Reactivity of β-Methylamino-l-alanine in Complex Sample Matrixes

Aug 20, 2012 - β-methylamino-l-alanine (BMAA) is a naturally occurring nonprotein amino acid originally ... Neurotoxicity Research 2018 33 (1), 184-1...
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Reactivity of β‑Methylamino‑L‑alanine in Complex Sample Matrixes Complicating Detection and Quantification by Mass Spectrometry W. Broc Glover,† Caitlyn M Liberto,† W. Stephen McNeil,† Sandra Anne Banack,‡ Paul R. Shipley,† and Susan J. Murch*,† †

Department of Chemistry, University of British Columbia, Okanagan Campus, Kelowna, British Columbia, Canada, V1V 1V7 Institute for Ethnomedicine, Jackson, Wyoming, 83001 United States

‡

S Supporting Information *

ABSTRACT: β-methylamino-L-alanine (BMAA) is a naturally occurring nonprotein amino acid originally discovered in cycad seeds and traditional foods of the Chamorro people of Guam. Recent research has implicated BMAA as a potential factor in neurodegenerative disease and described the production of BMAA in cyanobacteria, but conflicting results have complicated the interpretation of data. We hypothesized that the reactivity of BMAA with metal ions in the sample matrix and the formation of metal adducts in electrospray ionization mass spectrometry (MS) analysis confound results. Dilute solutions of TCA, MgCl2, NaCl, CuCl2, ZnCl2 (0.01 M), or artificial ocean water (Instant Ocean, 3.5 g/L) reduced the signal attributable to the BMAA M + H+ peak by 78−99.7%. The degree of adduct formation was significantly affected by MS settings such as induction voltage. A number of the detected ion peaks in BMAA standards were consistent with the formation of metal−BMAA complexes in addition to the adduct formation. A standard of Zn(BMAA)2 was synthesized, and the effects of sample preparation, derivatization, column chromatography, pH, and interactions with serine were determined. Together, these data demonstrate that sample matrix, formation of adducts, and mass spectrometry settings complicate analysis of BMAA, that analysis by detection of the parent ion and daughter ion fragmentation patterns are highly susceptible to false negative findings, and that failure to detect BMAA cannot be considered proof of absence of the compound.

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1970s and 1980s evaluated BMAA toxicity in a variety of experimental animal models including monkeys,7,8 but it was eventually concluded that the amount of BMAA quantified using the methods of the time in traditionally prepared, washed cycad seed flours was not sufficiently high enough to induce the effects seen in model animals.9 It was concluded that a person would need to consume huge quantities of BMAA-containing cycad seed flour to receive an effectively toxic dose.7,9 Thus, research into BMAA as a potential environmental neurotoxin was largely suspended until the publication of a medical hypothesis proposing that BMAA could be biomagnified through the ecosystem and exposure could occur through a variety of traditional foods.10 A study of the distribution of BMAA in cycads found that the amino acid was concentrated in the outermost layer of the seeds11 and Whiting3 had reported “In Guam, the green outer husks of seeds of Cycas circinalis are chewed to relieve thirst and, when dried, are considered a tasty sweet”. Detailed studies of the cycads found that BMAA was produced by symbiotic cyanobacteria in the roots, and studies of isolated cultures discovered that 90% of cyanobacteria produced BMAA.12,13 More detailed studies of traditionally prepared cycad flours revealed that BMAA was associated with protein and was retained in the washed flour at much higher concentrations than previously quantified.14,15 Further studies in Guam and around the world demonstrated higher exposures

he Chamorro people of Guam experienced an epidemic of amyotrophic lateral sclerosis (ALS) and parkinsonismdementia complex (PDC) from the end of World War II to the late 1990s.1 At the height of the epidemic, death rates attributed to ALS and PDC were 50 to 100 times greater than in other parts of the world.2 Research investigated many different potential causes of the Guam disease such as: genetic factors, calcium, aluminum, and magnesium deficiencies or toxicities, viruses or other potential infections, environmental causes, and eventually traditional foods.2,3 The Chamorro knew that the cycad (Cycas circinalis; now Cycas micronesica) seeds that they used for food and medicine could be toxic, and they associated the incidence of ALS/PDC with the handling and consumption of cycads (Whiting 1954; 1957; 1958 field notes3,4). Japanese soldiers who lived in the Guam hills from the end of World War II until their surrender in 1962 described acute illness after eating improperly prepared cycad seeds (Kurland JT, 1962 field notes3). Chamorro traditional knowledge concerning the preparation of flour from cycad seeds included grating or shredding the gametophyte of the seeds and soaking in water for several weeks. Water used to soak the seeds was then fed to chickens and if the chickens lived for the time it took for seeds to dry, it was determined that sufficient toxin had been removed.2 These field observations led to a search for a neurotoxin in cycad seeds from Guam and the discovery of α-amino-βmethylaminopropionic acid (β-methylamino- L -alanine; BMAA).5 In early studies, BMAA was shown to be acutely toxic to chicks and mice.6 Further research throughout the © 2012 American Chemical Society

Received: June 21, 2012 Accepted: August 20, 2012 Published: August 20, 2012 7946

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ionization interface at 101 psi with desolvation at 500 L/h. The source temperature and desolvation temperature were 130 and 100 °C, respectively. Voltages were as follows: capillary, 2.9 kV; extractor, 2 V; RF lens, 0.7 V. The MS scan ranged from 80 to 1000 Da with a span of 0.2 Da, a dwell time of 0.5 s, and a scan frequency of 1 s. Each minute, the cone voltage increased by 5 V/min until a cone voltage of 100 was achieved after 20 min. Data was exported to Excel for quantification of BMAA and its adducts. To determine whether the phenomena observed with the formation of ion adducts in the single quadrupole EMD1000 MS was specific to this instrument or indicated a larger issue with all mass spectrometers, the same solutions were tested for ion adduct contents using the same infusion protocols on a second MS. Standard solutions were filtered and directly infused into a time of flight mass spectrometer (TOF-MS; Waters LCT Premier) by continuous flow via a Waters 1525 Binary HPLC pump at 100 μL/min. The sample cone voltage was initially set to 5 V and as above increased by 5 V/min, using 20 consecutive 1 min MS Scan Functions in V+ ESI mode, until a voltage of 100 V was reached. Data was collected in continuum mode over the m/z range of 50−1000. The capillary voltage was set to 3500 V. A desolvation temperature of 450 °C was used, with a corresponding N2 gas flow of 800 L/ h. The source temperature was set to 120 °C, and the cone gas flow was 50 L/h. Postacquisition analysis of the data was completed using MassLynx software (V4.1). All analyses were completed in triplicate. A total ion chromatogram (TIC) for each MS scan method was summed, and the resulting spectrum data was exported to Microsoft Excel for untargeted mining to identify the major adducts associated with the BMAA standard. Adducts were identified using a predicted m/z tool for ESI (Huang et al.41 ; http://fiehnlab.ucdavis.edu/staff/kind/ Metabolomics/MS-Adduct-Calculator/). Synthesis of a BMAA−Zinc Complex. To determine whether metal complexes of BMAA could form in complex sample matrixes, a Zn(BMAA)2 complex was synthesized and its behavior in common analytical methods evaluated. Details of the synthesis of preparation of Zn(II) and the Zn(BMAA)2 are found in the Supporting Information. Effect of Solvent pH on the Zn(BMAA)2 Complex. The Zn(BMAA)2 complex (10 mg) was dissolved in 30 mL of water, and 0.5 M HCl was added dropwise to the solution until the pH dropped by one unit (1−2 drops), as indicated by pH strips (ColorpHast, EM-Reagents). The mass spectrum was obtained by ESI-TOF-MS, and the process was repeated for each pH unit until a pH of 1 was achieved. Competition of BMAA and Serine. To determine the reactivity and potential interactions between serine and BMAA, serine was added to four different Zn(BMAA)2 solutions at molar ratios of 38:1, 191:1, 394:1, and 1972:1 to mimic physiological samples. The resulting solutions were infused directly into the TOF-MS using the same instrument parameters described above. AQC Derivatization of Zn(BMAA)2. To determine the effects of derivatization on metal−BMAA complexes in samples, Zn(BMAA)2 standard solutions were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) using protocols described previously.14,20 Amino acids were separated on a Waters Amino Acid Analysis column (100 × 2.1 mm; 1.7 μm, C18) over a gradient of 0.1% (v/v) aqueous formic acid (eluent A) and acetonitrile (eluent B). The gradient was changed linearly between all points: 0 min (99.1% A), 0.50

to BMAA from multiple food sources such as animals that fed on cycad seeds,12,14,16,17 fish, mussels, and oysters in the Baltic Sea18 and crabs, oysters, and shrimp from South Florida.19 Interest in BMAA increased when the amino acid was found in autopsy samples of Chamorro patients who died of ALS/PCD and two Canadian control patients who died of Alzheimer’s disease.14,20 Since the beginning of BMAA research, methods of identification and quantification of the molecule have been controversial (recently reviewed21). Four different laboratories undertaking quantification of BMAA traditionally prepared flours from Guam reported concentrations spanning nearly 6 orders of magnitude in replicate analysis of identical samples.9,14,15,22−24 The production of BMAA in cyanobacteria cultures was confirmed,25 but other studies failed to detect BMAA in cyanobacteria.26,27 The presence of BMAA in human brain tissues was confirmed,28 but other studies failed to detect BMAA in autopsy samples.29,30 Overall variability in methods and the accuracy of identification and quantification have complicated the interpretation of data. The majority of methods have used standard amino acid analysis techniques modified for the low concentrations including: paper chromatography, amino acid analyzers, HPLC with absorbance or fluorescence detection, GC/MS, LC-MS, LC-MS/MS, and NMR (reviewed21,31). The most frequently used method for detection of BMAA in cyanobacteria and other complex sample matrixes utilizes 6aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization12−14,19,20,28,31 and has been validated by several orthogonal techniques including mass spectrometry.14,31 A separate method has also identified BMAA in cyanobacteria using propyl chloroformate derivatization (EZ-faast) with MS detection of the derivatized compound at m/z 333.25 It has previously been suggested that matrix effects may be complicating factors in BMAA analysis32,33 or that isomers may be mistaken for BMAA.26,34 Most specifically, mass spectrometry based methods relying on chromatographic separation using a HILIC column and interface have most commonly reported a failure to detect BMAA.31 BMAA is a highly reactive compound that reacts with CO2/ bicarbonate to form carbamates,35,36 with aldehydes to form imines,37,38 and with some transition metal ions to form very stable chelates.39 We hypothesized that the formation of adducts and complexes masks the BMAA signal in electrospray ionization/mass spectrometry analysis and can distort chromatographic separations. To examine this possibility, we analyzed standard solutions of BMAA in various ionic solutions with varying mass spectrometry parameters. Further, we synthesized a complex of Zn(BMAA)2 to determine the fate of BMAA complexes in the analytical systems and we re-examined the effects of salt matrixes on derivatization protocols.

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EXPERIMENTAL SECTION Determination of BMAA Adducts in Mass Spectrometry. A 6.5 mM stock solution of L-BMAA HCl was made up in 0.1 M trichloroacetic acid (TCA). A 1:10 dilution was made in 0.01 M solutions of TCA, MgCl2, NaCl, CuCl2, ZnCl2, or Instant Ocean (3.5 g/L; composition in Supporting Information). BMAA standard solutions were introduced by constant direct infusion by syringe pump (0.025 mL/min; Hamilton Co., Reno, NV) into a single quadrupole mass spectrometer (Waters EMD 1000). Nitrogen gas (NitroFlow nitrogen generator; Parker Balston, Haverfill, MA) was supplied to the electrospray 7947

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Time of Flight Mass Spectrometry. Standard solutions of BMAA in 0.1 N TCA were found to have 3 prominent ions corresponding to BMAA (M + H+), a dimer of BMAA (2M + H+), and a sodium adduct of the BMAA dimer (2M + H + Na) (Figure 2A). Sodium adducts of the BMAA parent molecule and the dimer were found across all of the BMAA solutions indicating a constant low level of Na+ in the system but are not depicted in those graphs where a relatively low constant signal was detected. Increasing the ion induction cone voltage significantly decreased the signal of the parent compound and increased the proportion of signal attributed to m/z Na(BMAA)2 (Figure 2A). When the standard solution contained NaCl, the majority of the BMAA standard signal was seen in m/ z values corresponding to (BMAA)2 and Na-(BMAA)2 with a significant effect of induction cone voltage (Figure 2B). In a standard solution composed of MgCl2, the parent compound BMAA was completely undetected and the various Mg2+ and Cl− adducts were quantified (Figure 2C). One of the more interesting ion interactions was the formation of Zn(II) complexes. Signals were detected that corresponded to the predicted ions of BMAA and four different Zn−BMAA complexes including 64Zn(BMAA)2 and 67Zn(BMAA)2. The relative intensity of the complexes varied with induction cone voltage (Figure 2D). When the BMAA standard solution was made in a matrix of CuCl2, the parent compound with m/z 119 appeared only as a minor constituent and the spectra were dominated by the BMAA dimer (Figure 2E). When a BMAA standard was made in a solution of Instant Ocean simulating a marine environment, less than 3% of the total standard signal was found as the parent M + H+ (m/z 119), but complexes of HCl, Mg2+, and Na+ dominated the spectrum (Figure 2F). Investigation of BMAA−Metal Complexes. In addition to adduct formation, mass spectrometry observations indicated a number of unidentified peaks in the spectra of BMAA standards that we hypothesized could be BMAA−metal complexes. To investigate this potential, we synthesized a standard of Zn(BMAA)2 using a 2 step synthesis. The synthesis initially produced a fine powder which was analyzed by ESITOF-MS (Figure 3) and by 1H and 13C NMR. The 13C NMR spectrum of the coordinated BMAA showed all carbons being deshielded relative to the hydrochloride salt of free BMAA (Supporting Information, Figure S2) and an upfield shift in the proton environments (Supporting Information, Figure S3), consistent with the zinc coordinating via the carboxylate and likely both nitrogens. No signal was observed at the predicted parent m/s for Zn(BMAA)2 , but the Na+ adduct of Zn(BMAA)2 at m/z 321 and the expected Zn isotope pattern were observed (Figure 3). Preliminary computational modeling of various possible isomers of a Zn(BMAA)2 complex42 via density functional theory and a polarized continuum model (PCM)43 indicated the possible formation of a distorted tetrahedral geometry in solution (Supporting Information, Figure S1) in which the amino acid binds through both N atoms but could not definitively discount the possibility that the carboxyl group also participates in binding the metal center. Taken together, the computational, MS, and NMR data could not unambiguously determine the conformation of the Zn(BMAA)2 complex and several possibilities exist (Figure 4). It should be noted that Cu(BMAA)2 is believed to form a four-coordinate complex in which each amino acid binds through both N atoms.39 Attempts to isolate crystals and further characterize the complex by crystallography were not

min (99.1% A), 2.00 min (95.0% A), 3.00 min (95.0% A), 10.00 min (80% A), 10.50 min (15% A), 10.60 min (15% A), 11.00 min (99.1% A), and 12.00 min (99.1% A). BMAA was detected by TOF-MS scanning to collect spectra every 0.5 s across an m/ z range of 100−5000 Da. Data was collected in V+ mode, with the cone and aperture voltages set to 30 and 5 V, respectively. Leucine enkephalin was used as the lockmass reference solution to ensure accurate mass measurement.

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RESULTS The overall objective of the study was to determine whether analytical methodology and sample matrix effects could significantly alter the detection and quantification of BMAA. Since many of the reports investigating cyanobacteria involved fresh water and marine cultures, the synthetic marine environment “Instant Ocean” was used to mimic seawater. Two different styles of single quadrupole mass spectrometers were selected specifically to provide information on the ionization and reactivity of the parent BMAA ion in solution. All of the reports of multiple reaction monitoring (MRM) quantification of underivatized BMAA by MS/MS systems relied on the same parent BMAA ion with m/z 11926,27,30,33,40 but did not account for BMAA adduct ions in solution or electrospray. Huang et al.41 provided guidelines for identification of expected adducts in mass spectrometry analysis that were used to generate predicted ions (see Supporting Information). EMD 1000 Single Quadrupole Mass Spectrometry. The first hypothesis was that sample solutions reduce the overall signal attributable to a BMAA standard at the M + H+ peak. Our data show that the overall signal attributable to the BMAA M + H+ peak is reduced by 78−99.7% by the presence of Na+ in the solutions. Likewise, the overall signal attributable to the BMAA M + H+ peak is reduced by 86−93% by the presence of Mg2+ ions in solution. It is further interesting to note that the introductory voltage significantly affected the major ion adducts detected in a BMAA standard (Figure 1). For example, in the NaCl solution, the signal attributable to the BMAA M + H+ peak was 27% of the total BMAA signal at a cone voltage of 5 V but was reduced to 4−5% of the total BMAA signal at cone voltages between 25 and 100 V.

Figure 1. Confirmation of effects of varying cone voltage and the presence of sodium chloride on detection of BMAA in an EMD1000 mass spectrometer with electrospray ionization. 7948

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Figure 2. Effects of salt solutions and varying cone voltages on detection of BMAA by time of flight mass spectrometry. (A) Trichloroacetic acid standard. (B) Sodium chloride standard solution. (C) Magnesium chloride standard solution. (D) Zinc chloride standard solution. (E) Copper chloride standard solution. (F) Standard dissolved in Instant Ocean.

successful, similar to previous reports,39 and other methods will need to be developed to fully characterize the complex. Chemistry of Zn(BMAA)2. Part 1: pH Stability. To determine the stability of Zn(BMAA)2 in solution and common steps in sample preparations, we quantified changes in the concentration of the complex with pH change. As the pH was decreased, the signal associated with the sodium adduct of the complex (m/z 321) decreased (Figure 5) while signals corresponding to the m/z of BMAA and its’ adducts increased. The slope of the line may putatively indicate a pKa consistent with a pendent carboxyl group but is not definitive. Part 2: Displacement of BMAA in Zinc Complexes by Serine. To determine whether other amino acids in solution could interact with the BMAA−metal complexes such as Zn(BMAA)2, we reacted the complex with serine in solution at similar ratios as previously reported for serine and BMAA in

physiological samples. The addition of serine to the Zn(BMAA)2 solution at high levels displaced BMAA from the metal center and the m/z 404 indicating Zn−serine complex was observed (Figure 6). In addition, only a small portion of the BMAA released from the complex was observed as the m/z 119 signal. Part 3: Effect of Derivatization on the Zn(BMAA) 2 Complex. One of the issues with many previous reports has been a difference in determined concentrations by several orders of magnitude, and the formation of BMAA−metal complexes in samples may account for some of these differences. The underivatized Zn(BMAA)2 complex was eluted from chromatography columns at a significantly different retention time and m/z than single BMAA molecules and was not eluted from the HILIC column using the previously published conditions (data not included). We hypothesized 7949

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underlying the m/z 459 peak revealed m/z reflecting increasing lengths of BMAA−AQC polymer chains (Figure 7). Therefore, while measure of the fluorescence signal of the peak at this elution time would reflect the BMAA measurement, the amount of BMAA determined as the m/z 459 would be an underestimation (Figure 7).

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DISCUSSION One of the biggest challenges for understanding the prevalence and persistence of BMAA in food systems, ecological samples, and biological samples is the need for accurate methods for identification and quantification of the amino acid in complex mixtures.21 Recent advances in the technologies for MS have led to increased confidence in results from these systems. However, many of the MS methods for analysis of BMAA have relied on identification and fragmentation of a single, selected underivatized parent (M + H+) m/z 119 → 102.1,26 m/z 119 → 102, 88, and 76,40 m/z 119 → 102, 101, 88, and 79,33 or m/z 119 → 102, 88, 76, 73, and 44.27 The reliance on m/z 119 (M + H+) as the only quantified signal and the discard of all other data is only valid if m/z 119 is the main form of the parent ion in solution. Our data indicates that, even under optimal circumstances, m/z 119 accounts for less than 10% of the total BMAA ion in solution and that, under the most likely circumstances of the presence of Na+, Mg+, Zn+, and other ions, the m/z 119 accounts for less than 3% of the total BMAA. One of the interesting methodological quandaries to arise from these data is the basic role of standard curves in quantification. If the selected defining peak, in this case m/z 119, accounts for only a small percentage of the mass of the applied standard, then the standard curve used for determination of limits of detection, limits of quantification and quantification of analyte in the sample matrix all rely on the ratio of the selected m/z and the various other peaks remaining consistent for accuracy. Our data indicate that the relative amount of adducts and complexes vary with the composition of ions in solution. As a result, it seems unlikely that the ratio of m/z 119 to other ions would be consistent in complex sample matrixes. In addition, the external standard curves most often used for quantification do not take matrix effects into account or utilize synthetic matrixes, creating a discrepancy between the behavior of BMAA in isolated standards and complex biological matrixes. While recovery experiments can help with this problem, the spike is often added immediately before the sample is prepared, allowing limited time for reaction with the ions in the sample matrix and giving falsely high rates of recovery. As a result, application of these recovery data may lead to an extreme underestimation of the amount of BMAA present in a sample. This is particularly important for determining the potential for contamination of foods and environmental samples. BMAA has been quantified in isolated cyanobacteria from plant tissues and fresh water and marine environments as well as cyanobacteria cultures grown in BG11 culture media. Each of these solutions and sample matrixes contains high concentrations of metal ions in solution. Previous researchers39 have described the formation of Cu−BMAA and Zn−BMAA complexes and estimated that BMAA binding to Zn is 23 times more likely than the formation of other amino acid Zn complexes. Interestingly, Duncan22 also found high concentrations of Zn in the most neurotoxic traditionally prepared cycad flour samples from Guam but was able to detect only low levels of BMAA in those samples. The authors hypothesized that Zn from the galvanized pails used to prepare the samples

Figure 3. Mass spectra identification of the Zn(BMAA)2 complex in solution.

Figure 4. Proposed structures of a Zn(BMAA)2 complex in physiological samples.

Figure 5. Effect of changing pH on Zn(BMAA)2 quantification in solution.

that the process of derivatization could release BMAA from metal complexes thereby increasing the amount available for quantification in derivatization-based sample preparation methods. We subjected the Zn(BMAA)2 standard to the standard protocol for AQC derivatization14,20 and found that all of the BMAA signal was detected at m/z 459 and m/z 230 (M + 2H) signals, characteristic of double derivatized BMAA (Figure 7). However, further examination of the spectra 7950

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Figure 6. Displacement of BMAA from the Zn(BMAA)2 complex by serine in solution. From bottom to top, the molar ratio of serine to Zn(BMAA)2 was 1:1; 38:1; 191:1; 394:1, and 1972:1.

accounted for the observed neurotoxicity,22 but an equally likely hypothesis for this data could be that the high level of Zn in the sample led to a false low quantification of BMAA. Recent results indicate that Zn, Cu, and Fe are present at quite high concentrations in brain tissues.44 Zinc was found at the highest concentrations in the cerebral cortex and hippocampal regions of mouse brain,40 and the Zn2+ concentration in these regions can be elevated to high micromolar concentrations during synaptic activity45 so it seems that the neurotoxicity would be a direct result of Zn contamination. We were unsuccessful in our attempts to identify a peak corresponding to Zn(BMAA)2 in a HILIC chromatography system using the identical column and eluents of a previously published method,33 and there was no ion signal corresponding to BMAA detected in the Zn(BMAA)2 standard. Overall then, our data shows that the reactivity of BMAA with metal ions in solution may complicate analysis of many different types of samples and can mask the BMAA signal.

Methods need to be developed that take into account the reactivity of the amino acid in complex sample matrixes. Appropriately validated, rugged, and reproducible methods are required, and the relatively small size, potential confusion with isomers, and reactivity of the molecule necessitate that any methods should: (1) use an optimized sample preparation protocol and derivatization procedure to release BMAA from metal complexes that form in the samples; (2) use adequate reverse phase chromatographic separation from other amino acids; (3) use at least two orthogonal methods of detection such as fluorescence and mass spectrometry for both accurate qualification and quantification; (4) use multiple ions in mass spectra for detection and confirmation of the presence and persistence of BMAA. Together, our data indicate that it is possible to fail to detect BMAA in a BMAA standard and, therefore, failure to detect the analyte is not proof of absence of BMAA from any sample. In addition, our data show that BMAA can form complexes with metal ions and that these complexes are detected as different compounds in underivatized analysis. Overall, the formation of adducts and complexes of BMAA significantly complicates the detection and quantification of the molecule. One interesting possibility suggested by these data that should be investigated in future work is the potential for formation of BMAA−metal complexes within proteins. It is also possible that inclusions of a metal chelating agent in the sample preparation may improve the accuracy of BMAA analytical

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CONCLUSIONS AND FUTURE DIRECTIONS Together, these data indicate that interpretation of analytical results can be complicated by methodological choices and the presence of metal ions in samples, sample eluents, and instrumentation. Over-reliance on a single parent ion and MRM analysis may discard important spectral information, and failure to detect BMAA as the parent ion m/z 119 does not indicate the absence of BMAA in the tissue or solution. 7951

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Figure 7. Peak areas of multiple ions under the m/z 459 peak of derivatized Zn(BMAA)2. Note that the presence of metal ions in solution may result in short chains of amino acid and fluorescent tag. (3) Whiting, M. G. Econ. Bot. 1963, 17, 271−302. (4) Laqueur, G. L.; Whiting, M. G.; Mickelsen, O.; Kurland, L. T. J. Natl. Cancer Inst. 1963, 31, 919−951. (5) Vega, A.; Bell, E. A. Phytochemistry 1967, 6, 759−762. (6) Vega, A.; Bell, E. A.; Nunn, P. B. Phytochemistry 1968, 7, 1885− 1887. (7) Garruto, R. M.; Yanagihara, R.; Gajdusek, D. C. Lancet 1988, 332, 1079. (8) Spencer, P. S.; Nunn, P. B.; Hugon, J.; Ludolph, A. C.; Ross, S. M.; Roy, D. N.; Robertson, R. C. Science 1987, 237, 517−522. (9) Duncan, M. W.; Steele, J. C.; Kopin, I. J.; Markey, S. P. Neurology 1990, 40, 767−772. (10) Cox, P. A.; Sacks, O. W. Neurology 2002, 58, 956−959. (11) Banack, S. A.; Cox, P. A. Bot. J. Linn. Soc. 2003, 143, 165−168. (12) Cox, P. A.; Banack, S. A.; Murch, S. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13380−13383. (13) Cox, P. A.; Banack, S. A.; Murch, S. J.; Rasmussen, U.; Tien, G.; Bidigare, R. R.; Metcalf, J. F.; Morrison, L. F.; Codd, G. A.; Bergman, B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5074−5078. (14) Murch, S. J.; Banack, S. B.; Cox, P. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12228−12231. (15) Cheng, R.; Banack, S. A. ALS 2009, 10, 41−43. (16) Banack, S. A.; Murch, S. J.; Cox, P. A. J. Ethnopharm. 2006, 106, 97−104. (17) Banack, S. A.; Murch, S. J. ALS 2009, 10, 34−40. (18) Jonasson, S.; Eriksson, J.; Berntzona, L.; Spacil, Z.; Ilag, L. L.; Ronnevid, L. O.; Rasmussen, U.; Bergman, B. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9252−9257. (19) Brand, L. E.; Pablo, J.; Compton, A.; Hammerschlag, N.; Mash, D. C. Harmful Algae 2010, 9, 620−635. (20) Murch, S. J.; Cox, P. A.; Banack, S. A.; Steele, J. C.; Sacks, O. W. Acta Neurol. Scand. 2004, 110, 267−269. (21) Cohen, S. A. Analyst 2012, 137, 1991−2005.

methods. Finally, the need for validated, accurate analytical methods for BMAA has recently been suggested,46 but our data indicate that particular attention should be focused on improved methods for analysis of BMAA by mass spectrometry. Understanding the complexity of this analysis will improve our overall understanding of ionization, adducts, and the separation of ions and will have implications for other analytical methods.

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ASSOCIATED CONTENT

* Supporting Information S

Figure S1: Protocol for synthesis of Zn(BMAA)2. Figure S2: 13 C NMR of BMAA HCl (top) and Zn(BMAA)2 (bottom) demonstrating downfield shift of all carbon resonances. Figure S3: 1H NMR of HCl (top) and Zn(BMAA)2 (bottom). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: University of British Columbia, Okanagan Campus, 3333 University Way, Kelowna, British Columbia, Canada, V1V 1V7. Tel: 250-807-9566. Fax: 250-807-9249. E-mail: susan. [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Waring, S. C.; Esteban-Santillan, C.; Reed, D. M.; Craig, U. K.; Labarthe, D. R.; Peterson, R. D.; Kurland, L. T. Neuroepidemiology 2004, 23, 192−200. (2) Kurland, L. T. Trends Neuro. Sci. 1988, 11, 51−54. 7952

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dx.doi.org/10.1021/ac301691r | Anal. Chem. 2012, 84, 7946−7953