Liquid Chromatographic Determination of the Cyanobacterial Toxin β-N-Methylamino-L-Alanine in Algae Food Supplements, Freshwater Fish, and Bottled Water

Abstract

Beta-N-Methylamino-L-alanine (BMAA) is a neurotoxin originally found in cycad seeds and now known to be produced by many species of freshwater and marine cyanobacteria. We developed a method for its determination in blue-green algae (BGA) food supplements, freshwater fish, and bottled water by using a strong cation-exchange, solid-phase extraction column for cleanup after 0.3 M trichloroacetic acid extraction of BGA supplements and fish. Bottled water was applied directly onto the solid-phase extraction column. For analysis of carbonated water, sonication and pH adjustment to 1.5 were needed. To determine protein-bound BMAA, the protein pellet left after extraction of the BGA supplement and fish was hydrolyzed by boiling with 6 M hydrochloric acid; BMAA was cleaned up on a C18 column and a strong cation-exchange, solid-phase extraction column. Determination of BMAA was by liquid chromatography of the fluorescent derivative formed with 9-fluorenylmethyl chloroformate. The method was validated by recovery experiments using spiking levels of 1.0 to 10 microg/g for BGA supplements, 0.5 to 5.0 microg/g for fish, and 0.002 microg/g for bottled water; mean recoveries were in the range of 67 to 89% for BGA supplements and fish, and 59 to 92% for bottled water. Recoveries of BMAA from spiked extracts of hydrolyzed protein from BGA supplements and fish ranged from 66 to 83%. The cleanup developed provides a useful method for surveying foods and supplements for BMAA and protein-bound BMAA.

Assessment of the mutagenic and genotoxic activity of cyanobacterial toxin beta-N-methyl-amino-L-alanine in Salmonella typhimurium

“…The reported L-BMAA concentrations in the aquatic environment vary widely between studies ranging from ng/g dry weight to low mg/g dry weight range in phytoplankton (Jonasson et al, 2010;Faassen, 2014). Concentrations similar to those for phytoplankton were reported also in animals used for human consumption, such as crabs, oysters, mussels, fish and algae food supplements (Scott et al, 2009;Jonasson et al, 2010;Niedzwiadek et al, 2012;Jiang et al, 2014b;Reveillon et al, 2015;Salomonsson et al, 2015). Considering that L-BMAA biosynthesized by symbiotic or/and freeliving cyanobacteria can accumulate in organisms and can biomagnify in the food web, both in free amino acid and proteinbound forms, concentrations in organisms can increase significantly.…”

The neurotoxin BMAA in aquatic systems : analysis, occurrence and effects

“…For sixteen methods, most data needed for method validation (detection limits, linear range, precision and recovery) were provided [34,76,79,82,93,94,100,105,111,121,122,135,145,148]. However, unvalidated methods (or methods for which no sufficient validation data were provided) were repeatedly referred to as 'validated'.…”

“…The two studies in which no BMAA was detected in higher aquatic organisms (fish and shrimp) have used LC-FLD analysis (LOD 0.21 µg/g DW for fish and 0.3 µg/g DW for shrimp [147,148]). Although it is shown that BMAA can be present in aquatic ecosystems, this conclusion is only based on a narrow selection of articles ( Figure 5.4).…”

“…GC-MS Y + + + + ---+ + + + + -+ ---~ + ~ ~ ~ [76] LC-MS/MS N ~ ---~ x + + + + + + + -+ + -+ + + + + [111] 1 H-NMR X ~ ---~ x ~ ~ + + + + + + -+ + + + -+ + [148] LC-FLD Y ~ ---+ + + + + x + + + + + -+ + + + + + [102] LC-MS/MS N + + + + + x + + + + + -+ + + -+ + + + -+ [157] LC…”

“…However, reported BMAA concentrations in aquatic systems vary widely between studies. Several studies have detected BMAA in all tested cyanobacteria samples, whereas others have not detected it in any sample ( [147,148]. Bioaccumulation of BMAA in higher aquatic organisms has been reported [83].…”