MS Method for the Determination of Cyanobacteria Toxins in

of 5000; and (d) recoveries of the five toxins from reagent water, river waters, ... for the Determination of Extracellular Hepatotoxins in Irish ...
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Anal. Chem. 2004, 76, 1342-1351

A LC/MS Method for the Determination of Cyanobacteria Toxins in Water Mila Maizels†

Oak Ridge Institute for Science and Education, 26 West Martin L. King Jr. Drive, Cincinnati, Ohio 45268 William L. Budde*

U.S. Environmental Protection Agency, 26 W. Martin L. King Jr. Drive, MS-593, Cincinnati, Ohio 45268

The cyanobacteria toxins anatoxin-a, microcystin-LR, microcystin-RR, microcystin-YR, and nodularin were separated in less than 30 min on several 1 mm × 15 cm reversed-phase liquid chromatography (LC) columns, and their electrospray mass spectra were measured using injections of 50 ng or less with a benchtop time-of-flight (TOF) mass spectrometer. New data from this work include the following: (a) the impact of acetic acid concentrations in the methanol-water mobile phase on measured ion abundances; (b) the performance of the electrospray-TOF mass spectrometer as an LC detector; (c) the accuracy and precision of exact m/z measurements after LC separation with a routinely used mass spectrometer resolving power of 5000; and (d) recoveries of the five toxins from reagent water, river waters, and sewage treatment plant effluent samples extracted with C-18 silica particles enmeshed in thin Teflon membrane filter disks. This technique has the potential of providing a relatively simple and reasonable-cost sample preparation and LC/ MS method that provides the sensitivity, selectivity, reliability, and information content needed for source and drinking water occurrence and human exposure studies. Several classes of toxic compounds are produced by various genera, species, and strains of cyanobacteria, and these substances have been investigated for about the last 15-20 years.1 Results to date include the isolation of many specific substances from both bacterial cells and water, the evaluation of toxicities, structure determinations, analytical methods development, occurrence surveys, and measurements of toxin stabilities in the aqueous environment. Besides the desire for basic knowledge of natural products, this research is stimulated by a concern for the impacts of these toxins on wildlife, pets, and farm animals and by the potential for human exposure through drinking and recreational waters, with subsequent adverse health effects. Structures have been proposed for over 20 alkaloid neurotoxins, including the * Corresponding author. E-mail: [email protected]. Fax: 513-569-7757. Voice: 513-569-7309. † Present address: Quintiles, Inc., 10245 Hickman Mills Drive, Kansas City, MO 64137. (1) Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, Monitoring and Management; Chorus, I., Bartram, J., Eds.; E & FN Spon: London, England, 1999.

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saxitoxin paralytic shellfish poisons,1,2 and over 60 microcystin and nodularin cyclic peptide hepatotoxins.1,3-7 Most of these structures are reasonably well established. During the spring of 2001, The U.S. Environmental Protection Agency (USEPA) convened a panel of scientists to assist in identifying a target list of cyanobacteria toxins that are likely to pose a health risk in source and finished waters of the drinking water utilities in the United States. The meeting summary contains background information on the USEPA drinking water contaminant candidate list, known toxins and the cyanobacteria species that produce them, health effects, occurrence, treatment options for removal of cyanobacteria and toxins from water, the stabilities of toxins, and analytical methods.8 The toxins recommended for further research with highest priority are the neurotoxins anatoxin-a (1) and cylindrospermopsin (2) that are shown in Scheme 1 and the cyclic heptapeptide microcystin hepatotoxins.8 Within the latter group, microcystin-LR (3a) is shown in Scheme 1 and Table 1, and it is the most frequently reported microcystin.1 Microcystin-LA (3b), microcystin-RR (3c), and microcystin-YR (3d) in Table 1 are also often reported. To conduct large-scale occurrence surveys and human exposure studies, reliable, sensitive, and quantitative analytical methods are required for the priority toxins. Analytical costs would be minimized if the priority toxins could be determined in a single efficient analytical method. Further benefits would accrue if the single analytical method was capable of accurately recognizing other cyanobacteria toxinssfor example, those in Table 1sthat may occur in unexpected frequencies and concentrations in water. A variety of analytical techniques for cyanobacteria toxins have been explored. These include mouse and invertebrate bioassays,1 protein phosphatase inhibition assays,1,9 immunoassays,1,9-11 and (2) Locke, S. J.; Thibault, P. Anal. Chem. 1994, 66, 3436-3446. (3) Eckart, K. Mass Spectrom. Rev. 1994, 13, 23-55. (4) Lawton, L. A.; Edwards, C.; Codd, G. A. Analyst 1994, 119, 1525-1530. (5) Namikoshi, M.; Sun, F.; Choi, B. W.; Rinehart, K. L.; Carmichael, W. W.; Evans, W. R.; Beasley, V. R. J. Org. Chem. 1995, 60, 3671-3679. (6) Namikoshi, M.; Yuan, M.; Sivonen, K.; Carmichael, W. W.; Rinehart, K. L.; Rouhiainen, L.; Sun, F.; Brittain, S.; Otsuki, A. Chem. Res. Toxicol. 1998, 11, 143-149. (7) Robillot, C.; Vinh, J.; Puiseux-Dao, S.; Hennion, M.-C. Environ. Sci. Technol. 2000, 34, 3372-3378. (8) Creating a Cyanotoxin Target List for the Unregulated Contaminant Monitoring Rule; U.S. Environmental Protection Agency Technical Service Center, 26 W. Martin Luther King Drive, Cincinnati, OH 45268, May 17-18, 2001. 10.1021/ac035118n CCC: $27.50

© 2004 American Chemical Society Published on Web 01/24/2004

Scheme 1

Table 1. Names, Monoisotopic Molecular Masses (MM), and Abbreviated Structures of 3a-3d and 10 Analogous Microcystins That Differ from 3a or 3d by Substitution of the Indicated Variable Amino Acid Residues

3a

3b 3c 3d

a

name

MMa

abbreviated structure

refs

microcystin-LR microcystin-LW microcystin-LF microcystin-LM microcystin-LA microcystin-LY microcystin-LL microcystin-RR microcystin-YR microcystin-FR microcystin-AR microcystin-WR microcystin-YA microcystin-YM

994.548 77 1024.526 97 985.516 07 969.488 15 909.484 77 1001.510 99 951.531 72 1037.565 82 1044.528 04 1028.533 12 952.501 82 1067.544 02 959.464 04 1019.467 41

cyclo(Ala-Leu-MeAsp-Arg-Adda-Glu-Mdha) -Leu-Trp-Leu-Phe-Leu-Met-Leu-Ala-Leu-Tyr-Leu-Leu-Arg-Arg-Tyr-Arg-Phe-Arg-Ala-Arg-Trp-Arg-Tyr-Ala-Tyr-Met-

3-5 4, 19 4, 19 19 3, 4 3, 4 7 3-5, 18 3, 4, 18 3-5, 18 3, 5, 18 3, 4 3 3

Monoisotopic molecular mass calculated using the masses of the isotopes with the largest natural abundances.

reversed-phase liquid chromatography (LC) separations combined with various detection techniques.1 The LC detection techniques include ultraviolet (UV) absorption at a fixed frequency,12,13 (9) Metcalf, J. S.; Bell, S. G.; Codd, G. Appl. Environ. Microbiol. 2001, 67, 904909. (10) Rivasseau, C.; Racaud, P.; Deguin, A.; Hennion, M.-C. Environ. Sci. Technol. 1999, 33, 1520-1527. (11) Fischer, W. J.; Garthwaite, I.; Miles, C. O.; Ross, K. M.; Aggen, J. B.; Chamberlin, A. R.; Towers, N. R.; Dietrich, D. R. Environ. Sci. Technol. 2001, 35, 4849-4856. (12) Wicks, R. J.; Thiel. P. G. Environ. Sci. Technol. 1990, 24, 1413-1418. (13) Bateman, K. P.; Thibault, P.; Douglas, D. J.; White, R. L. J. Chromatogr., A 1995, 712, 253-268.

continuous measurement of UV spectra with a photodiode-array detector,4 continuous measurement of complete mass spectra,13,14 selected ion monitoring (SIM) mass spectrometry (MS),13,14 and several tandem (MS/MS) mass spectrometric techniques. Tandem techniques include product ion scans,13,15 precursor ion scans,13 and selected reaction monitoring (SRM) or, as it is sometimes called, multiple reaction monitoring (MRM).13,15 Separation by capillary electrophoresis with UV and MS detection has (14) Barco, M.; Rivera, J.; Caixach, J. J. Chromatogr., A 2002, 959, 103-111. (15) Pietsch, J.; Fichtner, S.; Imhof, L.; Schmidt, W.; Brauch, H.-J. Chromatographia 2001, 54, 339-344.

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also been reported.13 Generally these analytical techniques also require sample preparation to eliminate or minimize interferences and to concentrate the sample to achieve desirable detection limits. All the reported methods have deficiencies that limit their applicability to large-scale occurrence surveys and human exposure studies. Briefly, the time-consuming mouse bioassay cannot distinguish among a large number of potential toxins and requires sacrificing many animals. The phosphatase inhibition assay is only applicable to the microcystin hepatotoxins and, like immunoassays, cannot distinguish among the more than 60 known structures. Separate enzyme inhibition and immunoassays are required for the neurotoxins and microcystins. While LC is capable of separating the cyanobacteria toxins, the UV detector has many deficiencies. It is not sufficiently sensitive, many natural aquatic interferences absorb in the UV region, the 60 microcystins all have very similar UV spectra, and the neurotoxins either have weak UV spectra or do not absorb in the UV. Acquisition of complete mass spectra as compounds elute from an LC column allows reliable identifications of many analytes. However, this approach is less useful when few ions are produced, as in electrospray, and sensitivity is limited because some ions are not detected during scanning with magnetic deflection and quadrupole mass spectrometers. The SIM and SRM/MRM data acquisition techniques provide great signal/noise enhancement, selectivity, and quantitative capabilities, but are limited to targeted compounds whose ions are designated for monitoring during various elution periods. The purpose of the research reported in this paper was to explore and demonstrate a relatively simple and reasonable-cost sample preparation and LC/MS approach that has the potential of providing the reliability, sensitivity, selectivity, information content, and quantitative capabilities needed for large-scale occurrence and exposure studies. Toxins are partitioned from water samples with extraction disks containing very fine C-18 silica particles enmeshed in a thin Teflon matrix.16 The eluate from the disks is analyzed with LC/MS using electrospray ionization, as do the other LC/MS techniques that have been reported. However, in contrast to previous studies, benchtop time-of-flight (TOF) mass spectrometry is employed with continuous fullspectrum data acquisition and real-time exact m/z measurements. With this LC/MS technique, there is no need to specify target analyte ions for SIM or SRM/MRM and no need for optimization of collision energies for each target analyte ion monitored. Excellent sensitivity is provided by the high duty cycle of the TOF analyzer, that is 10 000 ion pulses/s, which ensures that few ions are lost or discarded in the ion pulsing region. Furthermore, no ions are deliberately discarded or discharged in the TOF analyzer. Selectivity and confirmation of an ion’s composition is supported by routine exact m/z measurements of all ions at a consistently available and used resolving power of ∼5000. The goal of this research is a method that will allow the assessment of an environmental water sample, or a related sample such as urine, in ∼1 h including sample preparation, chromatography, and data processing time. For this report, the approach was tested with the priority toxins anatoxin-a and microcystins LR, RR, and YR. However, other cyanobacteria toxinssfor example, nodularins that may occur in water samples can be recognized as compounds

of interest and often identified because of the high-sensitivity fullspectrum data acquisition with exact m/z measurements of all ions. The priority toxin microcystin-LA was not available for this work but very likely will be amenable to the same technique. Only very small quantities of microcystins LF and LW and the alkaloid cylindrospermopsin were available; these amounts were sufficient for the estimation of LC/MS retention data but not sufficient for the evaluation of recoveries from water.

(16) Hagen, D. F.; Markell, C. G.; Schmitt, G. A.; Blevins, D. D. Anal. Chim. Acta 1990, 236, 157-164.

(17) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 26422646.

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EXPERIMENTAL SECTION Materials and Apparatus. Anatoxin-a fumarate was obtained from Sigma-Aldrich (St. Louis, MO). Microcystins LF, LR, LW, RR, and YR and nodularin were obtained from Calbiochem (San Diego, CA). The supplier stated 95+% purity by high-performance liquid chromatography, and all were used without further purification. A sample of cylindrospermopsin was a gift from Professor Wayne Carmichael of Wright State University, Dayton, OH. Methanol, Optima grade with a 99.9+% purity, and glacial acetic acid, Optima grade with a 99.5% purity, were obtained from Fisher Scientific (Fair Lawn, NJ). Water was from a Millipore (Bedford, MA) Milli-Q water system. Empore 47-mm C-18 extraction disks and an apparatus to support the disks and provide vacuum assist during the extraction were obtained from Varian Instruments (Walnut Creek, CA). Standard Solutions. Single-analyte solutions of anatoxin-a and microcystins LR, RR, and YR and nodularin were prepared in 50% (v/v) methanol-water at a concentration of 250 µg/mL. Similar solutions of microcystins LF and LW were prepared at a concentration of 25 µg/mL. Aliquots of 100-400 µL of the individual analyte solutions were mixed and diluted with 50% (v/v) methanol/ water to prepare standard solutions of multiple analytes at concentrations of 5-50 µg/mL of each analyte. All the analyte standard solutions were stored in 10-mL vials equipped with Teflon-lined caps in a freezer at -20 C. Liquid Chromatography. Separations were conducted with a Micro-Tech Scientific (Sunnyvale, CA) Ultra-Plus II liquid chromatograph with either a Phenomenex (Torrance, CA) 1.0 mm (i.d.) × 150 mm C18 LUNA 3-µm column or a phenyl-hexyl 3-µm column with the same dimensions from the same supplier. The mobile-phase composition generally was held at 95% water-5% methanol for 2 min, linearly programmed to 95% methanol in 25 min, and then held at 95% methanol for 5 min; however, minor variations of this gradient elution were also used. For most experiments, both the water and the methanol contained 0.006% (v/v) acetic acid (1 mM); however, a range of acid concentrations was also evaluated. The pH of the water phase containing 0.006% acetic acid was 4. The injection volume was 10 µL, generally containing 50 ng of each analyte, and the flow rate was 35 µL/ min. At the conclusion of an LC/MS analysis, the column was flushed with 95% water-5% methanol for 15 min before the next injection. Mass Spectrometer. The TOF mass spectrometer was an Applied Biosystems (Framingham, MA) Mariner benchtop instrument equipped with an electrospray ion source. The electrospray design is similar to that described by Bruins et al.17 A high voltage (HV) is applied to an inner stainless steel tube containing the

Figure 1. Diagram of the electrospray interface to the TOF mass spectrometer showing the positions of the electrospray needle, nozzle, first skimmer, ion focusing quadrupole, and the flows of nitrogen gas.

mobile phase, and nitrogen gas flows through an outer concentric tube to provide pneumatic assistance (Figure 1). The HV was +3000, and the nebulizing gas flow rate was 0.4 L/min. A flow of heated nitrogen was also maintained at 0.6 L/min countercurrent to the aerosol spray to promote evaporation of solvent molecules from the charged droplets and desolvation of the positive ions (Figure 1). The spray needle was on a ∼45° horizontal angle to the plane of the face plate that contained an orifice leading to the mass spectrometer, and the tip of the needle was located ∼1 cm from the orifice. The exact angle and distance was adjusted for optimum ion detection and spray stability. Ions are injected orthogonal to the flight tube through a series of orifices and ion focusing lenses. The nozzle, first skimmer, and an ion focusing quadrupole are shown in Figure 1 with typical dc voltage ranges. The nozzle voltage was usually either 180 or 300 V, but 90 and 230 V were used during an evaluation to find the optimum values. The nozzle and quadrupole temperatures were 140 °C. Inside the high vacuum of the spectrometer (not shown in Figure 1), push and pull electrodes accelerate the ions in batches at 10 000 pulses/s into the flight tube. The field free flight tube is 0.6 m followed by a second-order electrostatic mirror and a return path to a microchannel ion detector giving an effective path length of 1.3 m for the TOF analyzer. A resolving power of ∼5000 based on the full peak width at half peak height definition was routinely available and used in these experiments. The analyzer mass range was set at 50-1100 Da, and individual spectra were accumulated over a period of 2 s each. The mass spectrometer was calibrated for exact mass measurements by flow injectionsusing the spectrometer’s internal syringe pumpsof a solution containing the peptides angiotensin I, bradykinin, and neurotensin (Sigma Chemical) in 50% (v/v) acetonitrile-water. The injection flow rate was 35 µL/min, and the other conditions were the same as in the LC/MS experiments. The [M + 3H]3+ ions of the peptides at m/z 432.8998, 354.1944, and 558.3105, respectively, were used to calibrate the instrument, and the instrument calibration was stable for many months. No lock mass or internal standard was used to stabilize the exact m/z calibration. Environmental Samples. Samples were collected by a sampling team from the U.S. Geological Survey during September 2002 from the west fork of the Little River near Clermont, GA (labeled GA river), the Washite River near Dickson, OK (labeled OK river), and the treated effluent of a sewage treatment plant located on the South Platte River above Clear Creek near

Commerce City, CO (labeled CO STP). The samples were stored at 5 °C until used. Fortification of Water Samples. To 100 mL of reagent watersor an environmental water sampleswas added 200 µL of a solution containing 50 µg/mL anatoxin-a, microcystins LR, RR, and YR, and nodularin to give a water sample with a concentration of 100 µg/L for each analyte. Samples were allowed to acclimatize to the water matrix at room temperature for varying periods ranging from several hours for the reagent water samples to ∼24 h for the environmental water samples. Extraction Procedure for Aqueous Samples. The water samples were adjusted to pH 10 with pH paper by addition of a few drops of 1 M sodium hydroxide in water. The extraction disks were conditioned by soaking in 10 mL of methanol for ∼5 min, and the methanol was flushed from the disks with 10 mL of water. Before the disks dried, the 100-mL water samples were poured into the sample reservoir and drawn through the extraction disks with a slight vacuum assist. Extraction times varied from ∼5 min for the reagent water samples to ∼20 min for the river waters and the treated sewage treatment plant effluent. The disks were dried by drawing laboratory air through them for ∼15 min, and the analytes were eluted with 5-10 mL of methanol. The eluate volumes were reduced to 2 mL with a gentle stream of dry nitrogen. Assuming 100% recovery, the concentrations of the analytes in the eluates would be 5 µg/mL. Recovery Measurements. Recoveries were determined after calibration of the LC/MS with a standard solution containing 5 µg/mL of each analyte. Most analytes were not available in sufficient quantities to allow a multipoint calibration or the determination of the linearity and dynamic range of the measurements. This information will be acquired in future studies. RESULTS AND DISCUSSION Anatoxin-a (1), molecular weight 165, and cylindrospermopsin (2), molecular weight 415, are shown in Scheme 1. Microcystins have molecular weights in the 900-1100 range and are cyclic peptides containing seven amino acid residues. Besides some common natural amino acids, and some closely related structures, the cyclic peptide toxins contain the unusual amino acid 3-amino9-methoxy-2,6,8-trimethyl-10-phenyldecadienoic acid (Adda). Microcystin-LR (3a) shown in Scheme 1 is the prototype of the cyclic heptapeptide toxins and the most frequently reported microcystin.1 About one-fifth of the more than 60 known microcystins are the result of substitutions of the amino acids Leu (variable AA1) and Arg (variable AA2) in 3a by common natural amino acids. Table 1 contains the names, monoisotopic molecular masses, and abbreviated structures of 3a and 13 reported microcystins that differ from 3a by substitution of the Leu or the Arg residues, or both, with the indicated amino acids. Usually the Leu or the Arg is conserved in these structures, but a few known toxins have both of the variable residues replaced. Most of the other reported microcystins have structures that are minor modifications of the most common microcystinssfor example, there are 19 reported variations of 3a, 5 of 3c, and 8 of 3d. These minor modifications are often methylations or demethylations such as substitution of a dehydroalanine residue for an N-methyldehydroalanine (Mdha), substitution of an aspartic acid residue for the β-methylaspartic acid (MeAsp) residue, substitution of a homotyrosine for a tyrosine, or methyl esterification of the Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Chart 1

glutamic acid. In some microcystins, a serine or methylserine is substituted for the Mdha or Ala residues, and various combinations of the minor modifications are also reported. Nodularins have similar structures but are pentapeptides containing the amino acids Adda, Glu, and Ala. Only a few of these are known and they are found predominately in brackish water which is not often used as a source for drinking water. Nodularin (4) shown in Chart 1 was available for this study and was included as a surrogate for other cyanobacteria toxins that could be present in unexpected quantities in various water samples. Analytical Approach. The procedure used in this work to extract toxins from water samples differs from those procedures reported previously. Most investigators have used solid-phase extractionssmore precisely liquid-solid extractionssto separate toxins from water and concentrate them in small volumes of an organic solvent eluate, usually methanol. Commercially available 0.5-6-mL plastic cartridges typically packed with 0.1-1.0 g of bonded C-18 silica, or other liquid chromatography stationaryphase materials, have been used by several investigators for extractions and concentrations prior to LC/UV, LC/MS, and even immunoassay determinations.4,10,11,13,14 These procedures work reasonably well, but sample flow rates through the packed cartridges can be fairly low. Up to 1 h or more may be required to process a 500 mL-1 L water sample even with a vacuum assist. Water samples containing particulates or high molecular weight humic materials often clog those cartridges and that can prevent the successful completion of a sample analysis. Traditional liquidliquid extractions are not effective because of the low solubility of some toxins in water-immisible organic solvents. A cloud-point extraction has been applied to microcystins; however, the surfactants used in this procedure would be a significant limitation with electrospray and mass spectrometry detection.20 Aqueous methanolmodified supercritical carbon dioxide has been used to extract microcystins from freeze-dried bacterial cells, but that procedure is not applicable to water samples.21 (18) Namikoshi, M.; Rinehart, K. L.; Sakai, R.; Stotts, R. R.; Dahlem, A. M.; Beasley, V. R.; Carmichael, W. W.; Evans, W. R. J. Org. Chem. 1992, 57, 866-872. (19) Lawton, L. A.; Edwards, C.; Beattie, K. A.; Pleasance, S.; Dear, G. J.; Codd, G. A. Nat. Toxins 1995, 3, 50-57. (20) Man, B. K.-W.; Lam, M. H.-W.; Lam, P. K. S.; Wu, R. S. S.; Shaw, G. Environ. Sci. Technol. 2002, 36, 3985-3990. (21) Pyo, D.; Shin, H. Anal. Chem. 1999, 71, 4772-4775.

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In this exploratory study, toxins were partitioned from water samples using 47-mm-diameter circular extraction disks containing very fine, bonded C-18 silica particles enmeshed in a thin Teflon matrix.16 This technique is an option in EPA Method 525.2 that has been evaluated statistically with 118 small-molecule analytes including pesticides, phenols, plasticizers, and polycyclic aromatic compounds.22 With a modest vacuum assist, 500 mL-1 L of lowparticulate water sample can be processed in as little as 10 min, and most other water samples are extracted with minimal impedance to sample flow rate. An accurate drawing of the commercially available apparatus used to support the disk during a vacuum-assisted extraction has been published.23 No other types of extraction disks were evaluated because sufficient quantities of some analytes were not available. Liquid Chromatography/Mass Spectrometry. The LC/MS approach used in this work is microbore LC with 1-mm-inside diameter (i.d.) reversed-phase columns, electrospray, and TOF mass spectrometry. The 1-mm columns were selected to provide a higher sample peak concentration than conventional wider bore LC columns, and to allow microliter per minute LC flow rates that provide efficient introduction of ions into the mass spectrometer with electrospray. At a flow rate of 35 µL/min, no splitting of the effluent is necessary, and the entire LC flow is directed into the electrospray needle interface to the mass spectrometer. The TOF spectrometer is a relatively low-cost benchtop instrument with a routinely available and used resolving power, that is m/∆m, of ∼5000 that provides exact m/z measurements to support routine identifications when one or only a few ions are observed in a mass spectrum. The toxins were partially or completely separated with either a C-18 or a phenyl-hexyl column using a gradient elution with a methanol/water mobile phase containing a small amount of acetic acid. The linear gradient is from 95% water to 95% methanol in 25 min at constant acetic acid concentration. Anatoxin-a elutes in ∼3 min and three microcystins (RR, LR, YR) and nodularin elute in the range of 20-30 min. Figure 2 shows total ion chromatograms (TICs) for the separations on the two columns using the same methanol/water gradient elution. The C-18 column gives a better separation with the conditions used, but nodularin does not elute until ∼28.7 min. The phenyl-hexyl column did not give a complete separation of microcystins YR and LR and nodularin with the conditions used, but their amounts can be readily determined by extracting the integrated abundances of their characteristic ions from the TICs. Mean retention times and mean peak widths for anatoxin-a and four cyclic peptide toxins on the C-18 column are given in Table 2. The relative standard deviations in Table 2 are from three measurements and indicate excellent precision for these LC data. The retention of anatoxin-a was most variable, probably because it is not well retained on the column, and the mean peak widths of the toxins in Table 2 generally increase with retention time. Only very small quantities of microcystins LF and LW and cylindrospermopsin were available, which precluded other experiments with them. Their retention times in Table 2 were determined from a single measurement. Mass Spectra of Toxins. The electrospray mass spectra of the toxins depend on interface conditions, including the potential (22) Budde, W. L. Analytical Mass Spectrometry: Strategies for Environmental and Related Applications; Oxford University Press: New York, 2001. (23) Ferrer, I.; Barcelo´, D.; Thurman, E. M. Anal. Chem. 1999, 71, 1009-1015.

Figure 2. Total ion chromatograms of the separation of anatoxin-a and four cyclic peptide toxins on C-18 silica and phenyl-hexyl silica columns with the same amounts of each analyte injected (50 ng) and the same methanol/water gradient elution Table 2. Mean Retention Times, Mean Peak Widths, and Corresponding Relative Standard Deviations (RSD) for Anatoxin-a and Four Cyclic Peptide Toxins on a C-18 Column with a Methanol-Water Gradient Elution analyte anatoxin-a cylindrospermopsin microcystin-RR microcystin-YR microcystin-LR nodularin microcystin-LW microcystin-LF a

mean retention time (min)

RSDb (%)

mean peak width (s)

RSDb (%)

1.95

27

3.76

0.93 0.95 0.93 0.60

22 46 36 54

3.09 3.39 4.17 5.23

2.96 10.5a 21.6 26.5 26.9 28.7 39.4a 41.0a

From a single measurement. b n ) 3.

difference between the nozzle and first skimmer (Figure 1). Relative abundances of the major ions in the electrospray mass spectra of anatoxin-a and four cyclic peptide toxins as a function of the nozzle voltage, with the other LC/MS conditions constant, are shown in Table 3. Generally, the [M + H]+ ions are the base peaks except for microcystin-RR, which has two basic arginine residues, and gives a base peak [M + 2H]2+ ion. Increasing the nozzle potential from 90 to 300 V tends to decrease the population of [M + 2H]2+ ions of the microcystins, although this effect is small with microcystin-RR. In addition, some fragment ions are produced through collision-induced dissociation. With a nozzle voltage of 300, anatoxin-a gives a 10-50% relative abundance [M - 17]+ ion at m/z 149 that is postulated to form by the loss of NH3 from the [M + H]+ ion at m/z 166 as shown in Scheme 2. Cylindrospermopsin produces a 25% relative abundance [M - 80]+ ion at m/z 336 that is formed by the loss of SO3 from the [M + H]+ ion at m/z 416. Microcystins give an ion at m/z 135 that has been identified as the [phenyl-CH2 - CH(OCH3)]+ ion that is formed by fragmentation of the [M + 2H]2+ ion by cleavage

of the Adda residue between C-8 and C-9.24 The spectrum of microcystin-RR contained a 40% relative abundance m/z 135 ion; however, the spectra of microcystins LR and YR had m/z 135 ions less than 10% relative abundance, and nodularin did not give a m/z 135 ion. Some other low-abundance (∼5% or less) ions were observed in the spectra of microcystins RR and LR when measured with a nozzle potential of 300 V. These were generally fragment ions formed by cleavages of ring peptide bonds, and they have been identified previously and used to determine the sequences of amino acid residues in the cyclic peptides.24 For subsequent work, nozzle potentials of 180 or 300 V were generally used. When it was desired to observe higher abundances of [M + 2H]2+ ions, the lower potential was used. When it was desired to promote fragmentation of molecular ionssfor example, to ensure the presence of fragment ions to support identifications of the specific compounds in environmental samplessthe higher nozzle potential was used. Mobile-Phase Matrix Effects. The test analytes generally give [M + H]+ ions, and some also give [M + 2H]2+ ions with electrospray. Therefore, it would be expected that, by increasing either the strength or the concentration of an acid in the mobile phase, the equilibrium concentrations of these ions in solution would be increased, and the abundances of these ions in the gas phase would also be increased. An exception is anatoxin-a that has a measured pKa of 9.36 and would be 99% protonated at a pH of 7.2.25 However, some strong organic acids, for example, trifluoroacetic acid (TFA), can have the opposite effect and effectively neutralize analyte ions through the formation of ion pairs with the trifluoroacetate anion.26 Ion pairing also can reduce the mean charge state of multiple-charged peptide and protein (24) Yuan, M.; Namikoshi, M.; Otsuki, A.; Rinehart, K. L.; Sivonen, K.; Watanabe, M. F. J. Mass Spectrom. 1999, 34, 33-43. (25) Koskinen, A. M. P.; Rapoport, H. J. Med. Chem. 1985, 28, 1301-1309. (26) Kuhlmann, F. E.; Apffel, A.; Fischer, S. M.; Goldberg, G.; Goodley, P. C. J. Am. Soc. Mass Spectrom. 1995, 6, 1221-1225.

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Table 3. Relative Abundances of the [M + H]+ and [M + 2H]2+ Ions in the Electrospray Mass Spectra of Anatoxin-a and Four Cyclic Peptide Toxins as a Function of the Nozzle Voltage nozzle ) 90 V

nozzle ) 180 V

nozzle ) 230 V

nozzle ) 300 V

analyte

[M+H]+

[M+2H]2+

[M+H]+

[M+2H]2+

[M+H]+

[M+2H]2+

[M+H]+

[M+2H]2+

anatoxin-a microcystin-RR microcystin-YR microcystin-LR nodularin

100 4 100 100 100

0.4 100 23 35 0

100 2.5 100 100 100

0 100 42 43 0

100 3.5 100 100 100

0 100 27 60 0

100 15 100 100 100

0 100 0 0 0

Scheme 2

analytes.27 Anions of weaker acids, such as acetic and formic acids, have a lower ion-pairing tendency than the trifluoroacetate ion, and therefore, these acids are favored mobile-phase components in electrospray LC/MS. In addition to reduced mean charge states, the abundances of positively charged peptide and protein ions in the gas phase are reduced at higher concentrations of acids in the mobile phase.27 This general tendency is documented in a study of the effect of the acetic acid concentration on the [M + 1]+ ion abundances in the electrospray LC/MS spectra of a group of carbamate, urea, and thiourea pesticides28 and has been noted by other investigators. The impact of the acetic acid concentration in the mobile phase on selected ion abundances of the five cyanobacteria toxins was examined. The concentration of acetic acid in the methanol and water phases was varied from 0.001 (v/v) to 0.1%, and the abundances of selected ions were measured after injection of a mixture containing 50 ng of each analyte into the phenyl-hexyl column. As the acetic acid concentration increased, the abundances of most ions decreased by factors of 30-90% as shown in Figure 3. This decrease is most noticeable with the anatoxin [M + H]+ ion, and the microcystin-RR and -YR [M + 2H]2+ ions. At higher acetic acid concentrations, which are very large compared to the analyte concentrations, the acetate ion concentration is greatly increased, especially during the early stages of the separation when the proportion of water in the mobile phase is still high. Although acetate is weakly ion pairing, its effect is noticeable in Figure 3. For all subsequent experiments, the concentration of acetic acid in both solvents was 0.006% (v/v) or ∼1 mM. This acetic acid concentration, which is relatively low compared to acid concentrations typically used in LC/electrosprayMS studies of mildly basic compounds, gave optimum electrospray ion abundances for most of the available cyanobacteria toxins. In verification of previous conclusions about TFA, the same range (27) Mirza, U. A.; Chait, B. T. Anal. Chem. 1994, 66, 2898-2904. (28) Wang, N.; Budde, W. L. Anal. Chem. 2001, 73, 997-1006.

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Figure 3. Effect of acetic acid concentration in the methanol/water mobile phase on the ion abundances of anatoxin and nodularin [M + H]+ ions and microcystin [M + 2H]2+ ions.

of concentrations of this acid in the mobile phase gave either no ions or barely discernible ion abundances with the same amounts of toxins injected and with very similar LC conditions. The range of acetic acid concentrations studied had a negligible impact on the chromatographic resolution of the toxins; however, the selected 0.006% (v/v) may not give the optimum separation of the analytes. Exact m/z Measurements. A limitation of electrospray is that often only one or a few ions are produced even with conditions that favor collision-induced dissociation in the ion source. For example, even at the highest nozzle potential available, the only ion of significant abundance produced by nodularin is the [M + H]+ ion (Table 3 and related discussion). The integer m/z of one or a few ions and the retention time may not be sufficient to unequivocally identify a target analyte, identify a known toxin that is not a target analyte, or identify an unexpected unknown substance in a sample. One of the advantages of the TOF mass spectrometer is the routine availability of exact m/z measurements to support identifications of substances. Exact m/z measurements with an accuracy of 10 parts-per-million (ppm) or better have been used for more than 40 years to determine ion compositions.22,29 (29) Russell, D. H.; Edmondson, R. D. J. Mass Spectrom. 1997, 32, 263-276.

Table 4. Mean Errors and Precision of Exact m/z Measurements of Selected Ions during LC/MS of Toxins

analyte

measd ion

anatoxin-a microcystin-RR microcystin-YR microcystin-LR nodularin

[M+H]+ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+H]+

mean measd mean error, m/z (n ) 3) calcd m/z ppm (RSD) 166.1253 519.7906 523.2710 498.2782 825.4533

166.1227 519.7902 523.2713 498.2817 825.4505

16 (26) 0.77 (1.7) -0.57 (2.0) -7.0 (4.7) 3.4 (1.2)

However, exact m/z measurements in real time during LC/MS is a recent development that is just beginning to be used routinely.30 The accuracy and precision of exact m/z measurements of the [M + H]+ ions produced by a group of small-molecule pesticides during electrospray LC/MS were recently evaluated with the same LC/TOF-MS used in this work.30 As in the current work, a resolving power of ∼5000 was routinely available and used for the measurements. The mean errors from three exact m/z measurements of each ion from three replicate LC/MS injections of the 10 test analytes were in the range of 0-5.4 ppm. Most single measurement errors were less than 10 ppm; however, occasional random errors larger than 10 ppm led to the conclusion that the mean of three measurements provided consistently reliable results. For routine analyses of samples containing only wellcharacterized target analytes, a single measurement is generally sufficient. For samples containing unexpected or unknown substances, two additional injections provide the accuracy and precision necessary for reliable exact m/z measurements of the unknown ions. In this work, the TOF instrument was calibrated for exact m/z measurements with a solution of the peptides angiotensin I, bradykinin, and neurotensin. The [M + 3H]3+ ions of the peptides at m/z 432.8998, 354.1944, and 558.3105, respectively, were used for the calibration that was generally stable for many weeks. The mean errors and RSDs of the exact m/z measurements from three LC/MS injections of the five test toxins are shown in Table 4. These measurements were conducted with a nozzle potential of 180 V so that the microcystin-YR and -LR [M + 2H]2+ ions would be available for measurement (Table 3). The mean errors and RSDs from measurements of the microcystin and nodularin ions are similar to those obtained for the small-molecule pesticides.30 Measurements with this level of accuracy and precision would provide valuable information for the identification of nontarget but known toxins and unexpected and unknown substances in an environmental samples. The mean error of the anatoxin-a [M + H]+ measurement is outside the generally useful 10 ppm limit, and replicate measurements were more variable than for the larger toxin ions (Table 4). This is attributed to a nonlinearity at the low-m/z end of the calibration that has been previously identified31 and that is expected to be corrected in future instrument models. Recoveries from Water Samples. Recoveries of the five test toxins from deionized water, two river waters, and a treated (30) Maizels, M.; Budde, W. L. Anal. Chem. 2001, 73, 5436-5440. (31) Debre´, O.; Budde, W. L.; Song, X. J. Am. Soc. Mass Spectrom. 2000, 11, 809-821.

Figure 4. Percentage recoveries of the five test toxins from three 100-mL aliquots of reagent water with each toxin fortified at 100 µg/L

Figure 5. Single-measurement percentage recoveries of the five test toxins from fortified environmental water samples.

sewage treatment plant effluent with the disk extraction procedure and LC/TOF-MS were determined at a fortified concentration of 100 µg/L. This concentration was selected to determine whether the analytes are efficiently retained by the C-18 silica particles at a concentration that is representative of the higher reported concentrations of the toxins in water. Microcystin-LR was measured by LC/MS in a water sample from a reservoir in Spain at 270 µg/L, although most reported measurements are below this level.14 Breakthrough of the analytes during a fairly rapid disk extraction process is a concern, especially at the higher concentration levels that are also the levels of most risk for adverse effects on humans, pets, farm animals, and wildlife. Figure 4 shows the percentage recoveries of the five test toxins from three 100-mL aliquots of deionized reagent water with each toxin fortified at 100 µg/L. Mean recoveries ranged from an acceptable low of 68% for anatoxin-a to a high of 98% for microcystin-YR, and the grand mean recovery of the five toxins was 87%. The water samples were made pH 10 or greater just prior to extraction to ensure that anatoxin-a and the other analytes were present in water as the free bases. As a free base, anatoxin-a may be strongly retained at acidic Si-OH groups on the C-18 silica surface, and this may account for its somewhat lower recovery. The RSDs of the mean recoveries ranged from a low of 5% for nodularin to a high of 19% for microcystin-YR, and the grand mean RSD was 13%. River water samples were collected from the west fork of the Little River near Clermont, GA (labeled Clermont, GA River in Figure 5) and the Washite River near Dickson, OK (labeled Dickson, OK River in Figure 5). A sample was also obtained from the treated effluent of a sewage treatment plant (STP) located on Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 6. Complete mass spectrum TIC of the separation of anatoxin-a and four cyclic peptide toxins on a phenyl-hexyl silica column with 5 ng of each analyte injected and a methanol-water gradient elution.

the South Platte River above Clear Creek near Commerce City, CO (labeled Commerce City, CO STP in Figure 5). These samples, which had agricultural and treated municipal waste background components, respectively, were fortified at the 100 µg/L level with the five test toxins. The samples were not filtered before extraction to remove particulate matter, but the added analytes were acclimatized to the water matrix at room temperature and at their natural pH for ∼24 h before the disk extraction. The types of water samples, the 100 µg/L concentration, and the acclimatization process were selected to test the extraction procedure under conditions that probably would be encountered during highvolume routine analyses of a wide variety of water matrixes. The pH of the samples was adjusted to 10 or slightly above just before extraction. The flow rates of the fortified environmental water samples through the extraction disks were ∼20 min, which was slower than the flow rates of reagent waters (Figure 4), and no clogging of the disks was observed. Aliquots of the three water samples were also analyzed without fortification with toxins to determine whether interfering background ions from toxins or other components were present, but none were detected. Figure 5 shows the single-measurement percentage recoveries of the five toxins from the fortified environmental samples. The recoveries of anatoxin-a from the Dickson, OK River sample and the Commerce City, CO STP are somewhat improved compared to those obtained with reagent water. This may be due to a positive impact from the background matrix that contains basic compounds that reduce anatoxin-a retention at acidic SiOH sites on the silica surface. Curiously, the recoveries of microcystin-RR from the three environmental samples were essentially identical at 45-46%, which was lower than expected given its 85% mean recovery (17% RSD) from reagent water. It is reasonable to conclude that, during the 24-h acclimatization period, microcystin-RR may have been slightly degraded or affected in some other constant way in all three samples; however, the near50% recoveries are within the useful analytical range. The recoveries of the other toxins from the three fortified environmental water samples are generally within the ranges that would be predicted from the data in Figure 4 and the corresponding RSDs. There appears to be a trend in Figure 5 that indicates lower recoveries of all analytes except microcystin-RR in the Dickson, OK, river water sample compared to the Clermont, GA, river water sample and still lower recoveries from the Commerce City, CO STP effluent compared to the river water samples. Although the number of samples is too small to establish a 1350

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statistical trend, it is not unreasonable to expect lower recoveries from a STP effluent matrix, which, while treated, still has a higher background of organic substances than the river waters. These background substances compete with analytes for sorption sites in the extraction disks, and this may lower recoveries of analytes somewhat. It is clear that the disk extraction technique combined with the LC/TOF-MS full-spectrum data acquisition technique can accurately distinguish the test analytes from the varied background of organic substances in a treated STP effluent. Detection Limits. Previous work has demonstrated submicrogram per liter detection limits for some cyanobacteria toxins with LC/MS and LC/MS/MS techniques, typically by using 500mL water samples and the SIM and SRM/MRM data acquisition techniques, which are limited to target analytes, to increase analyte signal/noise ratios.14,15 Instrument and method detection limits32,33 were estimated for the full-spectrum LC/TOF-MS technique used in this work with 100-mL water samples. A solution of the five test toxins was diluted by a factor of 10 to a concentration that corresponds to 10 µg/L in a water sample with the extraction and concentration procedure used in this work. Figure 6 shows the total ion chromatogram of the separation and measurement of the five test toxins with 5 ng of each injected on the phenylhexyl column. The resolution of microcystins YR and LR and nodularin is improved in Figure 6 compared to the resolution of the same analytes on the same column, but with 50 ng of each injected, as shown in Figure 2. The measured signal/noise ratios of the peaks in Figure 6 are 161/1 for anatoxin-a, 35/1 for microcystin-RR, 13/1 for microcystin-YR, 42/1 for microcystinLR, and 86/1 for nodularin. The widely published and generally accepted definition of the lower limit of instrument detection is a signal/noise ratio of 3/1.22 Therefore, with the exception of microcystin-YR, instrument detection limits are estimated to be generally less than 0.5 ng injected, which corresponds to method detection limits (MDLs) of generally less than 1 µg/L with the extraction and concentration procedure used in this work. This estimate does not take into account the variable recoveries from environmental samples shown in Figure 5, but even with a slight increase in method detection limits to account for less than 100% recovery, the estimated MDL for microcystin-LR is below the World Health Organization’s provisional guideline of 1 µg/L for microcystin-LR in drinking water.1 Sufficient quantities of most (32) Glaser, J. A.; Foerst, D. L.; McKee, G. D.; Quave, S. A.; Budde, W. L. Environ. Sci. Technol. 1981, 15, 1426-1435. (33) Code of Federal Regulations, Title 40, Part 136, Appendix B.

analytes were not available to determine statistical MDLs that are used by the USEPA in some environmental regulations.32,33 The cyanobacteria toxins are not addressed in current USEPA regulations. Extraction of a 100-mL water sample with the C-18 silica particles enmeshed in Teflon disks, concentration of the eluate to 2 mL, and analysis of a 10-µL aliquot with full-spectrum microbore column LC/TOF-MS provides the broad spectrum analytical capability, qualitative reliability, and quantitative information needed for the determination of the cyanobacteria toxins in water. Analysis of a single sample requires ∼1 h, assuming an instrument is calibrated and ready for analysis. Qualitative reliability and the ability to recognize unexpected or unknown toxins and other substances in water samples is provided by the acquisition of complete mass spectra during the chromatographic separation and by precisely measured retention times for target analytes and other substances. The mass spectra include fragment ions formed by collision-induced decomposition at a nozzle potential of 300 V and an exact m/z measurement for every ion detected. The precision and accuracy of exact m/z measurements can be improved further, if required, by several additional injections and statistical analysis of the data. Quantitative analysis

recoveries and precision are similar to those established with similar techniques for many small-molecule environmental pollutants including pesticides, products of incomplete combustion, and other industrial products and byproducts. ACKNOWLEDGMENT The authors thank Dr. Susan Glassmeyer of this laboratory and her associates at The U.S. Geological Survey for the samples of river water and sewage treatment plant effluent and for the basic information about these samples. The authors also express appreciation to Professor Wayne Carmichael of Wright State University, Dayton, OH, for the sample of cylindrospermopsin. This research was supported in part by the appointment of M.M. to the postgraduate research program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Environmental Protection Agency.

Received for review September 23, 2003. Accepted December 19, 2003. AC035118N

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