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.

LC-MS/MS Analysis of Cyanotoxins in Bivalve Mollusks—Method Development, Validation and First Evidence of Occurrence of Nodularin in Mussels (Mytilus edulis) and Oysters (Magallana gigas) from the West Coast of Sweden

“…As natural products of bacteria that typically inhabit eutrophic water bodies, cyanotoxins pose a risk to animals, domestic as well as wild and marine, and humans [ 27 , 28 , 29 , 30 ]. Except through the drinking water, the risk for humans arises upon consumption of aquatic organisms or products that have been contaminated with cyanotoxins such as fish, shellfish or dietary supplements based on cyanobacteria [ 31 , 32 , 33 , 34 ], and through other types of food [ 25 ].…”

A singleMicrocoleusspecies causes benthic cyanotoxic blooms worldwide

“…The presence of ATX and dhATX was confirmed by analyzing the samples by Ultra-high-performance liquid chromatography (UHPLC) coupled with a high-resolution mass spectrometer (HR-MS) [32] or a triple quadrupole mass spectrometer (TQMS) [33]. The extract from scum dominated by Microcoleus contained 0.1 % and 5% of ATX and dhATX respectively (11 µg of anatoxin-a and 241 µg of dihydroanatoxin-a in a 8.2 mg of extract).…”

“…The column was pre-equilibrated with 0% B for 1min, the gradient increased to 60% B over 3.5 min and to 100% over 0.05 min, and was kept to 100% B for 1.24 min. The detection was achieved using the SRM mode as previously reported [33] and a fragmentation from 166.2 to 130 was selected. For the quantification of dihydroanatoxin-a, the calibration curve was based on the SIM of anatoxin-a at 166.2 m / z and the concentration of dihydroanatoxin-a in the sample was calculated using the SIM mode at 168.2 m / z .…”

“…The sample collected in early August was analyzed by Ultra-high-performance liquid chromatography (UHPLC) coupled with a high-resolution mass spectrometer (HR-MS) [32]. The samples collected in September (STable 1) were analyzed using a UHPLC coupled with a triple quadrupole MS (TQMS) and with a selected reaction monitoring strategy for ATX as published previously [33]. Briefly, the cyanobacteria samples were centrifuged (30 min, 4000 g) and the supernatant was discarded.…”

Detection of Anatoxins in Human Urine by Liquid Chromatography Triple Quadrupole Mass Spectrometry and ELISA

“…ATX exposures have been linked to the deaths of multiple animal species and associated with human illness [ 5 , 21 , 22 , 23 ]. A recent report of dog death in the UK occurred only 45 min after direct exposure to ATX-contaminated water, algal mat, and fish [ 23 ]. Reports of human intoxication remain anecdotal due to a lack of diagnostic testing.…”