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LC/MS/MS Analysis of Lipophilic Marine Toxins in Shellfish According to EU Regulations on Ascentis Express C18

By: Emily Barrey, and Olga Shimelis, Reporter US Volume 34.1


Marine biotoxins can be found worldwide in a variety of saltwater environments. They consist of lipophilic and hydrophilic compounds. These toxins in shellfish have recently come under increased scrutiny. The European Union (EU) has issued regulation changes requiring all shellfish commodities to be tested for levels of these toxins. The current method for the detection of these compounds is a mouse bioassay (MBA) where the effects of the toxins are noted on living mice.1 These toxins pose a health risk to humans with complications such as headaches and gastrointestinal side effects. Current efforts are now being undertaken to update the Codex standard to consider other methods for toxin detection. These methods include LC/MS/MS-based methods. This class of compounds presents a challenge to LC/MS/MS analysis because of the complex nature of matrices as well as the positive and negative ionization techniques needed. Resolution of several isobaric compounds also adds to the difficulty of the method. A robust and highly sensitive method is needed in order to reliably detect and quantitate these compounds from marine food products.

This study will demonstrate a rapid and reproducible method for the analysis of lipophilic marine toxins using certified reference materials.


Mussel tissue homogenate was purchased from National Research Council Canada and spiked at 10 ng/g with 10 different lipophilic marine toxins. After spiking, a methanolic extraction (1:10 w/v) was performed and the samples were analyzed by LC/MS/MS.

Table 1. Marine Toxins and Mass Spec Parameters

Peak No. Compound Polarity (ES) Precursor Ion Product Ion
1 Gymnodimine B (GYM-b) + 508.3 489.3
2 13-Desmethyl spirolide C (SPX1) + 692.5 443.7
3 Yessotoxin (YTX-b) - 1140.6 1060.6
4 Homo-yessotoxin (hYTX) - 1155 1074.5
5 Okadaic acid (OA-c) - 803 254.5
6 Dinophysistoxin-2 (DTX2) - 803 254.7
7 Azaspiracid-3 (AZA3) + 828 658.5
8 Pectenotoxin-2 (PTX2-b) + 875.9* 213.1
9 Azaspiracid-1 (AZA1) + 842.3 653.4
10 Dinophysistoxin-1 (DTX1) - 816.8 254.7

*Ammonium adduct.

Sample Analysis

Analytical separation was performed using an Ascentis® Express C18, 10 cm × 2.1 mm, 2.0 μm HPLC column (Product No. 50813-U). Figure 1 depicts a chromatogram of the analytes. Quantitation was performed using matrix matched calibration standards ranging from concentrations of 0.5 ng/mL to 10 ng/mL corresponding to a concentration range of 5 to 100 ppb in shellfish.

LC/MS/MS Analysis of Marine Toxins on Ascentis Express C18

Figure 1. LC/MS/MS Analysis of Marine Toxins on Ascentis Express C18

Results and Discussion

The HPLC method was developed using Ascentis Express C18 column with 2.0 μm particles with a UHPLC system. In this case the separation was done under acidic mobile phase conditions which showed better compatibility with the stability of the silica-based HPLC column. The Fused-Core® particle contributed to the fast run time with low back-pressure. The separation of the toxins was achieved in less than 4.5 minutes. Utilizing fast MS polarity switching, separation of analytes corresponding to species detected under negative and positive polarities (e.g. DTX2 and AZA3) was achieved. Also, baseline resolution was observed for the isobaric compounds OA-C and DTX2.

The peak shapes for GYM-b and SPX1 were broad under acidic run conditions. Although optimal peak shape was not observed for these compounds, quantitation was reproducible. Figure 2 shows a typical calibration curve. Recoveries for the 10 marine toxins are given in Figure 3. All of the analytes had recoveries ≥ 80% with %RSD’s less than 15% for n=5.

Calibration Curve for Matrix Matched Standards for GYM-b

Figure 2. Calibration Curve for Matrix Matched Standards for GYM-b


Marine Toxin Recoveries in Mussel Homogenate Spiked at 10 ng/g

Figure 3. Marine Toxin Recoveries in Mussel Homogenate Spiked at 10 ng/g


A simple and sensitive method for the analysis and quantification of lipophilic marine toxins in mussel homogenate has been developed using LC/MS/MS analysis. Acidic mobile phase conditions that were compatible with the Fused-Core 2.0 μm Ascentis Express column provided sufficient resolution for the detection of the marine toxins. The HPLC method, with a total run time under 7 minutes, produced separation of the isobaric species and of the toxins that required polarity switching during detection. This method was able to successfully obtain recoveries of more than 80% for all analytes using matrix-matched calibration standards.




  1. Chapela, M.J., et al., Lipophilic toxins analyzed by liquid chromatography-mass spectrometry and comparison with mouse bioassay in fresh, frozen, and processed molluscs. J Agric Food Chem, 2008. 56(19): p. 8979-86.


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