A METHOD FOR DETERMINATION OF PARALYTIC SHELLFISH POISONS IN WATER BY CAPILLARY ELECTROPHORESIS

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1 Cao J., Chandrasena N. & Hawkins P. (1999) A Method for determination of Paralytic Shellfish Poisons in Water by Capillary Electrophoresis. Proc. 18 th AWWA Federal Convention, Adelaide, A METHOD FOR DETERMINATION OF PARALYTIC SHELLFISH POISONS IN WATER BY CAPILLARY ELECTROPHORESIS EXECUTIVE SUMMARY Jing Cao, Nimal Chandrasena, Peter Hawkins, CRC for Water Quality & Treatment Capillary Electrophoresis (CE) is regarded as a powerful analytical tool that combines many advantages of modern liquid chromatography (HPLC) with those of electrophoresis. The major advantages are: (a) high efficiency (related to decreased zone-broadening in a capillary column, fully automated instrumentation, on-line detection connected to computers supported by high efficiency software for rapid data acquisition, analysis, display, and storage,); (b) analysis speed (usually in the rage of 3-10 minutes for a complete separation); (c) operational simplicity (i.e. no pumps, column seals, guard columns or large quantities of organic solvent); (d) low to very low sample volume requirements (usually nl of submitted sample); (e) applicability to polar and non-polar substances equally well with very little modifications; and (f) accuracy and precision in quantitative results. Among major drawback of CE is short optical length of capillary which causes a raising of detection limits. However, detection limits of an application may be improved by either improved clean-up procedures, pre-concentration of the sample, by adjustment of (larger) injection volume, or by special separation techniques such as on-column sample stacking. Paralytic shellfish poisoning (PSP) is a severe form of seafood poisoning, which results from the ingestion of the filter feeding shellfish which contaminated with neurotoxins. At least 18 PSP toxins, which include saxitoxin (STX) and neosaxitoxin (NEO), a complex suite of sulfate and N-sulfate analogies, and their decarbamoyl derivatives, have been identified mainly produced by marine dinoflagellates and accumulated in the shellfish. The PSP toxins are poisonous because of their high-affinity binding to the sodium-ion channel of nerve cell membranes. This interferes with the propagation of electrical impulses along nerve fibres, causing muscular paralysis and death by respiratory failure. In CE, separation of analytes relies on the difference of movement of molecules under the influence of an electrical field, which means the technique is most suited for separation of charged molecules. As many of the neurotoxic paralytic shellfish poisons (PSP toxins) are charged molecules, CE has a high potential as a separation technique for their analysis in water or other matrices. Although these compounds do not contain a chromophore with significant absorptivity in the wavelength range nm, the high sensitivity of CE combined with a UV detector (CE-UV) or a diode array detector (CE-DAD) permits detection limits of part per million levels. Development of CE-based analytical procedures to characterise and quantify neurotoxins is attractive to water industry, particularly to supplement currently available HPLC methods.

2 A CE method for separation of six Paralytic Shellfish Poisons (PSP toxins) was developed using a Beckman P/ACE 5050 instrument and a CE-DAD. The experimental conditions with respect to buffer, ph, ionic strength and voltage were modified from previously used conditions to better separate and detect the underivatized toxins using a wavelength of 200 nm. Morpholine buffer (150 mm), at an operational ph of 5.0, under an applied field of 15 kv voltage provided good separation of Saxitoxin (STX), Neosaxitoxin (NEO) and four Gonyautoxins (GTX1, GTX2, GTX3 and GTX4) with stability and reproducibility. Improved selectivity enabled subsequent stable quantitation of these toxins from a mixture. Field Amplified Sample Stacking (FASS), a relative new technique in CE, was successfully applied to reduce detection limits. The increased sensitivity facilitated detection of toxins at low concentration from a water matrix. The results of the separation method developed and the potential for its adoption as a routine protocol for water quality monitoring are discussed. KEYWORDS Paralytic Shellfish Poisoning toxins (PSPs), Algal Toxins, Capillary Electrophoresis. INTRODUCTION In the last decade, Capillary Electrophoresis (CE), has developed into a powerful technique for rapid, high resolution separation and trace-level analysis of complex polar molecules, small ions, peptides, carbohydrates, oligonucleotides, and pharmaceutical drugs from complex matrices (Grossman et al., 1989; Kuhn & Hoffstetter-Kuhn, 1993). Separations approaching 10 6 theoretical plates in less than 20 min have been readily demonstrated, based on the differences in electrophoretic mobilities of charged molecules under an applied electric field. CE applications are limited only by the necessity for solubility and non-zero electrophoretic mobility of analytes. Although high-performance-liquid chromatography (HPLC) is currently the most commonly used technique for the analysis of algal toxins (Van Egmond et al. 1994), CE is rapidly becoming an important analytical tool, complementary to HPLC for similar analysis (Thibault et al., 1991; Laycock et al., 1994). CE-based methods for algal toxin analysis, with their potential for greater selectivity and sensitivity, would provide the means for an independent assessment of the "risks" associated with trace levels of algal toxins in drinking water supplies. CE is particularly attractive because of high efficiency separations. In CE, liquid flow caused by electroosmosis (EOF) shows a plug profile because the driving force is uniformly distributed along a small diameter capillary. Consequently, a uniform flow velocity vector occurs across the tube, except in the double layer region very close to the capillary surface where flow velocity approaches zero. Zone broadening caused by the laminar flow profile, which is one of the major sources of band broadening in HPLC, is therefore negligible in CE. The nearly flat flow profile provided by EOF allows high separation efficiencies to be realized, often as high as 100-fold compared with HPLC. Small diameter capillaries (<150 m i.d.) are necessary in CE to reduce zone-broadening effects of convection caused by radial temperature gradients (Joule heating) through the electrolyte. Separation selectivity is manipulated by changing the electrophoretic medium: typically the ph and buffer composition, as well as the conditions of equilibria under which electrophoretic migration occurs. Although the use of small diameter capillaries in CE effectively reduces zone-broadening effects from Joule heating, small sample sizes are required, which demand a sensitive detection method. 2

3 Other major advantages of CE are: analysis speed, based on EOF (usually 5-20 minutes for a complete separation) coupled with on-line detection; operational simplicity (i.e. no pumps, column seals, guard columns or large quantities of organic solvents); very low sample volume requirements (usually nl of submitted sample); applicability to polar and non-polar substances equally well with little modifications; and accuracy and precision in quantitative analyses. A CE instrument typically consists of anode and cathode reservoirs with corresponding electrodes, the capillary column where separation occurs, the injection system and the detector (Figure 1). Analytes are injected at the sample site and migrate to the waste reservoir. Separation is based on the different migration velocities of the analytes, which is the sum of the velocity of the electroosmotic flow and the electrophoretic movement. Hence, as given in equation (1): v = v EOF + v ep (1) where v [cm/s] is the apparent velocity of analytes, v EOF [cm/s] the velocity of electroosmotic flow and v ep [cm/s] the velocity of the electrophoretic movement. Equations 2-5 (Kuhn & Hoffstetter-Kuhn, 1993) provide the basis for calculating the differences in electrophoretic mobilities of different analytes in a CE separation. The electrophoretic mobilities of analytes ( ep ) [cm 2 /(10 5 V s)] (2) are calculated by dividing the apparent velocity, v [cm/s] (3) by the field strength, E [V/cm] (4), and subtracting the electroosmotic mobility, EOF [cm 2 /(10 5 V s)] (4). The field strength is simply the voltage, V [V], divided by the total capillary length (L t ) [cm], where L d [cm] is the length of capillary from injection point to detector, t m [s] is the migration time of analytes from injection point to detection. ep + EOF = L t d m L t V (2) L v = d t m (3) v ep = E - EOF (4) Figure 1 A schematic diagram of a CE set up V/volt Cathode Detector Capillary Anode Used buffer Fresh buffer solution Sample The magnitude of electrophoretic mobility depends on the charges carried by the analytes and their sizes in the buffer, which are expressed as (5): ep = Z (5) 6 r 3

4 where Z is the effective charge of the analytes [C] which depends on the degree of the dissociation of the analytes and therefore their pk a values, in the buffer, is the viscosity of the electrolyte system [C V s/cm 3 ], and, r is the hydrodynamic radius of the analytes [cm]. Paralytic Shellfish Poisoning (PSP) is a severe form of seafood poisoning, which results from the ingestion of contaminated shellfish. Toxic phytoplankton blooms, which occur sporadically along many coasts around the world, are the primary source of PSP toxins. Symptoms of PSP are primarily neurological and can vary from nausea and vomiting to death resulting from respiratory paralysis. The toxins include Saxitoxin (STX), and Neosaxitoxin (NEO) and a complex suite of sulfate and N-sulfonate derivatives (Shimuzu, 1988). At least 18 PSP toxins have been identified (Table 1) mainly produced by marine dinoflagellates and accumulated in the shellfish that filter-feed on these organisms such as mussels, clams and oysters. More recently, cyanobacteria, such as Anabaena circinalis, have been found to produce PSP toxins. Based on toxicity, PSPs fall into three primary categories. The carbamate toxins (STX, NEO, GTX 1-4) are the most toxic. The N-sulfocarbamoyl derivatives (B 1, B 2, and C1-4) are much less toxic. The toxicity of decabamoyl toxins is intermediate between that of carbamate and sulfocarbamoyl toxins. This heterogeneity of PSP toxins and their occurrence as mixtures of compounds with different ionisable functionalities presents difficulties in the development of chemical analytical techniques. However, as many of the PSP variants are charged molecules, CE has great potential as a separation technique (Thibault et al., 1991). In fact, the purity of commercially available PSP standards is routinely demonstrated by CE separations (Laycock et al., 1994). Table 1 Structures of Paralytic Shellfish Poisoning (PSP) Toxins PSP R1 R2 R3 R4 MW STX H H H CONH B1 H H H CONHSO GTX2 H OSO 3- H CONH C1 H OSO 3- H CONHSO GTX3 H H OSO 3- CONH C2 H H OSO 3- CONHSO NEO OH H H CONH B2 OH H H CONHSO GTX1 OH OSO 3- H CONH C3 OH OSO 3- H CONHSO GTX4 OH H OSO 3- CONH C4 OH H OSO 3- CONHSO dcstx H H H H dcgtx2 H OSO 3- H H dcgtx3 H H OSO 3- H dcneo OH H H H dcgtx1 OH OSO 3- H H dcgtx4 OH H OSO 3- H CE-based PSP toxin separations have been previously done with a range of electrolytes including sodium citrate (Thibault et al., 1991), sodium borate (Sciacchitano and Mopper, 1993), TRIS-HCl (Laycock et al., 1994), Trisma (Pleasance et al., 1992), and morpholine (Buzy et al., 1994) using low wavelength (>200 nm) for detection. A rapid CE-based analytical method of characterising and quantifying PSPs is very attractive to the water industry, particularly to supplement currently available HPLC methods. In this paper, such a method to separate and quantify six common PSPs is described. To achieve high sensitivity, an on-column concentration procedure (i.e. sample stacking) is introduced, which increases the sensitivity of the analysis by approximately 10-fold. 4

5 EXPERIMENTAL METHODS PSP analysis was carried out using a conventional CE separation method and a sensitivity-enhanced, new technique of field-amplified sample stacking (FASS) described below. Instrumentation and Operational Conditions A Beckman P/ACE Model-5510 CE was used with a diode array detector (CE-DAD), operated by P/ACE System 5000 series software. The separation was performed in a fused-silica capillary (47 cm x 50 m i.d.) with an effective length of 40 cm to the detection window. The capillary was thermostated in a water bath-cooling system at 30 o C, with the detector placed on the cathode side. For the conventional CE method without sample stacking, the instrument operated under a voltage of 15 kv, and nl samples were loaded into the capillary by a 20s automated pressure injection. All electrolytes (running buffer) used in the separations were degassed in a vacuum filter bottle and filtered through a 0.45 m filter before use. Capillary preparation and regeneration involved initial purging with 0.1 M NaOH for 5 min, followed by Milli-q water and running buffer purging for 5 min each. Between sample runs, the capillary was purged with 0.1 M NaOH for 1 min, followed by Milli-q water for 0.5 min, and then with the running buffer for 1 min. PSP Standards PSPs standards were purchased from National Research Council of Canada. These were a set of four ampoules, which contained individual solutions of (1) STX (0.14 mg/ml), (2) NEO (0.14 mg/ml), (3) a mix of Gonyautoxin-2 (GTX 2, 0.12 mg/ml) and Gonyautoxin-3 (GTX 3, mg/ml), and (4) a mix of Gonyautoxin-1 (GTX 1, mg/ml) and Gonyautoxin-4 (GTX 4, mg/ml). Diluted stock solutions of these were made in 0.1-M acetic acid. Development of Separation Conditions Preliminary experiments investigated three buffer systems, namely, Tris-HCL (ph 7.2) with 25 mm sodium dodecyl sulfate (SDS), 25 mm sodium phosphate (ph 7) with 15 mm SDS and 25 mm sodium borate (ph 8.85) with 25 mm SDS, for suitability in establishing a conventional CE method for PSP analysis. All three buffers performed reasonably (data not presented), but were not highly efficient. Calibration curves of peak areas against concentrations of STX indicated that the most linear detector response (at 200 nm) was with the phosphate buffer system, with a detection limit of 10 g/ml of STX, achieving 11,000 theoretical plates. This high detection limit and insufficient efficiencies led us to investigate morpholine as the buffer, which had been earlier used by Buzy et al. (1994) at 100 mm and ph 4. After several experiments, the low conducting morpholine buffer system (150 mm morpholine, adjusted to ph 5 with formic acid), was found suitable for a complete separation of the six PSP standards within 8-10 minutes. Head-Column Field-Amplified Sample Stacking (FASS) method Zhang et al. (1996) described the successful application of a relatively new technique, i.e. head-column field-amplified sample stacking (FASS), to analyse positively chargeable, hydrophobic compounds by CE. This method, which provides a >10-fold sensitivity enhancement, required an initially introduced low-conductivity zone (water plug) of >1 mm length, injected by an electrostatic injection over a time period under 60 s (Zhang et al. 1996). Given that the six PSP toxins are positively charged at ph 5, the FASS method was applied with the morpholine buffer. A water plug, approximately 1 mm, was initially introduced into the column by inserting the capillary inlet into a vial of water. This was followed by an electrostatic 5

6 GTX min GTX min GTX1 6.4 min GTX min NEO 4.60 min STX 4.49 min injection for 20s of the sample (40-60 nl) at voltage 10 kv. The separation was run at 15 kv. The diode-array detector, using the absorption maximum at 200 nm detected PSPs. RESULTS AND DISCUSSION Identification of PSP Toxin Peaks and Quantitative Analysis The detection of PSPs provides a considerable challenge to development of analytical techniques, because of lack of a strong UV chromophore absorbing above 220 nm, low volatility, small molecular weight and the highly polar nature of these compounds. In our study, the DAD operating in the range nm identified PSP toxins which have their highest absorption around 200 nm. Without a mass-spectrometric attachment (i.e. CE-MS), identification of a PSP toxin by CE-DAD (or CE-UV) separations is based on comparing the electrophoretic mobility of the analyte with that of its authentic standard, under the same experimental conditions. This requires highly constant experimental conditions since even small changes in operational parameters can alter the mobility of analytes. The separation, optimised in this work, with or without FASS, achieved such a stability of migration times. Additionally, PSP peaks were identified unequivocally by the method of co-migration and peak enhancement, whereby a test sample is spiked with the reference compound(s) prior to injection. An electropherogram depicting the conventional CE separation of the six PSP toxins in morpholine buffer (150 mm, ph 5) is given in Figure 2. Compared to the preliminary work with phosphate buffer (data not presented), the detection limit of STX was improved by a factor of 140. Application of FASS to an optimised Morpholine buffer-based separation consistently and reliably separated the suite of toxins. The detection limits of STX, NEO, and GTX 1-4 were reduced by 9, 8 and 3 fold compared with that without the FASS method as Figure 2, respectively, in this separation (Figure 3). Figure 2 An electrophoregram showing the separation of six PSPs with 150-mM morpholine buffer at ph 5 under 15 kv applied voltage without FASS. m A U Minutes 6

7 GTX min GTX min GTX min GTX min STX 4.62 min NEO 4.88 min The electrophoretic mobilities of six PSPs and EOF calculated for the two separations, with and without FASS, are given in Table 2. One reason for the increased sensitivity of detecting peaks by 3-9 fold is the electrical injection, which introduces higher amounts of positive PSP toxin ions. A second reason is related to the introduction of the water plug in FASS, which lowers the electrical strength applied on the supporting buffer. As a consequence, there is less Joule heating in the buffer, which leads to higher viscosity and lower EOF (Table 2), while the amount of electrical strength on the sample is increased, leading to higher electrophoretic mobilities. Table 2 Electrophoretic mobilities of six PSP standards after separation using method with or without head-column FASS. Operational Conditions, as given above. Method Electrophoretic mobility (cm 2 /v s) EOF STX NEO GTX2 GTX3 GTX1 GTX4 (cm 2 /v s) Without FASS With FASS Figure 3 An electrophoregram showing separation of PSP toxin standards with 150-mM morpholine buffer, ph 5, 15 kv applied voltage, using on-column FASS method m A U Minutes Quantitative analysis was by the external standard method. The unknown concentration of PSP toxins in a test sample was determined by correlating its peak areas to calibration curves developed with reference samples of known concentrations. Linearity of the detector response allowed the curves to be used accurately for quantitation of PSPs in an unknown sample. The lowest concentrations detected and integrated for STX, NEO and GTX 1-4 were 140, 140, and 360 g/l (ppb) respectively, below which the detector s response was not distinguishable from background. 7

8 Since peak areas of consecutive runs are compared, highly reproducible and accurate injection volumes are obviously required, which are achieved in the CE through automation. Analytical Precision Precision and accuracy of the CE separations were demonstrated by reproducibility of migration time and peak area, in multiple injection experiments. In the methods described, migration times for PSP toxin standards varied only slightly between different independent sample injections, indicating highly stable separations. The accuracy of quantitation was proved using known amounts of toxin standards spiked into 0.1 M acetic acid. Over many experiments, the concentrations of reference compounds in spiked samples were obtained with <5% variation only (data not presented). Application of the CE method for PSP analysis in an Anabaena circinalis sample Application of the CE method for PSP analysis was trialled with freeze dried samples of an algal bloom dominated >95% by Anabaena circinalis. Samples of freeze dried material (100 mg) were suspended in 5 ml of 0.1 M acetic acid and the cells were broken up by ultrasonication for 5 min in ice. The homogenate was centrifuged at 40,000g for 30 minutes until most of the suspended material sedimented. The pellet was re-extracted twice more with ultrasonication and centrifugation as before, and the combined extracts were subject to CE analysis using FASS. Identification of toxins was by comparison of migration times, and by co-migration combined with peak enhancement. Whilst highly reproducible migration times and quantitative results were easily achieved during sample runs on a given day, the results (Table 3) indicated some variation in migration times and quantitative results from different freeze-dried bloom samples which were the same but non-homogenized bloom samples. CONCLUSIONS This work establishes an efficient method for analysis of STX, NEO and GTX1-4, which can be applied to water and algal bloom material in a single CE-DAD experiment within 10 min, using detection at 200 nm. The method reported yielded reproducible quantitative results in terms of both migration times and sensitivity. Owing to the small volume of sample that can be injected onto the capillary (a few nanolitres), the concentration detection limit is of the order of 140 g/l for STX and NEO and 360 g/l for GTX toxins, without any sample pre-concentration. In comparison with existing techniques such as HPLC using derivatization and fluorescence detection, the determination of PSP toxins directly by CE holds promise for routine screening in natural extracts and water. Particularly attractive are its ease of operation, small sample consumption, speed of analysis, separation efficiency and the potential for full automation. Table 3 Migration times (minutes, t m ) and Concentration ( g/g) of PSPs detected in freeze-dried samples of Anabaena circinalis from Lake Belvedere, Homebush Bay. STX NEO GTX2 GTX3 Toxicity Sample No./ (STX eq) Analysis Date t m Conc. t m Conc. t m Conc. t m Conc. Sample 1; 3/8/ Sample 2; 10/9/ Sample 3; 28/9/98-Run Sample 3; 28/9/98-Run Sample 3; 28/9/98-Run

9 It is well known that increased sensitivity in on-line detection of analytes can be achieved by either improved clean-up procedures, pre-concentration of the sample, by adjustment of (large) injection volume, or by special techniques such as sample stacking (Kuhn & Hoffstetter-Kuhn, 1993). These possibilities are exciting opportunities for development of relatively inexpensive, rapid analysis of PSP toxins for water industry needs. ACKNOWLEDGEMENTS The authors thank the CRC for Water Quality & Treatment Research Grant No: under Program 2 for funding this research. Special thanks are due to Drs. Brenton Nicholson and Dennis Steffensen for their interest, support and administration of the Program. REFERENCES Buzy A, Thibault P & Laycock M V (1994) Development of a Capillary Electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins. Journal of Chromatography A Grossman P D, Colburn J C, Lauer H H, Nielsen R G, Riggin R M, Sittampalam G S, and Rickard E C (1989) Application of Free-Solution Capillary Electrophoresis to the Analytical scale separation of Proteins and Peptides. Analytical Chemistry 61, Kuhn R and Hoffstetter-Kuhn S (1993) Capillary Electrophoresis: Principles and Practices. Springer-Verlag, Berlin. Laycock M V, Thibault P, Ayer S W and Walker J A (1994) Isolation and Purification Procedures for the preparation of Paralytic Shellfish Poisoning Toxin Standards. Natural Toxins 2, Pleasance S, Ayer S W, Laycock M V and Thibault P (1992) Ionspray Mass Spectrometry of Marine Toxins, III. Analysis of Paralytic Shellfish Poisoning Toxins by Flow-injection Analysis, Liquid Chromatography/Mass Spectrometry and Capillary Electrophoresis/Mass Spectrometry. Rapid Communications in Mass Spectrometry 6, Sciachhitano C and Mopper B (1993) Analysis of Paralytic Shellfish Toxin (Saxitoxin) in Mollusks by Capillary Electrophoresis. Journal of Liquid Chromatography 16 (9&10), Shimuzu, Y (1988) The Chemistry of Paralytic Shellfish Toxins. In Handbook of Natural Toxins. Vol. 3. Marine Toxins and Venoms. (Ed. A. T. Tu) pp Marcel Dekker Inc. New York. Thibault P, Pleasance S and Laycock M V (1991) Analysis of paralytic shellfish poisons by capillary electrophoresis. Journal of Chromatography 542, Van Egmond H P, Van Den Top H J, Paulsch W E, Goenaga X and Vieytes M R (1994) Paralytic Shellfish Poison reference materials: an intercomparison of methods for the determination of Saxitoxin. Food Additives and Contaminants 11 (1), Zhang C-X and Thormann W (1996) Head-Column Field-Amplified Sample Stacking in Binary System Capillary Electrophoresis: A Robust Approach providing over 1000-Fold Sensitivity Enhancement. Analytical Chemistry 68,