Abstract
β-N-methylamino-L-alanine (BMAA) is a difficult molecule to detect, primarily due to its presence in low concentrations in complex matrices. This has resulted in contradictory reports on the presence of BMAA in cyanobacteria. We report improved sensitivity of detection using propyl chloroformate derivatisation, liquid chromatographic (LC) separation, andsingle quadrupole mass spectrometry (MS) detection. Triple quadrupole mass spectrometry (MS/MS) was used to confirm the identity of BMAA in cyanobacteria based on product ions. We show a 10-fold increase in sensitivity with the LC-MS method compared to the previously published gas chromatography mass spectrometry (GC-MS) method with pre-columnderivatised BMAA using a commercially available amino acid derivatisation kit. Clear chromatographic separation of BMAA from 2,4-diaminobutyric acid (DAB), as well as the 20 standard amino acids, was achieved. The analytical method was validated by multiple derivatisation of samples, multiple users, and multiple injections, as well as in various matrices.The quantifier ion used was [M + H]+ = 333 m/z. The MS/MS product ions 273 m/z and 245 m/z were used in identification and peak confirmation. Additionally, we confirm the presence of BMAA in cyanobacteria previously screened with GC-MS as well as the presence of BMAA in newly isolated cultures.
Highlights
The non-proteinogenic amino acid β-N-methylamino-L-alanine (BMAA, Fig. 1) is an excitatory neurotoxin, causing neurodegeneration as a result of selective motor neuron death, at concentrations as low as 30 μM (Rao et al, 2006), and damage to motor neurons at concentrations of 10 μM in the presence of other neurotoxic compounds (Lobner et al, 2007)
BMAA was first identified in the seeds of Cycas micronesica, an indigenous cycad on the island of Guam (Vega and Bell, 1967), where it was initially implicated in the high incidence of amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS/PDC) (Cox et al, 2005)
BMAA produced by symbiotic cyanobacteria (Nostoc) in Cycas micronesica coralloid roots (Cox et al, 2003) was consumed by Chamorro people in the form of flour products made from the cycad seeds
Summary
The non-proteinogenic amino acid β-N-methylamino-L-alanine (BMAA, Fig. 1) is an excitatory neurotoxin, causing neurodegeneration as a result of selective motor neuron death, at concentrations as low as 30 μM (Rao et al, 2006), and damage to motor neurons at concentrations of 10 μM in the presence of other neurotoxic compounds (Lobner et al, 2007). Post-column derivatisation with ninhidrin and analysis by amino acid analyser (Banack et al, 2007), and methods employing no derivatisation with HILIC LC-MS/MS, have been reported (Rosén and Hellenäs, 2008; Kubo et al, 2007; Moura et al, 2009; Krüger et al, 2010; Faassen et al, 2009). For LC-MS/MS, samples of free and protein-associated BMAA extracts were derivatised using propyl chloroformate as previously described and separated by HPLC (Water Acquity Ultra Performance LC) on a Phenomenex EZ:Faast AAA-MS column (250 x 2.0 mm) by gradient elution (0.00 min = 68% B, 13.00 min = 83% B, 13.01 min = 68% B, 17.00 min = 68% B) with a mobile phase composition of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B) (flow rate: 0.25 ml∙min-1;column temperature: 35°C). Accuracy was calculated as a percentage variation relative to the nominal concentration of each point
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