Analytical and Bioanalytical Chemistry

, Volume 397, Issue 6, pp 2245–2252 | Cite as

Development of a fast and selective method for the sensitive determination of anatoxin-a in lake waters using liquid chromatography–tandem mass spectrometry and phenylalanine-d 5 as internal standard

  • Ioannis K. Dimitrakopoulos
  • Triantafyllos S. Kaloudis
  • Anastasia E. HiskiaEmail author
  • Nikolaos S. ThomaidisEmail author
  • Michael A. Koupparis
Original Paper


Anatoxin-a is a potent alkaloid neurotoxin produced by a number of cyanobacterial species and released in freshwaters during cyanobacterial blooms. Its high toxicity is responsible for several incidents of lethal intoxications of birds and mammals around the world; therefore anatoxin-a has to be regarded as a health risk and its concentration in lakes and water reservoirs should be monitored. Phenylalanine is a natural amino acid, also present in freshwaters, isobaric to anatoxin-a, with a very similar fragmentation pattern and LC retention. Since misidentification of phenylalanine as anatoxin-a has been reported in forensic investigations, special care must be taken in order to selectively determine traces of anatoxin-a in the presence of naturally occurring phenylalanine. A fast LC tandem MS method was developed by using a 1.8 μm 50 × 2.1 mm C18 column for the separation of anatoxin-a and phenylalanine, achieving a 3-min analysis time. Isotopically labelled phenylalanine-d 5 was employed as internal standard to compensate for electrospray ion suppression and sample preconcentration losses. Both compounds were preconcentrated 1,000-fold on a porous graphitic carbon solid-phase extraction (SPE) cartridge after adjustment of sample pH to 10.5. The method was validated by using lake water spiked at four different levels from 0.01 to 1 μg L−1. Anatoxin-a recovery ranged from 73 to 97%, intra-day precision (RSD%) ranged from 4.2 to 5.9, while inter-day precision (RSD%) ranged from 4.2 to 9.1%. Limits of detection and quantification were 0.65 and 1.96 ng L−1 respectively. The method was successfully applied for the detection of anatoxin-a in Greek lakes at concentrations ranging from less than 0.6 to 9.1 ng L−1.

A novel LC-MS/MS method has been developed for the determination of anatoxin-a in lake waters during cyanobacterial blooms, using phenylalanine-d5 as internal standard


Anatoxin-a Cyanotoxins Water analysis LC-ESI-MS/MS Isotope-labelled internal standard Porous graphitic carbon SPE 


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ioannis K. Dimitrakopoulos
    • 1
    • 2
  • Triantafyllos S. Kaloudis
    • 3
  • Anastasia E. Hiskia
    • 2
    Email author
  • Nikolaos S. Thomaidis
    • 1
    Email author
  • Michael A. Koupparis
    • 1
  1. 1.Laboratory of Analytical Chemistry, Department of ChemistryUniversity of Athens, Panepistimioupolis ZografouAthensGreece
  2. 2.Institute of Physical Chemistry, Laboratory of Catalytic-Photocatalytic Processes (Solar Energy-Environment)National Center of Scientific Research “Demokritos”AthensGreece
  3. 3.Organic Micropollutants Laboratory, Water Quality Control DepartmentAthens Water Supply and Sewerage Company (EYDAP SA)AthensGreece

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