals. This technique has been used to monitor the release of growth hormone from various co-polymeric devices made of vinylpyrrolidone-hydroxyethyl methacrylate. has demonstrated promise using online SPE coupled with HPLC–MS/MS [68]. and we would like to highlight two of them. CE with amperomeric detection was used by Ha et al. Crit. from fish to mammals.D. The second method. These methods may offer appealing options to analytical laboratories wishing to avoid complex and lengthy sample work-ups. are the current targets of such research [88]. and phthalates from 0. 1998. environmental waters. J. which are appealing when sample availability or storage space is limited. Yoo. that have been found to impact the endocrine system appear surprisingly low. Endocrine Disrupting Compounds: A Report to the Minnesota Legislature. Golden. The concentrations of a single EDC. Stuyvaert. 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. 35 (2005) 435–458.A. phenols. . J. foods or medicines for human consumption. we found the majority of them involve mass spectrometry detection. efficiency. however. A vast amount of research has been conducted in this field and a number of elegant methods have been proposed for analysis of EDCs. Vollmer. 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. such as biochemistry. Health B (2004) 1–32. This system is simple. G. In addition. such as SPME. and phthalates at the ng L−1 level. S. Toxicol. as well as the financial bottom line.6 ng L−1 using UV detection [78]. Environ. such as charged aerosol detection (CAD) or TOF-. estrogen conjugates. and polymer science. In light of this study. systems that offer the flexibility and sensitivity to be used for other types of analyses. 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. Other areas available for exploration and improvements involve the use of the newer types of detectors. W. the focus is to make the complicated simple by finding the most efficient path to the end goal. When comparing the methods reported in Tables 3 and 4. an HPLC method. and methods that have the capability of being fully automated with high sample throughput. as well as the higher pressure rapid resolution HPLC and UHPLC systems that exist. beverages. Rev. Also in 2005. It may be possible to fully automate the sample preparation and analysis for this method. Gandy.A. While this novel approach is not yet commercially available. Lee. Eertmans. sensitive. K. alkyl phenolss. Automation is also highly desirable and. food science. professional opinions.

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