Determination of Paralytic Shellfish Poisons Using Liquid Chromatography Roger Guevremont National Research Council Canada, Institute for Environmental Chemistry, Ottawa KlAOR6, Canada Michael N. I2uigley1 Chevron Science Center, University of Pittsburgh, Pittsburgh, PA 15260 A recently published article in this Journal drew attention to the interest created among students when links were drawn between chemistry and the James Bond spy novels written by Ian Fleming (1).Tetrodotoxin, a poison derived from sources such a s the Japanese puffer fish (genus Fugu), was mentioned in several stories. I n fact, tetrodotoxin is just one of a large number of natural poisons found in fish and shellfish. Because toxic shellfish are often associated with the occurrence of so-called "red-tides" (2), the Old Testament 'Author to whom correspondence should be addressed. Current address: Bradfield Hall, Cornell University, Ithaca, NY 14853.
(a) GTX I
Thus says the LORD,"By this you shall know that I am the LORD.Behold, I will strike the waters which are in the Nile with the rod that is in my hand, and they shall he turned to hload 0. And the fish that are in the river shall die, the river shall stink, and the Egyptians will loathe to drink the water of the river.
The Increasing Problem of Toxic Shellfish Since Biblical times, public attention has increasingly focussed on the illness produced from eating toxic shellfish. The most serious mass poisoning in recent years occurred in Eastern Canada in late 1987 (4-6). Around the world, harvests of clams, mussels, scallops, and others are closely monitored for the presence of various toxins, including t h e paralytic shellfish poisons (PSP's). (See Fig. 1.) Numerous instances of PSP infestation have been reported along the west coast of Canada and the USA, the Maritime Provinces of Canada (Nova
(b) GTX I 1
(c) GTX I l l
(d) GTX IV
Figure 1. Molecular structures for the PSP's. GTX is the generic name, gonyautoxin. GTX I is neosaxitoxin H-a-sulfate ester, and GTX I I is saxitoxin 11-a-sulfateester. GTX I l l is saxitoxin 11-gsulfate ester, and GTX IV is neosaxitoxin 11-$-sulfateester. STX is saxitoxin, and NED is neosaxitoxin.
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may contain the earliest known account of what has become a world-wide problem (3. Exodus 7:17-18).
Journal of Chemical Education
Fioure 2. Distribution of PSP outbreaks in North America. See text for fdier oescrtptlon Insets show areas of general ponoetaleo) occurence InroJgnoLl the word
ods involves the use of reverse-phase high-performance liquid chromatography (12). Separation is achieved by injection of shellfish extracts onto a reverse-phase column and elution with a mobile phase containing ion-pairing reagents. Detection of individual toxins depends on formation of fluorescent derivatives (131, a s shown in Figure 3. The techniaue can be adawted for use bv senior students who are interested in enviionmental prohems and have access to shoreline waters or contaminated shellfish. Alkaline oxidation
Experimental Procedure Harvesting the Shellfish Staff of local government environmental health offices should he able to help locate beaches that have yielded toxic shellfish. Figure 2 indicates those parts of the U S . and Canadian coastlines subject to frequent beach closure due to PSP infestation of local shellfish. Insets show that the problem is world-wide. Collect approximately 2 kg of shellfish, and keep them frozen until needed. Extraction of PSP's from Shellfish Meat
This procedure should be carried out in a fume hood, and gloves should be warn.
.Caution:
Add isolation
Carefully break open (shuck) the shellfish, and remove the meat. The toxins are found in greatest concentration in the hepatopancreas. For those able to identify this organ, remove and clean them (otherwise use all the meat), by washing with distilled water. After removing excess water with a sieve, homogenize the meat in a blender with 50% aqueous ethanol solution. Filter the meat-alcohol mixture. Preparation of the Shellfish Extract
Figure 3. Oxidation of saxitoxin (a)to 8-amino-6-hydroxymethyl-2-iminopurine-3(2H)-propionicacid (b),followed by acid isolation to fluorescent 2-amino-8,9-dihydro-4-hydroxy-meihyl- pyrimido [2,1-b]purin-7(1H)-one (c).Adapted from ref 13. Scotia, New Brunswick, Prince Edward Island), and along the Gulf of Mexico Coast from Brownsville, Texas, to Fort Myers, Florida. (See Fig. 2.) Detecting Saxitoxin
One of the most important PSP's is saxitoxin (CA Reg. no. 35523-89-10) which has a n L D 6 of ~ &I0 pg kg-' (intraperitoneal injection, mouse). As the generic name implies, PSP's are neurotoxins, and in amounts greater than 1mg can easily cause death in humans (7). As a result, eating just two or three PSP-infested shellfish (even when cooked) can he fatal. Saxitoxin is reported to be 50 times more potent than curare, a more commonly known neurotoxin i2,. Interestingly, the presence of I'SP'S in shelllish can bc traced hack throuch toxic dinoflagellates ofthe two genera, Protogonyaular and ~ y r w ~ i n i u(8, m 91, which the filter-feeding shellfish concentrate, to toxic bacteria, which had in turn infested the dinoflagellates (10,ll). Due to the dire economic imulications for manv coastal communities dependent on sh'ellfish harvesting,"interest in the PSP ~ r o b l e mis intense and has resulted in manv lnnovatlve tnchnques for the detect~onand determination of the toxlns The most popular of the ~n.;rrnmentalmeth-
Add the shellfish extract to the top of a column of catiouexchange resin, for example, hydrogen form Bio-Rex 70 (Bin-Rad), and elute the retained toxins with 1mol L-' hydrochloric acid solution. Transfer the eluate to a column of gel filtration media, for example, Bio-Gel P2 (Bio-Rad), and elute with 0.05 mol L-' acetic acid solution. Collect the eluate in 10-mL fractions. Stock Standard Solutions
The recommendations of Sullivan (10) were followed in preparation of 1L of 5 mol L-' sodium hydroxide solution 1L of 0.5 ma1 L-' ammonia solution 50 mL of 500 mmol L-' sodium hexane sulfonate
(ion-pairreaeent) solution
1 L of 50 mmal L-' pkriodic acid solution
acid soMobile Phase A. Add 3.0 mL each of .uhosuhoric . lut~on,sodium hrx;inrsuIfonate solut~on,and sodium heprancsulfonatc solutim to 990 ml. ol'de~onrzcdwater. Adiust the pH to 6.7 with ammonia solution. Mobilephase B. Add 3.0 mL each of sodium hexanesulfonate solution and sodium heptanesulfonate solution and 12.5 mL of phosphoric acid solution to 720 mL of deionized water. Adjust the pH to 7.0 with ammonia solution. Finally, add 250 mL of acetonitrile. Oxidant Solution. Add 100 mL of periodic acid solution and 200 mL of phosphoric acid solution to 600 mL of deionized water. Adjust the pH to 7.8 with sodium hydroxide solution, and finally dilute to l L:with deionized water. Volume 71
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0
TIME (MIMTTES) Figure 4. Schematic diagram of instrumentation required for liquid chromatoaraohic determination of PSP's. Comoonents: helium sparg ng gas tar. nign-presswe recprocat ng p~mpsand agrao ent conlro er ,o):lneclor ,C gJara w L m n (d),ana ytca col~rnn(e,, ox aant so JI on p m p I,: reacllon co a1 90 'C g ; aco so ton pLmp ,h : f dorescence aeteclor ,; integrator (,, 0
,
,
Nitric Acid Solution. Dilute 47 mL of concentrated nitric acid (70% wlv) to l L with deionized water. Use two high-pressure reciprocating pumps with a gradient controller to pass mobile phases through a guard column and 150 x 4.1 mm analytical column of PRP-1 resin (Hamilton) a t a flow rate of 1.3 mL m i d . Post-column reaction of the separated toxins is achieved by mixing a stream of oxidant solution a t a flow rate of 1.3 mL min? with the eftluent from the column a t 90 'C. In order to optimize the fluorescent intensity, mix the combined stre& with nitric acid solution a t a flow rate of 0.3 mLmin-'. Use a n excitation wavelength of 340 nm, and monitor the fluorescent emission a t 460 nm. Determine peak heights or areas of the separated components using a strip chart recorder or electronic integrator. A schematic diagram is given in Figure 4. Analysis Iniect a suitable volume of filtered shellfish extract into the sample loop valve, and use a 20-min (0% mobile phase B to 100% mobile phase B) nonlinear gradient to separate
Figure 5. Chromatogram of a shellfish extract, showing separated PSP's. The curved line is the associated gradient profile.
the toxins. A typical chromatogram of n shellfish extract with the associated gradient profile is shown in Figure 5. Compare the peak height5 or arras on the chromatogram with those obtained for standard toxin solutions. Standard solution^ uf the PSP's are commercially available: Acknowledgments The authors are grateful to Dr. Sherwood Hall, FDA, Washington, DC, for providing the shellfish extract, and to M. Ruth Bailey, David W. North, and David J. Myatt, Health and Welfare Canada, Halifax, Nova Scotia, for the chromatogram. The authors are also grateful to Ms. Lisa A. Also, University of Pittsburgh, for assistance in the preparation of the manuscript. Literature Cited
15.
4. ~ i ~ S h o u f i .hhz i n k m . Canadian Chemical Nem, 1988.40, 5. QuiUim, M.A.; Wlight, J. L. C.And. Chem. 1989,61,1053A. 6. Wrkht, J. L. C. Conodlon ChamleolNews 7. Halatead, B. Pokomus and Venomous Merino Animals ofthe
1990.42.18.
W. World: Danvin: Enreton, 1978. 8. Kodams. M.; Ogata T. Mar Follut Bull. 1988, 19,559. 9. Ceme1la.A. D:Sullivan. J. J.;Boyer, 0. L.; Tay1or.F.J.R.;Andersen,R. JBiochom. Sysf E d 1387, 15, 171. 10. Sullivan, J. J.;Wekell,M. M.:Kentda L. L. J F m d S d . 1985.50, 26. 11. Kodama,M.;Ogafa,T:Shigem.S.;S&smoto,S.Mor EcolPlog. Ser. 1990.61.203, sutavm,J. J.; waken. M. M.I" sparood QU~EV ~ ~ b ~ ~ i ~P-. t r ~o" tnsymp.; . . Kramer, D. E.; Liston. J.. Eds.: University ofAlasks, November 1986; pp 357371. 13. Bates, H. A : Rapoprt. H. JAgapiC. Food Chem. 1975,23,237, 12.
'Saxitoxin, neosaxitoxin, and gonyautoxins l-IV are available from Calbiochem, P.O. Box 12087,San Diego. CA92112-4180.
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