Selective LC-MS/MS method for the identification of BMAA from its isomers in biological samples

Abstract

Algal blooms are well-known sources of acute toxic agents that can be lethal to aquatic organisms. However, one such toxin, β-N-methylamino-L-alanine (BMAA) is also believed to cause amyotrophic lateral sclerosis, also known as Lou Gehrig's disease. The detection and identification of BMAA in natural samples were challenging until the recent introduction of reliable methods. However, the issue of potential interference from unknown isomers of BMAA present in samples has not yet been thoroughly investigated. Based on a systematic database search, we generated a list of all theoretical BMAA structural isomers, which was subsequently narrowed down to seven possible interfering compounds for further consideration. The seven possible candidates satisfied the requirements of chemical stability and also shared important structural domains with BMAA. Two of the candidates, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG) have recently been studied in the context of BMAA. A further isomer, β-amino-N-methyl-alanine (BAMA), has to be considered because it can potentially yield the fragment ion, which is diagnostic for BMAA. Here, we report the synthesis and analysis of BAMA, together with AEG, DAB, and other isomers that are of interest in the separation and detection of BMAA in biological samples by using either high-performance liquid chromatography or ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. We detected for the first time BAMA in blue mussel and oyster samples. This work extends the previously developed liquid chromatography-tandem mass spectrometry platform Spacil et al. (Analyst 135:127, 2010) to allow BMAA isomers to be distinguished, improving the detection and identification of this important amino acid.

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

“…This method was subsequently used for a food web study [83]. In 2012, the same group published two more methodological articles, one on separation of BMAA from isomers [97] and one describing quantification by LC-MS/MS [135]. These articles have greatly contributed to aquatic BMAA research, but on the same time give rise to some questions.…”

“…For instance, why was an optimum ratio between sample protein and derivatisation reagent advised in the first study [125], and this ratio by far exceeded in the second study [94]? Furthermore, the LC-MS/MS method was adjusted in 2012 because the ratio between product ions used for BMAA identification in samples did not always correspond to the ratio in a BMAA standard [97]. If these ratios do not correspond it is uncertain whether BMAA is present in the samples.…”

“…Commonly used derivatisation techniques in combination with LC-MS/MS analysis are AQC (e.g. [34,97,98]), propyl chloroformate (e.g. [91,99]) and dansyl chloride [100,101]), see Box 1.2 for explanation of AQC derivatised LC-MS/MS analysis.…”

“…When using LC-MS/MS based methods, it is important to distinguish between BMAA and its structural isomers such as 2,4-diaminobutyric acid (2,4-DAB, mostly referred to as DAB), N-(2-aminoethyl) glycine (AEG), ß-amino-N-methylalanine (BAMA) and 3,4-diaminobutyric acid (3,4-DAB) [97,104]. Mostly, BMAA is chromatographically resolved from its structural isomers, but additional selectivity can be obtained by more advanced techniques such as differential mobility spectrometry [104].…”

“…BMAA has also been detected in (some samples of) higher aquatic organisms [83,94,97,100,145], at concentrations between 4.7 and 14.1 µg/g DW in crabs [145], 6.8 and 46.9 µg/g DW in oysters [145] and 0.63 and 1.6 µg/g wet weight in mussels [100] by LC-MS/MS based studies. 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]).…”