High-Performance Liquid Chromatographic Method Applied to

Chapter 4 High-Performance Liquid Chromatographic Method Applied to Paralytic Shellfish Poisoning Research 1

John J. Sullivan

Downloaded by PURDUE UNIV on July 7, 2016 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch004

U.S. Food and Drug Administration, 22201 Twenty-Third Drive, SE, Bothell, WA 98021 T h e use of high performance liquid chromatography (HPLC) for t h e study o f paralytic shellfish poisoning (PSP) has facilitated a greater understanding o f the biochemistry a n d chemistry o f t h e toxins involved. H P L C enables the determination o f the type a n d quantity o f t h e P S P toxins present i n biological samples. A n overview o f the H P L C m e t h o d is presented that outlines t h e conditions for b o t h separation a n d detection o f the P S P toxins. Examples o f the use o f the H P L C method i n toxin research are reviewed, including its use i n the determination o f the enzymatic conversion o f the toxins and studies o n the movement o f the toxins u p the marine food chain. Advances i n o u r understanding o f the biochemistry a n d chemistry o f natural products are necessarily tied to the availability o f analytical methods for their detect i o n . W i t h o u t t h e means to rapidly a n d accurately detect a c o m p o u n d i n biological samples, research i n t o its biogenesis and metabolic pathways is severely limited. This has been t h e case i n the marine toxins field a n d especially for paralytic shellfish p o i s o n i n g ( P S P ) where t h e development o f rapid and accurate analytical methods for the toxins involved has paralleled t h e growth i n o u r understanding o f the p h e n o m e n o n . Analytical methods for P S P range from t h e somewhat inaccurate and non-quantitative mouse bioassay developed i n the 1930's to more efficient and sophisticated methods based u p o n separation techniques such as T L C , electrophoresis, and H P L C . A historical perspective o n these methods is the subject o f a recent review (1). I n most cases, the identification o f the P S P toxins and advances in o u r knowledge o f t h e biochemistry o f P S P were a direct result o f the development o f new analytical techniques, particularly the H P L C method. W i t h the development o f H P L C , a new dimension was added to t h e tools available for the study o f natural products. H P L C is ideally suited to the analysis o f non-volatile, sensitive compounds frequently found i n biological systems. U n l i k e other available separation techniques such as T L C and electrophoresis, H P L C methods provide b o t h qualitative and quantitative data and can be easily automated. The basis for the H P L C method for the P S P toxins was established i n the late 1970's when Buckley et a l . (2) reported the post-column derivatization o f t h e P S P toxins based o n an alkaline oxidation reaction described by Bates and R a p o p o r t (3). Based o n this foundation, a series o f investigations were conducted to develop a rapid, efficient H P L C method to detect the m u l t i p l e toxins involved i n P S P . O r i g i nally, a variety o f silica-based, bonded stationary phases were utilized with a lowpressure post-column reaction system ( P C R S ) (4,5). Later, with improvements i n toxin separation mechanisms and the utilization o f a high efficiency P C R S , a 1

Current address: Varian Associates, Inc., 2700 Mitchell Drive, Walnut Creek, CA 94599 This chapter not subject to U.S. copyright Published 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

4. SULLIVAN

Paralytic Shellfish Poisoning Research

67

routine method for separation o f the 12 most c o m m o n P S P toxins was achieved (6,7). T h e H P L C method (7) for the P S P toxins has a variety o f applications i n both research and i n public health m o n i t o r i n g programs. A number o f advances i n o u r understanding o f the biochemistry o f P S P are a direct result o f this technique. F o l l o w i n g is a brief overview o f the H P L C method with a couple o f examples o f its utility i n P S P research.


Overview o f the H P L C M e t h o d T h e P S P toxins represent a real challenge to the analytical chemist interested i n developing a method for their detection. There are a great variety o f closely related toxin structures (Figure 1) and the need exists to determine the level o f each individually. They are totally non-volatile a n d lack any useful U V absorption. These characteristics coupled with the very l o w levels found i n most samples (subp p m ) eliminates most traditional chromatographic techniques such as G C and H P L C with U V / V I S detection. However, by the conversion o f the toxins to fluorescent derivatives (5), the problem o f detection o f the toxins is solved. It has been found that the fluorescent technique is highly sensitive a n d specific for P S P toxins and many o f the current analytical methods for the toxins utilize fluorescent detection. W i t h the toxin detection problem solved, the development o f a useful H P L C method was possible and somewhat straightforward. P S P Toxin

Separations

A variety o f H P L C separation strategies have been reported using silica, C-18, and i o n exchange columns (8,9,10). Additionally, toxin separations were developed i n the author's laboratory utilizing amino and cyano bonded phase H P L C columns (4,5). A l l o f these techniques achieved some degree o f toxin separation but either c o u l d not separate a l l o f the toxins desired o r the columns were found to be somewhat unstable under the conditions used. T h e most efficient and reliable toxin separations that have been reported utilize ion-interaction chromatography o n porous polymer columns (6,7). I n this separation strategy, the c o l u m n packing acts as a support for the formation o f a dynamic ion-exchange mechanism between the toxin molecules and a counter i o n contained i n the m o b i l e phase. A s i n conventional ion-exchange, p H and ionic strength c o n t r o l toxin e l u t i o n . However, the primary advantage o f ion-interaction chromatography is that the number and characteristics o f the exchange sites o n the c o l u m n can be varied by adding an organic solvent to the m o b i l e phase and choosing the proper type and concentration o f counter i o n . This greatly increases the efficiency o f the separations, particularly for a series o f compounds such as the P S P toxins where net charges for the various toxins are between -1 and + 2 at p H ca. 7.0. T h e H P L C conditions utilized for the P S P toxin separations (7) are o u t l i n e d i n Table I. Since there is such a range i n net charge for the toxins, conditions have not been found i n which a l l o f the toxins could be separated i n a single H P L C run. M e t h o d 2 can be utilized to separate toxins C I , C 2 , C 3 , and C 4 , while M e t h o d 1 is used to separate the remainder o f the toxins (Figure 2). T h e separat i o n mechanism operating i n M e t h o d 1 is by far the most complex, with a gradient being performed over the course o f the H P L C r u n that varies p H , i o n i c strength, and acetonitrile concentration. It is critical, particularly for this separation, that great care be taken i n mobile phase preparation to achieve consistent toxin separations; details o f these procedures are outlined elsewhere (7). Nevertheless, with care, these H P L C separations are reproducible and c o l u m n lifetimes exceeding 1000 sample analyses are c o m m o n .



MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY

Carbamate Toxins

Sulfamate Toxins

Decarbamoyl Toxins

FM

R2

R3

H

H

H

STX

B1

dc-STX

OH

H

H

NEO

B2

dc-NEO

OH

H

oso;

GTX I

C3

dc-GTX I

H

H

OSOJ

GTX II

C1

dc-GTX II

H

OSOJ

H

GTX III

C2

dc-GTX

OH

OSO-

H

GTX IV

C4

d c - G T X IV

R4: H

2

N

R4: H Y

° "

R4:

0

- /V ""

Figure 1. Structures o f the P S P toxins. Several o f the decarbamoyl toxins have not been reported but are postulated to occur i n nature based o n presence o f the others.


H

°~

III

SULLIVAN


Table I. HPLC Conditions for Separation of the PSP Toxins


Post Column Reaction System (PCRS): Oxidant - 0.5 m L / m i n o f 5 m M periodic acid i n 100 m M phosphate buffer, p H 7.80 A c i d - 0.3 m L / m i n o f 0.75 M nitric acid R e a c t i o n C o i l - 1 m L v o l u m e held at 90 ° C Fluorescence detector at 340 nm excitation, 400 n m emission

Method 1: H P L C c o l u m n - H a m i l t o n P R P - 1 , 15 cm x 4.1 m m , 10 /im packing R o w R a t e - 1.3 m L / m i n , C o l u m n Temperature - 35 ° C M o b i l e Phase A * Water with 1.5 m M each hexane and heptane sulfonate, and 1.5 m M a m m o n i u m phosphate (as P 0 ) , p H 6.70 M o b i l e Phase B : 2 5 % A c e t o n i t r i l e with 1.5 m M each hexane and heptane sulfonate and 6.25 m M a m m o n i u m phosphate, p H 7.00 Gradient: 4

T i m e (min) 0 4 11 17 17.5

A(%) 100 100 70 10 0

B(%) 0 0 30 90 100

Method 2: (Same P C R S M o b i l e Phase phosphate, M o b i l e Phase phosphate, Gradient:

conditions, c o l u m n , and flow rate as M e t h o d 1) A : 3 m M tetrabutylammonium phosphate, 15 m M a m m o n i u m p H 7.50 B : 3 m M tetrabutylammonium phosphate, 30 m M a m m o n i u m p H 6.00

T i m e (min) 0 2.5 6 7

A(%) 100 100 80 0

B(%) 0 0 20 100


70


Detection of the Toxins


D e t e c t i o n o f the P S P toxins has proven to be o n e o f the largest hurdles i n the development o f analytical methods. T h e traditional means, and still i n wide use today, is determination o f mouse death times for a 1 m L injection o f the test solut i o n . There are a variety o f drawbacks to utilization o f this technique i n routine analytical methods, that have prompted the search for replacements. I n 1975 Bates and R a p o p o r t (3) reported the development o f a fluorescence technique that has proven to be highly selective for the P S P toxins, and very sensitive for many o f them. This detection technique has formed the basis for analytical methods involving T L C (11), electrophoresis (72), c o l u m n chromatography (13), autoanalyzers (14), and H P L C (5,6,7). The application o f the fluorescence derivatization technique i n an H P L C m e t h o d involves utilization o f a post c o l u m n reaction system ( P C R S ) as shown i n Figure 3 to carry o u t the wet chemistry involved. T h e reaction is a 2-step process with oxidation o f the toxins by periodate at p H 7.8 followed by acidification with nitric acid. A m o n g the factors that influence toxin detection i n the P C R S are periodate concentration, oxidation p H , oxidation temperature, reaction time, and final p H . B y far, the most important o f these factors is oxidation p H and, unfortunately, there is not o n e set o f reaction conditions that is o p t i m u m for a l l o f the P S P toxins. T h e reaction conditions outlined i n Table I, while not o p t i m i z e d for any particular toxin, were developed to allow for adequate detection o f a l l o f the toxins involved. Care must be exercised i n setting up an H P L C for the P S P toxins to duplicate the conditions as closely as possible to those specified i n order to achieve consistent adequate detection limits. Research Applications of H P L C B y utilizing the H P L C method, it is possible to determine the level o f each individual toxin i n sample solutions. This provides a "toxin profile" that can be very useful i n P S P toxin research studies. T h e ability to examine relative changes i n toxin concentration and profile has greatly facilitated studies relating to toxin production by dinoflagellates, metabolism o f toxins i n shellfish, and movement o f toxins u p the food chain. Since the H P L C method is easily automated and requires only very small sample sizes ( < 1 g tissue), it has clear advantages over other analytical procedures for the toxins i n many research situations. T w o examples o f the utilization o f H P L C for the study o f the P S P toxins follow. Metabolism of the PSP Toxins in Clams O n e o f the first applications o f the H P L C method was the investigation o f differences i n toxin profiles between shellfish species from various localities (4). It became apparent immediately that there were vast differences i n these toxin profiles even among shellfish from the same beach. There were subtle differences between the various shellfish species, and butter clams had a completely different suite o f toxins than the other clams and mussels. It was presumed that a l l o f the shellfish fed o n the same dinoflagellate population, so there must have been other factors influencing toxin profiles such as differences i n toxin uptake, release, o r metabolism. These presumptions were strengthened when toxin profiles i n the littleneck clam (Prototheca Staminea) were examined. It was found that, i n this species, none o f the toxin peaks i n the H P L C chromatogram had retention times that matched the n o r m a l P S P toxins. It was evident that some alteration i n toxin structure had occurred that was unique i n this particular shellfish species. Based

on

this

observation,

a

series

o f investigations

were

conducted


to

SULLIVAN


B

ci


,C2

IJJ 0

4

8

12

16

0

4

8

Figure 2. Chromatogram o f the P S P toxin standards. ditions from Table I: (A) M e t h o d 1; (B) M e t h o d 2.

12

16

Separation c o n -

ACID DATA SYSTEM

DETECTOR

Figure 3. F l o w diagram o f the P C R S utilized i n the H P L C separation o f the P S P toxins.



72


characterize the toxins i n littleneck clams (4, 15). These studies involved determining the toxin profiles i n various clam tissues, incubating k n o w n P S P toxins w i t h tissue preparations, and conducting chemical stability and modification studies o n the u n k n o w n toxins. A l l o f these investigations utilized H P L C separations to characterize the changes taking place i n toxin structure. Figure 4 illustrates some o f these studies w h i c h involved conversion o f the k n o w n toxins to their u n k n o w n form and then back to the k n o w n form again.

t

0

i

l

5

l

10

i

15

0

l

l

5 10 Minutes

i

0

l

5

l

10

Figure 4 A . Chromatogram illustrating the studies conducted o n the enzymatic conversion o f the P S P toxins to decarbamoyl metabolites (appended w i t h an M " i n these figures). Conversion o f C I to d c G T X I I ( G T X I I M ) i n a homogenate o f littleneck clam tissue after 4 and 48 nr. n



SULLIVAN


o

10 min

F i g u r e 4 B . Chromatogram illustrating the studies conducted o n the enzymatic conversion o f the P S P toxins to decarbamoyl metabolites (appended w i t h an " M " i n these figures). Conversion o f a mixture o f C I and C 2 by the clam enzymes was chromatographically distinct from conversion to G T X II and G T X III w i t h weak acid.

i 0

i 5

i 10

i 15

» 20

Minutes

i 25

i 0

i 5

i 10

i 15

r 20

i 25

Minutes

F i g u r e 4 C . Chromatogram illustrating the studies conducted o n the enzymatic conversion o f the P S P toxins to decarbamoyl metabolites (appended w i t h an " M " i n these figures). H P L C was used to confirm that the toxin metabolites (labeled " M " ) were the decarbamoyl form through conversion back to the carbamate form w i t h chlorosulfonyl isocyanate. (See Ref. 15 for H P L C conditions.)


74


Results o f these investigations indicated that there were enzymes present i n the littleneck clam that facilitated decarbamoylation o f either the carbamate o r sulfocarbamoyl toxins to their decarbamoyl form (see Figure 5). These enzymes may be unique to this particular species o f shellfish since, although the decarbamoyl toxins have been found i n small quantities i n other species, n o shellfish species examined to date contains the predominance o f the decarbamoyl toxins found i n the littleneck clams. These studies w o u l d not have been possible without the availability o f the H P L C method that enabled both qualitative and quantitative determination o f the toxins i n extremely small amounts o f biological samples.


Tracing Toxins Up the Food Chain In order to be a hazard to humans, o r to marine vertebrates, the P S P toxins have to be transferred up the food chain from the producing dinoflagellates. B y gaining insights into this transport, the nature and extent o f the impact o f the toxins o n sensitive organisms can be determined. U t i l i z a t i o n o f H P L C i n studies such as these has the advantage o f providing information o n both toxin c o m p o s i t i o n and concentration. A number o f investigations have utilized H P L C to study the transport o f the toxins u p the food chain. Ideally, i n a study such as this, toxin analyses need to be performed o n both the causative dinoflagellates and the accumulating organisms. This is often very difficult to accomplish due to the transient nature o f most dinoflagellate blooms. Consequently, many investigations have relied o n culturing dinoflagellates from the same locality i n which the b l o o m occurred along w i t h collecting the accumulating organism from the vicinity. A third experimental design is to conduct the entire study i n the laboratory, feeding cultured dinoflagellates to the accumulating organisms. A l l o f these approaches have been utilized along w i t h H P L C detection o f the toxins. O n e o f the first uses o f the H P L C method was i n the study o f a Protogonyaulax catenella b l o o m i n Quartermaster Harbor, Washington (4). This incident provided some o f the first evidence that toxin metabolism was occurring i n littleneck clams; chromatograms from these studies are illustrated i n Figure 6. Jonas-Davies et a l . (16) studied a variety o f organisms during a b l o o m several years later i n the same bay and determined that the toxins were accumulated i n a variety o f intertidal organisms besides bivalve molluscs. H P L C was used to determine that Gymnodinium catenation was the causative organism i n P S P outbreaks i n Tasmania (17) and S p a i n (Sullivan, unpublished). G. catenation produces a suite o f toxins predominated by the low-toxicity sulfamate toxins, C I to C 4 , and while shellfish feeding o n G. catenation contain predominantly these toxins, they also contain some carbamate and decarbamoyl toxins. Currently, it is not k n o w n i f the carbamate and decarbamoyl toxins are present due to chemical conversion from the sulfocarbamoyl form o r due to selective retention o f trace quantities already present i n the dinoflagellates. T h e H P L C played a key role i n these studies by providing a rapid, quantitative means to differentiate the various toxins present. H P L C has also been utilized i n more complex food chain transfers. It has been k n o w n for some time that the toxins can be responsible for fish kills i n the Bay o f F u n d y (18). T h e vectors for these fish kills are z o o p l a n k t o n that feed o n toxic dinoflagellates. In two related studies (19; Sullivan, unpublished), H P L C was utilized to investigate the transport o f toxins from dinoflagellates to z o o p l a n k t o n and then to fish. T h e H P L C method is ideally suited for this since only very small sample sizes (ca. 100,000 dinoflagellate cells) are required.



4. SULLIVAN


STX: X=H GTX II: X=«OS0 GTX III: X=0OSO3

3

Figure 5. Enzymatic transformation o f the P S P toxins i n the littleneck clams. H P L C was used extensively to determine the presence and characteristics o f these conversions. (Reproduced with permission from Ref. 15. Copyright 1983 A c a d e m i c Press, Inc.).


75


GTX II -

} GTX III

C2


I o

10 20 Minutes

GTX in

C I 0

GTX IIM \ , GTX HIM

10 20 Minutes

STX

10 20 Minutes

STX Bl I

0

I

I

10 20 Minutes

30

F i g u r e 6. H P L C separation o f the toxins present i n various organisms during a dinoflagellate b l o o m i n Quartermaster Harbor, Washington: (A) dinoflagellates, (B) mussels, (C) littleneck clams, (D) butter clams. T h e toxin metabolites ("M") were later found to be the decarbamoyl form. (See Ref. 4 for H P L C conditions.)


4. SULLIVAN


77

Conclusion H P L C is a key t o o l i n the study o f P S P . A l t h o u g h this technique has only been utilized for approximately 5 years i n P S P research, a number o f important discoveries can be linked directly to it. It is unlikely that these studies w o u l d have been possible without the availability o f H P L C . A s H P L C becomes m o r e widely available to researchers i n the toxin field, progress w i l l continue to be made i n elucidating the nature o f P S P , particularly i n an understanding o f m a m m a l i a n uptake, distribution, and metabolism, an area that, to date, has not been investigated.

Acknowledgment


I w o u l d like to thank Sherwood H a l l these studies.

for the supply o f toxin standards used i n

Literature Cited 1. Sullivan, J. J.; Wekell, M. M.; Hall, S. In Handook of Natural Toxins, Vol. 4, Marine Toxins and Venoms; Tu, A. T., Ed.: Marcel Dekker, Inc., 1988, in press. 2. Buckley, L. J.; Oshima, Y.; Shimizu, Y. Anal. Biochem. 1978, 85, 157-64. 3. Bates, H. A.; Rapoport, H. J. Agric. Food Chem. 1975, 23, 237-39. 4. Sullivan, J. J. Ph.D. Thesis, Univ. of Washington, Seattle, 1982. 5. Sullivan, J. J.; Iwaoka, W. T. J. Assoc. Offic.Anal.Chem. 1983, 66, 297-303. 6. Sullivan, J. J.; Wekell, M. M. In Seafood Toxins; Ragelis, E. P., Ed.; ACS Symposium Series No. 262; American Chemical Society: Washington, DC, 1984; pp 197-205. 7. Sullivan, J. J.; Wekell, M. M. In Seafood QualityDetermination;Kramer, D. E.; Liston, J., Eds.; Univ. of Alaska, Sea Grant College Program, Anchorage; 1987; pp 357-71. 8. Boyer, G. L. Ph.D. Thesis, Univ. of Wisconsin, Madison, 1980. 9. Robinson, K. A. Biochem. Biophys. Acta 1982, 687, 315-20. 10. Onoue, Y.; Noguchi, T.; Nagashima, Y.; Hashimoto, K.; Kanoh, S.; Ito, M.; Tsukada, K. J. Chrom 1983, 257, 373-79. 11. Hall, S.; Reichardt, P. B.; Neve, R. A. Biochem. Biophys. Res. Comm. 1980, 97, 649-53. 12. Oshima, Y.; Fallon, W. E.; Shimizu, Y.; Noguchi, T.; Hashimoto, Y. Bull. Jap. Soc. Sci. Fish. 1976, 42, 851-56. 13. Ikawa, M.; Wegener, K.; Foxall, T. L.; Sasner, J. J.; Noguchi, T.; Hashimoto, K. J. Agric. Food Chem. 1982, 30, 526-28. 14. Jonas-Davies, J.; Sullivan, J. J.; Kentala, L. L.; Liston, J.; Iwaoka, W. T.; Wu, L. J. Food Sci. 1984, 49, 1506-09. 15. Sullivan, J. J.; Iwaoka, W. T.; Liston, J. Biochem. Biphys. Res. Comm. 1983, 114, 465-72. 16. Jonas-Davies; J.; Liston, J. In Toxic Dinoflagellates; Anderson, D. M.; White, A. W.; Baden, D.G., Eds:, Elsevier Science Publishing: New York, 1985; pp 467-72. 17. Oshima, Y.; Hasegawa, M.; Yasumoto, T.; Hallegraeff, G.; Blackburn, S. Toxicon 1987, 25, 1105-11. 18. White, A. W. J. Fish. Res. Board Canada 1977, 34, 2421-24. 19. Boyer, G. L.; Sullivan, J. J.; LeBlanc, M.; Anderson, R. J. In Toxic Dinoflagellates; Anderson, D. M.; White, A. W.; Baden, D. G., Eds.; Elsevier Science Publishing: New York, 1985; pp 407-12. RECEIVED July 10, 1989


High-Performance Liquid Chromatographic Method Applied to

Recommend Documents