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ANALYSIS OF PARALYTIC SHELLFISH TOXINS IN BIVALVE MOLLUSKS USING HPLC METHOD WITH POST-COLUMN DERIVATIZATION AND FLUORESCENCE DETECTION

The paralytic shellfish toxins are a group of 18 secondary metabolites deposited in bivalve mollusks by dinoflagelates. Dinoflagelate blooms are seasonal, occurring during warm months. Since it is unpredictable whether the infestation will occur, the shellfish population should be regularly monitored for toxins. Ingestion of contaminated shellfish can lead to paralytic shellfish poisoning: a life-threatening illness. The Mouse Bioassay method used to detect dinoflageiate-derived neurotoxins has major drawbacks, which led to exploration of chromatographic methods of analysis. Recently, a HPLC method that utilizes post-column oxidation of the toxins under alkaline conditions has been approved as a new official AOAC method - OMA 2011.02. Three groups of toxins are separated on C18 column using a single step-gradient method. The products of post-column derivatization of the toxins can be detected with high sensitivity using a fluorescence detector, leading to the determination of toxin type and concentration. We describe the use of Pickering Laboratories post-column derivatization system for analysis of paralytic shellfish toxins according to AOAC Method 2011.02.

Method

Sample Preperation:

- Transfer 5 g of homogenized shellfish tissue into a 50 mL centrifuge tube and add 5 mL of 0.1 N HCI

- Vortex the mixture, adjust the pH to be in range of pH 2-4 as necessary

- Heat the mixture in the boiling water bath for 5 min, cool to room temperature,
   and recheck the pH, adjusting if necessary

- Centrifuge the mixture and transfer 500 uL of supernatant into a microcentrifuge tube

- Add 25 uL of 30% trichloroacetic acid (TCA) to deproteinate the extract, mix well and centrifuge

- Adjust pH with I M NaOH to optimum range of pH 2-4

- Filter through 0.2 um filter and inject

Analytical Conditions

Column: Zorbax Bonus RP column, 3.5 urn, 4.6 x 150 mm (Agilent Technologies)

Flow rate: 0.8 mL/min

Mobile Phase:  A. 11 mM heptane sulfonate, 5.5 mM phosphoric acid, adjusted to
                           pH 7.1 with ammonium hydroxide.
                       B. 11 mM heptane sulfonate, 16.5 mM phosphoric acid, 11.5 % acetonitrile,
                           adjusted to pH 7.1 with ammonium hydroxide.

Post-column Conditions

Post-Column System: Pinnacle PCX or Vector PCX

Reactor Volume: 1.0 mL

Reactor Temperature: 85·C

Reagent 1: 100 mM phosphoric acid, 5 mM periodic acid, adjusted to pH 7.8 with 5 M sodium hydroxide

Reagent 2: 0.75 M nitric acid

Reagent Flow Rates: 0.4 mL/min

Detection: Fluorescence detector, Aλ_cx : 330 nm, Aλ_cm: 390 nm

ACKNOWLEDGEMENTS:

We would like to thank Jeffery van de Riet and Ryan Gibbs (Canadian Food Inspection Agency) for their help.

REFERENCES:

(1) van de Riet, J.M., Gibbs, R.S., Chou, F.W., Muggah, P.M., Rourke, W.A., Burns, G., Thomas, K, Quilliam, M.A.
(2009) J. AOAC Int. 92, 1690-1704.

(2) AOAC Official Method 2011.02. Paralytic Shellfish Toxins in Mussels, Clams, Oysters, and Scallops. Post-Column Oxidation (PCOX) Method.