Confirmation Using Triple Quadrupole and High-Resolution Mass Spectrometry of a Fatal Canine Neurotoxicosis following Exposure to Anatoxins at an Inland Reservoir

Abstract

Cyanobacterial blooms are often associated with the presence of harmful natural compounds which can cause adverse health effects in both humans and animals. One family of these compounds, known as anatoxins, have been linked to the rapid deaths of cattle and dogs through neurotoxicological action. Here, we report the findings resulting from the death of a dog at a freshwater reservoir in SW England. Poisoning was rapid following exposure to material at the side of the lake. Clinical signs included neurological distress, diaphragmatic paralysis and asphyxia prior to death after 45 min of exposure. Analysis by HILIC-MS/MS of urine and stomach content samples from the dog revealed the detection of anatoxin-a and dihydroanatoxin-a in both samples with higher concentrations of the latter quantified in both matrices. Detection and quantitative accuracy was further confirmed with use of accurate mass LC-HRMS. Additional anatoxin analogues were also detected by LC-HRMS, including 4-keto anatoxin-a, 4-keto-homo anatoxin-a, expoxy anatoxin-a and epoxy homo anatoxin-a. The conclusion of neurotoxicosis was confirmed with the use of two independent analytical methods showing positive detection and significantly high quantified concentrations of these neurotoxins in clinical samples. Together with the clinical signs observed, we have confirmed that anatoxins were responsible for the rapid death of the dog in this case.

Keywords: anatoxin-a; cyanobacteria; cyanotoxins; dihydroanatoxin-a; dog poisoning; reservoir.

Figure A1

Maps showing (a) area in relation to SW England and Wales and (b) close up of reservoir with red arrow showing location where dog poisoning occurred (map images courtesy of Google Earth).

Figure 1

Chemical structures of example neurotoxic cyanotoxins including anatoxin-a and associated analogues, dihydroanatoxin-a, homoanatoxin-a and epoxyanatoxin-a, together with an interfering compound Phenylalanine and saxitoxin.

Figure 2

SRM chromatograms obtained following the analysis of (a) ATX calibration standard (b) urine extract (c) Stomach content extract and (d) blood extract, labelling SRM transitions and chromatographic retention times for ATX, dhATX and Phe. ATX = anatoxin-a; dhATX = dihydro anatoxin-a; Phe = phenylalanine; M = matrix interference peak.

Figure 2

SRM chromatograms obtained following the analysis of (a) ATX calibration standard (b) urine extract (c) Stomach content extract and (d) blood extract, labelling SRM transitions and chromatographic retention times for ATX, dhATX and Phe. ATX = anatoxin-a; dhATX = dihydro anatoxin-a; Phe = phenylalanine; M = matrix interference peak.

Figure 2

SRM chromatograms obtained following the analysis of (a) ATX calibration standard (b) urine extract (c) Stomach content extract and (d) blood extract, labelling SRM transitions and chromatographic retention times for ATX, dhATX and Phe. ATX = anatoxin-a; dhATX = dihydro anatoxin-a; Phe = phenylalanine; M = matrix interference peak.

Figure 2

SRM chromatograms obtained following the analysis of (a) ATX calibration standard (b) urine extract (c) Stomach content extract and (d) blood extract, labelling SRM transitions and chromatographic retention times for ATX, dhATX and Phe. ATX = anatoxin-a; dhATX = dihydro anatoxin-a; Phe = phenylalanine; M = matrix interference peak.

Figure 3

LC-HRMS identification of ATX. (A) Base peak (BP) Extracted Ion Chromatograms at m/z 166.1226 and (B) product ion scans for anatoxin standard, urine extract and stomach extract.

Figure 4

LC-HRMS identification of dhATX. (A) Base peak (BP) Extracted Ion Chromatograms at m/z 168.1383 and (B) product ion scans for urine extract (top traces) and stomach extract (bottom traces).

Figure 5

LC-HRMS identification of ATX analogues. Extracted Ion Chromatograms for anatoxin analogues obtained in stomach extract showing base peaks (BP) and associated accurate masses.