A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Abstract

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Toxic cyanobacteria and cyanotoxins in European waters – recent progress achieved through the CYANOCOST Action and challenges for further research

“…Differences in analytical approaches were assumed to be one of the main causes of these diverging results, and in 2012, two independent studies recommended suitable analytical methods for BMAA determination (Cohen 2012;Faassen et al, 2012a). Although some analytical challenges remain (Lage et al, 2015;Faassen et al, 2016), a more consistent picture of the presence of BMAA in aquatic systems appears when only the studies that used recommended analytical approaches (Cohen, 2012;Duncan, 2012;Faassen et al, 2012a) are considered (Faassen, 2014).…”

“…While at first cyanobacteria were hypothesised to be the source of BMAA in aquatic systems, BMAA has now also been detected in diatoms (Jiang et al, 2014a;Lage et al, 2015;Réveillon et al, 2015), and a diatom strain has been shown to contain BMAA during different growth phases (Réveillon et al, 2016a). Recently, a major fraction of BMAA was found to be present in a soluble, bound form in a diversity of samples (Faassen et al, 2016;Rosén et al, 2016), where traditionally BMAA was considered to be present either as a free molecule or in a protein-associated form (Murch et al, 2004). Only if more consensus is created on the presence of BMAA in aquatic systems, and human exposure levels can be assessed with confidence, will it be possible to determine whether BMAA does indeed play a role in the globally-occurring neurodegenerative diseases Alzheimer's, Parkinsonism and amyotrophic lateral sclerosis, as is currently hypothesized (Bradley and Mash, 2009).…”

Specificity of the metabolic signatures of fish from cyanobacteria rich lakes

“…The free and bound β-Methylamino-L-alanine (BMAA) were also measured in the phytoplankton biomass according to the hydrophobic interaction liquid chromatography (HILIC)/MS-MS based methods described previously (Combes et al, 2014;Faassen et al, 2016). This method used solid-phase extraction based on mixed mode sorbent to concentrate and clean up the phytoplankton extract that contained free BMAA.…”

Is there a BMAA transfer in the pelagic and benthic food webs in the Baltic Sea?

“…The method that was employed involves a clean-up procedure based on the liquid-liquid extraction followed by a solid phase extraction, which can introduce a significant loss of BMAA during the work-up. As shown by the inter-laboratory evaluation, the recovery of this clean-up procedure might be as low as <10% (Faassen et al, 2016). Therefore, the addition of an internal standard before the clean-up is required to control for the losses during the sample work-up.…”