Cyclic imines, including spirolides (SPXs) and pinnatoxins (PnTXs), are structurally similar neurotoxins that antagonize the nicotinic receptors. The toxins produce rapid, all-or-nothing responses in mice; exposed animals either suffer acute trauma to the central nervous system and die within 20 minutes or present no symptoms at all. While no direct link between these compounds and human intoxication has emerged to date, the detection of cyclic imines in shellfish and sea water provoked the European Food Safety Authority to call for sufficient exposure data to conduct public health risk assessments. In this paper, García-Altares et al.1 applied liquid chromatography mass spectrometry (LC-MS) for rapid screening of trace cyclic imines in marine products.
In order to avoid false positives, the researchers detected the compounds using low resolution 3200QTrap followed by high resolution (HR) MS on an LTQ Orbitrap Discovery instrument (Thermo Scientific) with inspection for the precursor ion and three product ions within a fit-for-purpose mass window. Using solid phase adsorption toxin tracking (SPATT) devices to assess 34 passive seawater samples, they reported unequivocal detection of PnTX-G (0.3 to 0.9 μg/kg-resin) in 17 samples. They also analyzed samples of mussels and oysters and reported positive detection of PnTX-G (2 to 60 μg/kg) and SPX-1 (2 to 16 μg/kg) in 13 of the 22 samples. Five of these samples were positive for both cyclic imines.
The researchers report that nine of the shellfish samples presented indicators of PnTX-G and/or SPX-1 in the Orbitrap but did not meet the standards for positive identification. They observed a similar occurrence for SPX-1 indicators in SPATT samples. This suggests either an overloaded C-trap with reduced sensitivity for low abundance species or that the broad dynamic range compromised the mass accuracy measurements for ions at both extremes of the intensity spectrum.
The team also interrogated for acyl ester derivative analogs of common cyclic imines. They compared free pinnatoxins after alkaline hydrolysis with the concentration present in the crude extract and reported only a 20% increase in PnTX-G, which was below the threshold for analogs. Precursor ion scans confirmed the absence of acyl esters, in agreement with other studies. The researchers note that future studies should include species- and state-specific screening for these derivatives.
García-Altares et al. report that high resolving power and precise mass accuracy, coupled with the ability to screen unlimited precursor ions in full scan mode, render Orbitrap HRMS ideal for decreasing reliance on reference standards. Indeed, the measurement of the precursor and three diagnostic product ions meets the standard for confirming positive identification of pinnatoxin and spirolide analogs, according to existing guidelines.2 In this study, they present excellent reliability for mass accuracy measurements (<2 ppm in all cases). Even at very low concentrations (2.5 ng/mL SPX-1, 0.95 ng/mL PnTX-G), the team noted robust mass accuracy (<1 ppm).
The researchers posit that, even in the absence of specific regulations, shellfish safety monitoring programs should include cyclic imines in their assessment protocols. They note that benchtop HRMS platforms and suitable techniques like the one described in this paper could ensure identification of emerging marine toxins and provide robust risk assessment for public health and consumer awareness.
References
1 García-Altares, M. et al. (2014) ‘Confirmation of Pinnatoxins and Spirolides in Shellfish and Passive Samplers from Catalonia (Spain) by Liquid Chromatography Coupled with Triple Quadrupole and High-Resolution Hybrid Tandem Mass Spectrometry.’ Marine Drugs, 12, 3706-3732, doi:10.3390/md12063706
2 European Commission (2002) 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Union, L221, 8–36.
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