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.sub.5] as internal standard

• Published in: Analytical and Bioanalytical Chemistry 2010, Vol.397 (6), p.2245
• Main Authors: Dimitrakopoulos, Ioannis K, Kaloudis, Triantafyllos S, Hiskia, Anastasia E, Thomaidis, Nikolaos S, Koupparis, Michael A
• Format: Report
• Language: English
• Subjects: Analysis; Health aspects; Hostages; Liquid chromatography; Mass spectrometry; Methods; Phenylalanine; Water
• ISSN: 1618-2642
• DOI: 10.1007/s00216-010-3727-3

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 50x2.1 mm C18 column for the separation of anatoxin-a and phenylalanine, achieving a 3-min analysis time. Isotopically labelled phenylalanine-d5 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.sup.-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.sup.-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.sup.-1]. Keywords Anatoxin-a * Cyanotoxins * Water analysis * LC-ESI-MS/MS * Isotope-labelled internal standard * Porous graphitic carbon SPE