the domoic acid peak, with the characteristic 242 nm absorbance maximum. A very sensitive .... (c) HPLC mobile phase: Mix 100 mL acetonitrile with ca. 400 ml ...
M. A. Quilliam. Chemical methods for domoic acid, the amnesic shellfish poisoning (ASP) toxin. In: G.M. Hallegraeff, D.M. Anderson & A.D. Cembella (Eds.), Manual on Harmful Marine Microalgae, Monographs on Oceanographic Methodology, Vol. 11, Chapter 9. Intergovernmental Oceanographic Commission (UNESCO), Paris, 247-266 (2003).
Chemical methods for domoic acid, the amnesic shellfish poisoning (ASP) toxin M. A. Quilliam ___________________________________________________________________________ 9.1 INTRODUCTION The amnesic shellfish poisoning (ASP) toxin, domoic acid, was originally isolated from a red macroalga Chondria armata by Japanese researchers studying the insecticidal properties of algal extracts (Takemoto and Daigo, 1958). The structure was later revised by Ohfune and Tomita (1982) (see Fig. 9.1). Domoic acid belongs to a group of amino acids called the kainoids, which are classed as neuroexcitants or excitotoxins that interfere with neurotransmission mechanisms in the brain. The original member of the group, kainic acid (Fig. 9.1), was isolated earlier from another red macroalga Digenea simplex (Murakami et al., 1953). Other members of the group include acromelic acids A and B, isolated from mushrooms (Konno et al., 1988). The ability to perform quantitative analyses for domoic acid became very important after the 1987 incident of ASP in eastern Canada (Wright et al., 1989). Domoic acid was shown to be the causative agent for that incident and was found to be present in the shellfish at levels as high as 1000 mg kg-1 in tissues that caused the illness. This contamination level was easily detected by the mouse bioassay for paralytic shellfish poisoning (PSP) (AOAC, 2000a), with symptoms of domoic acid toxicity being very characteristic and clearly distinct from those for PSP toxins. It quickly became apparent, when the regulatory level was set at 20 mg kg-1 by Canadian regulatory authorities, that the mouse bioassay with a detection limit for domoic acid of only 40 mg kg-1, could not be used for routine monitoring. High performance liquid chromatography with ultraviolet detection (HPLC-UV) was the first chemical analytical method for domoic acid and is still the most commonly used for monitoring shellfish. This method has the advantages of being relatively simple, fast, reproducible and accurate. It has also been validated through the AOAC International Official Methods Program (AOAC, 2000b), which gives it increased acceptance by the regulatory community. A number of other analytical approaches, including thin layer chromatography (TLC), capillary electrophoresis (CE) and liquid chromatography with detection by mass spectrometry (LC-MS), have also been developed and will be presented in this chapter. CH3
CH3
COOH
COOH H COOH H3C
N
COOH
N
H
H
Domoic Acid
Kainic acid
COOH
Figure 9.1 Structures of domoic acid and kainic acid.
Studies immediately after the ASP incident in 1987 led to the discovery that domoic acid was produced by the diatom Pseudo-nitzschia pungens f. multiseries (= Nitzschia pungens f. multiseries) (Subba Rao et al., 1988; Bates et al., 1989). A laboratory culture of this diatom produced domoic acid at levels ranging from 1 to 20 pg cell-1, with 99% purity) trifluoroacetic acid (TFA). (d) 9-Fluorenylmethylchloroformate (FMOC-Cl): Available from Aldrich Chemical Co. (Milwaukee, WI) (e) FMOC-Cl Reagent Solution (15 mM): Dissolve 38.7 mg FMOC-Cl in 10.0 ml acetonitrile. Store in 2-mL glass screw-cap vials with teflon-lined septa in a dessicator at 20oC. All vials should be purged with nitrogen prior to storage. Once the septum has been pierced, any unused reagent should be discarded. (f) Borate buffer (1M, pH 6.2): Dissolve 6.18 g orthoboric acid (BH3O3) in 95 ml deionized water, adjust the pH to 6.2 with 2 M sodium hydroxide, and dilute with water to 100 ml. (g) Domoic acid calibration solutions: DACS-1C (certified 100 µg ml-1, see Appendix 1). Prepare working solutions daily by accurately diluting DACS-1C in seawater to give 5, 10, 25, 50, 100, 200 and 500 ng ml-1. (h) Dihydrokainic acid(DHKA): Available from Sigma Chemical Company (St. Louis, MO) (i) DHKA internal standard solution: Prepare a stock solution (100 µg ml-1) by dissolving 1.0 mg DHKA in 10.0 ml acetonitrile-water (1:9). Prepare a working solution (2 µg ml-1) by diluting the stock solution 50-fold in seawater. (j) HPLC mobile phase: (i) Isocratic system: 40% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid in water. (ii) Gradient system: Solvent A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile. Degas with ultrasonication and gentle vacuum. (k) Liquid Chromatograph: Isocratic or preferably gradient system equipped with a dual monochromator fluorescence detector set for excitation at 264 nm and emission at 313 nm protected by a 280 nm cut-off filter. A filter fluorometer equipped with the appropriate filters may be substituted. (l) HPLC Column: 25 cm long x 4.6 mm i.d. packed with 5 µm C18 bonded silica gel (Vydac 201TP, Supelco LC-PAH, or equivalent); use of a guard column is recommended. Operating conditions: ambient column temp. for isocratic, 55 oC for gradient; 1.0 ml min-1 mobile phase flow rate; 10 µl injection volume for isocratic, up to 100 µl for gradient. If the HPLC is millibore compatible, a 25 cm long x 2.1 mm i.d. column can be substituted, using a 2 µl injection volume for isocratic or 20 µl for gradient, and a mobile phase flow rate of 0.2 ml min-1. Gradient elution: programmed linearly from 30% to 50% B over 15 min, followed by an increase to 100% B over 2 min which is maintained for 5 min before programming back to initial conditions over 2 min. Maintain initial conditions for a further 12 min before the next injection.
Preparation of Samples 1. For determination of domoic acid dissolved in seawater or culture media, filter a 1 ml subsample through a 0.22-µm disposable filter (Millex-GS, Millipore Corp., Bedford, MA). Take the filtrate through the following derivatisation and HPLC procedures. 2. For determination of overall concentrations of domoic acid in plankton culture samples or suspensions of plankton in seawater, first determine the cell density in representative subsamples. Sonicate a homogenous subsample (10 ml) for 1 min at 100 W using a 1-cm diameter probe to disrupt the cells. Then filter 1 ml of sample through a Millex-GS 0.22-µm disposable filter. Take the filtrate through the following derivatisation and HPLC procedures. 3. For determination of concentrations of domoic acid in phytoplankton cells as well as in the medium, first determine the cell density in representative sub-samples. Centrifuge a 10-ml aliquot of sample in a conical tube for 5 min at approximately 900 x g. Remove 5 ml of supernatant and treat as in step 1. Centrifuge the remaining material for another 5 min at 900 x g. Remove all but approximately 0.2 ml supernatant, make the volume to 10 ml with seawater. Sonicate for 1 min at 100 W using a 1-cm diameter probe to disrupt the cells. Then filter the solution through a Millex-GS 0.22-µm disposable filter. Take the filtrate through the following derivatization and HPLC procedures. 4. For other samples, such as plankton biomass (fresh or freeze-dried) or plankton cells isolated on filters, first suspend the cells in seawater and then proceed with step 3. Derivatisation Mix the following in a glass test tube (10 x 75 mm) using a vortex mixer for 10 s: 200 µl sample, calibration solution (g) or seawater blank; 50 µl borate buffer (f); and 10 µl DHKA internal standard solution (i). Then add 250 µl FMOC-Cl reagent solution (e) and mix. After exactly 45 s, add 500 µl ethyl acetate and mix for an additional 45 s. After the mixture has settled, two distinct phases will be visible. Remove the upper organic layer with a disposable glass pipette and discard. This is repeated with two more 500 µl portions of ethyl acetate and 20 s mixing. Transfer most of the aqueous bottom layer to a vial for HPLC analysis. The control of contamination is important when working with samples having low domoic acid concentrations. All glassware, syringes and vials should be rigorously washed (water, methanol, acetone). Deliver all reagents using glass syringes dedicated to each solution, and deliver the sample with an air displacement pipette with disposable tips. HPLC Analysis Inject derivatised calibration solutions (g) over the concentration range 10-300 ng ml-1. If good linearity of response and a zero intercept are evident, single point calibration may then be used routinely. Replicate injections should have a CV
