Reversed-phase HPLC/FD method for the quantitative analysis of the neurotoxin BMAA (β-N-methylamino-l-alanine) in cyanobacteria

A method has been developed and optimized in order to detect and quantify the non-protein amino acid β-N-methylamino-l-alanine(BMAA) in cyanobacteria. The novelty of the method is that we have used methanol instead of acetonitrile as the eluent. The method includes extraction with 0.1 M trichloroacetic acid (free BMAA) or protein hydrolysis with 6 M hydrochloric acid (total BMAA), derivatization with AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and reversed-phase high-performance liquid chromatography analysis with fluorescence detection (HPLC/FD). Detection limits ranged from 0.35 to 0.75 pg injected, while quantification limits ranged from 1.10 to 2.55 pg injected for total and free BMAA hydrolysis, respectively. The linear response range was up to 850 pmol in both methods, embracing three orders of magnitude. The method was successfully applied to a lyophilized estuarine species of Nostoc (LEGE 06077). All previous published methods for BMAA quantification, using HPLC/FD, have reported the usage of acetonitrile. This is the first report using methanol as the mobile phase. Although the elution strength differs with both solvents, the final method proved efficient for the quantification of BMAA in this complex sample. The method resulted effective, low-priced, and simple, being suitable for routine monitoring of BMAA in cyanobacteria.

► This is, to our knowledge, the first report of BMAA measurement in cyanobacteria using methanol as the eluent.
► This is the first report that BMAA was detected in Nostoc sp from estuarine Portuguese water.
► We presented here an optimized and simple method for BMAA measurement.