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Published online 8 August 2008
Published in J Environ Qual 37:1817-1824 (2008)
DOI: 10.2134/jeq2007.0368
© 2008 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Organic Compounds in the Environment

New Measurements of Cyanobacterial Toxins in Natural Waters Using High Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry

Curtis J. Hedmana, William R. Kricka, Dawn A. Karner Perkinsa, Elisabeth A. Harrahyb and William C. Sonzognia,*

a Wisconsin State Lab. of Hygiene, 2601 Agriculture Drive, Madison, WI, 53718
b Dep. of Biological Sciences, 320 Upham Hall, Univ. of Wisconsin- Whitewater, Whitewater, WI 53190

* Corresponding author (sonzogni{at}wisc.edu).

Received for publication July 13, 2007.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results
 Discussion
 Conclusions
 REFERENCES
 
The presence and levels of the cyanobacterial toxins microcystin-LR, anatoxin-a, and cylindrospermopsin were measured in various Wisconsin waters where algal nuisance or bloom conditions were noted. Out of 74 samples analyzed, 36 had detectable levels of microcystin-LR (49%), and four had detectable levels of anatoxin-a (5%). Cylindrospermopsin, the toxin produced by Cylindrospermopsis (a warm water species that has been moving its range northward, including to Wisconsin), was not detected in the field samples tested. Concentrations of microcystin-LR ranged from 1.2 to 7600 µg L–1. Anatoxin-a ranged from 0.68 to 1750 µg L–1, which is the highest concentration reported from around the world. Cyanobacterial toxins, because of their high potency, deserve continued scrutiny by resource managers and public health officials responsible for recreational waters.

Abbreviations: HPLC-MS/MS, high performance liquid chromatography coupled to tandem mass spectrometry • MRM, multiple reaction monitoring • nU, natural units • USEPA, United States Environmental Protection Agency


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results
 Discussion
 Conclusions
 REFERENCES
 
CYANOBACTERIA, also known as blue-green algae, often proliferate under eutrophic conditions to extremely high levels (termed blooms in lakes). In addition to the aesthetic concerns caused by these blooms, chemicals produced by certain cyanobacterial species have been shown to elicit toxic effects on aquatic and mammalian organisms that have come into contact with them (Falconer, 1999). Incidents of cyanobacterial toxin–induced illness or death involving livestock, pets, or even humans have occurred worldwide. In Wisconsin, the Dane County (south central Wisconsin) Coroner attributed the death of a young boy to complications caused by the ingestion of cyanotoxins (Dane County Coroner's Office, 2002). Although the presence of algal toxins in Wisconsin lakes is not new (Sonzogni et al., 1988; Repavich et al., 1990; Karner et al., 2001), interest among public health officials has recently increased in Wisconsin and elsewhere around the world as reports of algal toxin poisoning increase (Kuiper-Goodman et al., 1999).

Of the fresh water cyanobacteria, Microcystis sp. and Anabaena sp. are probably best known for producing potent toxins. The planktonic Microcystis aeruginosa is common in Wisconsin lakes and produces microcystins, a large group of cyclic heptapeptide hepatotoxins with many structural variants. There have been a number of HPLC-MS/MS methods published for the analysis of microcystin variants (Rivasseau, et al., 2000; Hummert, et al., 2001; Dell'Aversano, et al., 2004; Zhang, et al., 2004). In this study, where a method for several different types of algal toxins (not just microcystin toxins) is developed and used, the variant microcystin-LR (Fig. 1a ) was focused on because it is the most frequently detected cyanobacterial toxin in environmental monitoring studies (Kotak et al., 2000; Chorus 2001). Microcystin-LR is relatively stable in the environment (Sivonen and Jones 1999). Anatoxin-a (Fig. 1b) is a toxin produced by Anabaena flos-aquae and other species and is a potent neurotoxin (Mahmood and Carmichael, 1987; Fitzgeorge et al., 1994). It is much less stable in the environment than microcystin-LR once released from the cell (Smith and Sutton, 1993).


Figure 1
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  		Fig. 1. Cyanobacterial toxin structures, empirical formulas, and molecular weights (mw).

 
Cylindrospermopsis raciborskii is a tropical cyanobacterial species that has recently been detected in temperate regions (Padisák, 1997). Its range is extending northward at an alarming rate. The species produces a toxin called cylindrospermopsin (Fig. 1c), which can affect the liver, kidneys, heart, thymus, spleen, and intestines (Hawkins et al., 1985). Once released from the cell, the toxin is relatively stable, although not as stable as microcystin-LR (Chiswell et al., 1999). Cylindrospermopsis raciborskii is of special concern because it is reported to produce toxins more frequently than other cyanobacterial species (St. Amand, 2002). Unlike many other cyanobacteria, C. raciborskii tends to not form a scum layer on lake surfaces; rather, it is distributed throughout the water column (Hawkins et al., 1997). Without the appearance of an unsightly scum, water users may be more likely to be exposed to the toxin. Thus, the spread of this exotic species has garnered the attention of public health officials.

Although techniques to measure the presence of cyanotoxins have been known for many years, a relatively rapid, accurate, and reproducible multi-toxin procedure that can be used by health officials to quickly assess whether toxins are present in a lake or reservoir has not been available. With the advent of high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS), such measurements are now possible at levels less than 1 µg L–1 (Oehrle and Westrick, 2003). The purpose of this paper is to present results of measurements of three cyanobacterial toxins: microcystin-LR, anatoxin-a, and cylindrospermopsin. Measurements were made using an analytical method that used HPLC-MS/MS to quickly and simultaneously identify and quantify the toxins. Results are discussed with respect to their importance to public health.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
 Discussion
 Conclusions
 REFERENCES
 
Chemicals and Materials
Anatoxin-a-fumerate (CAS No. 64285–06–09) was obtained from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). Microcystin-LR (CAS No. 101043–37–2) was purchased from Alexis Biochemicals (San Diego, CA). Cylindrospermopsin (CAS No. 143545–90–8) was supplied by James Sinclair of USEPA (Cincinnati, OH) and Geoffrey Eaglesham of Queensland Heath Scientific Services (Archerfield, Australia). D-phenylalanine, L-phenylalanine, and formic acid were purchased from Sigma/Aldrich Chemical Company (St. Louis, MO). Methanol was used as the solvent for the stock standards. Calibration standards were prepared by adding the stock standard solution to reagent-grade water. Reagent-grade water (<6 x 10–3 mS m–1) was obtained by use of a Purelab Plus UV/UF Laboratory Water Purification System (US Filter Co., Lowell, MA).

Sample Collection and Preparation
Surface water samples (n = 421) were collected throughout Wisconsin from late spring through early autumn during 2004 and 2005 and in late spring in 2006 by the Wisconsin Department of Natural Resources. Sampling was generally in response to an algal bloom or nuisance condition thought to be due to cyanobacteria, and the samples were brought to the Wisconsin State Laboratory of Hygiene where cyanobacteria genera were identified and enumerated using light microscopy. Table 1 lists the predominant organisms identified and their concentration range (natural units per liter [nU L–1]). The focus of this paper is on a subset of these samples that were tested for the presence of toxins. Only samples with high levels (>5 x 106 nU L–1) of cyanobacterial genera capable of producing toxins were selected for toxin analysis. These samples were from the southern part of the state, where lakes receive high nutrient inputs from agricultural and urban watersheds and are typically quite eutrophic. The resulting data should not be inferred to represent the spatial distribution of toxins in Wisconsin but rather are examples of the occurrence of cyanotoxins.


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  		Table 1. Toxin-producing cyanobacteria species found in Wisconsin surface waters, concentrations observed during the study, and toxins known to be associated with the organisms (Sivonen and Jones, 1999).

 
Samples were collected near the shoreline from the top 6 inches of water. Separate samples were collected for microscopic identification/enumeration and cyanobacterial toxin analysis. Samples (approximately 0.3 L) for toxin analysis were collected in 1-L amber glass bottles with Teflon-lined lids and immediately placed on ice for transport to the laboratory. Upon receipt at the laboratory, the samples were stored frozen at approximately –20°C until analyzed. Although samples were stored on ice and kept in a dark cooler during transport, anatoxin-a is unstable and can degrade during storage (Stevens and Krieger, 1991). Therefore, anatoxin measurements may underestimate the amount originally in the sample.

Cyanobacterial toxins were extracted from cyanobacterial cells as described previously (Eaglesham et al., 1999). All samples were subjected to three freeze/thaw cycles, which released the cyanobacterial cell contents by cell lysis. After the first freeze, the sample was thawed, and 0.025-L aliquots were transferred to 0.060-L amber glass bottles. Spike solutions were added at this time to all matrix spike samples. All aliquots were then subjected to two additional freeze/thaw cycles so that all samples were subjected to three freeze/thaw cycles. Samples were then filtered using a 0.45-µm syringe filter (Whatman 25 mm Acrodisc GHP; Pall Gelman Sciences, Ann Arbor MI). Approximately 0.0015 L of each filtrate was placed into 0.002-L amber glass HPLC autosampler vials for subsequent instrumental analysis.

HPLC-MS/MS Analysis
Cyanobacterial toxins were separated for MS/MS analysis by use of an Agilent Technologies 1100 Series high-performance liquid chromatography (HPLC) system in reversed phase mode using a 150 x 2.0 mm YMC ODS AQ 5-µm analytical column (Waters Corporation, Milford, MA). Binary gradient mobile phase conditions were used as follows: solvent A = 0.1% formic acid in reagent grade water; solvent B = 0.1% formic acid in acetonitrile (Burdick and Jackson, Morristown, NJ); flow rate = 0.3 x 10–3 L min–1; gradient program = hold at 98% A for 4 min, followed by a linear decrease to 75% A for 5 min, a further decrease to 45% A at 20 min, and then a step change to 98% A for a 10-min column re-equilibration before the next injection. A sample injection volume of 20 µL was used. Tandem mass spectrometry analysis was performed using an Applied Biosystems/MDS SCIEX API 4000 triple quadrupole mass spectrometer operating with TurboIonSpray in the positive ionization mode. Source conditions were as follows: ion spray voltage at 5500 V; curtain gas at 25 psi; nebulization gas GS1 at 20 psi; desolvation gas GS2 at 60 psi; source temperature at 500°C; and declustering potential settings at 56, 96, and 146 V for anatoxin-a, cylindrospermopsin, and microcystin-LR, respectively. Table 2 shows the molecular ion to quantitative and confirmatory daughter transitions used for the target compounds. Figure 2 shows a representative chromatogram for this method. Samples were run in batches, with the samples per batch varying to a maximum of 20.


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  		Table 2. Quantitative/confirmatory multiple reaction monitoring (MRM) transitions and ion intensity ratios (IIR) used for an initial demonstration of capability study.

 

Figure 2
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  		Fig. 2. Chromatogram of toxin standards using high-performance liquid chromatography coupled to tandem mass spectrometry.

 
A five-point linear external calibration, prepared by spiking reagent grade water with the target analytes, was used for quantitation. Nodularin, a cyanobacterial toxin produced by the brackish water cyanobacterium Nodularia, was initially evaluated as a possible internal standard (Namikoshi et al., 1994). Although nodularin is somewhat similar in structure to microcystin-LR, it is not a good surrogate for anatoxin-a or cylindrospermopsin. A comparison of results calculated with and without incorporation of the nodularin internal standard showed better check standard and matrix spike recovery performance without the internal standard. No other candidates for an internal standard were obvious, so an internal standard was not used for quantitation.

Following quality control procedures outlined in USEPA (1996) and American Public Health Association (2005), the method was first subjected to an initial demonstration of capability study. Other quality control procedures included the analysis of reagent blanks, five-point calibration line correlations, calibration verification standards, reagent water spikes, field sample matrix spikes (laboratory-fortified matrix samples), and field sample matrix spike duplicates with each batch of samples analyzed. Reporting limits of 1.0, 0.5, and 0.5 µg L–1 were used for microcystin-LR, anatoxin-a, and cylindrospermopsin, respectively. These limits were identical to the lowest standard used for calibration purposes. Criteria for the standards was a baseline to noise ratio of about 10:1 and recovery (calculated using the calibration line) of 70 to 130%. Results less than the reported limit were reported as "not detected." The current World Health Organization drinking water guideline for microcystin-LR is 1.0 µg L–1 (Gupta, 1998), the same as the reporting limit used. Drinking water guidelines for anatoxin-a and cylindrospermopsin have not been established. The method was optimized for the analysis of three toxins in one run; lower detection or reporting limits could have been obtained if the instrumental conditions were optimized for any one toxin.

All analyses were performed in a laboratory (Wisconsin State Laboratory of Hygiene) with a quality system in place. The laboratory is accredited by the National Environmental Laboratory Accreditation Conference Institute and the U.S. Environmental Protection Agency's drinking water program (among other accreditations).

Different variants exist for two of the three cyanobacterial toxins studied. There are many known variants of microcystin, and there are three known variants of anatoxin (Furey et al., 2003). The target analytes that we used for this study represent the most commonly studied variants for each cyanobacterial toxin.


   Results
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
Discussion
 Conclusions
 REFERENCES
 
HPLC-MS/MS Evaluation
The quality control tests performed indicated that the results of the toxin analysis are reliable. The mean percent recovery results from four replicate spikes of reagent-grade water, performed as part of an initial demonstration of capability, were 99.6, 104, and 97.5 for microcystin-LR, anatoxin-a, and cylindrospermopsin, respectively. Results of various quality control tests are given in Table 3 . All blank samples analyzed produced no detectable peaks for the three target analytes. All standard curves, which were run with each of eight batches of approximately 3 to 20 samples per batch, had correlation coefficients of greater than 0.9953. Spiked reagent water showed recoveries of 82 to 130%. Samples were spiked between the first and second freeze/thaw cycles. The three analytes' field sample matrix spike recoveries (including recoveries of the spiked duplicate) ranged from 77 to 124%, and duplicate analyses had differences of 0.2 to 16%. Overall, the direct aqueous injection HPLC-MS/MS method performed well.


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  		Table 3. Quality control data for the simultaneous analysis of three cyanotoxins by direct-injection high-performance liquid chromatography coupled to tandem mass spectrometry. Data were generated on each batch of samples analyzed.

 
Cyanotoxin Analysis
Out of 74 field samples analyzed for cyanobacterial toxins, 49% had reportable levels of microcystin-LR, and 5% had reportable levels of anatoxin-a. Cylindrospermopsin was not detected in any of the field samples tested for this study. The concentrations detected ranged from 1.2 to 7600 µg L–1 for microcystin-LR and from 0.7 to 1750 µg L–1 for anatoxin-a. Table 4 gives examples of toxin results over the 3-yr study. Hundreds of samples of nuisance algal conditions were collected from around the state, but not all were analyzed for toxins.


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  		Table 4. Selected high-performance liquid chromatography coupled to tandem mass spectrometry cyanotoxin results from various Wisconsin waters collected in 2004–2006 as part of this study.

 

   Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
Conclusions
 REFERENCES
 
The use of direct aqueous injection HPLC-MS/MS proved to be a selective means of monitoring for cyanobacterial toxins. The mass filtering capacity of the triple quadrupole MS/MS detector avoided problems typically experienced with UV detection or single quadrupole MS. The mass filtering capacity also resulted in a reduction in baseline noise that allowed for reporting levels of 0.5 to 1 µg L–1 (with signal to noise ratios >10:1) to be achieved by direct aqueous injection (no sample cleanup other than syringe filtration). In addition, the use of gradient HPLC elution allowed for the analysis of mixtures of analytes that vary widely in polarity and size, which is the case with microcystin-LR, anatoxin-a, and cylindrospermopsin (Fig. 1).

HPLC-MS/MS allowed for the determination of an unknown compound that was detected in both anatoxin-a multiple reaction monitoring (MRM) transitions in one of the field samples. The retention time of this compound was approximately 2.7 min later than that observed for anatoxin-a. In addition, although the suspect peak produced signals from both (quantitative/confirmatory) of the anatoxin-a MRM channels monitored, these signals were present in different ratios compared with the quantitative/confirmatory peaks for the anatoxin-a standards. Re-analysis of the sample, performed by switching from MRM MS/MS detection to Product Ion Scan mode, allowed for acquisition of daughter fragment MS/MS spectra from the unknown compound peak. Using first principles and MS/MS identification protocols published previously (McLafferty, 1980; Willoughby et al., 2002), a tentative identification of L-phenylalanine (an essential amino acid) was made. This identification was later confirmed by running a reference standard. L-phenylalanine has been reported to occur in the presence of lake microflora under aerobic conditions (Otsuki and Takahisa, 1972), although its half-life is on the order of days. Given that L-phenylalanine was detected in freshly extracted biological cell lysates, the presence of the amino acid seems reasonable. Shortly after our observation, another group studying anatoxin-a in fresh water reported finding L-phenylalanine (Furey et al., 2005). The possibility that L-phenylalanine can exist in lake samples with anatoxin-a is important because it could cause misinterpretation of mass spectral results.

The HPLC-MS/MS method developed for this study was designed to give a rapid, selective, and sensitive response for public health inquiries. It uses freeze/thaw cycling to lyse cells and release toxins. However, although this process is rapid, it may not fully solubilize the entire cyanobacterial toxin present. Microcystins remain in the cyanobacterial cells and are released from the cells as the bloom dies off (Berg et al., 1987; Watanabe et al., 1992; Sivonen and Jones 1999). Some microcystins are more lipophilic and could have a tendency to adhere to the lipids of the cell membranes. Although it was found that cyanotoxins do not bind well to sediment (Rapala et al., 1994), sorption could also be a source of low bias. A more thorough extraction using sonication and/or solvents may increase the recovery of cyanotoxins from cellular components and dissolved organic matter. Some researchers filter the surface water sample and extract the cells with sonication and solvent (Nicholson and Burch, 2001). By analyzing the filtrate and the extracted cells, the intra- versus extracellular amounts of cyanotoxins present can be quantified.

With respect to the cyanotoxins measured, our results are especially interesting when compared with measurements made by other investigators. Table 5 shows the highest cyanotoxin levels from other fresh water studies that reported toxin levels on a µg L–1 basis, along with results from this study. Although overall measurements of fresh water cyanotoxins are limited, microcystin measurements are more common than those of other cyanotoxins. The highest value for microcystin reported here is within the range of values reported elsewhere. The levels of microcystin reported in this study could contain low bias in comparison to the previous studies listed because microcystin-LR was the only variant tested in our method. To add an anecdote, two samples tested for microcystins by HPLC-MS/MS were also tested using a microcystin ELISA kit (EnviroGard Microcystins Plate Kit; Strategic Diagnostics Inc., Newark, DE) that the laboratory was coincidentally evaluating. This immunoassay was reported to be responsive, to varying degrees, to microcystin-LR and several other microcystin variants (as well as some nodularins). The immunoassay gave similar results to the mass spectral analysis, suggesting that, at least for these samples, microcystin-LR was the predominant variant.


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  		Table 5. Maximum concentrations of microcystin, anatoxin-a, and cylindrospermopsin reported from various field studies (some microcystin studies included more variants than microcystin-LR, the only variant measured in the current study).

 
To the best of our knowledge, the highest concentration of anatoxin-a observed in this study is higher than reported elsewhere by an order of magnitude. This high level came from a small pond (about 0.2 ha) located a few meters from the shores of Lake Kegonsa (near Madison, Wisconsin), a eutrophic lake that is part of the Yahara chain of lakes (Sonzogni et al., 1975; Carpenter et al., 2007). The pond is hydraulically connected to Lake Kegonsa via a short channel. The pond was sampled due to reports of dogs swimming in the pond that showed signs of poisoning by cyanotoxins. Algal counts showed the pond harbored dense populations of Anabaena sp., along with other species such as Aphanizomenon sp. Although this pond was sampled frequently over the course of the study, anatoxin-a was not always present. It is not known whether the high value reported in Table 4, which came from a sample taken in late June of 2006, represents a peak amount or is a common occurrence. Continued study and monitoring is important because the concentration measured far exceeds the unofficial health guideline of 3 µg L–1 (Carmichael, 2001). Moreover, a human death occurred in Madison, Wisconsin when an individual swallowed golf course pond water containing cyanobacteria capable of producing anatoxin-a (Dane County Coroner's Office, 2002). Although the concentration of anatoxin-a in the water at the time of the victim's exposure is not known, Anabaena sp. cells were found in the victim's stool, so concern about anatoxin-a seems warranted.

For anatoxin-a, the primary concern for recreational exposure is acute toxicity because it is rapidly metabolized and does not bioaccumulate (Fawell et al., 1999), whereas microcystins accumulate quickly in the liver and are not readily metabolized or degraded within the body (Robinson et al., 1991; Kuiper-Goodman et al., 1999). After review of the "no observable adverse effect" level and "lowest observed adverse effect" level data from animal toxicity experiments (Falconer et al., 1994), the World Health Organization determined a tolerable daily intake level of 0.04 µg kg–1 of body weight for microcystin (Gupta, 1998). For the highest microcystin-LR concentration detected during this study (7600 µg L–1), this tolerable daily intake level would be exceeded if a child weighing 10 kg ingested only 0.052 x 10–3 L of this water. This information, coupled with the fact that microcystins can persist for weeks or longer in the environment (Tsuji et al., 1993; Jones et al., 1995; Lahti et al., 1997), raises concern for repeated low-level ingestions that could cumulatively cause liver damage via recreational exposure (Fitzgeorge et al., 1994). The environmental and/or genetic factors that cause cyanobacterial strains to produce toxins are under study (Lehtimäki et al., 1997; Rapala and Sivonen, 1998; Kotak et al., 2000; Dittmann and Wiegand, 2006), but cyanobacterial toxin production cannot be predicted simply by observation of a bloom event. Many samples in our study with high cyanobacterial counts were not associated with correspondingly high cyanotoxin concentrations.

Cylindrospermopsin was not found in any samples. Although C. raciborskii was only recently reported to occur in Wisconsin, this species was identified in a number of Wisconsin lakes in this study. For example, Twin Valley Lake, a popular recreational lake located in southern Wisconsin, was found to have concentrations of 1 x 107 nU L–1 of Cylindrospermopsis sp. in the summer of 2004. It is not known why the toxin was not detected where high concentrations of Cylindrospermopsis sp. were observed. It is possible it was produced but at concentrations below our reporting limit (0.5 µg L–1). It is also possible the environmental conditions or genetic make-up necessary for toxin production were not present. However, given the establishment of Cylindrospermopsis sp. in several Wisconsin surface waters, the nonsurface forming nature of Cylindrospermopsis sp., and the continued possibility of cylindrospermopsin production, the spread of Cylindrospermopsis sp. remains a concern.

Samples were analyzed as part of a long-term investigation of nuisance conditions. Cyanotoxins were measured for only a small proportion of the hundreds of samples that were obtained for algal identification and enumeration (Microcystis sp., Anabaena sp., and Aphanizomenon sp. were the most common cyanobacteria counted). Also, sampling was not random but was targeted at lakes experiencing nuisance bloom conditions. Nevertheless, most lakes in the southern half of Wisconsin are eutrophic, so the data confirm that cyanotoxins can exist at relatively high concentrations in these lakes.

The results of cyanotoxin analyses in this study were sometimes used by Wisconsin public health officials in deciding whether to close a swimming beach or post warnings during bloom conditions. The analytical technique described here has utility for public health assessment with respect to the presence of cyanobacterial toxins. Clearly, cyanobacteria toxins at levels reported represent a threat to public health, so it is important to know when toxins are present and at what concentration.


   Conclusions
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
 Conclusions
REFERENCES
 
1. Microcystin-LR was found more often than the other two cyanotoxins in the Wisconsin waters studied (sampled during nuisance bloom conditions). Levels were in the range reported by other investigators worldwide.

2. A sample from a small pond was found to contain more anatoxin-a than previously reported by other investigators. The public health significance of such levels of anatoxin-a, a potent neurotoxin, should not be underestimated.

3. Although Cylindrospermopsis sp. was found in several Wisconsin lakes, one of the toxins produced by this exotic cyanobacterium, cylindrospermopsin, was not detected in any of the samples tested.

4. Analysis of water samples for the presence of cyanotoxins is important from a public health perspective. Direct aqueous injection (no sample cleanup) HPLC-MS/MS analysis can be useful for rapidly detecting three different cyanobacterial toxins—microcystin-LR, anatoxin-a, and cylindrospermopsin—particularly when rapid results are needed to assess safety. More information is needed on what triggers toxin production so predictive models can be developed to forecast the presence of toxins and the public can be warned about possible exposure. Additional cyanotoxins, such as other microcystin variants, lyngbyatoxins, and representative saxitoxins, should be considered in monitoring programs.


   ACKNOWLEDGMENTS
 
We thank Geoffrey Eaglesham of Queensland Health Sciences, and James Sinclair of the USEPA for supplying our laboratory with cylindrospermopsin standard solutions used for this study. The we also thank Chris Kaisershot of MDS/SCIEX for his assistance in instrument operation.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
 Conclusions
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
 Conclusions
 REFERENCES
 





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