Abstract
The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.
Highlights
Licensee MDPI, Basel, Switzerland.Marine biotoxins are naturally occurring compounds mostly produced by microalgae—namely, diatoms and dinoflagellates, which accumulate in bivalves as a result of their filtering-feeding activity [1,2]
An European Union (EU) Standard Operation Procedure is available for the analysis of these compounds, which involves the extraction with methanol followed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-electrospray ionization (ESI)-MS/MS)
The Liquid chromatography coupled with mass spectrometry (LC-MS) quantitation based on the accurate mass-extracted ion chromatogram (AM-XIC) has been widely used [9,22,23]
Summary
Licensee MDPI, Basel, Switzerland.Marine biotoxins are naturally occurring compounds mostly produced by microalgae—namely, diatoms and dinoflagellates, which accumulate in bivalves as a result of their filtering-feeding activity [1,2]. The pectenotoxin group (PTXs), and sulphated cyclic polyether brevetoxin-like compounds, known as yessotoxins (YTXs). Another group of lipophilic toxins are the azaspiracids (AZA group), which lead to azaspiracid poisoning (AZP) and show symptoms similar to those induced by DSP toxins [4,5]. Lipophilic toxins of OA, YTX and AZA groups are currently regulated by the European Union (EU) by setting their maximum levels in marketed shellfish products [6,7]. An EU Standard Operation Procedure is available for the analysis of these compounds, which involves the extraction with methanol followed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS)
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