18
D e t e r m i n a t i o n of P a r a l y t i c S h e l l f i s h by
High
Pressure
Liquid
Poisoning
Toxins
Chromatography
JOHN J. SULLIVAN and MARLEEN M. WEKELL
Downloaded by UNIV OF MINNESOTA on May 13, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch018
Department of Health and Human Services, Public Health Service, Food and Drug Administration, Seafood Products Research Center, Seattle, WA 98174
An improved high pressure liquid chromatographic (HPLC) procedure for the PSP toxins is described. The method involves separation of the toxins on a polystyrene divinylbenzene resin column (Hamilton, PRP-1) in the reversed phase mode using heptane and hexane sulfonic acids as ion-pairing reagents. Detection of the toxins is by fluorescence following post-column alkaline periodate oxidation. The sensitivity of the HPLC method is better than the standard mouse bioassay by at least a factor of four for each of the individual toxins.
Paralytic s h e l l f i s h poisoning or PSP i s a s i g n i f i c a n t t o x i c o l o g i c a l problem a f f e c t i n g s h e l l f i s h e r i e s i n many of the temperate regions of the world. PSP problems arise following the accumulation in bivalve molluscs of a number of d i n o f l a g e l l a t e derived neurotoxins (Figure 1). T r a d i t i o n a l l y , analyses for the presence of the PSP toxins in both s h e l l f i s h and the causative d i n o f l a g e l l a t e s have been performed using the mouse bioassay (j_). Due to the non-specific nature o f the bioassay and a number of factors a f f e c t i n g i t s precision and accuracy ( 2 ) , there i s a need for a l t e r n a t i v e assay procedures. Since the PSP toxins lack native fluorescence, useful UV absorption or adequate v o l a t i l i t y , more t r a d i t i o n a l a n a l y t i c a l procedures such as gas chromatography or spectrometry have proven i n e f f e c t i v e in assaying for the toxins. A number of chemical assays for the toxins have been developed though with the fluorometric method of Bates and Rapoport (3) proving to be the most useful to date. This assay i s based on oxidation o f the PSP toxins under alkaline conditions to fluorescent d e r i v a t i v e s . The assay i s highly sensitive, f a i r l y s p e c i f i c for the PSP toxins and was incorporated into a detection method in the column chromatographic separation o f the toxins described by Buckley et a l 0 0 . Following the incorporation o f a number of modifications, the fluorometric method was u t i l i z e d in a post column reaction system (PCRS) with separation of the toxins by high pressure l i q u i d chromatography (HPLC) ( 5 , 6 ) . While t h i s system proved quite useful T h i s chapter not subject to U . S . copyright. Published 1984, A m e r i c a n C h e m i c a l Society
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
198
SEAFOOD TOXINS
for routine toxin assays, a number of problems were evident. Using the procedure as described, i t was not possible to achieve complete separation of a l l the PSP toxins on a single HPLC column. In addition, while the HPLC method was more sensitive than the mouse bioassay for most of the toxins, the detection l i m i t s for neosaxitoxin and gonyautoxins I and IV were too high to be useful due to the poor fluorescence of the N-1 hydroxy toxins (see Figure 1). In this paper i s described a modified HPLC procedure in which marked improvements i n toxin separation and detection can be achieved. The alterations to the previously published method include u t i l i z a t i o n of an alternate HPLC column and an improved reaction/detection system to achieve adequate s e n s i t i v i t y .
Downloaded by UNIV OF MINNESOTA on May 13, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch018
Materials and Methods Apparatus. The major components i n the HPLC system (Figure 2) include a Varian HPLC pump (model 5000, Varian Assoc., Walnut Creek, CA), Altex i n j e c t i o n valve (model 210, Beckman Inst., F u l l e r t o n , CA), PRP-1 HPLC column (15 cm χ 4.1 mm; Hamilton Co., Reno, NV), Kratos post column reaction system (PCRS) (model URS-051, Kratos Analytical Inst., Westwood, NJ), and a Perkin Elmer fluorescence detector (model LS-4, Perkin Elmer Corp., Norwalk, CT). The PCRS was modified s l i g h t l y to accomplish the two step reaction necessary to achieve oxidation of the toxins and a flow diagram i s i l l u s t r a t e d in Figure 3. Pump A i n the PCRS d e l i v e r s the oxidant (periodic acid) and base (ammonium hydroxide) with two elevated chromatography columns (60 cm χ 1.9 cm) used as reagent reservoirs. Either the oxidant or wash solution (water) i s directed to pump A via the six port valve. It i s important that the reservoirs and connecting tubing are well matched to assure that both oxidant and base drain at exactly the same rate. The reaction c o i l (1 ml volume, which i s normally mounted i n t e r n a l l y i n the PCRS) was removed from the PCRS and submerged i n a thermostatically controlled water bath maintained at 75 C. The HPLC conditions which have proven to be e f f e c t i v e i n achieving e f f i c i e n t separation and detection of the PSP toxins are i l l u s t r a t e d i n Table I. Carbamate Toxins
Ν R1
R2
Η
Ν
R3 Ν
ON Ν ON
Ν
(R4-N) STX
N-Sulfocarbamoyl Toxins (R4-S0â) Β1
ΝΕΟ
Β2
GTXI
C3
Η
OSO3 0
Ν
Ν
OSOâ GTXII 0
C1
Ν
0S0 Ν
GTX III
C2
GTX IV
C4
3
ON OSO3 Ν
F i g u r e 1. PSP n e u r o t o x i n s : s a x i t o x i n - S T X , gonyautoxin-GTX, n e o s a x i t o x i n - N E O , and B1-C4 are s u l f o c a r b a m o y l t o x i n s .
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
Determination of PSP Toxins by
Downloaded by UNIV OF MINNESOTA on May 13, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch018
SULLIVAN AND WEKELL
HPLC
Post Column Detector
JJIUJL
Reaction
Recorder
System
\f
F i g u r e 2.
Oxidant IJ
Flow diagram f o r t h e HPLC system.
Base
1
_χ i f 6 Port Valve
u
Column
Bath
fr^
ο
Heating
>
Reagent Pump A Coil Reagent
Λ
>•
