Screening for multiple classes of marine biotoxins by liquid chromatography–high-resolution mass spectrometry

Abstract

Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography-mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7 min prior to MS data acquisition at 2 Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1 ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4 ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4 min, and MS data was collected at 1 Hz in positive mode, yielding mass accuracy of less than 1 ppm error at a resolving power of 100,000 for the analytes studied (m/z 300-500). Data were processed by extracting 5 ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10 μg/L (parts per billion) for the positive ions, 1.6-5.1 μg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14 μg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.

Identification of Pinnatoxins and Discovery of Their Fatty Acid Ester Metabolites in Mussels (Mytilus edulis) from Eastern Canada

“…The instrument was calibrated and operated as previously described. 29 The LC conditions described above for the direct analysis of pinnatoxin acyl esters (NRC Method 2) were used. Data were acquired in positive ion mode with a 3.3 kV spray voltage, sheath and auxiliary gas flows of 45 and 10 (arbitrary units), respectively, 250°C capillary temperature, and 300°C heater temperature.…”

The current role of high-resolution mass spectrometry in food analysis

“…Their limited availability as reference material and the assumption that toxicity varies significantly from analogue to analogue made the development of LC-MS/MS methods difficult. This is probably the reason why HRMS seems to be the preferred technique for the analysis of marine toxins [10][11][12].…”

“…Currently, only a few articles have reported screening approaches based on 50,000 or more FWHM HRMS approaches. These include screening for marine toxins [11,12], water pollutants [18], pesticides [27], and veterinary drugs [23,24] and the combined detection of veterinary drugs, mycotoxins, and pesticides [20].…”

Differential Mobility Spectrometry for Improved Selectivity in Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Analysis of Paralytic Shellfish Toxins

“…Compared with the lipophilic classes of toxins that are routinely quantitated by LC-MS/MS [11,12], the analysis of PSTs remains a significant instrumental challenge because of their polarity, the large number of structurally similar analogues, including several epimeric pairs (Figure 1), as well as chemical interferences and matrix effects in ESI. Hydrophilic interaction liquid chromatography (HILIC) has emerged as the LC mode of choice and is able to separate all PST analogues with potential to interfere with one another in MS/MS [6][7][8][9][10]. Equally important as selectivity between interfering PST analogues is selectivity between PSTs and interfering matrix components that co-elute in complex biological samples.…”

“…Analysis of PSTs is most often carried out with ion-pair reverse phase LC (RPLC) with pre-or post-column oxidation and fluorescence detection [4,5]. More recently, LC-tandem mass spectrometry (LC-MS/MS) methods using electrospray ionization (ESI) have been developed [6][7][8][9][10]. Compared with the lipophilic classes of toxins that are routinely quantitated by LC-MS/MS [11,12], the analysis of PSTs remains a significant instrumental challenge because of their polarity, the large number of structurally similar analogues, including several epimeric pairs (Figure 1), as well as chemical interferences and matrix effects in ESI.…”