Anal. Chem. 2002, 74, 2719-2742
Environmental Mass Spectrometry: Emerging Contaminants and Current Issues Susan D. Richardson
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30605 Review Contents General Reviews Contaminant Candidate List Endocrine Disrupting Chemicals and Pharmaceuticals Chiral Contaminants Polybrominated Diphenyl Ethers Algal Toxins Drinking Water Disinfection Byproducts (Including NDMA) Methyl tert-Butyl Ether Organotins Perchlorate Arsenic Natural Organic Matter Microorganisms Miscellaneous Environmental Applications and New Technologies Literature Cited
Table 1. List of Acronyms 2721 2722 2723 2727 2728 2729 2729 2731 2732 2733 2733 2735 2736 2737 2739
This review covers developments in environmental mass spectrometry over the period of 2000-2001. A few significant references that appeared between January and February 2002 are also included. The previous Environmental Mass Spectrometry review was very comprehensive, including more than 500 references. This year, however, Analytical Chemistry has changed its approach to include only 100-200 significant references and to mainly focus on trends in analytical methods. As a result, this year the review will limit its focus to new, emerging contaminants and environmental issues that are driving most of the current research. Even with a more narrow focus, only a small fraction of the quality research publications could be discussed. Thus, this review will not be comprehensive, but will highlight new areas and discuss representative papers in the areas of focus. Numerous abstracts were consulted before choosing the best ones to present here. Abstract searches were carried out using the Web of Science. A table of acronyms is provided (Table 1) as a quick reference to the acronyms of analytical techniques and other terms discussed in this review. A table of useful websites is also provided (Table 2). The overall trends in analytical methods for environmental analysis include a greater use of solid-phase microextraction (SPME); increased use of fast-gas chromatography/mass spectrometry (GC/MS); increased use of chiral separations (usually with chiral GC columns or using capillary electrophoresis (CE)); and more on-line coupling of extraction and separation with detection, such as solid-phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS) or GC/MS and ion chromatography (IC) coupled to inductively coupled plasma mass spectrometry (IC-ICPMS). The use of matrix-assisted laser de10.1021/ac020211h Not subject to U.S. Copyright. Publ. 2002 Am. Chem. Soc.
Published on Web 05/03/2002
AAS AES AOC APCI API ASE CCL CDC CE CID CLSA DBPs DNPH ECD ECNI EDCs EI ELISA EPA ESI FAIMS FT GAC GC GPC HAAs HG IC ICP ICR IPC IR LC MALDI MCL MIMS MIP MS MTBE MX NCI NDMA NMR NOM PAA PCBs PCR PBDEs PFBHA RSD SPE SPME THMs TOF TOX
atomic absorption spectrometry atomic emission spectrometry assimilable organic carbon atmospheric pressure chemical ionization atmospheric pressure ionization accelerated solvent extraction Contaminant Candidate List Centers for Disease Control and Prevention capillary electrophoresis collisionally induced dissociation closed-loop stripping analysis disinfection byproducts 2,4-dinitrophenylhydrazine electron capture detection electron capture negative ionization endocrine disrupting chemicals electron ionization enzyme-linked immunosorbent assay Environmental Protection Agency electrospray ionization high-field asymmetric waveform ion mobility spectrometry Fourier transform granular activated carbon gas chromatography gel permeation chromatography haloacetic acids hydride generation ion chromatography inductively coupled plasma ion cyclotron resonance ion pair chromatography infrared spectroscopy liquid chromatography matrix-assisted laser desorption/ionization maximum contaminant level membrane introduction mass spectrometry microwave-induced plasma mass spectrometry methyl tert-butyl ether 3-chloro-(4-dichloromethyl)-5-hydroxy2(5H)-furanone negative chemical ionization nitrosodimethylamine nuclear magnetic resonance natural organic matter peracetic acid polychlorinated biphenyls polymerase chain reaction polybrominated diphenyl ethers pentafluorobenzylhydroxylamine relative standard deviation solid-phase extraction solid-phase microextraction trihalomethanes time of flight total organic halide
sorption/ionization (MALDI)-MS and electrospray ionization (ESI)-MS has also increased for the analysis of microorganisms. Research in this area is beginning to go beyond simple fingerprinting and empirical matching of MALDI or ESI mass spectra Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 2719
Table 2. Useful Websites website
comments
http://www.epa.gov http://www.epa.gov/ogwdw/methods/methods.html http://www.gpo.gov/su•docs/aces/aces140.html http://www.epa.gov/ogwdw/ccl/cclfs.html http://www.chbr.noaa.gov/CoastalResearch http://www.dhs.ca.gov/ps/ddwem/chemicals/NDMA/NDMAindex.htm http://ehis.niehs.nih.gov/roc/ninth/rahc/nnitrosodimethylamine.pdf http://www.epa.gov/safewater/arsenic.html http://www.epa.gov/ncerqa/grants http://www.epa.gov/scipoly/oscpendo/overview.htm http://www.psr.org/tedfs.htm
to the organisms; papers are reporting increased development of modeling and algorithms for improving identifications, complete sequencing of protein biomarkers, and techniques to explore the structure and function of these microorganisms. MALDI- and ESIMS are also being used to probe the structures of high molecular weight natural organic matter (i.e., humic materials). Previously, mass spectral analysis of humic material was only possible through the use of chemical and thermal degradative techniques, such as pyrolysis-GC/MS, which did not permit the analysis of the original, intact molecule. The availability of MALDI- and ESI-MS, along with the use of high-resolution Fourier transform (FT)-ion cyclotron resonance (ICR)-MS and MS/MS, is allowing the analysis of intact humic materials for the first time by mass spectrometry. A few new analytical techniques have also been developed during the last 2 years, including the development of a singlesided membrane introduction mass spectrometry (MIMS) technique that allows the on-line determination of semivolatile organics in air (see discussion under the Miscellaneous Environmental Applications and New Technologies section) and the use of collisional cooling with MALDI-MS to permit the probing of viral structures (see Microorganisms section). The formation of stable association complexes to allow the ESI-MS detection of contaminants is also a relatively new development worthy of noting. Of all the organic environmental contaminants, pesticides continue to be studied the most. However, current research is focusing more on those pesticides that are considered to be endocrine disrupting, on pesticide degradation products, and on occurrence/degradation of chiral isomers. Alachlor (and other acetanilide pesticides), triazine, and their degradation products can be found on the current Contaminant Candidate List (CCL), a list of unregulated contaminants that are to be monitored in drinking water systems and considered for future regulation (based on their occurrence and health effects). Chiral chromatography (using either chiral GC columns or CE with mass spectrometry) is being used to study the occurrence and environmental fate of pesticides that are chiral. Typically, one pesticide enantiomer is the active one, and the other is inactive. In addition, one pesticide enantiomer is typically degraded differently in the environment (their fate is not the same). Therefore, with the manufacture and use of pesticides containing racemic mixtures, there was the potential for one form of the pesticide to accumulate in the environment and cause unintended effects on nontarget 2720 Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
U.S. EPA’s website; provides a searchable link to U.S. EPA regulations and methods link to the U.S. EPA’s Office of Ground Water and Drinking Water’s analytical methods direct link to the Federal Register link to the Contaminant Candidate List (CCL) link to NOAA’s website for algal toxin information link to California Department of Health Services site for NDMA information NTP report (2000) on NDMA link to the U.S. EPA’s website for arsenic link to the U.S. EPA’s STAR Grants solicitations link to the U.S. EPA’s EDC screening program useful website for EDC information
species. Because earlier fate research studied racemic mixtures, there was also the potential for an incorrect assessment of the pesticide’s half-life in the environment; i.e., the rate of degradation may give the impression that the pesticide would completely degrade, when only one form may be degrading. It is also interesting that the ability to separate pesticide enantiomers has also led pesticide manufacturers to offer a particular enriched chiral isomer commercially. Thus, there is expected to be increased use of single chiral forms of pesticides. Endocrine disrupting chemicals (EDCs) are also an important issue. Although EDCs can hardly be considered an “emerging” issue (there has been concern about EDCs since the early 1990s), most EDC research has been conducted only in the last 5 years, and the last 2 years have seen substantial growth. As time goes on, more chemicals are being discovered to be endocrine disrupting. In the United States, the Food Quality Protection Act and the Safe Drinking Water Act Amendments (published in 1996) helped to promote new research on EDCs. These two legislative acts require that the U.S. Environmental Protection Agency (EPA) develop a screening and testing strategy for estrogenic substances and other EDCs. Publication of the book Our Stolen Future in 1996 also helped to publicize this area of concern, much as Rachel Carson’s book, Silent Spring, helped to launch the beginnings of the environmental movement in the 1960s. One area of very recent interest related to this area is the study of pharmaceuticals in water. In addition to concern about potential estrogenic effects to wildlife, there is also concern about potential estrogenic effects in humans, through the introduction of pharmaceuticals into drinking water sources. Due to improved analytical methods (typically LC/MS) that can measure highly polar pharmaceuticals at the low levels required, there has been an explosion of research in this area, with researchers not only measuring their occurrence in waters but also studying their fate in wastewater treatment plants. Several studies are, in fact, showing that there has been incomplete removal at wastewater treatment plants, and therefore, there is the possibility that pharmaceuticals could enter source waters for drinking water. As a result, various pharmaceuticals are currently being considered as possible future CCL drinking water contaminants for further monitoring. The discovery of nitrosodimethylamine (NDMA) as a disinfection byproduct (DBP) in drinking water treatment (and also as a contaminant) has received much interest due to its known cancer potency. Other recently identified DBPs are also receiving
attention. Lower detection limits, improved analytical instrumentation and methods, and new derivatization procedures are allowing significant advances in an area that has active for almost 30 years. Organotins are receiving renewed attention partly because of a new study showing that they can leach out of poly(vinyl chloride) (PVC) pipe into drinking water at continuous ppb levels. Organotins were previously only thought to be an environmental water contaminant, mainly from the use of organotins as antifouling paints on ships. New research is indicating that there is a potential threat of human exposure through drinking water. Although not considered as great a toxicological risk, methyl tert-butyl ether (MTBE) is also still receiving significant study, due to its impact on groundwater sources (and entry into drinking water) from leaking underground gasoline storage tanks. Perchlorate contamination in groundwater has also recently been shown to be significant, and its use in some fertilizers has been a recent concern. Arsenic research has also increased exponentially, with the development of improved analytical methods permitting the study of specific species of arsenic in water, foods, and biological samples (including human urine). The ability of mass spectrometry to measure polar, higher molecular weight compounds has also permitted the analysis of algal toxins in environmental samples. Many algal toxins are peptide related; e.g., microcystins are cyclic peptides produced by blue green algae. Algal toxins have been responsible for large fish kills, poisoning of shellfish, other animal deaths, and illness in people, and they are being considered as possible future CCL candidates. A final group of emerging contaminants included in this review are polybrominated diphenyl ethers (PBDEs). These compounds are widely used as flame retardants in furniture (particularly in the foam cushions used in chairs), textiles, plastics, paints, and electronic appliances. PBDEs are environmentally persistent, having been found in human milk, human blood, birds, fish, marine mammals, air, and sediments, and there is concern about potential adverse developmental effects from exposure to PBDEs. The majority of PBDE studies have been from Europe, which has a Directive to control emissions of these compounds. Although there were a few early measurements of PBDEs in the United States in the late 1970s and early 1980s, they have not been regulated in the United States. However, interest appears to be increasing in the United States. GENERAL REVIEWS In addition to a focus on emerging contaminants, general reviews are also included. The previous Environmental Mass Spectrometry review published in 2000 contained 532 references and covered developments in analytical methods and research for air and particulate matter, water (drinking water, surface water, groundwater, seawater, and wastewater), soils and sediment, natural organic matter, biological samples, foods, microorganisms, and also the development of field-portable/mobile mass spectrometers (1). Budde, a leader in environmental mass spectrometry for many years, also published a book, Analytical Mass Spectrometry: Strategies for Environmental and Related Applications (2). This book begins with a wonderful historical overview of environmental legislation and environmental and technical developments that contributed to the widespread use of mass spectrometry. The development of the “Priority Pollutant List” is
discussed, as are the development of EPA Methods. Later in the book, analytical strategies are discussed for the quantification of target analytes and the identification of unknowns, with detailed descriptions of GC/MS, LC/MS, exact mass measurements, and other mass spectrometry techniques, as well as strategies for enhancing analyte selectivity and lowering detection limits. It is impossible to do this book justice in the small space allotted here. Suffice it to say that this book is an excellent reference for the practicing mass spectrometrist (not just for those involved in environmental measurements) and also for students and others who want to learn about quantitative analysis and other techniques involving mass spectrometry. Niessen edited a book on the Current Practice of Gas Chromatography/Mass Spectrometry (3), which provides a perspective on how GC/MS is used by researchers in a wide variety of different applications, including environmental applications. A review article published in a thematic issue on Mass Spectrometry in Chemical Reviews chronicled the use of mass spectrometry in environmental research (4). This comprehensive review included more than 1100 references and discussed the early, historical use of mass spectrometry in the mid-1970s and advances made over the next 30 years, focusing mostly on significant analytical methods and studies that occurred over the last 8-10 years. The analysis of a wide variety of analytes (both organic and inorganic and including microorganisms) is discussed. Clement et al. published their biennial review on Environmental Analysis, which included 636 references and covered environmental review articles, applications involving SPME, air monitoring, and analysis, water analysis, solid sample analysis, biological sample analysis, radionuclides, quality assurance topics, and biomarkers (5). In Richardson’s 2001 review on Water Analysis, trends in analytical methods for water analysis were detailed (which included drinking water DBPs, pesticides, EDCs, pharmaceuticals, surfactants, textile dyes, microorganisms, algal toxins, natural organic matter, and other organic and inorganic pollutants), as well as discussions about new regulatory methods and new regulations (6). Noble and Prather reviewed the use of real-time single-particle mass spectrometry for the analysis of aerosols (7). This review details the historical development of the technique from 1973 through 1998, with discussions of the recent use of time-of-flight (TOF) mass spectrometers for real-time analyses. Reemtsma reviewed the use of LC-atmospheric pressure ionization (API)MS for water analysis (8, 9). In part I of this review (8), the achievements of LC/MS for advancing the types of water constituents that can be studied are detailed, as well as the use of separation techniques, such as LC, IC, CE, and size exclusion chromatography. In part II, obstacles for LC/MS are discussed, including the difficulty in identifying unknown compounds and the difficulty in quantitation of target analytes in complex samples (9). Suggestions are offered to some of these problems through the use of tandem mass spectrometry, TOF-MS, and improved chromatographic separations or sample cleanup procedures. Bacon et al. published annual reviews of Atomic Mass Spectrometry, covering developments from 1999 to 2000 (10) and 2000 to 2001 (11). The growing interest in speciation studies is discussed, as well as the renaissance of GC/MS for metal speciation analysis. Cave et al. also published an annual review on Atomic Spectrometry (for 2000-2001), with a focus on Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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environmental analysis (12). This review includes 677 references and concludes that last year’s developments in atomic spectrometry have focused mainly on the improvement of existing techniques to produce more reliable and robust methods. Baude et al. published a review of glow discharge atomic spectrometry for the analysis of environmental samples (13), and Seubert discusses the on-line coupling of IC with ICP-atomic emission spectrometry (AES) and ICPMS (14). Johnson et al. detailed the trends and applications of MIMS (15). In this review, new semipermeable membranes are discussed, including recently developed membranes that enable the analysis of polar compounds. Limitations of this technique are discussed, along with potential solutions for overcoming some of these. Reviews also focused on specific analytes/contaminant groupss for example, pesticides (16, 17), surfactants (18, 19), agrochemicals (20), and organic aerosols (21). Sherma provided a review of pesticide analysis over the period of 1999-2000sincluding new methods and studies of exposure, monitoring, pesticide degradation, persistence, leaching, mobility, and metabolism (16). Hogendoorn and van Zoonen discussed recent and future developments of LC and LC/MS for pesticide analysis (17). Included in this review are recent major developments in LC column packing materials, including immunoaffinity sorbents, restricted access materials, and molecularly imprinted polymers. Morelli and Szajer published a two-part review of surfactant analysis that covered developments from 1995 to 1998 (18, 19). Menzinger et al. reviewed the analysis of agrochemicals (mainly pesticides) using CE and CE/MS (20). This review covered the literature from 1990 to 2000 and includes a discussion of preconcentration and separation methods. Finally, Jacobson et al. reviewed the state of the science of organic atmospheric aerosols (21). Due to the complexities in obtaining complete chemical information on aerosols, this review mainly focused on presenting a basis for defining what data are needed. An overview of the major environmental issues involving organic aerosols is presented, followed by a description of the distribution, sources, and chemical and physical properties that are known. Analytical methods that are used to study aerosols are presented (which include mass spectrometry), and important unanswered scientific questions and suggestions for future research priorities are discussed. CONTAMINANT CANDIDATE LIST The Contaminant Candidate List is a recent development that is associated with a new regulatory rule, called the Unregulated Contaminants Monitoring Rule (22). This Rule resulted from the Safe Drinking Water Act Amendments of 1996, which requires the U.S. EPA to establish a monitoring program for unregulated contaminants and to publish a list of contaminants to be monitored. Specifically, the U.S. EPA must publish a Contaminant Candidate List every five years to identify potential substances for future regulation. Monitoring data will be collected from drinking water utilities to determine whether a contaminant occurs at a frequency and in concentrations to warrant further analysis and research on potential health effects and possible regulation. From the CCL, a minimum of five candidates must be selected to be considered for regulation within a five-year period. The first CCL was published in March 1998 and contains both chemical and microbial contaminants. Included are many pesticides (such as triazine and 2722 Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
Table 3. CCL Chemicals 1,1,2,2-tetrachloroethane 1,2,4-trimethylbenzene 1,1-dichloroethane 1,1-dichloropropene 1,2-diphenylhydrazine 1,3-dichloropropane 1,3-dichloropropene 2,4,6-trichlorophenol 2,2-dichloropropane 2,4-dichlorophenol 2,4-dinitrophenol 2,4-dinitrotoluene 2,6-dinitrotoluene 2-methyl-phenol (o-cresol) acetochlor alachlor ESA and other acetanilide pesticide degradation products aldrin aluminum boron bromobenzene DCPA monoacid degradate DCPA diacid degradate DDE diazinon dieldrin disulfoton diuron EPTC (s-ethyl-dipropylthiocarbamate) fonofos hexachlorobutadiene p-isopropyltoluene (p-cymene) linuron manganese methyl bromide methyl tert-butyl ether (MTBE) metolachlor metribuzin molinate naphthalene nitrobenzene organotins perchlorate prometon RDX sodium sulfate terbacil terbufos triazines and their degradation products (including, but not limited to cyanazine and atrazine-desethyl) vanadium
its degradation products), volatile contaminants (such as tetrachloroethane), metals (such as aluminum, boron, manganese, and vanadium), chemical warfare agents (such as RDX), and other chemical contaminants, such as organotins, perchlorate, methyl bromide, and MTBE (a complete list of chemical contaminants is provided in Table 3). Included among the microbial contaminants are Acanthamoeba, adenoviruses, Aeromonas hydrophila, caliciviruses, coxsackieviruses, cyanobacteria (blue-green algae), other freshwater algae and their toxins, echoviruses, Helicobacter pylori, microsporidia, and Mycobacterium avium intracellulare. The complete CCL list, along with identified research priorities, can be found at http://www.epa.gov/ogwdw/ccl/cclfs.html. As a result of establishing the first CCL and identifying research needs regarding these contaminants, significant efforts have begun with regard to methods development, occurrence, health effects, and treatment research. Funding for much of this initial CCL research was provided through external grants offered
by the U.S. EPA (STAR Grants), and this funding is expected to continue. An example of one of the areas of interest last year involved the neurotoxicity of aluminum, where research proposals were solicited to determine factors that influence the occurrence of various aluminum complexes, the relative influence of such complexes on the distribution of aluminum in the body, and the dose-dependent contribution of such complexes to neurotoxic effects. The STAR Grants program has four solicitation periods during the year (January, April, August, October); announcements can be found at http://es.epa.gov/ncerqa/grants/. Because the CCL is a relatively new regulatory development, there has not been time for the completion of new projects or for a large number of publications to appear in print. The articles that have appeared include ones discussed below. The main purpose for introducing this emerging environmental issue is to inform researchers of this large effort and make them aware of research needs and the potential for new research. An overview of the drinking water standards program in the United States, which included a discussion of the CCL, can be found in an article published by Brass in 2000 (23). Published research efforts on CCL analytes/issues include new methods development work and studies on fate and transport of specific CCL pesticides. Winslow et al. discussed considerations necessary for collecting occurrence data for particular CCL analytes (24). Two technical hurdles discussed involved the preservation of target analytes and the selection of a suitable solidphase extraction material. CCL analytes that were studied included diazinon, disulfoton, fonofos, terbufos, prometon, 1,2-diphenylhydrazine, nitrobenzene, acetochlor, 2,4,6-trichlorophenol, 2,4dichlorophenol, and cyanazine (a potential degradation product of triazine herbicides). From this study, an analytical method was developed, which involved the use of a chelating agent to bind metals that can cause hydrolysis, a pH 7 buffer to minimize hydrolysis, the use of ascorbic acid to quench residual chlorine in the drinking water (and prevent degradation of many of the analytes), the use of styrene divinylbenzene as the solid-phase material for extraction, and analysis by GC/MS. In another CCL study, Magnuson et al. determined that a triazine degradation product (deisopropylatrazine) can interfere with the GC/ion trap-MS analysis of deethylatrazine (DEA, another atrazine degradation product) and outlined a procedure to overcome this (25). Panshin et al. developed a new method for analyzing atrazine and four of its degradation products using solid-phase extraction on a graphitized carbon black cartridge, derivatization with methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and analysis by GC/MS (26). This method was used to study atrazine and its degradation products in pore water in Indiana. Following application of atrazine onto a field, results showed that atrazine and its degradation products were transported rapidly through the vadose zone and that the degradation products were more persistent than atrazine in the pore water. Maximum concentrations of atrazine occurred 1557 days after application, and maximum concentrations of the degradation products occurred 11-140 days after atrazine application. A new method for determining cyanuric acid (a potential degradation product of triazine herbicides) was developed by Magnuson et al. (27). This method involved the extraction of
cyanuric acid by a microscale liquid-liquid extraction, evaporation to dryness, addition of an aqueous solution of a quaternary ammonium cationic surfactant, and analysis by ESI-MS. The surfactant and cyanuric acid form a stable association complex, which enables the quantification of cyanuric acid. Aelion and Mathur studied the biodegradation of atrazine to deisopropylatrazine and deethylatrazine in various coastal sediments with different land uses (28). Using GC/MS to quantify the analytes, it was found that less degradation was observed in golf course sediments, with a greater degradation occurring on undeveloped land. Finally, Acero et al. studied the degradation kinetics of atrazine and its degradation products with ozone and OH radicals in order to predict their behavior in drinking water treatment (with ozonation and advanced oxidation processes) (29). Besides dealkylated and amide degradation products, two new degradation products with an imine group were identified. Results also showed that the ethyl group was more reactive than the isopropyl group and that the acetamido and imino groups were resistant to chemical oxidation. ENDOCRINE DISRUPTING CHEMICALS AND PHARMACEUTICALS EDCs are not a new issue, necessarily, but have recently become a major area of concern worldwide. In addition to the U.S. Food Protection Act and the Safe Drinking Water Act Amendments, there is a European Union Directive (2001) that addresses many of the EDCs as priority substances. The endocrine system is an intricate system of hormones that regulate development, growth, reproduction, and behavior. Certain synthetic and natural chemicals have the ability to mimic these hormones and, thus, can interfere or disrupt the normal function. EDCs can alter hormonal function by binding to hormone receptors directly or by indirectly relaying molecular messages through a complex array of cellular proteins that activate genes and alter cell growth and division. In wildlife, EDCs are suspected of being responsible for the decline in certain species (e.g., possible increased sterility with the American alligator), change of sex in fish and shellfish, and other problems. EDCs are also suspected in declining sperm counts in humans, although this has not been proven. Both natural estrogens and anthropogenic EDCs can reach the aquatic environment through wastewater discharges. Fish and wildlife can be exposed, and humans can become exposed through the intake of this water into drinking water treatment plants. Chemicals that have been determined to be estrogenic include synthetic estrogens (such as commonly used birth control pills), steroids, pesticides, phthalates, alkylphenol ethoxylate surfactants, dioxins, polychlorinated biphenyls (PCBs), and natural estrogens, such as phytoestrogens, that are found in many plants, including soybeans, wheat, rice, carrots, beans, potatoes, cherries, and apples. Trussell published a nice review in 2001 on EDCs in the Journal of the American Water Works Association, where he explains in detail the endocrine system and specifically how EDCs interfere with natural receptors (30). Trussell also makes recommendations on how the water industry should actively respond to this issue. New Analytical Methods for EDCs. A variety of different analytical mass spectrometry methods are used to measure EDCs, including GC/MS, LC/MS, CE/MS, and MS/MS techniques. Petrovic et al. published an overview of analytical methods for Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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EDCs in freshwater sediments (31). EDCs discussed include five groups of compounds considered as priorities within the European Union and the United States: alkylphenols, polychlorinated compounds (dioxins, furans, biphenyls), polybrominated diphenyl ethers, phthalates, and steroid sex hormones. Castillo and Barcelo used toxicity-based fractionation methods with LC/MS and GC/ MS to characterize several organic pollutants (including phthalates, alkylphenol ethoxylates, and other EDCs) in textile wastewaters and landfill leachates (32). Most of the polar organic pollutants were identified using a sequential solid-phase extraction protocol followed by LC/ESI-MS and ion pair chromatography (IPC)-ESI-MS. Ingelse et al. developed a direct injection LC/MS/ MS method for determining organophosphorus pesticides in water, which permitted detection limits of 0.01-0.03 µg/L (33). Takeda et al. published a CE/MS method for analyzing dichlorophenols (one of theses2,4-dichlorophenolsis a suspected EDC) (34). Because of their widespread use and potential for endocrine disruption, alkylphenol ethoxylate surfactants have become a focus of interest in environmental analytical chemistry. Several LC/MS methods have been developed for their measurement in a variety of environmental matrixes, including surface water, drinking water, wastewater, sludges, and sediment. Detection limits are generally quite low, in the nanogram per liter range. Petrovic et al. developed a SPE-LC/MS method for simultaneously measuring halogenated derivatives of alkylphenol ethoxylates and their metabolites in sludges, river sediments, and surface, drinking, and wastewaters (35). Detection limits ranged from 20 to 100 ng/L. Using this method, bromononylphenols and bromononylphenol ethoxylates were among the many halogenated derivatives found in sludges from a Barcelona drinking water treatment plant. This study is particularly important because it provides evidence that alkylphenol ethoxylate surfactants can be halogenated during chlorine disinfection in the presence of bromide ion. An LC/MS method was also developed for determining anionic and nonionic surfactants, their degradation products, and EDCs in sewage sludge (36). Ultrasonic solvent extraction with 7:3 (v/v) methanol/dichloromethane allowed recoveries of 86-100% for polyethoxylates and 84-94% for polar degradation products. Following solid-phase extraction, the less polar analytes were analyzed using LC/atmospheric pressure chemical ionization (APCI)-MS in the positive ion mode, and the more polar analytes were analyzed using ion pair-LC/ESI-MS. In a subsequent analysis of sewage sludge, nonylphenol was found at high levels, ranging from 25 to 600 mg/kg, with polyethoxylates being found at 2-190 mg/kg levels. EDC Measurements in Water and Sediments. Petrovic et al. carried out an occurrence study in Spain for alkylphenol ethoxylates, their degradation products, and linear alkylbenzenesulfonates in coastal waters and sewage sludge (37). As Spain is one of the European countries that still discharges untreated wastewaters and sewage sludge to the sea, many of these compounds were detected in the coastal waters and marine sediments. Two types of alkylphenol ethoxylate degradation products, alcohol ethoxylates and coconut diethanolamides, were reported for the first time. The alcohol ethoxylates were found to accumulate in the bottom sediment. Bester et al. reported the detection of nonylphenols, nonylphenol ethoxylates, linear alkyl2724
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benzenesulfonates, and bis(4-chlorophenyl) sulfone in the German Bight of the North Sea (38). LC/MS/MS was used to confirm their identities; water concentrations were in the low- to midnanogram per liter range. Barber et al. reported results from a study involving the measurement of EDCs in treated municipal wastewater and river water samples taken from Chicago, IL, Minneapolis/St. Paul, MN, Detroit, MI, Milwaukee, WI, and Des Plaines, IL (39). Analytes included alkylphenol ethoxylates, 17β-estradiol, bisphenol A, and caffeine. In this case, GC/MS was used instead of LC/MS; the derivatization of the alkylphenol ethoxylates to their corresponding methyl esters allowed the use of GC/MS. Detection limits were 0.06-35 µg/L. Azevedo et al. used SPE with GC/MS to analyze nonylphenol and bisphenol A in surface waters from Portugal (40). Following preconcentration of a 200-mL water sample, 0.01 and 0.002 µg/L detection limits were achieved. In two of the river samples measured, concentrations of the 4-nonylphenol isomers and bisphenol A were present at sufficient levels (above 10 µg/L and above 2 µg/L, respectively) that could cause estrogenic effects in fish. Degradation products of EDCs were the focus of several studies. Castillo et al. reported the identification of photodegradation products of alkylphenol ethoxylate surfactants using LC/ APCI-MS, as studied under controlled conditions in the laboratory and also in actual wastewater (41). Degradation products included fatty alcohols, fatty acids, and poly(ethylene glycol)s. Photodegradation in wastewater was found to be more efficient than in deionized water for one of the ethoxylates studied, with a half-life of 48 and 29 h, respectively. Maruyama et al. followed the seasonal changes in ethylene oxide chain length of alkylphenol ethoxylate surfactants in rivers in Tokyo using LC/ESI-MS (42). Variations were seen in the ethylene oxide chain lengths at different times of the year, with lower chain length polyethoxylates dominant in the summer when higher temperatures and greater microbial activity were present. Jonkers et al. carried out a laboratory aerobic biodegradation study of nonylphenol ethoxylates in river water using LC/ESI-MS to monitor the kinetics of degradation (43). Results showed a relatively fast primary decomposition of the nonylphenol ethoxylates, with >99% degradation observed after 4 days. Contrary to a generally proposed degradation pathway of ethylene oxide chain shortening, these results show that initiation involved the ω-carboxylation of the individual ethoxylate chains. Further degradation led to short-chain carboxylated ethylene oxides and oxidation of the nonyl chain, resulting in metabolites that have both a carboxylated ethoxylate and an alkyl chain of varying lengths. Fragmentation produced by LC/ESI-MS/MS was used to identify the metabolites. In this study, nonylphenol was not found as a metabolite. Jenkins et al. reported the identification of androstenedione in a Florida river containing paper mill effluent (44). The effluent was causing the female fish (eastern mosquitofish) in the river to masculinize, with the production of elongated anal fins (a typically male-specific trait). To identify the androgenic components in the effluent, river water was collected and fractionated using solid-phase extraction and LC. Androstenedione was identified in the two fractions that induced androgenic activity in the cell culture assays. EDCs in Human and Biological Samples. Research has also been conducted on human exposure to EDCs, which include
measurements of EDCs in human serum, urine, and tissue. Frias et al. developed a sensitive GC/MS/MS method to determine organochlorine pesticide EDCs in human serum (45, 46). This method was used to measure lindane, vinclozolin, aldrin, p,p′-DDT, o,p′-DDT, and p,p′-DDE (45) and PCBs (46) in serum samples from women living in agricultural areas of Almeria, Spain. GC/ MS/MS was found to be superior to GC/electron capture detection (ECD) for these measurements because matrix interferences could be distinguished from responses from the EDC analytes. Researchers from the U.S. Centers for Disease Control and Prevention (CDC) developed a high-throughput and sensitive LC/APCI-MS/MS to quantify phthalate metabolites in human urine (47). Human urine samples were processed using enzymatic deconjugation of the glucuronides, followed by solid-phase extraction and LC/APCI-MS/MS analysis, and quantification using isotope dilution. This method permitted the rapid detection (7.7 min total run time) of eight urinary metabolites of the most commonly used phthalates, with low-nanogram per milliliter detection limits. Precision of the method was optimized by incorporating 13C-labeled internal standards for each of the eight analytes, as well as a conjugated internal standard to monitor deconjugation efficiency. This method is being used to elucidate potential associations between human exposure to phthalates and adverse health effects. A similar LC/MS/MS method also using enzymatic deconjugation and SPE was developed for measuring phytoestrogens in human serum and urine (48). Smeds and Saukko used GC-ECD and GC/MS to measure organochlorine pesticides and PCBs in human adipose tissue (49). The samples consisted of abdominal, mammary, and perirenal fat tissues from 27 men and women in Finland. Lipids were separated from the low molecular weight compounds using preparative gel permeation chromatography (GPC), and the extracts were further cleaned up using Florisil chromatography. Of the 23 pesticides analyzed, only 7 were detected in the samples. All samples contained the DDT metabolite, 4,4′-DDE; hexachlorobenzene, and PCBs. Other compounds identified were 4,4′-DDT, 4,4′-DDD, pentachlorobenzene, and β- and γ-hexachlorocyclohexane (HCH). 4,4′-DDE is antiandrogenic and was the most abundant of the analytes measured, with levels ranging from 3.5 to 3229 ng/g (ppb) lipids. Statistical analyses showed a correlation of DDE levels with age in females and a correlation of hexachlorobenzene with age in males. Sandau et al. reported the identification of a new compound, 4-hydroxyheptachlorostyrene, in polar bear plasma that was found to be a potential EDC (50). This new compound was a major component in the chlorinated phenolic fraction of the plasma and was identified using high-resolution electron ionization (EI)-MS. This compound was hypothesized to be a likely metabolite of octachlorostyrene, and it was shown to have the capacity to bind to circulating proteins, suggesting an endocrine-disrupting mechanism similar to OH PCBs. This study is particularly important because it shows that phenolic metabolites of relatively minor contaminants can possess the capacity to bind circulating proteins, and their significance as potential EDCs may have been underestimated. Pharmaceuticals. Pharmaceuticals have become a major issue in environmental chemistry, due to their presence in environmental waters (following incomplete removal in wastewater
treatment) and concern about possible estrogenic effects, both to wildlife and to humans (through entry of these compounds into source waters used for drinking water). Although environmental concentrations are generally very low (low ng/L), these levels would be sufficient to induce estrogenic responses and cause reproductive and developmental effects in wildlife. Many pharmaceuticals are highly polarswhich necessitates the use of either LC/MS or an efficient derivatization procedure combined with GC/MS for their analysis. Interest in this area is evidenced by reviews that were published in the last 2 years. Ternes reviewed analytical methods for determining pharmaceuticals in aqueous environmental samples (51). Methods include those using SPE, derivatization, detection, and confirmation by GC/MS, GC/MS/ MS, and LC/ESI-MS/MS. A wide variety of pharmaceuticals can be determined in the nanogram per liter range. Ternes discusses the advantages and disadvantages of GC/MS and LC/MS methods. Most of the time, LC/MS methods demonstrated lower relative standard deviations (RSDs) than the derivatization-GC/ MS methods, and they permitted the analysis of extremely polar pharmaceuticals (such as β-blockers) that do not work as well by GC/MS because of incomplete derivatization. LC/ESI-MS methods, however, can suffer from ion suppression when samples (such as sewage samples) are highly contaminated, which necessitates the use of an effective sample cleanup or the use of surrogate standards. De Alda and Barcelo reviewed analytical methods for determining estrogens and progestogens in wastewater and discuss the difficulty in measuring these compounds at nanogram per liter levels in complex matrixes (such as wastewater) (52). Because of these complexities, usually a complicated, timeconsuming extraction and purification process is needed before final determination by various detection methods used (LC, LC/ MS, GC/MS, or immunoassay). This review covers all of the analytical methods used for determining these compounds in wastewater and discusses key procedural stepssfrom sampling to analysissand the techniques most commonly used in those measurements. In 1999, Daughton and Ternes published a review on pharmaceuticals and personal care products in the environment (53). Compounds discussed in this review include prescription drugs and biologics, nutraceuticals, fragrances, sun screen agents, and others. A very recent major article has also appeared just in time to be included in this review. A team of U.S. Geological Survey researchers (Kolpin et al.) has published results from a U.S. Nationwide Reconnaissance Survey of pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams from 1999 to 2000 (54). Although previous research has shown that antibiotics, other prescription drugs, and nonprescription drugs can be present in streams, this study was the first to examine their occurrence in a wide variety of hydrogeologic, climatic, and land-use settings across the United States. Five newly developed analytical methods were used for these measurements, including methods using SPE with LC/ESI-MS and liquid-liquid extraction with GC/MS. Ninety-five organic wastewater contaminants were measured in water samples from a network of 139 streams across 30 states, including sites that are susceptible to contamination (i.e., downstream of intense urbanization and livestock production). The motivation for this study was that there are little data on the occurrence, transport, and fate of many synthetic organic Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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chemicalssparticularly for hormonally active compounds, personal care products, and pharmaceuticals. Target contaminants were present in 80% of the streams sampled and represented a wide range of residential, industrial, and agricultural origins and uses, with 82 of the 95 target compounds found in this study. The most frequently detected compounds were coprostanol (fecal steroid), cholesterol (plant and animal steroid), N,N-diethyltoluamide (insect repellant), caffeine, triclosan (antimicrobial disinfectant), tris(2-chloroethyl)phosphate (fire retardant), and 4-nonylphenol (nonionic detergent metabolite). Measured concentrations were generally low (ng/L-µg/L range) and rarely exceeded drinking water guidelines, health advisories, or aquatic life criteria. There are not guidelines established, however, for many of the target compounds. Several papers described the development and use of LC/MS methods for determining pharmaceuticals and estrogens in environmental samples (55-62). Examples of pharmaceuticals include the following: analgesic drugs, such as ibuprofen, ketoprofen, naproxen, diclofenac, aspirin, and genifibrozil; natural estrogens, such as estradiol, estriol, and estrone; synthetic estrogens, such as ethynylestradiol, mestranol, and diethylstilbestrol; progestogens, such as norethindrone and levonorgesterel; antibiotics, including sulfonamides, macrolids, and penicillins; a natural hormone, progesterone; and others, such as antiphlogistics, psychiatric drugs, tranquilizers, antineoplastic drugs, vasodilators, antiepileptics, antirheumatics, broncholytics, lipid-lowering agents, antidiabetics, and β-blockers. La Farre et al. studied analgesic drugs (common over-the-counter ones) in wastewater using LC/MS and also developed a toxicity protocol by determining the toxicity units for the individual pharmaceuticals and then applying this to the analysis of actual wastewater samples (55). In another study involving the removal of natural and synthetic estrogens by wastewater treatment (activated sludge), Baronti et al. discovered that estriol, estradiol, and estrone could be effectively removed (85-95%), but not ethynylestradiol (61%) (57). Further, concentrations of ethynylestradiol in the effluent from some wastewater plant measurements were actually higher than in the influent to the plant. All four estrogens were also detected in river waters sampled downstream of small towns whose sewage is either treated by percolating filter treatment or was directly discharged to the river. Croley et al. reported the development of three optimized mass spectrometric protocols for the trace determination of steroid hormones in environmental samples; these protocols involved the use of GC/MS/MS, LC/MS with selected ion monitoring, and LC/MS/MS (61). Sacher et al. reported the use of a SPE-LC/ESI-MS method (along with a derivatization-SPE-GC/MS method) for measuring pharmaceuticals in groundwater, as part of a monitoring program in Germany (62). Pharmaceuticals measured included analgesics, antiphlogistics, antirheumatics, β-blockers, broncholytics, lipidlowering agents (and their metabolites), antiepileptics, vasodilators, tranquilizers, antineoplastic drugs, iodinated X-ray contrast media, and different types of antibiotics. Several of these pharmaceuticals were detected in groundwaters during this monitoring effort, and their occurrence could be tracked to an impact of municipal or industrial wastewater. Golet et al. used fluorescence spectroscopy and LC/MS/MS to quantify antibacterial compounds (synthetic antibiotics) used 2726
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in human and veterinary medicine in wastewaters (63). Using this method, nine fluoroquinolones and the quinolone pipemidic acid were quantified in effluents of urban wastewater treatment plants. The two most abundant human-use fluoroquinolonessciprofloxacin and norfloxacinsoccurred in primary and tertiary wastewater effluents at concentrations ranging from 249 to 405 ng/L and 45 to 120 ng/L, respectively. Schiffer et al. developed a method to study the fate of the steroid growth promoters, trenbolone acetate and melengestrol acetate, after application in cattle (64). These steroids were determined in solid cow dung, liquid manure, and soil using SPE-LC, followed by quantitation using a sensitive enzyme immunoassay. Mass spectrometry was used for validation of the method. Results of this study showed that, during storage of the liquid manure, the level of trenbolone decreased from 1700 to 1100 µg/g, corresponding to a half-life of 267 days. Before storage, the concentrations of trenbolone and melengestrol in the dung hill ranged from 5 to 75 and 0.3 to 8 ng/g, respectively; after storage, 10 and 6 ng/g were detected. In soil samples, trenbolone was measurable up to 8 weeks after fertilization, and melengestrol was detected even longer. CE/MS (60, 65) methods have also been developed for measuring pharmaceuticals in environmental samples. In a CE/ MS method developed by Ahrer et al., liquid-liquid extraction was used, followed by SPE and CE/MS analysis of pharmaceuticals in river water samples (60). CE/MS was useful for measuring the pharmaceuticals in the aqueous samples, but the additional sample pretreatment (use of liquid-liquid extraction prior to SPE) was necessary to improve detection limits. By using liquid-liquid extraction prior to SPE, low detection limits between 4.9 and 19 ng/L could be achieved; however, standard addition was recommended due to relatively high standard deviations (16-30%). Using SPE-LC/MS, detection limits between 0.04 and 1.1 ng/L were achievedswithout the additional pretreatment required for CE/MS. One important step taken during the sample workup involved the silanization of all glassware that contacted either the water sample or the extract. This small precaution led to much improved extraction efficiencies for the pharmaceuticals. New SPE-GC/MS methods have also been developed. Sacher et al. reported a method using SPE followed by GC/MS (after derivatization of the acid compounds) (62). Zwiener et al. used SPE with on-line derivatization and GC/ion trap-MS/MS to study the biodegradation of pharmaceutical residues (clofibric acid, ibuprofen, diclofenac) (66). In this study, a pilot sewage plant and biofilm reactors operating under oxic and anoxic conditions were used as model systems with synthetic sewage water containing 10-50 mg/L dissolved organic carbon and pharmaceuticals in concentrations of 10 µg/L. Diclofenac and ibuprofen were partially degraded, but clofibric acid was persistent. Under oxic conditions, a high degree of degradation was found especially for ibuprofen, and two metabolites were identified using mass spectrometry: hydroxyibuprofen and carboxyibuprofen. Moeder et al. developed a SPME-GC/MS method for determining nonylphenols and pharmaceuticals including ibuprofen, paracetamol, phenazone, and carbamazepine in water (67). Polyacrylate and Carbowaxdivinylbenzene SPME coatings were found to be the best for extracting these compounds. Detection limits ranged from 0.2 to 50 µg/L, and low concentrations of organic matter did not affect these limits.
GC/MS methods were also used for other interesting environmental studies of pharmaceuticals (68-73). Spengler used standard addition with GC/MS to quantify several natural and synthetic estrogens including 17-β-estradiol, 17-R-ethynylestradiol, and mestranol; phytoestrogens, genistein and β-sitosterol; and xenoestrogens in effluents of several sewage treatment plants in Germany (68). Except for R-endosulfan and mestranol, all analytes were detected in the majority of samples collected from 18 sewage treatment plants. Median concentrations of steroidal estrogens were between 0.4 and 1.6 ng/L. These results were compared to results from other investigations in Europe and the United States. Hemming et al. examined the toxicity and estrogenicity of wastewater effluent flowing through a constructed wetland system and used GC/MS to measure targeted estrogenic compounds (including 17-β-estradiol, ethynylestradiol, and nonpharmaceutical estrogenic compounds (69). Kuch used GC-negative chemical ionization (NCI)-MS with derivatization (conversion to pentafluorobenzoylate esters) and SPE to determine estrogens (estrone, 17R-estradiol, 17-β-estradiol, 17-R-ethynylestradiol) and other estrogenic compounds (bisphenol A, 4-tert-octylphenol, 4-nonylphenol) in surface and drinking water (70). This method permitted detection limits of 20-200 pg/L, which are quite low compared to other methods (including LC/MS) that are published. In drinking water, bisphenol A was found in concentrations ranging from 300 pg/L to 2 ng/L, 4-nonylphenol from 2 to 15 ng/L, 4-tertoctylphenol from 150 pg/L to 5 ng/L, and steroids from 100 pg/L to 2 ng/L. River water samples contained somewhat higher concentrations of these contaminants. Xiao et al. used a similar derivatization-GC/NCI-MS method to analyze estrogens in water from the Thames River in the U.K.; detection limits were in the 0.2 ng/L range, and estrone was the most abundant estrogen measured (71). Cathum and Sabik developed a pentafluorobenzyl bromide derivatization-GC/MS method to determine steroids (17-β-estradiol, estrone, testosterone) and coprostanol in surface waters, effluents, and mussel tissue (72). Coprostanol was not derivatized in this method, so it was measured in its original form. Method detection limits were 2 ng/L for surface water and effluent and 3 ng/g for mussel tissue. Using this method, 17-β-estradiol, estrone, and coprostanol were detected in surface water in the St. Lawrence River at concentrations ranging from 2 to 67 ng/L; only coprostanol was found in effluent and mussel samples, at concentrations of 14 667 ng/L and 32 252 ng/g, respectively. Niven et al. used a silylation derivatization-GC/MS method to investigate the origins of the estrogenic aromatic-ring steroids in U.K. sewage treatment plant effluents (73). This study was prompted by earlier findings that increased fish feminization could be attributed to the presence of aromatic-ring steroids. One hypothesis was that cholesterol could be converted to an aromatic ring intermediate and then to estrone. But, because this intermediate could only be detected in solid particles associated with the effluents, it was concluded that this hypothesized pathway is probably not a major one. Finally, immunoaffinity techniques were used with GC/MS for determining steroid estrogens. Huang and Sedlak used GC/MS/ MS to confirm a new enzyme-linked immunosorbent assay (ELISA) method for analyzing estrogenic hormones in municipal wastewater effluent and surface water (74). Detection limits were approximately 0.1 ng/L in wastewater and 0.05 ng/L in surface
water. Ferguson et al. used immunoaffinity extraction coupled with LC/ESI-MS to determine the steroid estrogens β-estradiol, estrone, and R-ethynylestradiol in wastewater (75). The use of immunosorbents allowed the removal of interfering wastewater compounds that can cause severe ionization suppression problems during electrospray. The immunoextration also helped to increase the signal-to-noise ratios for the analytes, allowing detection levels of 0.18 and 0.07 ng/L for R-ethynylestradiol and estrone, respectively. In an actual wastewater effluent, recoveries were >90%; concentrations of R-ethynylestradiol ranged from 0.77 to 6.4 ng/L and estrone ranged from 1.6 to 18 ng/L. CHIRAL CONTAMINANTS A major development, particularly in pesticide research, is the use of chiral chromatography (often with mass spectrometry) to analyze individual chiral isomers. Chemically, chiral isomers are very similar, having the same boiling points, melting points, and typically the same solubility, reactivity, and other chemical properties. Microbially and biologically, however, they can behave very differently. Typically, one form is active against the insects and pests that the pesticide is designed to attack, and the other form is inactive. Likewise, in the environment, one form can be actively degraded by microbes, and the other form can accumulate. It was not until recent developments allowed the separation and low-level detection of these isomers that their environmental behavior could be studied. However, early research is showing that the environmental behavior of chiral compounds is not straightforwardsit is not always possible to predict the enantiospecific transformation. Microbial populations in environmental matrixes can change, and even reverse, the enantiomeric ratios (so microbial processes may not always show selective degradation of the same enantiomer). Some environmental processes are not enantioselective toward a particular chemical, even if microorganisms are involved. Sometimes microbial degradation rates are sufficiently rapid for both enantiomers, so that enantioselective degradation is not important. Some compounds are degraded much faster chemically (abiotically) than microbially, so that enantioselective degradation is not important, and sometimes enantiomerization can occur, where one enantiomer is microbially converted to the other (76). The ability to separate enantiomers and produce a single enantiomeric isomer has not been lost on pesticide manufacturers. This ability has allowed manufacturers to sell a new, patented enantiomeric form of a pesticide, creating new markets for their products. The development of enantiomerically enriched pesticides may actually be a benefit for the environment, as less material could potentially be applied to crops, less may be accumulated in the environment, and there may be fewer unintended side effects on nontarget species. Ward published a 2000 Analytical Chemistry review on chiral separations (77). While the focus was not on mass spectrometry per se, the review provided details on the types of chiral phases used for separations and various separation techniques (including CE, supercritical fluid chromatography, GC, and LC) that are often used with mass spectrometry. Chiral selectors now include cyclodextrins, proteins, crown ethers, polysaccharides, polyacrylamides, polymeric chiral surfactants, macrocyclic antibiotics, and Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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ergot alkaloids. Cyclodextrins still remain the most popular chiral selectors used for environmental applications. Garrison reviewed the analysis of chiral pesticides and PCB congeners in environmental samples (78). This article provides a brief background on chiral chemistry in general and the importance of chirality to the drug industry, followed by a summary of examples of the enantioselectivity of pesticides and PCB occurrences and degradation in the environment. Although there are now numerous papers in the literature studying the environmental fate and effects of chiral compounds, only a few representative examples are provided here. Buser et al. used chiral GC/MS to determined whether a change in enantiomer ratios had occurred since an enantiomerically enriched product of metolachlor (the biologically active 1′-S isomer) was introduced to the market (79). In fact, a clear excess of the 1′-S isomer was apparent in surface waters from two lakes in Switzerland collected in 1998 and 1999. In a separate study, the environmental behavior of the chiral acetamide pesticide, metalaxyl, was studied in soils using chiral GC/MS (80). As with the metolachlor, there is now a new, commercially available form of metalaxyl that is enriched with the biologically active (R) enantiomer (called metalaxyl-M). The degradation of this fungicide, along with its primary carboxylic acid metabolite, was measured in soils investigated under controlled laboratory conditions. The degradation of metalaxyl was shown to be enantioselective, with the biologically active R enantiomer being degraded faster than the inactive S enantiomer, resulting in residues enriched with (S)-metalaxyl. The degradation of the carboxylic acid metabolite was also enantioselective. Mattina et al. developed a new chiral/GC-ion trap MS method to quantitatively measure both achiral and chiral components of technical chlordane in soil, plant, and air compartments in the soil (81). Using this method, differences were noted in both the absolute and the relative amounts of the (+) and (-) enantiomers of transand cis-chlordane, indicating the presence of enantioselective processes in compartments of soils, plant roots, and aerial plant tissues. This study represents the first comprehensive report of enantioselective processes into and through plant tissues for a variety of field-grown food crops. Chiral PCBs have also been the focus of research (82, 83). Nineteen of the 209 possible PCB congeners are chiral, and these were targeted in measurements of river and riparian biota samples (fish, bivalves, crawfish, water snakes, barn swallows) (82) and aquatic bed sediment samples (83) from selected sites throughout the United States. Nonracemic enantiomeric ratios were observed for four PCBs (PCBs 91, 95, 136, and 149) in aquatic and riparian biota samples from a reservoir heavily contaminated with PCBs and for four other PCBs (PCBs 132, 174, 176, and 183) from river fish and bivalves nationwide. Fish and bivalves showed marked differences in enantiomeric ratios as compared to sediment from the same sampling site, indicating that microbial degradation processes must be different for those biota as compared to those in sediment. Species-dependent patterns were also observed, and the presence of nonracemic PCBs in fish and bivalves suggests a greater metabolic degradation than had been indicated in previous achiral studies. In the sediment studies, nonracemic enantiomeric ratios were found for seven PCB congeners, and the enantioselectivity of one of these (PCB 91) was found to reverse between 2728
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the Hudson River and Housatonic River sites, indicating that these two sites must have different PCB biotransformation processes. POLYBROMINATED DIPHENYL ETHERS PBDEs have been used for many years as flame retardants in a variety of commercial products including the foam cushions in chairs and other furniture, plastics, textile coatings, electronic appliances, and printed circuit boards. Forty thousand tons were produced globally in 1992, and these compounds have been shown to be persistent and ubiquitous in the environment. Due to concerns about potential adverse development effects and the widespread presence of these compounds in environmental and human biological samples, there has been a Directive established to control emissions of these compounds in Europe. In the United States, however, most environmental studies are only beginning, and PBDEs have so far not been listed on the CCL. In the United States, Hites’ research group has published two investigations of PBDEs in Great Lakes air (84) and in fish from the northeastern United States (85). GC/MS with either EI or electron capture negative ionization (ECNI) was used for analysis. His group is also currently carrying out a study to measure PBDEs in the placentas of newborn babies (86). In the study of Great Lakes air samples collected from 1997 to 1999, PBDEs were found in all samples collected from urban, rural, and remote sites near the Great Lakes, and the total concentrations were similar to some of the organochlorine pesticides, ranging from 5 pg/m3 near Lake Superior to 52 pg/m3 in Chicago (84). The spatial trends were well correlated to those of PCBs, and levels of PBDEs were constant from 1997 to 1999. At ambient temperatures (20 (3 °C), 80% of the tetrabromo homologues were in the gas phase, and 70% of the hexabromo homologues were in the particulate phase. This particle-to-gas-phase partitioning in the atmosphere is believed to be important for these chemicals. In Hites’ study of PBDEs in fish, concentrations and spatial variations were investigated in fish from two small lakes in the northeastern United States and two of the Great Lakes (85). PBDE concentrations in fish were found to range from 6.9 to 18 ng/g wet weight, or 150 to 300 ng/g lipid, and were similar to measurements of organochlorine pesticides, such as total chlordane. Comparisons of fish and sediment concentrations indicated that tetra- through hexa-substituted congeners have a similar bioavailability, while the deca-substituted form did not appear to be bioavailable. Alaee et al. published a new analytical method for measuring PBDEs in fish (87). The authors found GC/highresolution-EI-MS to be superior to ECNI-MS, offering detection limits ranging from 0.1 to 4l8 pg for the 23 brominated diphenyl ethers studied. This method was demonstrated by analyzing PBDEs in commercially available certified reference materials of Lake Ontario lake trout, Pacific herring, and sockeye salmon. In an occupational exposure study in Norway, Thomsen et al. used diazomethane derivatization with GC/ECNI-MS to investigate how human exposure to brominated flame retardants is related to specific occupations (e.g., workers at an electronics dismantling facility, workers who produced printed circuit boards, or other laboratory personnel) (88). Nine brominated flame retardants, including many PBDEs, were quantified in human plasma samples, including tri-, tetra-, penta-, hexa-, and heptabrominated congeners. People working at the electronics dismantling plant had signifi-
cantly higher plasma levels of tetrabromobisphenol A and 2,2′,4,4′,5,5′-hexabromodiphenyl ether compared to other groups, and the heptabrominated congener, 2,2′3,4,4′,5′,6-heptabromodiphenyl ether, was only detected in plasma from this group. 2,4,6Tribromophenol was the most abundant flame retardant present, with plasma concentrations ranging from 0.17 to 81 ng/g lipids. 2,2′,4,4′-Tetrabromodiphenyl ether was the dominant PBDE in all of the individual samples, with levels ranging from 0.43 to 14.6 ng/g lipids. Total amounts of the seven flame retardants were 8.8, 3.9, and 3.0 ng/g lipids for the groups of electronic dismantlers, the circuit board producers, and the laboratory personnel, respectively. Large variations were found in the individual concentration levels found within these groups, especially for the electronics dismantlers, where RSDs for the flame retardant concentrations ranged from 23 to 164%. Levels could not be correlated with the age of the worker. ALGAL TOXINS The increase in frequency and intensity of harmful algal blooms has led to increased incidences of the poisoning of shellfish, large fish kills, and deaths of livestock and wildlife, as well as illness and death in humans. Toxins produced by these algae have been implicated in these adverse effects. Algal toxins that impact human health are generally categorized as neurotoxins or hepatotoxins that are produced from dinoflagellates, diatoms, or cyanobacteria (blue-green algae). Dinoflagellate and diatom toxins impact humans primarily through the consumption of seafood, and cyanobacteria generally impact humans through drinking water contamination. For example, saxitoxins, which have heterocyclic guanidine structures, are produced by dinoflagellates and cyanobacteria and cause paralytic shellfish poisoning. Anatoxins, which have heterocyclic structures, are produced by cyanobacteria and are neurotoxic. Microcystins and nodularins, which have cyclic peptide structures, are produced by cyanobacteria and are hepatotoxic. “Red tide” toxins, which have heterocyclic polyether structures, are produced by red tide dinoflagellates (mostly from Gymnodinium breve) and are neurotoxic. The National Oceanic and Atmospheric Administration (NOAA) has a wonderful website that provides the structures of these algal toxins and further details (www.chbr.noaa.gov/CoastalResearch/Diversityessay.htm). Algal toxins are currently being considered for inclusion in a future CCL list. Many of these toxins are peptide-related, have relatively high molecular weights, and are highly polar, which hindered their environmental measurements until the recent application of electrospray and APCI ionization techniques. Pierce and Kirkpatrick published a review of innovative techniques for studying algal toxins and the parent microorganisms (89). Zweigenbaum et al. developed a microbore-LC/ESI-ion trapMS/MS method for determining trace amounts of microcystins in environmental samples (90). Full-scan LC/ESI-MS data could be obtained on 250 pg of material, representing the detection limit of microcystin-LR. Hormazabal et al. developed a LC/MS method to simultaneously determine anatoxin-A and microcystin desmethyl-3, LR, RR, and YR in fish, with detection limits of 15, 2, 10, 1, and 10 ng/g, respectively (91). Pietsch et al. used ion pair SPE and LC/ESI-MS/MS to measure anatoxin-A, saxitoxin, microcystins, and nodularin in water samples (92). Limits of quantification were approximately 50 ng/L for microcystins
(microcystin-LR, -YR,-RR, and -LA), nodularin, and anatoxin-A and 630 ng/L for saxitoxin. This method would allow monitoring under World Health Organization guidelines. Ells et al. developed an analytical method for measuring microcystins in water using a relatively new analytical techniquesESI-high-field asymmetric waveform-ion mobility spectrometry (FAIMS)-MS (93). This new technology allowed rapid analyses and detection limits of 1-4 nM (∼1-4 µg/L) for microcystin-LR, -RR, and -YR. Robillot et al. used micro-LC/ESI-MS and a protein phosphatase bioassay to determine the hepatotoxin kinetics of microcystins produced from Microcystis aeruginosa PCC 7820 (94). In this study, three new variants were identified using collisionally induced dissociation (CID)/postsource decay-MALDI/TOF-MS: desmethylated microcystin-LW, desmethylated microcystin-LF, and microcystin-LL. Toxin production could be correlated to biomass increase up to the middle of the exponential phase of growth and ceased thereafter. Toxin release was found to occur during the stationary phase, and extracellular microcystin concentrations reached 0.25 mg/L. DRINKING WATER DISINFECTION BYPRODUCTS (INCLUDING NDMA) Drinking water disinfection byproducts continue to be of interest, particularly with the Stage 1 disinfectants (D)/DBP Rule taking effect in 2002, which lowered permissible levels of trihalomethanes (THMs) from 100 to 80 µg/L and regulated five of the haloacetic acids (HAAs) (monochloro-, dichloro-, trichloro-, monobromo-, and dibromoacetic acid), bromate, and chlorite for the first time. Recent reproductive and developmental epidemiologic studies are also suggesting an association of chlorinated DBPs with various effects, including spontaneous abortion (early-term miscarriage). DBPs are formed when disinfectants used to kill pathogens in the water react with organic matter or bromide naturally present in the source waters. Recent studies are showing that ingestion of water is not the only important exposure route to these chemicalssinhalation (from showers) and dermal absorption (baths, etc.) are also important, sometimes contributing to significantly greater exposures (particularly for volatile DBPs) than ingestion. Current areas of interest include the discovery and identification of new DBPs in drinking water, particularly for newer disinfectants where there is little known and for highly polar DBPs that are not amenable to commonly used GC/MS techniques. A new nationwide DBP occurrence study is also underway that is providing quantitative occurrence data on high-priority DBPs for which no data previously existed. There is also interest in higher molecular weight DBPs that are known to be present but have not been identified due to the lack of appropriate analytical methods for their identification. Due to potential toxicity and to taste-and-odor problems, there is renewed interest in iodinated DBPs, including iodo-THMs. In addition, the discovery of nitrosodimethylamine as a DBP (from chloramine or chlorine disinfection) and its detection in California groundwaters has spawned great interest, due to its listing as a probable human carcinogen. Discovery of New DBPs. Richardson published a tutorial review in 2002 on the role that GC/MS and LC/MS have played and are currently playing in the discovery of drinking water DBPs (95). Examples of how GC/MS has been used to identify the unknown chemicals were presented, as well as a summary of the Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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types of chemical DBPs that have been identified. Limitations of GC/MS were presented, including the difficulty in analyzing high molecular weight compounds and highly polar compounds. In fact, more than 50% of the total organic halogen (TOX) material formed by the chlorination of drinking water and more than 60% of the total assimilable carbon (AOC) formed in the ozonation of drinking water remain unidentified. The use of LC/MS for characterizing or identifying the highly polar or high molecular weight fraction has only recently begun but is showing early promise for providing additional information on the unidentified fraction. The author also identifies current research needs and gives recommendations for future research. In addition, Richardson et al. published a compilation of more than 200 DBPs that have been identified over the last 8 years for ozone, chlorine dioxide, chloramine, and chlorine disinfection by their research group at the U.S. EPA (96). These identifications were possible due to the combined use of GC/MS (including highresolution EI-MS and chemical ionization) and GC/infrared spectroscopy, as well as the use of different derivatizing agents. Monarca et al. reported the mutagenicity and identification of DBPs from surface waters disinfected with peracetic acid (PAA) and compared the results to disinfection with chlorine dioxide or hypochlorite (chlorine) (97). PAA is an experimental disinfectant that has not been used yet for drinking water disinfection. Results showed that a slight mutagenicity was observed in the PAA-treated waters but was similar to the mutagenicity already present in the untreated, raw waters. DBPs identified by GC/MS included mainly carboxylic acids, which are also observed as DBPs of other disinfectants. Taguchi reported the identification of two new DBPs in drinking water using GC/MS (including high-resolution EIMS and chemical ionization) and also tandem mass spectrometry (98). The two compounds were tentatively identified as 1-aminoxy1-chlorobutan-2-ol and 1-aminoxy-1-bromobutan-2-ol, respectively. Richardson et al. published the development of a 2,4-dinitrophenylhydrazine (DNPH) derivatization-SPE-LC/ESI-MS method for identifying highly polar carbonyl DBPs in drinking water (99). By using the DNPH derivatization and C18 solid-phase extraction, highly polar carbonyl compounds (uncharged) could be isolated from salts that would interfere with the electrospray process, and low concentrations of analytes could be concentrated and measured. Using a gradient LC program, aldehydes were easily distinguished from ketones by their different chromatographic behavior (syn and anti derivative isomers coeluted for aldehydes, and separated for ketones) and also by the presence of a key ion in the collisionally induced dissociation (CID) mass spectra of aldehydes that is not present for ketones. This method is not recommended as a replacement for the commonly used pentafluorobenzylhydroxylamine (PFBHA) derivatization-GC/MS method because detection limits are not as low, but it is shown to have potential as a tool for identifying carbonyl-containing DBPs that are missed by other methods. Using this method, several highly polar DBPs were identified in ozonated drinking waters, including pyruvic acid, glyoxylic acid, ketomalonic acid, 5-ketohexanal, 6-hydroxy-2-hexanone, and one new DBP, 1,3-dihydroxyacetone. New Nationwide DBP Occurrence Study. Scientists from the U.S. EPA, the University of North Carolina, and the Metropolitan Water District of Southern California are conducting a U.S. Nationwide DBP Occurrence study, with a focus on newer “high2730
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priority” DBPs for which little or no occurrence is available (100102). The entire group of DBPs (>500) that have been published in the literature were first prioritized by experts according to predicted adverse health effects, resulting in a manageable group of approximately 50 “high-priority” DBPs that were selected for this study. These high-priority compounds include iodo-THMs, bromo acids, bromoacetonitriles, bromoaldehydes, 3-chloro-(4dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), brominated forms of MX (the so-called BMXs), and other nonhalogenated compounds such as cyanoformaldehyde. Standards were obtained for these compounds (many of which had to be synthesized), rugged analytical methods were developed (mostly GC and GC/MS methods), and drinking water was sampled across the United States. The published proceedings papers cited here represent preliminary results obtained from the first few months of sampling. In addition to obtaining quantitative occurrence information for high-priority DBPs, new DBPs are also being identified through the use of derivatization and various GC/MS (high-resolution EI and CI) and LC/MS techniques. The fate and transport of DBPs is also being studied in drinking water distribution systems. This study is expected to fill a large knowledge gap regarding the occurrence of DBPs that have not been previously addressed. NDMA. NDMA has become a human exposure concern due to its occurrence in California drinking waters and the discovery that it can be formed as a DBP in drinking water treatment with chloramine or chlorine, as well as originate from point-source pollution (contaminant from rocket fuel, plasticizers, polymers, batteries, etc.). Its discovery as a DBP suggests that it may be much more widespread than previously thought. Concern arises because NDMA is listed as a probable human carcinogen, with a risk much greater than that posed by chloroform, another DBP that has been regulated in the United States since the Safe Drinking Water Act was passed in 1979. NDMA had been previously discovered in 1989 at ppb levels in Canadian drinking waters, but it had not been identified in U.S. waters until very recently. The observation of NDMA in U.S. waters is largely due to improved analytical techniques that have allowed its determination at low-nanogram per liter concentrations. Recent measurements have shown it is present at 10 ng/L or less in chlorinated drinking water, and it can be formed at 100 ng/L levels or higher in wastewater treated with chlorine (103). Following its discovery in California well water, the State of California issued an action level of 0.002 µg/L (2 parts per trillion) for NDMA, which was subsequently revised to 0.01 µg/L, due to the analytical difficulty in measuring it at the original proposed level. The California Department of Health Services has a nice website that provides further background and details about NDMA (www.dhs.ca.gov/ ps/ddwem/chemicals/NDMA/NDMAindex.htm). This site also provides a link to the U.S. National Toxicology Program (NTP) 2000 report on NDMA. NDMA is currently not regulated in the United States for drinking water and is not on the CCL. Raksit and Johri developed an analytical method using isotope dilution and GC/MS that can measure as little as 3 ng/L NDMA in water (104). Mitch and Sedlak used GC/MS and GC/MS/MS to study the formation of NDMA during chlorination (105). This study revealed that NDMA can form by the reaction of dimethylamine and other secondary amines (potential NDMA precursors in drinking water) and that this process may involve the slow initial
formation of 1,1-dimethylhydrazine by the reaction of monochloramine and dimethylamine, followed by rapid oxidation to NDMA and other products, including dimethylcyanamide and dimethylformamide. Other pathways, such as the reaction of sodium hypochlorite with dimethylamine, can also lead to NDMA formation. However, the rate of NDMA formation was 10-fold slower for chlorine than for monochloramine. This reaction showed a strong pH dependence due to competing reactions. In another paper, Choi and Valentine also studied the formation of NDMA, using dimethylamine as a model precursor (106). 15N-Labeled monochloramine was used to track the incorporation of nitrogen into the nitroso group, and mass spectrometry was used to analyze the products. This work showed that the nitrogen from monochloramine was the source of a nitrogen atom in the nitroso group of NDMA, and its formation increased with increasing monochloramine concentration. The proposed NDMA formation pathway involves the formation of 1,1-dimethylhydrazine (from the reaction of dimethylamine with monochloramine), followed by oxidation by monochloramine to NDMA. Trihalomethanes. Cancho et al. evaluated four different analytical techniques for analyzing iodo-THMs in drinking water: headspace-GC-ECD, purge-and-trap-GC/MS, closed loop stripping analysis (CLSA)-GC/MS, and liquid-liquid extraction with GCECD (107). The best method proved to be liquid-liquid extraction with GC-ECD, and it was subsequently used to evaluate the stability of iodo-THMs in water and also for measuring iodo-THMs in drinking water samples at different stages of treatment from drinking water plants in Barcelona, Spain. The procedures used to synthesize the iodo-THM standards (CHCl2I, CHClI2, CHBr2I, CHBrI2, CHBrClI) were also published in this paper. Only three (CHCl2I, CHBrClI, CHBr2I) of the six iodo-THMs were found in the drinking water samples, with average levels less than 1 µg/ L. THMs have also been the target of fast-GC and fast-GC/MS methods. For example, Chang and Her developed a MIMS-fastGC/MS that enabled 20 samples to be analyzed per hour (108). The use of GC separation with MIMS allowed CHCl3 and CHBrCl2 to be distinguished in chlorinated drinking water. Control of injection temperature and injection time overcame problems with water permeating across the membrane. Detection limits for CHCl3, CHBrCl2, CHBr2Cl, and CHBr3 were 2, 4, 4, and 8 ng/L, respectively. Human exposures to THMs continue to be of importance. Previously conducted epidemiologic studies have mostly considered exposures through ingestion (drinking) of water, and most have not considered other routes of exposure. Backer et al. published results of a human exposure study that examined individuals’ exposure to THMs through drinking, showering, or bathing in tap water (109). Thirty-one adult volunteers showered with tap water for 10 min, bathed for 10 min in a bathtub filled with tap water, or drank 1 L of tap water during a 10-min time period. Blood samples were collected immediately after exposure, 10 min later, and 30 min later and were analyzed along with tap water samples by purge-and-trap-GC/MS (with detection limits in the pg/L range). The highest levels of THMs were found in blood samples from people who took 10-min showers, whereas the lowest levels were found in blood samples from people who drank 1 L of water. This study demonstrates that household
activities, such as showering or bathing, can be very important routes of exposure to THMs. It should be noted, however, that inhalation through showering is probably not going to be an important route of exposure for nonvolatile DBPs, such as HAAs. Haloacetic Acids. Due to the recent regulation of five HAAs in drinking water (under the Stage 1 D/DBP Rule promulgated in 1998), HAAs continue to be the subject of new analytical methods. There are also human exposure studies and metabolism research being conducted to assess our exposure to them and understand their mechanism of toxicity (carcinogenicity). Urbansky published a review of techniques and methods for determining HAAs in drinking water (110). Techniques discussed included GC/MS, GC-ECD, CE, LC, IC, and ESI-MS, along with derivatization using different methylation techniques (i.e., diazomethane and acidic methanol). Urbansky also published a paper on the fate of HAAs in drinking water and their chemical kinetics (111). Magnuson and Kelty developed a microextraction-ESI-MS method for determining nine HAAs in drinking water at microgram per liter levels (112). This method involved the formation of stable association complexes between the HAAs and perfluoroheptanoic acid, which was added to the drinking water extracts. Loos and Barcelo developed a SPE-LC/ESI-MS method for determining HAAs in drinking water at low-microgram per liter levels (113). The use of triethylamine as an ion-pairing reagent enabled the complete separation of the HAAs by LC. Using this method, high-microgram per liter levels of chlorinated and brominated HAAs were detected in drinking waters from Barcelona, Spain. This method was also used to detect HAAs in swimming pool water samples from Spain (where mg/L levels were detected) and also surface river water from Portugal (where µg/L levels were detected). Sarrion et al. developed an in situ derivatization-SPME-GC/ion trap-MS method for determining HAAs in water (114). This method involved derivatization of HAAs to their methyl esters with dimethyl sulfate, headspace sampling using SPME, and GC/ion trap-MS determination. The addition of tetrabutylammonium hydrogen sulfate (ion pair reagent) improved the formation of HAA methyl esters (up to 90-fold) in the derivatization step. Detection limits ranged from 10 to 450 ng/L, which are among the lowest detection limits reported for HAAs using any method. Good precision was also obtained (RSD between 6.3 and 11.4%), the method was linear over 2 orders of magnitude, and good agreement was obtained when compared to EPA Method 552.2. Finally, Xie developed a new method for analyzing HAAs using liquid-liquid microextraction, acidic methanol derivatization, and GC/MS detection (115). Detection levels were microgram per liter, and in comparison to EPA Method 552.2 (which uses GC-ECD), cleaner baselines and fewer interfering peaks were evident. METHYL TERT-BUTYL ETHER MTBE remains a major concern in the environment, due to its widespread use as an oxygenate in gasoline and subsequent transport into groundwater from leaking underground storage tanks and other sources. MTBE contamination can also occur in surface water, which can be caused by fuel discharged by boats and other watercraft using two-stroke engines. MTBE has been used as a gasoline additive since its introduction in 1979 and, by 1998, was added to approximately 30% of all gasoline sold in the Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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United States. MTBE has been responsible for taste and odor problems in drinking water, and there are also concerns about possible adverse health effects. The U.S. EPA is now requiring monitoring of oxygenate compounds in groundwater at leaking underground storage tank sites, and it is considering the development of a formal secondary maximum contaminant level (MCL) for MTBE. This new standard would represent the first time that the U.S. EPA developed a secondary MCL based on taste and odor of a specific chemical. The U.S. EPA is continuing to study both the potential health effects and the occurrence of MTBE, and it is currently on the CCL for which EPA is considering setting health standards. Stocking et al. provided a background and perspective on the establishment of MCLs and secondary MCLs by states and the federal government. They also published the results of a consumer study designed to determine the odor threshold of MTBE in drinking water (determined to be 15 µg/ L) (116). New methods continue to be published for the trace analysis of MTBE. Cassada et al. published a SPME-GC/MS method to measure MTBE, ethanol, and related oxygenate compounds in water (117). This method, using a divinylbenzene/Carboxen/poly(dimethylsiloxane) SPME fiber for extraction, provided detection limits of 0.008 µg/L for MTBE (one of the lowest to date). Smallwood used GC/isotope ratio-MS to differentiate specific source markers for MTBE (118). The carbon isotopic compositions of MTBE for 10 gasoline samples from three different parts of the United States showed a wide range of carbon isotope compositions, which indicated that it may be possible to attribute a sample of MTBE in gasoline to a particular source. Halden et al. evaluated standard methods for analyzing MTBE and other oxygenates in contaminated groundwater (119). Methods tested include three purge-and-trap-GC and GC/MS methods. Consistently good results were obtained with EPA Method 8240B/ 60B, which uses MS detection, and also ASTM Method D4815, which uses flame ionization detection. Detection limits were shown to be sufficiently low to permit monitoring of MTBE under the primary and secondary action levels set by the State of California. Achten et al. used headspace-SPME and GC/MS to analyze MTBE in urban and rural precipitation in Germany (120). The detection limit of this method was 10 ng/L. Results showed that MTBE was detected more often in urban precipitation, and highest concentrations were observed in snow samples. Mean air equilibrium concentrations of 0.04 ppbV (urban) and 0.01 ppbV (rural) were observed. Urban runoff and corresponding precipitation sampling indicated that urban runoff might be composed of 20% MTBE that was transported by air and precipitation, whereas about 80% may be attributed to direct uptake of vehicle emissions and leakage near the road during precipitation. ORGANOTINS Organotins remain an important environmental contaminant and are currently on the CCL. Organotins are widely used in antifouling paints for ships, and there are dietary sources of organotins. Toxicity generally follows this order, trialkyl > dialkyl > monoalkyl, but the dialkyl form is much more neurotoxic, with an effect in brain cells as low as 30 ppb. Although there was already significant interest in organotins due to their potent 2732
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toxicity, there is increased concern due to possible introduction into drinking water. Jones-Lepp et al. published an important study in 2001 that shows the leaching of dibutyltin from PVC pipe at almost continuous levels of 1 µg/L (121). Dibutyltin is used as a heat stabilizer in PVC. The micro-LC/ESI-ion trap-MS method used for these measurements is described in a separate paper (122). This method was developed to avoid the use of derivatization and hydrolysis and to lower background interferences that are common with traditional methods. Takeuchi et al. published a review covering the variety of analytical methods that have been developed for the speciation of organotin compounds (123). Methods discussed included those based on GC coupled to atomic absorption spectrometry (AAS), flame photometry, MS, ICP, and microwave-induced plasma atomic emission spectrometry, as well as LC coupled to AAS, ICPMS, and fluorescence detection. Derivatization methods that are used to render the organotins volatile for GC analyses are also discussed. In addition to the micro-LC/MS method mentioned earlier, other researchers have also developed LC/MS techniques for measuring organotins in water. Gimeno et al. developed an online SPE-LC/APCI-MS method for determining diuron, irgarol 1051, folpet, and dichlofluanid in seawater samples (124). Recoveries of >85% were achieved for 100-mL seawater samples, with detection limits of 5 ng/L. This method was subsequently used to analyze water from different marina and fishing ports along the coast of Catalonia, Spain, over a 5-month period. Diuron and irgarol 1051 were detected in most samples at levels ranging from 27 to 420 and 15 to 511 ng/L, respectively. Wu et al. developed an automated in-tube SPME-LC/ESI-MS method for quantifying tributyltin in water samples (125). Detection limits of 0.05 µg/L and linearity over a range of 0.5-200 µg/L were achieved. Cardellicchio et al. developed a headspace SPME-GC/MS method for determining organotin species (monobutyl-, dibutyl-, and tributyltin) in marine sediments (126). This procedure involved extraction using a HCl/methanol mixture, in situ derivatization with sodium tetraethylborate, and headspace SPME extraction onto a poly(dimethylsiloxane) fiber. Detection limits ranged from 730 to 969 pg/g as Sn dry weight. New ICPMS methods were also published. Vercauteren et al. developed a headspace SPME-GC/ICPMS method for determining triphenyltin in water, potatoes, and mussels (127). Samples were digested with tetramethylammonium hydroxide or KOH/ ethanol, and sodium tetraethylborate was used to derivatize the triphenyltin to a more volatile compound that could be analyzed by GC/ICPMS. Monitoring of Sn-120 signals by ICPMS provided extremely low limits of detection in water (2 pg/L). A particularly clever method for measuring organotins was published by Vercauteren et al. in 2001 (128). In this method, a poly(dimethylsiloxane)-coated stir bar was used to extract organotin compounds from freshwater and seawater, with subsequent analysis by thermal desorption-GC/ICPMS. Extremely low detection limits of 0.1 pg/L were achieved. Leach et al. used TOF-MS with GCICP to measure organotin species (129). Using TOF-MS, many mass spectra could be acquired across each GC peak, which allowed a tin isotope ratio accuracy of 0.28% and a precision of 2.88%, with detection limits in the low-femtogram range and a dynamic range over 6 orders of magnitude. Mester et al. used
headspace-SPME with ICP-TOF-MS to enable the determination of organotin species without the need for chromatography (130). Two papers reported the MS/MS spectra for both organotin compounds and their hydrolysis products. Wei and Miller used ESI-MS/MS to investigate the fragmentation of organotin hydrolysis products (131), and Ostah and Lawson used EI-MS/MS to investigate the fragmentation of six organotin compounds (132). Several studies of organotins in the environment have been published, including occurrence studies, transport studies, and studies involving the measurement of organotins in biological samples, including human tissue samples. Amouroux et al. used GC/ICPMS to investigate the occurrence and speciation of volatile organotins in a contaminated area of the Arcachon Bay (southwestern France) and in the water column of the Scheldt (Belgium/ Netherlands) (133). The most ubiquitous species were found to be the methylated forms of butyltin derivatives, which suggested that biological or chemical methylation mechanisms may occur in sediments and lead to remobilization of tin species into the water column and into the atmosphere. Rajendran et al. used GC/ ICPMS to determine butyl-, octyl-, and tributylmonomethyltin compounds from two harbors in the Bay of Bengal (India) (134). As with the previously mentioned study, results showed that natural methylation was occurring. There was also evidence for an elevated rate of debutylation in estuarine environment, from the high concentrations of inorganic tin that were found in estuarine sediments. Finally, Jiang et al. used GC-flame photometric detection and ICPMS to determine organotin compounds (and other metals) in human organ samples from a victim who had died of organotin-contaminated lard (135). Organ tissues were first digested with CuSO4 and KBr/H2SO4 solutions, extracted with 0.1% tropolone.cyclohexane, derivatized with a Grignard reagent, and purified with Florisil. Results showed extremely high levels of methyltin compounds and smaller amounts of inorganic tin in the victim’s heart, kidney, liver, and stomach. PERCHLORATE Perchlorate has recently become an important environmental issue since its discovery in a number of U.S. water supplies, including Lake Mead and the Colorado River. Ammonium perchlorate has been used as an oxygenated in solid propellants used for rockets, missiles, and fireworks, and there is also possible contamination that can occur through the use of fertilizers (that contain Chilean nitrate). High quantities of perchlorate have been disposed of since the 1950s in Nevada, California, and Utah, which is believed to have contributed to contamination. Health concerns arise from perchlorate’s ability to disrupt the thyroid gland’s use of iodine in metabolic hormones, which could affect normal metabolism, growth, and development. Due to these concerns, the U.S. EPA has placed perchlorate on the CCL for further study. Current research needs include improved detection limits for fertilizer analysis, determination of perchlorate in biological samples, determination in fruit juices, uptake in citrus trees and fruit, and whether perchlorate enters the food chain through the use of fertilizer contaminated with perchlorate. Urbansky published a review of the practices and advances for quantifying perchlorate in environmental samples (136). This review discusses the strengths and weaknesses of gravimetry, spectrophotometry, electrochemistry, IC, CE, and MS for measur-
ing perchlorate and looks forward to where sample pretreatment and analysis methods are headed. A few methods have been published recently for quantifying perchlorate in water and urine samples. Koester et al. developed a ESI-MS/MS method for analyzing perchlorate in groundwater (137). Selective and sensitive detection was achieved by operating the mass spectrometer in the negative ion mode and by using MS/MS to monitor the ClO4- to ClO3- transition. Standard addition was used to overcome signal suppression caused by anions that are typically present in groundwater (e.g., bicarbonate and sulfate), and detection limits of 0.5 µg/L were achieved. This ESI-MS/MS method compared favorably to IC (statistically indistinguishable) for perchlorate measurements. In a method developed by Magnuson et al., an ion-pairing reagent was used to form an ion pair complex that could be measured by ESI-MS (138). A detection limit of 0.1 µg/L was achieved. Ells et al. published an ESI-FAIMS-MS method for the trace determination of perchlorate in water and in human urine (139). Detection limits were 0.050 µg/L and 0.37-4.8 ppb in water and certified urine samples, respectively. A few perchlorate studies are worthy of noting here. Urbansky et al. used ESI-MS (with ion pairing) and IC to carry out a survey of bottled waters for perchlorate contamination (140). For comparison, a finished potable water known to contain perchlorate was also tested. Results showed that none of the bottled waters were found to contain any detectable levels of perchlorate. In another study, Urbansky et al. measured perchlorate levels in samples of fertilizer that contained Chilean sodium nitrate (141). Using IC and ESI-MS, perchlorate was found to be homogeneously distributed in the fertilizer samples, with average levels ranging from 1.5 to 1.8 mg/g. Finally, a very recent study offered a solution to efficiently removing perchlorate from drinking water. In a February 2002 article, Brown et al. published results showing that perchlorate could be removed from drinking water using a granular activated carbon (GAC) filter (142). This could be done in a drinking water treatment plant with conventional contact times and without generating a concentrated perchlorate waste stream (as with ionexchange removal). When the filter was made biologically active, microorganisms in the filter could efficiently convert perchlorate to chloride, reducing 50 µg/L levels of perchlorate to below detection. Biologically active GAC filters have been used to remove (by chemical reduction) other inorganic contaminants, such as bromate, hypochlorous acid/hypochlorite, and chlorite. However, chlorate (which is more chemically related to perchlorate) is not biologically reduced by GAC, so the results of this important study were not necessarily intuitive. It is nice to see simple engineering solutions developed to remove potentially harmful contaminants. ARSENIC Arsenic has been a politically charged issue the last 2 years. Unlike many other contaminants that are anthropogenic, arsenic contamination of waters generally comes from natural sources, through the erosion of rocks, minerals, and soils. For several years, the U.S. EPA has conducted research on arsenic (occurrence, health effects, bioavailability) and was intending to lower the existing standard in drinking water of 50 µg/L to a level that would better protect human health. The general toxicity of arsenic is well known, but studies have also linked long-term exposure Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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of arsenic (at lower, nontoxic levels) to a variety of cancers in humans. In January 2001, the U.S. EPA lowered the standard (MCL) from 50 to 10 µg/L; however, two months later, the new EPA Administrator announced that EPA would withdraw this pending arsenic standard in order to seek independent reviews of the science behind the standard and the estimates of costs to communities to implement the Rule (http://www.epa.gov/safewater/arsenic.html). After seeking the advice of independent, expert panels convened by the National Academy of Sciences, the National Drinking Water Advisory Council, and the EPA Science Advisory Board regarding recommendations on the science, cost of compliance, and benefits analysis, the Rule was finally agreed upon and the original proposed standard of 10 µg/L was kept. This Rule became effective on February 22, 2002, and drinking water systems must comply with this new standard by January 23, 2006. On the nonpolitical front, arsenic research issues that have become important are determining individual species of arsenic (rather than total arsenic as had been done in the past) and occurrence of arsenic species in water, foods, and biological samples. Different arsenic species have different toxicities and chemical behavior in aquatic systems, so it is important to be able to identify and quantify the individual species of arsenic. Jain and Ali presented a nice review of the occurrence, toxicity, and speciation techniques for arsenic (143). Several new analytical methods have also been developed for measuring different arsenic species. These methods include SPME and SPE used with GC/MS, LC/ESI-MS, LC/ICPMS, and ICICPMS. The number of papers involving the development or use of LC/ICPMS techniques has grown significantly in the past 2 years. Mester and Pawliszyn developed a SPME-GC/ion trap-MS method to measure dimethylarsenic acid and monomethylarsonic acid in human urine samples (144). This method first involved thioglycol methylate derivatization, followed by SPME extraction (a poly(dimethylsiloxane) coating worked best) and GC/MS analysis. Detection limits of 0.12 and 0.29 ng/mL were obtained, and the method was linear over a 1-200 ng/mL range. Wu et al. published an on-line, in-tube SPME-LC/ESI-MS method that allowed the direction extraction and concentration of organoarsenic species from water by repeated aspiration/dispension steps (145). The polypyrrole capillary provided the best extraction efficiency. Gallagher et al. used accelerated solvent extraction (ASE) with IC-ICPMS for measuring arsenic compounds in seaweed (kelp) (146). IC-ESI-MS/MS was also used to tentatively identify the structure of an arsenosugar (molecular weight of 408) found in the seaweed. Martinez-Bravo et al. developed a new method using on-line, anion-exchange-LC/ICP-MS for measuring arsenic, selenium, and chromium(IV) species in water (147). Detection limits of 40-60 ng/L were obtained for the arsenic species, arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid. Bissen and Frimmel developed a LC/ICPMS method to determine the same arsenic species in soils (148). This method was used to identify arsenic species in contaminated soils from a tannery site and a former paint production site. Only 0.04% of the total arsenic from the tannery soil and 20% of the paint production site soil were released and extracted; results showed that the mobilization of 2734
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arsenic depended on the pH of the extraction solution and the type of soil. Wangkarn and Pergantis developed a high-speed separation method using narrow-bore-LC/ICPMS (149). An octadecyldimethylsilyl reversed-phase narrow-bore LC column was used, and separation of arsenite, dimethylarsinic acid, monomethylarsonic acid, and arsenate could be achieved in less than 2 min, using a mobile phase containing 5 mM tetrabutylammonium hydroxide as the ion-pairing reagent at pH 6.0. Detection limits were in the low-microgram per liter range. Chatterjee et al. developed a LC/ ultrasonic-high-power nitrogen-microwave-ICPMS method to measure arsenic species (150). The use of the ultrasonic nebulizer increased the ion signals a factor of 3-6 times for anionic arsenic compounds and 6-12 times for cationic species, as compared to traditional microwave-induced plasma (MIP)-MS methods. Compared to a standard LC/ICPMS coupling, levels using the ultrasonic nebulizer were 1.5-2 and 1.3-3.8 times higher for anionic and cationic arsenic species. Wei et al. used preoxidation of arsenite (to arsenate) with online photooxidation-membrane hydride generation-ICPMS to measure arsenic species in urine (151). The sample preoxidation eliminated As(III) and As(V) preservation concerns and simplified the chromatographic separations. Other arsenic species did not react (arsenobetaine, dimethylarsinic acid, monomethylarsonic acid, arsenate), and recoveries of 95-102% could be achieved for all arsenic species. Wei et al. also compared urinary arsenic speciation using either direct nebulization or hydride generation (HG) with IC-ICPMS detection (152). A gradient elution program provided the best resolution for the five arsenic species studied. In the membrane HG configuration, a photoreactor interface installed between the column and the HG device facilitated the detection of non-hydride arsenic species, with chloride interferences removed by a gas-liquid separator. Nakazato et al. developed an ion exclusion-LC/ICPMS method for determining eight inorganic and organic arsenic species in biological matrixes (153). Detection limits ranged from 0.067 to 0.34 µg/L, and this method was applied to human urine and fish samples. Important human exposure research is also being conducted to determine arsenic exposures in highly contaminated areas. Samanta et al. developed a LC/ICPMS method for determining five arsenic species (arsenite, arsenate, monomethyl arsonic acid, dimethyl arsenic acid, arsenobetaine) in urine samples and used this method to analyze arsenic from two groups of people in arsenic-affected villages in West Bengal, India (154). Drinking water treatment systems had been installed to remove the arsenic contamination; however, arsenic species found in the villagers’ urine indicates that they have exposures from surrounding areas. Mandal et al. also measured arsenic species in human urine from people in West Bengal, India (155). Using anion-exchange-LC/ ICPMS, both dimethylarsinous acid and monomethylarsonous acid were detected directly (without any pretreatment) for the first time in urine of people exposed to inorganic arsenic through their drinking water. Of the 428 subjects, monomethylarsonous acid was found in 48% and dimethylarsinous acid in 72%. These findings have important implications for the mechanism of metabolism, carcinogenicity, and detoxification of inorganic arsenic. Arsenosugars and other forms of organoarsenic have also been the subject of recent research. Pergantis et al. developed a
nanoelectrospray-TOF-MS/MS method for identifying arsenosugars at picogram levels (156). This method was used to generate structurally diagnostic fragment ions that can be used to identify arsenosugar unknowns without chemical standards, and it was applied to the identification of four trimethylarsonioribosides in an algal extract. McSheehy et al. also used ESI-MS/MS to identify arsenosugars in seaweed (157). A size exclusion LC step was used to purify the algal extracts prior to ESI-MS. Collisionally induced dissociation of the protonated molecular ions produced fragment ions that enabled the identification of specific organosugars. The identifications were confirmed by the analysis of arsenosugar standards. Francesconi and Edmonds reported the identification of a new arsenical in a clam kidney (158). Kidneys of clams can accumulate metabolic products from algae that grow in the mantle of the clams, and these metabolites include organoarsenic species that are biosynthesized by the algae from arsenate in seawater. Using LC/ESI-MS, the researchers identified this major component of the kidney (found to be 50% of the water-soluble arsenic) as 5-dimethylarsinoyl-2,3,4-trihydroxycarboxylic acidsa new natural product. Despite the number of studies on arsenic species, there are several research needs that remain. These include the development of analytical approaches that would better mimic the bioavailable fraction within dietary samplessdoes the arsenic fraction extracted with methanol in the laboratory represent the toxic forms of arsenic that are made available in the stomach/ gastrointestinal tract? There is also a need for techniques that provide simultaneous structural and atomic information at nanoto microgram per liter levels. Finally, improved extraction techniques for nonaqueous samples are needed to identify the fraction of organoarsenic that has not been identified and to ensure that aggressive extraction approaches are not changing the original form of arsenic present in the sample. It is expected that arsenic will remain an important issue and that research will be needed for several more years. NATURAL ORGANIC MATTER Natural organic matter (NOM) is a complex mixture of substances, such as amino acids, carbohydrates, lipids, lignins, waxes, organic acids, humic acids, and fulvic acids. Humic substances are complex macromolecular structures. Previous research using GPC has indicated that, of these humic substances, fulvic acids generally have molecular weight distributions of 2002000, and humic acids have much higher molecular weight distributions (1000-100 000). The understanding of NOM is important because it impacts such processes as the sorption or transformation/degradation of environmental pollutants, and it serves as precursor material to the formation of drinking water DBPs. In 1999, the American Chemical Society held a symposium on Natural Organic Matter and Disinfection By-products, which was later published in a book in 2000. In this book, Barrett et al. provides a nice overview of past research on NOM characterization and its relationship to DBP formation and control in drinking water (159). Also in this book, Leenheer et al. reported the comprehensive isolation of NOM from water for spectral characterization and testing (160). Although the focus was not on mass spectrometry, the comprehensive isolation techniques developed
are important to researchers using mass spectrometry to characterize these fractions of NOM. Prior to the development of API-MS techniques, fulvic and humic acids were studied using chemical degradation techniques, such as pyrolysis, hydrolysis, oxidation/reduction, and chemical derivatization techniques, with GC/MS for detection. However, these degradative methods alter the original form of the humic material and destroy certain functional groups. Other techniques, such as 13C nuclear magnetic resonance (NMR) spectroscopy and infrared (IR) spectroscopy, have offered nondestructive “looks” at humic and fulvic acids, but because of the complexities of the NOM mixture, these techniques have only been able to provide average “bulk” information on these molecules (such as relative contributions of carboxylic acids, aromatic functionalities, and aliphatic carbon chains). It has not been possible to obtain discrete chemical structures. However, the development of atmospheric pressure ionization techniques (APCI and ESI) provides another analytical tool that can be used to nondestructively probe NOM structure and potentially yield added structural information. Further, the use of Fourier transform ion cyclotron resonance (FTICR)-MS is enabling high resolution of the multitude of MS ions that are formed from these complex NOM mixtures. Brown and Rice used ESI-FT-ICR-MS to probe the structures of four different fulvic acids (International Humic Substances Society peat, soil, Suwannee River (GA), and Nordic aquatic fulvic acids) (161). In this study, the spray solution composition was found to have a dramatic effect on the ion distributions, with highmass aggregates (m/z 2000-4000) being formed in less polar spray solutions. Positive ion ESI-mass spectra showed average molecular weights ranging from 1700-1900, with extremely complex mass spectra (a peak at every mass). Negative ion ESIMS resulted in multiply charged ions, whose distributions were affected by the acidification of the spray solution. Klaus et al. used ESI- and APCI-MS/MS to probe the structure of humic substances and the interaction of pesticides with humic substances (162). Humic and fulvic acids were isolated from four sources in Germany for this study. In comparison to ESI, APCI produced average molecular masses a factor of 4-6 lower. This is possibly due to the breaking of charge-transfer bonds, hydrogen bonds, or weak covalent bonds between NOM molecules. Positive ionESI-MS showed average molecular masses for fulvic acids of approximately 1400; humic acids, approximately 1500. The Suwannee River humic acid reference standard was similar (1280). Kujawinski et al. used FT-ICR-ESI-MS to analyze humic and fulvic acids and resolved individual compounds within these mixtures with a resolving power of approximately 80 000 (163). Two different samples were analyzed: dissolved organic matter (primarily fulvic acids) from the Suwannee River and a humic extract from a degraded wood collected on Mt. Rainier, WA. Results showed a cluster of peaks at every nominal mass between m/z 400 and 1200 for Mt. Ranier humic acid and between m/z 300 and 900 for Suwannee River dissolved organic matter. The resolving power was high enough to separate four to eight peaks per nominal mass. Because there were few peaks observed at half the nominal mass, and isotope peaks were one nominal mass higher than the original compound, the ions appeared to be due to singly charged species. Molecular weight distributions were different for the two types of organic matter. A Kendrick mass Analytical Chemistry, Vol. 74, No. 12, June 15, 2002
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analysis applied to the data yielded additional information about the degree of oxidation and unsaturation in the molecules. The Kendrick mass deficit plot indicated that there was a significant fraction of Suwannee River humic matter with a larger aliphatic contribution, as compared to the Mt. Ranier sample; this was judged by the greater fraction with lower Kendrick mass defects. Because odd-m/z ions correspond to an even mass ([M + H]+ ions) in the positive ion mode, and because the nitrogen content is generally low in humic acids, it was more likely that odd-m/z compounds contained primarily C, H, and O. It could also be surmised from the data that the high molecular weight compounds were either more aromatic or more oxygenated (or a combination of both) than their lower molecular weight counterparts. Kendrick analysis was also used to determine the CH2 content. Many compounds fit a series of increasing CH2 units, with a small number of total units (generally 400) polyacids. In the negative ion mode, polyacids formed multiply charged species, with the number of charges dependent on the number of carboxyl groups and on molecular spacing between these groups. For the Suwannee River fulvic acid, the average molecular weight distribution was m/z 591 in the negative ion mode and m/z 617 in the positive ion mode. These distributions compared relatively well with various independent molecular weight determinations that used ultracentrifugation, vapor pressure osmometry, low-angle X-ray scattering, or GPC. MS/MS of several ions showed a major daughter ion resulting from the loss of CO2 from the (M - H)parent ion; successive losses of CO2 (two to five units) were observed, with an average CO2 unit loss of 3.4. The loss of water from the parent ion was also prominent in the MS/MS spectra. An obvious discrepancy between the newer ESI-MS data on humic acids and earlier data provided by size exclusion chromatography is the lower mass averages observed by mass spectrometry. Humic acids have long been thought to be in the 1000100 000 Da range, but ESI-MS data are showing molecular weight 2736
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ranges much lower (approximately 300-1200 Da range). There are two thoughts on this: one is that there is a low molecular weight bias inherent with ESI-MS, due to the difficulty in obtaining stable molecules with multiple charges (that there is a minimum charge separation required to enable the ion to be stable and detected) (164). The other thought is that earlier measurements involving size exclusion chromatography may have involved the measurement of noncovalently linked aggregates (possibly colloidal) of biologically derived molecules (such as fatty acids, alcohols, and sugars) (163, 165). Thus, humic acids may not be as large as size exclusion data had suggested. At this time, it is not obvious which (or both) hypothesis is correctsthere is much more research that is needed to make this determination and allow a more detailed characterization of humic material. MICROORGANISMS Recent outbreaks of waterborne illness in the United States and other parts of the world (including Escherichia coli-induced gastroenteritis in Walkerton, ON, Canada in 2000, cryptosporidiosis in Milwaukee in 1993, and cholera in Peru beginning in 1991) have necessitated improved analytical methods for detecting and identifying microorganisms in water and other environmental samples. Mass spectrometry had played a minor role in the past through the use of pyrolysis-GC/MS but is beginning to play a more important role, with increased research using MALDI-MS and ESI-MS techniques. MALDI- and ESI-MS were initially used for fingerprinting bacteria and other microorganisms, allowing different species and strains to be distinguished and identified through their characteristic protein biomarkers. In the last 2 years, however, researchers are beginning to go beyond simple fingerprinting and empirical matchingsmodeling and algorithm development, as well as complete sequencing of protein biomarkers, are now prominent elements of this research. A large number of papers has been published just within the last 5 years on the use of mass spectrometry for characterizing microorganisms, and increased interest in this area is evidenced by the number of reviews that have been published. Representative reviews are mentioned here. Fenselau and Demirev published a review of the characterization of intact microorganisms by MALDI-MS with 113 references (166). This review included a summary of the instrumentation, sample collection, sample preparation, and algorithms for data analysis. Lay published a review of the use of MALDI-TOF-MS for characterizing bacteria (167). This review included applications involving the analysis of RNA and DNA, the detection of recombinant proteins, the characterization of targeted or unknown proteins, bacterial proteomics, the detection of virulence markers, and the rapid characterization of bacteria at the genus, species, and strain level. Krishnamurthy et al. reviewed bacterial typing and identification by MS and discussed the use of automated sample processing algorithms for unambiguously identifying bacteria (168). Van Baar published a review with 159 references on the characterization of bacteria by MALDI and ESI-MS (169). Included in this review is a brief introduction to MALDI- and ESI-MS, a discussion of microorganism characterization capabilities, applications, the use of polymerase chain reaction (PCR) technology with MS, and the analysis of whole bacteria and their lysates. An outlook to future developments is also presented. Dalluge reviewed the use of mass
spectrometry for the direct determination of proteins in cells, highlighted the advantages of MS techniques over traditional biochemical methods, and provided a critical review of their utility and potential as standard tools (170). Thomas et al. published a review on mass spectrometry in viral proteomics, which included a discussion of the identification of viral capsid proteins, viral mutants, and posttranslational modifications (171). Mass spectrometry is contributing to an understanding of the dynamic domains of the viral capsid that may have significant value for developing new approaches for viral inactivation. Several authors have published papers on the development of algorithms or pattern recognition software for enabling rapid typing of bacteria and other microorganisms. Bright et al. developed a search engine to rapidly build and search databases of intact cell microorganism mass spectra (172). Jarman et al. developed an algorithm for bacterial identification using MALDIMS (173). This mass spectral fingerprint comparison algorithm is fully automated and statistically based and was demonstrated using a blind study, which showed a 90% correct identification rate. Harrington et al. constructed a neural network model to enable successful classification of targeted bacteria (174). Sensitivity and target transform factor analysis was used to validate the model to ensure that bacteria are classified by peaks in the mass spectrum that show a causal relationship to the bacteria. Demirev et al. coupled MALDI-TOF-MS to Internet-based proteome database search algorithms to aid in the identification of microorganisms (175). A procedure for using a specific and common posttranslational modification in the search algorithm is described. Accounting for posttranslational modifications in protein biomarkers improved the identification reliability by at least an order of magnitude. Mass spectrometry is also being used to obtain detailed sequence information for protein components of microorganisms and to probe their overall structure and function. She et al. used MALDI-TOF-MS to determine the complete amino acid sequence for the coat protein of brome mosaic virus (176). In this research, mutations were also noted in some isolates. Tito et al. used ESITOF-MS to analyze the intact bacteriophage MS2 virus capsid (177). A particularly clever analytical tool was used to maintain the structure of the intact capsid so that it could be studied by mass spectrometry. This was done by collisional cooling with dry nitrogen in the intermediate pressure region of the TOF mass spectrometer to reduce the internal energy of the ions. This had the effect of minimizing the explosive forces of the ESI process and cooling the ions so that they retained sufficient stability as they traversed the interior of the mass spectrometer, enabling them to reach the detector intact. In the absence of collisional cooling, no high-m/z ions could be detected. Controlled dissociations of the intact capsid could then be induced to obtain more specific structural information. In particular, a series of peaks could be assigned to the monomeric unit (13 726 Da), which is in close agreement with the calculated amino acid sequence. This procedure holds promise for the identification of unknown protein assemblies with high molecular weights and further probing of microorganism structure. Kim et al. used MALDI-TOF-MS to analyze viral membrane glycoproteins (178). Proteins having N-linked carbohydrate moieties were also observed for a particular virus.
Several other microorganism studies are worth noting. Madonna et al. developed an on-probe sample pretreatment for the detection of proteins >15 000 Da from whole cell bacteria using MALDI-TOF-MS (179). Using this method, protein signals of >20 000 Da were routinely produced from both Gram-positive and Gram-negative bacteria. In a separate paper, Madonna et al. combined immunomagnetic separation with MALDI-TOF-MS to identify bacteria (180). This method involved mixing a bacterial mixture suspension with commercially available immunomagnetic beads (coated with polyclonal antibodies) specific for the target microorganism, a short incubation period (20 min), washing off of microorganisms, resuspension in deionized water, and direct application to the MALDI probe. Liquid suspensions containing bacterial mixtures could be screened within a 1-h total analysis time. Magnuson et al. used MALDI-TOF-MS to investigate whole and freeze-thawed Cryptosporidium parvum oocysts (181). Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral marker patterns for C. parvum were distinguishable from those of Cryptosporidium muris. Demirev et al. used high-resolution tandem MS with fragmentation-derived sequence tags and sequence-similarity proteome database searching to unequivocally identify major biomarker proteins from spores (182). The combination of tandem MS of protein biomarkers with bioinformatics greatly improved the specificity of individual microorganism identification. The identification and analysis of bacteria has also been extended to on-line aerosol TOF mass spectrometers and to a new fieldable ion trap-MS (183, 184). Stowers et al. described the analysis of single biological aerosol particles using an aerosolTOF-MS (183). The inlet to the instrument was coupled to an evaporation/condensation flow cell that allowed the aerosol to be coated with matrix material as the sample stream entered the mass spectrometer. Mass spectra were generated for aerosolized spores of Bacillus subtilis and also for gramicidin-S and erythromycin. The on-line addition of matrix allowed a nearly real-time chemical analysis of the particles with an overall system residence time of
