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Corporate Overview

The Standard – August 2018

2. Analyzing Cyanotoxins Using LC-MS/MS with 15N-Stable Isotope-Labeled Internal Standards

Cyanotoxins are toxic bioactive compounds that are released from planktonic cyanobacteria (blue-green algae) under certain conditions1. This can result in harmful algal blooms (HABs) that contaminate water systems and bioaccumulate in aquatic vertebrates and invertebrates. When algal blooms are detected, further analytical testing must be performed to determine the presence and amount of cyanotoxins, which can have enormous impacts on municipal drinking water, and the use of public recreational waters.

Despite the risks cyanotoxins have not been regulated, but have been nonetheless identified as microbial contaminant candidates by the US EPA under the Safe Drinking Water Act (SDWA). The US EPA included cyanotoxins as part of the fourth Unregulated Contaminant Monitoring Rule (UCMR 4), which requires monitoring of contaminants between 2018 and 2020 to help determine future direction of regulatory action for these compounds.3
 
Historically ELISA (Enzyme-linked immunosorbent assay) tests have been used to screen the presence of cyanotoxins. But ELISA has limitations, including lack of specificity and relatively high detection levels, which create some difficulties when confirmation of recreational and drinking waters is needed. In recent years, analysts have increasingly been moving to liquid chromatography with tandem mass spectrometry (LC-MS/MS), which offers very high specificity, and far better sensitivity. Factors such as matrix effects and losses due to sample cleanup steps can lead to inaccuracies, but these discrepancies can be mitigated by the use of an internal standard(s) during LC- MS/MS analysis.4

There are several classes of cyanotoxins with varying degrees of physicochemical properties and toxicity (e.g., microcystins are established nephrotoxins, β-N-methylamino-L-alanine (BMAA) is a known neurotoxin).2  As part of the UCMR 4, the US EPA developed Method 544 for determination of microcystins in drinking water using LC-MS/MS, and included a new deuterated, ethylated MC-LR internal standard (MCLR-d5).5  While synthesis of isotopically labeled parent microcystin compounds remains prohibitively challenging, a process of growing cell cultures in fully enriched media, followed by isolation and purification of specific microcystin congeners, has yielded several high purity, fully enriched 15N- labeled standards.These 15N-Microcystin standards are ideally suited for LC-MS/MS analysis using isotope dilution mass spectrometry, having an identical chemical structure, and with seven or more mass units separation from the native analyte.  

β-N-methylamino-L-alanine (BMAA) is another cyanotoxin whose environmental exposure has been implicated as increasing the risk of neurodegenerative diseases. BMAA is found not only in HABs, but may be a contaminant in Spirulina, an edible form of cyanobacteria frequently used in dietary supplements. To improve analysis of this compound, CIL collaborated with researchers at North Carolina State University to offer a new 13C3/15N2 labeled BMAA standard. 

Chemical Structure of cyanotoxins MC and BMAA

 

Figure. Chemical structure of featured cyanotoxins – MC in a) and BMAA in b). The MC structure is cyclo-(D-Ala-L-X-D-isoMeAsp-L-Z-Adda-D-isoGlu-Mdha), where D-isoMeAsp is D-erythro-β-methyl-aspartic acid, Adda is 3-amino-9-methoxy- 2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, and MDha is N-methyl-dehydro-alanine.6 The X and Z positions are variable L-amino acids that determine the suffix in the MC nomenclature (e.g., MC-LR has L at X and R at Z). 

 

Featured Products
 
  Catalog No.     Description     Concentration     Amount  
DLM-10260-0.025MG Microcystin-LR, ethylated (D5, 98%) Neat 25 µg
NLM-10295-1.2 Microcystin-LR (15N10, 98%) 10 µg/mL in 1:1 methanol:water 1.2mL
ULM-10342-1.2 Microcystin-LR (unlabled) 10 µg/mL in 1:1 methanol:water 1.2mL
NLM-10340-1.2 Microcystin-RR (15N10, 98%) 10 µg/mL in 1:1 methanol:water 1.2mL
ULM-10341-1.2 Microcystin-RR (unlabeled) 10 µg/mL in 1:1 methanol:water 1.2mL
NLM-10343-1.2 Microcystin-YR (15N10,98%) 10 µg/mL in 1:1 methanol:water 1.2mL
ULM-10344-1.2 Microcystin-YR (unlabeled) 10 µg/mL in 1:1 methanol:water 1.2mL
NLM-10345-1.2 Microcystin-LA (15N7, 98%) 10 µg/mL in 1:1 methanol:water 1.2mL
ULM-10346-1.2 Microcystin-LA (unlabeled) 10 µg/mL in 1:1 methanol:water 1.2mL
CNLM-10424-1.2* β-N-Methylamino-L-alanine (BMAA) (13C3,99%;15N2,98%) 100 µg/mL in 0.1M HCI 1.2mL
ULM-10493-1.2 β-N-Methylamino-L-alanine (BMAA) (unlabeled) 100 µg/mL in 0.1M HCI 1.2mL

*US Patent Pending: 62/368,562

Look for updates as we continue to develop and expand our labeled cyanotoxin standards line!

References
1. Dittmann, E.; Fewer, D.P.; Neilan, B.A. 2013. Cyanobacterial toxins: biosynthetic routes and evolutionary roots. FEMS Microbiol Rev, 37(1), 23-43.
2. Merel, S.; Walker, D.; Chicana, R.; et al. 2013. State of knowledge and concerns on cyanobacterial blooms and cyanotoxins. Environ Int, 59, 303-327.
3. Monitoring Unregulated Drinking Water Contaminants: Fourth Unregulated Contaminant Monitoring Rule. https://www.epa.gov/dwucmr/fourth-unregulated-contaminant-monitoring-rule.
4. Stewart, A.K.; Strangman, W.K.; Percy, A.J.; Wright, J.L.C. 2018. The biosynthesis of 15N-labeled microcystins and the comparative MS/MS fragmentation of natural abundance and their 15N-labeled congeners using LC-MS/MS. Toxicon, 144, 91-102.
5. US EPA Method 544 - Determination of Microcystins and Nodularin in drinking water by solid phase extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) https://nepis.epa.gov/Exe/ZyPDF.cgi/P100LXUH.PDF?Dockey=P100LXUH.PDF
6. Beri, J.; Kirkwood, K.I.; Muddiman, D.C.; Bereman, M.S. 2018. A novel integrated strategy for the detection and quantification of the neurotoxin β-N-methylamino-L-alanine in environmental samples. Anal Bioanal Chem, 410(10), 2597-2605.
 






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