4-Nitro-DL-phenylalanine

We developed a simple and effective analysis procedure that includes pretreatment and determination methods for beta-N-methylamino-l-alanine (BMAA), a cyanobacterial neurotoxin. BMAA may be produced by all known groups of cyanobacteria living in freshwater as well as marine environments. In this paper, we report a novel determination method for BMAA. A cation-exchange resin was effective for the selective concentration of BMAA from cyanobacterial extracts and yielded a high recovery rate. Moreover, liquid chromatography (LC)-electrospray ionization mass spectrometry with a hydrophilic LC column was effective for determining BMAA levels. The quantitation limit for BMAA based on selected ion monitoring (SIM) was determined as 0.5 ng at a signal/noise ratio of 5.

Since diverse taxa of cyanobacteria has been linked to biosynthesis of BMAA, a controversy has arisen about the detection of neurotoxic amino acids in cyanobacteria. In this context, a novel LC-MS/MS method was developed for the unambiguous determination of beta-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) in cyanobacteria and selected plant seeds. Both neurotoxic and non-proteinogenic amino acids were analyzed without derivatization considering the total concentration of the free and protein-bound form. The investigation of overall 62 cyanobacterial samples of worldwide origin by application of this method revealed the absence of BMAA, whereas seeds of Cycas revoluta contained 6.96 microg g(-1) of free BMAA. In contrast, the isomer DAB was confirmed in 16 cyanobacterial samples in concentrations of 0.07-0.83 microg g(-1),whereof one sample is distributed as nutritional supplement. In addition, seeds of Lathyrus latifolius contained 4.21 microg g(-1) of free DAB. Limits of detection were for BMAA<1.0 microg g(-1) in the cyanobacterial matrix and<0.14 microg g(-1) in angiosperm seeds. DAB exhibits higher sensitivities of <0.06 microg g(-1) in cyanobacteria and <0.008 microg g(-1) in angiosperm seeds. The highly specific analysis method with increased detection sensitivity eliminates the disadvantages of derivatization-based methods to be discussed.

We aimed to determine concentrations of the neurotoxic amino acids beta-N-methylamino-L-alanine (BMAA) and alpha-,gamma-diaminobutyric acid (DAB) in mixed species scum material from Dutch urban waters that suffer from cyanobacterial blooms. BMAA and DAB were analysed in scum material without derivatization by LC-MSMS (liquid chromatography coupled to tandem mass spectrometry) using hydrophilic interaction chromatography (HILIC). Our method showed high selectivity, good recovery of added compounds after sample extraction (86% for BMAA and 85% for DAB), acceptable recovery after sample hydrolysation (70% for BMAA and 56% for DAB) and acceptable precision. BMAA and DAB could be detected at an injected amount of 0.34 pmol. Free BMAA was detected in nine of the 21 sampled locations with a maximum concentration of 42 microg/g DW. Free DAB was detected in two locations with a maximum concentration of 4 microg/g DW. No protein-associated forms were detected. This study is the first to detect underivatized BMAA in cyanobacterial scum material using LC-MSMS. Ubiquity of BMAA in cyanobacteria scums of Dutch urban waters could not be confirmed, where BMAA and DAB concentrations were relatively low; however, co-occurrence with other cyanobacterial neurotoxins might pose a serious health risk including chronic effects from low-level doses.