Determination of ß-N-methylamino-L-alanine, N-(2-aminoethyl)glycine, and 2,4-diaminobutyric acid in Food Products Containing Cyanobacteria by Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry: Single-Laboratory Validation.

A single-laboratory validation study was completed for the determination of ß-N-methylamino-L-alanine (BMAA), N-(2-aminoethyl)glycine (AEG), and 2,4-diaminobutyric acid (DAB) in bulk natural health product supplements purchased from a health food store in Canada. BMAA and its isomers were extracted with acid hydrolysis to free analytes from protein association. Acid was removed with the residue evaporated to dryness and reconstituted with derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor). Chromatographic separation and detection were achieved using RP ultra-performance LC coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode. Data from biological samples were evaluated for precision and accuracy across different days to ensure repeatability. Accuracy was assessed by spike recovery of biological samples using varying amino acid concentrations, with an average recovery across all samples of 108.6%. The analytical range was found to be 764-0.746 ng/mL prior to derivatization, thereby providing a linear range compatible with potentially widely varying analyte concentrations in commercial health food products. Both the U. S. Food and Drug Administration (FDA) and U. S. Pharmacopeia definitions were evaluated for determining method limits, with the FDA approach found to be most suitable having an LOD of 0.187 ng/mL and LLOQ of 0.746 ng/mL. BMAA in the collected specimens was detected at concentrations lower than 1 µg/g, while AEG and DAB were found at concentrations as high as 100 µg/g. Finding these analytes, even at low concentrations, has potential public health significance and suggests a need to screen such products prior to distribution. The method described provides a rapid, accurate, and precise method to facilitate that screening process.

The natural non-protein amino acid N-ß-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

N-ß-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

Reactivity of ß-methylamino-L-alanine in complex sample matrixes complicating detection and quantification by mass spectrometry.

ß-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, MgCl(2), NaCl, CuCl(2), ZnCl(2) (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.

Cyanobacteria and cyanotoxins are present in drinking water impoundments and groundwater wells in desert environments.

Desert environments and drylands experience a drastic scarcity of water resources. To alleviate dependence on freshwater for drinking water needs, countries have invested in infrastructure development of desalination plants. Collectively, the countries of the Arabian Gulf produce 45% of the world's desalinated water, which is stored in dams, mega-reservoirs and secondary house water tanks to secure drinking water beyond daily needs. Improper storage practices of drinking water in impoundments concomitant with increased temperatures and light penetration may promote the growth of cyanobacteria and accumulation of cyanotoxins. To shed light on this previously unexplored research area in desert environments, we examined drinking and irrigation water of urban and rural environments to determine whether cyanobacteria and cyanotoxins are present, and what are the storage and transportation practices as well as the environmental parameters that best predict their presence. Cyanobacteria were present in 80% of the urban and 33% of the rural water impoundments. Neurotoxins BMAA, DAB and anatoxin-a(S) were not detected in any of the water samples, although they have been found to accumulate in the desert soils, which suggests a bioaccumulation potential if they are leached into the aquifer. A toxic BMAA isomer, AEG, was found in 91.7% of rural but none of the urban water samples and correlated with water-truck transportation, light exposure and chloride ions. The hepatotoxic cyanotoxin microcystin-LR was present in the majority of all sampled impoundments, surpassing the WHO provisional guideline of 1 µg/l in 30% of the urban water tanks. Finally, we discuss possible management strategies to improve storage and transportation practices in order to minimize exposure to cyanobacteria and cyanotoxins, and actions to promote sustainable use of limited water resources.

Liquid chromatography and mass spectrometry for the analysis of N-ß-methylamino-L-alanine with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

Numerous studies in the past decade have identified N-ß-methylamino-L-alanine (BMAA) as a putative environmental neurotoxin. Produced by cyanobacteria and accumulated at different levels of the trophic system, BMAA has been detected in the brain tissue of human patients that died from progressive neurodegenerative disease. Research into the mechanism of neurotoxicity has been hampered by conflicting results and disagreement in the literature over analytical methods used for quantification and detection. While several research approaches have been tested, the use of the derivatizing reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate is presented here as an effective and selective means for the analysis of BMAA and two co-occurring biological isomers, DAB and AEG, by liquid chromatography and tandem mass spectrometry.