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Analysis of PSP toxins in Norwegian mussels by a post-column derivatization HPLC method
Toxicon 43 (2004) 319–327 www.elsevier.com/locate/toxicon
Analysis of PSP toxins in Norwegian mussels by a post-column derivatization HPLC method T.N. Aspa,*, S. Larsenb, T. Aunea a
Section of Food Hygiene, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., 0033 Oslo, Norway Section of Epidemiology, Norwegian School of Veterinary Science, P.O. Box 8146 dep., 0033 Oslo, Norway
b
Received 4 July 2003; accepted 6 January 2004
Abstract The aim of this study was to investigate the agreement between the AOAC mouse bioassay and an HPLC method for determining Paralytic Shellfish Poisoning (PSP) toxicity in blue mussels from the Norwegian coast. The AOAC mouse bioassay has traditionally been used for determining the toxin levels. Recently, an HPLC method for determining PSP toxins has been implemented and run in parallel with the bioassay. Four hundred mussel extracts from the last six years were analysed with both methods. A highly significant linear correlation between the methods was achieved ðr ¼ 0:84Þ: However, the relation between the two methods was best described by a second-degree polynom. Totally, this model explained 87% of the variation in the HPLC method. By agreement analysis, it was possible to establish cut-off levels for the HPLC method related to the AOAC mouse bioassay with kappa values .0.80 for toxicity levels below 500 MU/100 g. The HPLC method could substitute the mouse bioassay in determining PSP toxicity in Norwegian mussels. Additionally, about 70 mussel samples were also analysed by the MIST Alerte test kit. The kit did not give any false negative results compared with the regulatory limit, but 30% of the samples below the cut point were also determined as positive. q 2004 Elsevier Ltd. All rights reserved. Keywords: Paralytic shellfish poisoning toxins; AOAC mouse bioassay; Norwegian mussels
1. Introduction Toxins associated with Paralytic Shellfish Poisoning (PSP) are among the most acutely toxic substances known. They constitute a group of at least 21 structurally related neurotoxins produced mainly by dinoflagellates of the genus Alexandrium (Wright, 1995). They can be divided into four subgroups and the three most important are shown in Fig. 1. The toxins accumulate in shellfish feeding on the algae with no apparent negative effect on the shellfish. However, these contaminated shellfish pose a serious threat to human health and economic losses for shellfish industries worldwide. The different toxins vary significantly in their toxicity (Fig. 1) * Corresponding author. Tel.: þ47-22-96-48-32; fax: þ 47-2296-48-50. E-mail address: [email protected] (T.N. Asp). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.01.004
with the main toxin, saxitoxin (STX), as one of the most toxic (WHO, 1984). PSP has been known for centuries in other parts of the world, while one of the earliest documented incidents of PSP in Europe took place in the Oslo Fjord in Norway in 1901. This episode is thoroughly described in the Norwegian Medical Journal the same year (Thesen, 1901). All together, seven episodes of human intoxications have been reported in Norway with a total of 32 victims, including 2 fatalities back in 1901. The last two reported episodes took place in the Trondheim Fjord in Mid-Norway in 1991 and 1992 when the highest concentration of PSP toxins measured in mussel was 96.000 MU/100 g in the spring of 1992 (van Egmond et al., 1993). A survey program on PSP toxins started in 1962 in the Oslo Fjord area and a more comprehensive monitoring program was established in 1981 (Yndestad and Underdal, 1985) covering larger parts of the coast. During the last 20 years, this program has expanded to
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Fig. 1. The PSP toxins and their relative toxicities.
include the rest of the coastline. Even the most northern part has been monitored since 1997 and considerable levels of PSP toxins in shellfish from this area have been demonstrated some years. Mussels intended for commercial marketing have undergone regular monitoring and control for PSP toxins since 1986. PSP contaminated mussels are demonstrated every year from several locations along the coast, but the toxin levels show a very variable pattern and the areas with high levels shift with time. The farming of shellfish is a fast growing industry in Norway and the demand for toxin testing of the mussels before marketing increases accordingly. In accordance with international trade, the method used for this testing has conventionally been the official AOAC mouse bioassay (AOAC International, 1984). This is a non-selective bioassay with animal symptoms and time to death as the criterion of PSP toxicity. The toxicity has been expressed as mouse units (MU) per 100 g mussel meat, where 1 MU is the amount of toxin required to kill a 20 g mouse in 15 min (Sommer and Meyer, 1937; Hall et al., 1990). The sensitivity of the mouse population used is calibrated using reference standard STX, but often 1 MU is said to equal 0.20 mg STX. Most countries use 400 MU/100 g shellfish as action level for closure of the harvesting (Mons et al., 1998). The bioassay also serves as the reference method in the European Union (EU) and EU Council Directive 91/492/EEC states that the total PSP toxin content must not exceed 80 mg/100 g of mollusc flesh in accordance with the biological testing method (Council of the European Community, 1991). The compiled toxicity of the PSP toxins determined with chemical methods may be expressed as saxitoxin equivalents (STX eq.). Then, the amounts of the toxin analogues present are converted to STX equivalents according to their individual toxicities. In reality, it is
the dihydrochloride of STX that is used, since this was the form of the STX originally used in the mouse bioassay. This means that the regulatory limit is actually 80 mg STX-diHCl eq./100 g. This equals 65 mg STX eq./100 g when STX is calculated as the free base. Ethical considerations against sacrificing a large number of animals place an increasing pressure on regulatory bodies and researchers to provide alternative methods. Several other methods are described in the literature. They comprise both biochemical assays designed to estimate the total toxicity (Usleber et al., 1997; Chu et al., 1996; Jellett et al., 1992), and chemical analytical techniques capable of more selective detection of the individual toxin analogues (Pleasance et al., 1992; Locke and Thibault, 1994; Janecek et al., 1993; Lawrence et al., 1995; Sullivan and Wekell, 1986; Oshima, 1995b). The most commonly used chemical methods utilize reversed phase liquid chromatography with fluorescence detection (HPLC-FLD). The toxins are derivatized (oxidized) into fluorescent substances either before separation (Lawrence et al., 1995; Lawrence and Niedzwiadek, 2001), or after (Oshima, 1995b). This study describes the implementation of a postcolumn derivatization HPLC method described by Oshima (1995a), but with some modification regarding the sample preparation. The results from the chemical method are compared with results from the mouse bioassay. Furthermore, a rapid field test kit, MIST Alerte, designed to screen for PSP toxins (Laycock et al., 2000; Jellett et al., 2002) was tested, and the results are compared with the results from the mouse bioassay. The test strip is sensitive to the carbamates (STX, NEO, GTX 1 – 4) and dcSTX, C1/2 and B1.
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2. Material and methods
2.4. Sample preparation for HPLC-FLD analysis
2.1. Material
The acidic extracts prepared for the mouse bioassay were also used for the chemical analysis. The proteins in 500 ml extract were precipitated with 50 ml 5% tungstic acid. The supernatants were subjected to Oasis HLB solid phase extraction (SPE) cartridges (Waters, Milford, Massachusetts, USA) and filtered through Spin-X microcentrifuge 0.2 mm filters (Costar, USA). The SPE sorbent consists of a macroporous copolymer with a mix of divinylbenzene and N-vinylpyrrolidone.
All together 409 mussel samples were analysed with both the AOAC mouse bioassay and the HPLC method. A total of 72 samples analysed with the mouse bioassay were additionally examined with the MIST Alerte test kit. The PSP extracts were stored at 220 8C. 2.2. Chemicals and standards All chemicals and solvents used were of pro-analysis or HPLC grade and the water was grade 1 from a Milli-Q system (Millipore, Milford, USA). The acetic acid, hydrochloric acid, 85% phosphorous acid, 25% ammonium hydroxide, periodic acid and di-potassium hydrogen phosphate were from Merck (Darmstadt, Germany), the tungstic acid from Sigma (Steinheim, Germany), the sodium salt of 1-heptan sulfonic acid from Acros (Acros Organics, Geel, Belgium), while methanol and acetonitrile were from BDH (BDH Laboratory Supplies, Poole, England). The carbamate toxins (STX, NEO, GTX 2/3 together and GTX 1/4 together) as well as B1 of the PSP complex were purchased from National Research Council of Canada, Institute for Marine Biosciences (Halifax, Canada). Recently, standard mixtures containing some of the other toxins (dcSTX and C1 – 4) were received from Canada in connection with an AOAC collaborative study of a precolumn derivatization method. 2.3. Mouse bioassay The mouse bioassay was performed according to the AOAC method for paralytic shellfish poisons using white female BOM:NMRI mice weighing between 16 and 20 g. The mice are injected with 1 ml extracts obtained from fresh shellfish meat extracted with 0.1 M HCl and pH adjusted to 3.0 ^ 0.5. The mice (2 or 3 mice per extract) are observed for 1 h and the mean survival time is used to calculate the toxicities in MU. The dose-response curve for the amount of STX against mouse survival time is not linear so in order to use it quantitatively it is necessary to dilute the shellfish extract until the survival time is between 5 and 7 min. In our monitoring programs, this is regarded as an unnecessary sacrifice of animals and consequently dilutions are not done since the main purpose is protection of consumers. This leads to larger uncertainties in the estimates of toxin levels above 400 and below 250 MU/100 g. In addition, when just one out of 2 or 3 mice dies within an hour, the result is normally given as trace amount, but in this study the MU was calculated based on the mouse that died in order to obtain a number for comparison. Three extracts from 1996 and 1997 were reanalysed with the mouse bioassay in 2003, and no significant loss in toxicity was found.
2.5. Post-column HPLC-FLD The post-column HPLC-FLD analyses were carried out according to the method described by Oshima (1995a) using a Perkin– Elmer HPLC system with two extra micro pumps and an oven with a reaction coil between the column and the detector in order to perform the post-column oxidation. The toxins were separated on a silica based reversed phase column (150 £ 4.6 mm2 i.d., 5 mm particles, Chrompack C8). Using the Oshima method it is possible to detect at least 12 of the PSP toxins by utilizing three different mobile phases. In this study, only the six carbamates were determined, since they were commercially available as standards. The mobile phase used was 2 mM sodium 1heptanesulfonate in 30 mM ammonium phosphate buffer (pH 7.1): acetonitrile (96:4) (mobile phase 1). A mobile phase consisting of 2 mM sodium 1-heptanesulfonate in 10 mM ammonium phosphate buffer (pH 7.1) was used occasionally to confirm GTX 1 – 4 (mobile phase 2). After separation of the toxins on the column, they were oxidized by 7 mM periodic acid in 50 mM sodium phosphate buffer (pH 9.0) in a reaction coil (0.5 mm £ 10 m) at 85 8C. The reaction was stopped by adding 0.5 M acetic acid and the oxidized toxins were detected by a fluorescence detector with lex ¼ 330 nm and lem ¼ 390 nm: 2.6. Toxicity calculations from the HPLC-results The quantities of each of the carbamates in the shellfish extracts were determined from peak height measurements compared with a calibration curve based on spiked shellfish material. The overall toxicities of the samples were given in SXT equivalents (and not STX-diHCl equivalents) based on the amount of toxin and its relative toxicity compared to STX (Fig. 1). The relative toxicities were based on the specific toxicities given by Oshima (1995b). 2.7. MIST Alerte test kit MIST Alerte test kit (Jellett Biotek Ltd, Nova Scotia, Canada) was used according to the manufacturer. Again, the extracts intended for the mouse bioassay were used. Hundred microlitres of the shellfish extracts were diluted with 500 ml of a buffer provided with the test kit. Hundred
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microlitres of the resulting solutions were placed on the test strip and the results recorded after 20 min. If the colour intensity of the test line was 100 or 75% of the control line, the test was considered negative, and if the colour intensity of the test line was 50, 25% or less compared with the control line, the test was considered positive. The cut point for the kit was given by the manufacturer to be 200 MU/100 g. 2.8. Statistical methods In order to express the outcome of the chemical method as a function of the results from the mouse bioassay, a General Linear Model based on a second-order polynom was used (Kleinbaum et al., 1998). The determination of the cut-off levels between the chemical method and the mouse bioassay, and between the test kit and the mouse bioassay, were performed using agreement analysis for categorized variables (Agresti, 1990). This agreement is estimated by Kappa which is an inter-correlation coefficient for such variables. Kappa is defined by the formula k ¼ ðp0 2 pe Þ=ð1 2 pe Þ in which p0 denotes the observed proportion and pe the expected. The mouse bioassay and the chemical method express the toxin content in two different ways (MU and STX equivalents), but both methods are quantitative using continuously distributed variables. Cut-off levels from one measuring scale were compared with cut-off levels from the other by calculating the kappa value for each cross-table. Best agreement was decided from the highest kappa value with most equal sensitivity for the two methods. The mouse bioassay and the test kit do also express the toxin content in two different ways, but here the comparison is between a quantitative and a qualitative method since the test kit is either positive or negative. In this case, the agreement analysis gave the experimental cut-point of the MIST Alerte test kit compared to different cut-off levels of the mouse bioassay.
3. Results The six carbamates were separated in one chromatographic run using mobile phase 1. Complete separation of GTX 1 and 4 is not achieved with this mobile phase (Fig. 2A), but they could be quantified individually using peak heights. However, matrix interferences were often experienced in the time region where the GTX’s eluted when C18 SPE sorbent was used in the sample preparation. Alternative SPE sorbents were evaluated and the Oasis HLB sorbent gave a cleaner extract (Fig. 2B). The recovery was somewhat lower with the HLB clean-up compared with the C18, but the limits of detection were still satisfactory compared with the regulatory limit of 65 mg STX eq./100 g shellfish (Table 1). The standard solution of GTX 2/3 also contained small amounts of their
decarbamoyl counterparts enabling determination of their chromatographic retention times. With mobile phase 1, both dcGTX2/3 and B1 have retention times together with the GTX’s. However, when mobile phase 2 was used, they could be separated with the exception of B1 and dcGTX 3 which co-eluted (Fig. 2C). The chromatograms provided with the original Oshima method showed that dcSTX would elute between NEO and STX using mobile phase 1 and later this was confirmed with a standard mixture containing dcSTX. The standard mixture with the four C toxins revealed that two toxins (C1/2) eluted together with the matrix front and the other two (C3/4) together with the GTX’s when mobile phase 1 was used (data not shown). The linear correlation based on all 409 shellfish extracts was 0.79, while the correlation was 0.84 if the values above 800 MU/100 g were excluded. However, the relation between the two methods was best described by a second-degree polynom. The second degree of AOAC mouse bioassay contributed significant and totally, this method explained 87% of the variation in the HPLC method (Fig. 3A). The relation between the two methods was expressed by the equation: STX-equivalents=100 g ¼ 22:0 þ 0:23 MU=100 g þ 0:00024 ðMU=100 gÞ2 : By agreement analysis, it was possible to establish cutoff levels for the HPLC method related to the AOAC mouse bioassay (Table 2). The best agreement for the 150 MU/100 g shellfish was 20 mg STX eq./100 g, even though the kappa value is somewhat higher for 10 mg STX eq./100 g, but here the methods differed more in sensitivities. For the 200 MU/100 g level the kappa values are increasing with increasing levels of STX eq./100 g up to 0.85 and 0.86 for 40 and 50 mg STX eq./100 g, respectively, and then decreasing for higher levels indicating less agreement. Forty microgram STX eq./100 g was decided to provide the best agreement, also here on account of the most equal sensitivities for the methods. Next level on the MU scale was 300 MU/100 g. At this level, the kappa values increases up to 0.88 for 80 mg STX eq./100 g and then decreases. This pair of toxicity values did also have the most equal sensitivities and, therefore, offered the best agreement. The kappa values at 400 MU/100 g are high for several levels on the STX eq./100 g scale, but the most equal sensitivities are demonstrated at 120 mg STX eq./100 g with a kappa value of 0.91. Also 500 MU/100 g show kappa values above 0.80 for several levels on the STX eq./100 g scale, but the best conformity in their sensitivities appeared at 130 mg STX eq./100 g with a kappa value of 0.85. For the two last levels on the MU/100 g scale, the best agreement was shown for 550 MU/100 g and 170 mg STX eq./100 g and 600 MU/100 g and 180 mg STX eq./100 g based on kappa values of 0.81 and 0.75, respectively, and most equal sensitivities. The correlation between the two methods could then be presented graphically by plotting these best agreement pairs of toxicities against each other (Fig. 3B). Here,
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Fig. 2. Chromatograms of the separation of the PSP toxins, GTX4 (1), GTX1 (2), GTX3 (3), GTX2 (4), NEO (5), STX (6), B1 and dcGTX3 (7), and dcGTX2 (8). (A) Separation of a standard solution of the carbamates using mobile phase 1 (see text). (B) Sample clean-up with the HLB solid phase extraction. Spiked and blank mussel extract in the upper and lower trace, respectively. (C) Separation of a standard solution of the gonyautoxins using mobile phase 2 (see text).
the linear correlation is 0.98. The amount of STX eq./MU for each toxicity level is also presented graphically (Fig. 3C), revealing that this was not constant throughout the toxicity scale. It increased from 0.13 mg STX eq./MU at 150 MU and up to 0.30 mg STX eq./MU at 400 MU and then levelled off with the exception of a lower value at 500 MU.
The toxicity scale for the test kit was given as either positive or negative. This was compared with different cutoff levels on the continuous scale for the mouse bioassay method (Fig. 4). The best agreement between the test kit and the mouse bioassay was expressed by the kappa values detected for 200– 210 MU/100 g. The two methods differed
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Table 1 Limits of detection for the carbamates with the post-column HPLC method expressed as nmol/g shellfish meat and mg STX equivalents/100 g shellfish meat Toxin
cLOD (nmol/g)
cLOD (mg STX eq./100 g)
STX NEO GTX 1 GTX 2 GTX 3 GTX 4
0.13 0.20 0.05 0.04 0.03 0.15
4 6 2 ,1 ,1 3
in sensitivities with 10 samples positive on the test kit below 200 MU/100 g and three samples negative on the test kit above this toxicity level (but still below the regulatory limit of 400 MU/100 g).
Fig. 3. (A) Toxicity estimates from the mouse bioassay and the HPLC-method plotted against each other. The second-degree relation with its 95% confidence lines are included. (B) Graphic presentation of the pairs of toxicities showing the best agreement. (C) STX equivalents per MU throughout the MU toxicity scale.
4. Discussion One obvious objection against basing an analytical method for determination of PSP toxicity on only the carbamates (STX, NEO, GTX 1 – 4) is the possibility of underestimation of total toxicity when some of the other PSP toxins are present in the shellfish. However, the AOAC extraction procedure used in the sample preparation cause at least partial hydrolysis of sulfocarbamoyl toxins (C1 – 4, B1, B2) present into their corresponding carbamates (Hall et al., 1990; Laycock et al., 1995; Anderson et al., 1996). The carbamates are considerably more toxic than the corresponding sulfocarbamoyls, and consequently, this will increase the toxicity of the shellfish extract (Kao, 1993). In addition, unknown peaks in the same retention region as the GTX’s, originating from sulfocarbamoyls present in the extract would result in reanalysis of the sample utilizing mobile phase 2 in order to get a better separation of the early eluting toxins. However, only a few samples showed peaks that could be attributed to these toxins and even then in very small amounts. The decarbamoyl toxins (dcSTX, dcNEO, dcGTX 1– 4), on the other hand, will not be included in the toxicity estimate based on the carbamates. If any of the toxins dcSTX, dcGTX 2 and 3 are present in the extracts, they will appear in the chromatograms. Peaks with coinciding retention times with these toxins were very rare. Thus, it appeared that the dominating toxins in Norwegian PSP contaminated shellfish are the carbamates and possibly sulfocarbamoyls that hydrolyse to their respective carbamates during extraction with boiling 0.1 M HCl. When comparing the HPLC method with the mouse bioassay, the linear correlation was 0.84 when results lower than 800 MU/100 g were plotted in an XY-diagram. This can be considered as a fairly good linear correlation between the methods. However, the linear correlation dropped to 0.76 if only the results between 150 and 500 MU/100 g were included, leaving out the lowest and highest values with the largest error in the estimated MU/100 g. This suggests that the correlation between the two toxicity scales was actually non-linear, at least for toxicity levels around the regulatory limit and lower. The agreement analysis support this assumption by showing that there seemed to be an increasing amount STX eq./MU with increasing toxicities up to 400 MU/100 g. At the toxicity level of 200 MU/100 g, 0.20 mg STX equals 1 MU, and this increase to 0.30 mg STX eq./MU at 400 MU. In the literature, 0.20 mg STX diHCl equals 1 MU (equals 0.16 mg STX) is most often used. This figure is based on results showing that 400 MU/100 g shellfish equals the regulatory limit of 80 mg STX diHCl/100 g. The reason why the HPLC method used in this study gave consistently higher toxicity estimates than the mouse bioassay compared with the average figures from the literature is unclear. The toxicity values of the different toxins present will, however, greatly influence the toxicity estimates achieved from the chromatographic
Table 2 Cross-tables with kappa values (k) and their 95% confidence intervals from the agreement analysis between the mouse bioassay and the HPLC method
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Kappa-values in bold print show the best agreements and values below 0.50 are not included in the table.
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from the Norwegian Research Council is gratefully acknowledged.
References
Fig. 4. Comparison between the MIST Alerte test kit and the mouse bioassay by kappa values with 95% confidence limits for different toxicity levels.
method. In this study, the toxicity factors reported by Oshima were used. In the literature, there are also several references stating that the mouse bioassay tend to underestimate the amount of PSP toxicity present in low-toxicity shellfish compared with liquid chromatographic methods (Lawrence et al., 1995; Oshima, 1995b; Salter et al., 1989). Agreement analysis between the commercial test kit (MIST Alerte) and the mouse bioassay gave a cut-off value of 200– 210 MU/100 g for the test kit, and this is in agreement with the cut point of 200 MU/100 g given by the manufacturer. The test kit gave no false negative results compared with the regulatory limit determined by the mouse bioassay, but 30% of the samples with PSP levels below the cut point were also determined as positive. This is also demonstrated by the agreement analysis by the difference in sensitivities of the two methods.
5. Conclusions Based on the results with PSP positive mussel samples through several years, an HPLC method determining all six carbamates STX, NEO and GTX 1 – 4, gives good estimates of total PSP toxicity in shellfish. The method can safely substitute the biological assay. However, the mouse bioassay should be used at intervals to safeguard against major changes in the PSP toxin profile. The MIST Alerte test kit shows promising results in identifying clearly PSP positive samples and used in the field it could greatly reduce the number of samples in need for a more comprehensive analysis of PSP toxins.
Acknowledgements The authors are grateful to Brit Heidenreich for technical assistance with the mouse bioassay. Financial support
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Analysis of PSP toxins in Norwegian mussels by a post-column derivatization HPLC method T.N. Aspa,*, S. Larsenb, T. Aunea a
Section of Food Hygiene, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., 0033 Oslo, Norway Section of Epidemiology, Norwegian School of Veterinary Science, P.O. Box 8146 dep., 0033 Oslo, Norway
b
Received 4 July 2003; accepted 6 January 2004
Abstract The aim of this study was to investigate the agreement between the AOAC mouse bioassay and an HPLC method for determining Paralytic Shellfish Poisoning (PSP) toxicity in blue mussels from the Norwegian coast. The AOAC mouse bioassay has traditionally been used for determining the toxin levels. Recently, an HPLC method for determining PSP toxins has been implemented and run in parallel with the bioassay. Four hundred mussel extracts from the last six years were analysed with both methods. A highly significant linear correlation between the methods was achieved ðr ¼ 0:84Þ: However, the relation between the two methods was best described by a second-degree polynom. Totally, this model explained 87% of the variation in the HPLC method. By agreement analysis, it was possible to establish cut-off levels for the HPLC method related to the AOAC mouse bioassay with kappa values .0.80 for toxicity levels below 500 MU/100 g. The HPLC method could substitute the mouse bioassay in determining PSP toxicity in Norwegian mussels. Additionally, about 70 mussel samples were also analysed by the MIST Alerte test kit. The kit did not give any false negative results compared with the regulatory limit, but 30% of the samples below the cut point were also determined as positive. q 2004 Elsevier Ltd. All rights reserved. Keywords: Paralytic shellfish poisoning toxins; AOAC mouse bioassay; Norwegian mussels
1. Introduction Toxins associated with Paralytic Shellfish Poisoning (PSP) are among the most acutely toxic substances known. They constitute a group of at least 21 structurally related neurotoxins produced mainly by dinoflagellates of the genus Alexandrium (Wright, 1995). They can be divided into four subgroups and the three most important are shown in Fig. 1. The toxins accumulate in shellfish feeding on the algae with no apparent negative effect on the shellfish. However, these contaminated shellfish pose a serious threat to human health and economic losses for shellfish industries worldwide. The different toxins vary significantly in their toxicity (Fig. 1) * Corresponding author. Tel.: þ47-22-96-48-32; fax: þ 47-2296-48-50. E-mail address: [email protected] (T.N. Asp). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.01.004
with the main toxin, saxitoxin (STX), as one of the most toxic (WHO, 1984). PSP has been known for centuries in other parts of the world, while one of the earliest documented incidents of PSP in Europe took place in the Oslo Fjord in Norway in 1901. This episode is thoroughly described in the Norwegian Medical Journal the same year (Thesen, 1901). All together, seven episodes of human intoxications have been reported in Norway with a total of 32 victims, including 2 fatalities back in 1901. The last two reported episodes took place in the Trondheim Fjord in Mid-Norway in 1991 and 1992 when the highest concentration of PSP toxins measured in mussel was 96.000 MU/100 g in the spring of 1992 (van Egmond et al., 1993). A survey program on PSP toxins started in 1962 in the Oslo Fjord area and a more comprehensive monitoring program was established in 1981 (Yndestad and Underdal, 1985) covering larger parts of the coast. During the last 20 years, this program has expanded to
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Fig. 1. The PSP toxins and their relative toxicities.
include the rest of the coastline. Even the most northern part has been monitored since 1997 and considerable levels of PSP toxins in shellfish from this area have been demonstrated some years. Mussels intended for commercial marketing have undergone regular monitoring and control for PSP toxins since 1986. PSP contaminated mussels are demonstrated every year from several locations along the coast, but the toxin levels show a very variable pattern and the areas with high levels shift with time. The farming of shellfish is a fast growing industry in Norway and the demand for toxin testing of the mussels before marketing increases accordingly. In accordance with international trade, the method used for this testing has conventionally been the official AOAC mouse bioassay (AOAC International, 1984). This is a non-selective bioassay with animal symptoms and time to death as the criterion of PSP toxicity. The toxicity has been expressed as mouse units (MU) per 100 g mussel meat, where 1 MU is the amount of toxin required to kill a 20 g mouse in 15 min (Sommer and Meyer, 1937; Hall et al., 1990). The sensitivity of the mouse population used is calibrated using reference standard STX, but often 1 MU is said to equal 0.20 mg STX. Most countries use 400 MU/100 g shellfish as action level for closure of the harvesting (Mons et al., 1998). The bioassay also serves as the reference method in the European Union (EU) and EU Council Directive 91/492/EEC states that the total PSP toxin content must not exceed 80 mg/100 g of mollusc flesh in accordance with the biological testing method (Council of the European Community, 1991). The compiled toxicity of the PSP toxins determined with chemical methods may be expressed as saxitoxin equivalents (STX eq.). Then, the amounts of the toxin analogues present are converted to STX equivalents according to their individual toxicities. In reality, it is
the dihydrochloride of STX that is used, since this was the form of the STX originally used in the mouse bioassay. This means that the regulatory limit is actually 80 mg STX-diHCl eq./100 g. This equals 65 mg STX eq./100 g when STX is calculated as the free base. Ethical considerations against sacrificing a large number of animals place an increasing pressure on regulatory bodies and researchers to provide alternative methods. Several other methods are described in the literature. They comprise both biochemical assays designed to estimate the total toxicity (Usleber et al., 1997; Chu et al., 1996; Jellett et al., 1992), and chemical analytical techniques capable of more selective detection of the individual toxin analogues (Pleasance et al., 1992; Locke and Thibault, 1994; Janecek et al., 1993; Lawrence et al., 1995; Sullivan and Wekell, 1986; Oshima, 1995b). The most commonly used chemical methods utilize reversed phase liquid chromatography with fluorescence detection (HPLC-FLD). The toxins are derivatized (oxidized) into fluorescent substances either before separation (Lawrence et al., 1995; Lawrence and Niedzwiadek, 2001), or after (Oshima, 1995b). This study describes the implementation of a postcolumn derivatization HPLC method described by Oshima (1995a), but with some modification regarding the sample preparation. The results from the chemical method are compared with results from the mouse bioassay. Furthermore, a rapid field test kit, MIST Alerte, designed to screen for PSP toxins (Laycock et al., 2000; Jellett et al., 2002) was tested, and the results are compared with the results from the mouse bioassay. The test strip is sensitive to the carbamates (STX, NEO, GTX 1 – 4) and dcSTX, C1/2 and B1.
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2. Material and methods
2.4. Sample preparation for HPLC-FLD analysis
2.1. Material
The acidic extracts prepared for the mouse bioassay were also used for the chemical analysis. The proteins in 500 ml extract were precipitated with 50 ml 5% tungstic acid. The supernatants were subjected to Oasis HLB solid phase extraction (SPE) cartridges (Waters, Milford, Massachusetts, USA) and filtered through Spin-X microcentrifuge 0.2 mm filters (Costar, USA). The SPE sorbent consists of a macroporous copolymer with a mix of divinylbenzene and N-vinylpyrrolidone.
All together 409 mussel samples were analysed with both the AOAC mouse bioassay and the HPLC method. A total of 72 samples analysed with the mouse bioassay were additionally examined with the MIST Alerte test kit. The PSP extracts were stored at 220 8C. 2.2. Chemicals and standards All chemicals and solvents used were of pro-analysis or HPLC grade and the water was grade 1 from a Milli-Q system (Millipore, Milford, USA). The acetic acid, hydrochloric acid, 85% phosphorous acid, 25% ammonium hydroxide, periodic acid and di-potassium hydrogen phosphate were from Merck (Darmstadt, Germany), the tungstic acid from Sigma (Steinheim, Germany), the sodium salt of 1-heptan sulfonic acid from Acros (Acros Organics, Geel, Belgium), while methanol and acetonitrile were from BDH (BDH Laboratory Supplies, Poole, England). The carbamate toxins (STX, NEO, GTX 2/3 together and GTX 1/4 together) as well as B1 of the PSP complex were purchased from National Research Council of Canada, Institute for Marine Biosciences (Halifax, Canada). Recently, standard mixtures containing some of the other toxins (dcSTX and C1 – 4) were received from Canada in connection with an AOAC collaborative study of a precolumn derivatization method. 2.3. Mouse bioassay The mouse bioassay was performed according to the AOAC method for paralytic shellfish poisons using white female BOM:NMRI mice weighing between 16 and 20 g. The mice are injected with 1 ml extracts obtained from fresh shellfish meat extracted with 0.1 M HCl and pH adjusted to 3.0 ^ 0.5. The mice (2 or 3 mice per extract) are observed for 1 h and the mean survival time is used to calculate the toxicities in MU. The dose-response curve for the amount of STX against mouse survival time is not linear so in order to use it quantitatively it is necessary to dilute the shellfish extract until the survival time is between 5 and 7 min. In our monitoring programs, this is regarded as an unnecessary sacrifice of animals and consequently dilutions are not done since the main purpose is protection of consumers. This leads to larger uncertainties in the estimates of toxin levels above 400 and below 250 MU/100 g. In addition, when just one out of 2 or 3 mice dies within an hour, the result is normally given as trace amount, but in this study the MU was calculated based on the mouse that died in order to obtain a number for comparison. Three extracts from 1996 and 1997 were reanalysed with the mouse bioassay in 2003, and no significant loss in toxicity was found.
2.5. Post-column HPLC-FLD The post-column HPLC-FLD analyses were carried out according to the method described by Oshima (1995a) using a Perkin– Elmer HPLC system with two extra micro pumps and an oven with a reaction coil between the column and the detector in order to perform the post-column oxidation. The toxins were separated on a silica based reversed phase column (150 £ 4.6 mm2 i.d., 5 mm particles, Chrompack C8). Using the Oshima method it is possible to detect at least 12 of the PSP toxins by utilizing three different mobile phases. In this study, only the six carbamates were determined, since they were commercially available as standards. The mobile phase used was 2 mM sodium 1heptanesulfonate in 30 mM ammonium phosphate buffer (pH 7.1): acetonitrile (96:4) (mobile phase 1). A mobile phase consisting of 2 mM sodium 1-heptanesulfonate in 10 mM ammonium phosphate buffer (pH 7.1) was used occasionally to confirm GTX 1 – 4 (mobile phase 2). After separation of the toxins on the column, they were oxidized by 7 mM periodic acid in 50 mM sodium phosphate buffer (pH 9.0) in a reaction coil (0.5 mm £ 10 m) at 85 8C. The reaction was stopped by adding 0.5 M acetic acid and the oxidized toxins were detected by a fluorescence detector with lex ¼ 330 nm and lem ¼ 390 nm: 2.6. Toxicity calculations from the HPLC-results The quantities of each of the carbamates in the shellfish extracts were determined from peak height measurements compared with a calibration curve based on spiked shellfish material. The overall toxicities of the samples were given in SXT equivalents (and not STX-diHCl equivalents) based on the amount of toxin and its relative toxicity compared to STX (Fig. 1). The relative toxicities were based on the specific toxicities given by Oshima (1995b). 2.7. MIST Alerte test kit MIST Alerte test kit (Jellett Biotek Ltd, Nova Scotia, Canada) was used according to the manufacturer. Again, the extracts intended for the mouse bioassay were used. Hundred microlitres of the shellfish extracts were diluted with 500 ml of a buffer provided with the test kit. Hundred
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microlitres of the resulting solutions were placed on the test strip and the results recorded after 20 min. If the colour intensity of the test line was 100 or 75% of the control line, the test was considered negative, and if the colour intensity of the test line was 50, 25% or less compared with the control line, the test was considered positive. The cut point for the kit was given by the manufacturer to be 200 MU/100 g. 2.8. Statistical methods In order to express the outcome of the chemical method as a function of the results from the mouse bioassay, a General Linear Model based on a second-order polynom was used (Kleinbaum et al., 1998). The determination of the cut-off levels between the chemical method and the mouse bioassay, and between the test kit and the mouse bioassay, were performed using agreement analysis for categorized variables (Agresti, 1990). This agreement is estimated by Kappa which is an inter-correlation coefficient for such variables. Kappa is defined by the formula k ¼ ðp0 2 pe Þ=ð1 2 pe Þ in which p0 denotes the observed proportion and pe the expected. The mouse bioassay and the chemical method express the toxin content in two different ways (MU and STX equivalents), but both methods are quantitative using continuously distributed variables. Cut-off levels from one measuring scale were compared with cut-off levels from the other by calculating the kappa value for each cross-table. Best agreement was decided from the highest kappa value with most equal sensitivity for the two methods. The mouse bioassay and the test kit do also express the toxin content in two different ways, but here the comparison is between a quantitative and a qualitative method since the test kit is either positive or negative. In this case, the agreement analysis gave the experimental cut-point of the MIST Alerte test kit compared to different cut-off levels of the mouse bioassay.
3. Results The six carbamates were separated in one chromatographic run using mobile phase 1. Complete separation of GTX 1 and 4 is not achieved with this mobile phase (Fig. 2A), but they could be quantified individually using peak heights. However, matrix interferences were often experienced in the time region where the GTX’s eluted when C18 SPE sorbent was used in the sample preparation. Alternative SPE sorbents were evaluated and the Oasis HLB sorbent gave a cleaner extract (Fig. 2B). The recovery was somewhat lower with the HLB clean-up compared with the C18, but the limits of detection were still satisfactory compared with the regulatory limit of 65 mg STX eq./100 g shellfish (Table 1). The standard solution of GTX 2/3 also contained small amounts of their
decarbamoyl counterparts enabling determination of their chromatographic retention times. With mobile phase 1, both dcGTX2/3 and B1 have retention times together with the GTX’s. However, when mobile phase 2 was used, they could be separated with the exception of B1 and dcGTX 3 which co-eluted (Fig. 2C). The chromatograms provided with the original Oshima method showed that dcSTX would elute between NEO and STX using mobile phase 1 and later this was confirmed with a standard mixture containing dcSTX. The standard mixture with the four C toxins revealed that two toxins (C1/2) eluted together with the matrix front and the other two (C3/4) together with the GTX’s when mobile phase 1 was used (data not shown). The linear correlation based on all 409 shellfish extracts was 0.79, while the correlation was 0.84 if the values above 800 MU/100 g were excluded. However, the relation between the two methods was best described by a second-degree polynom. The second degree of AOAC mouse bioassay contributed significant and totally, this method explained 87% of the variation in the HPLC method (Fig. 3A). The relation between the two methods was expressed by the equation: STX-equivalents=100 g ¼ 22:0 þ 0:23 MU=100 g þ 0:00024 ðMU=100 gÞ2 : By agreement analysis, it was possible to establish cutoff levels for the HPLC method related to the AOAC mouse bioassay (Table 2). The best agreement for the 150 MU/100 g shellfish was 20 mg STX eq./100 g, even though the kappa value is somewhat higher for 10 mg STX eq./100 g, but here the methods differed more in sensitivities. For the 200 MU/100 g level the kappa values are increasing with increasing levels of STX eq./100 g up to 0.85 and 0.86 for 40 and 50 mg STX eq./100 g, respectively, and then decreasing for higher levels indicating less agreement. Forty microgram STX eq./100 g was decided to provide the best agreement, also here on account of the most equal sensitivities for the methods. Next level on the MU scale was 300 MU/100 g. At this level, the kappa values increases up to 0.88 for 80 mg STX eq./100 g and then decreases. This pair of toxicity values did also have the most equal sensitivities and, therefore, offered the best agreement. The kappa values at 400 MU/100 g are high for several levels on the STX eq./100 g scale, but the most equal sensitivities are demonstrated at 120 mg STX eq./100 g with a kappa value of 0.91. Also 500 MU/100 g show kappa values above 0.80 for several levels on the STX eq./100 g scale, but the best conformity in their sensitivities appeared at 130 mg STX eq./100 g with a kappa value of 0.85. For the two last levels on the MU/100 g scale, the best agreement was shown for 550 MU/100 g and 170 mg STX eq./100 g and 600 MU/100 g and 180 mg STX eq./100 g based on kappa values of 0.81 and 0.75, respectively, and most equal sensitivities. The correlation between the two methods could then be presented graphically by plotting these best agreement pairs of toxicities against each other (Fig. 3B). Here,
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Fig. 2. Chromatograms of the separation of the PSP toxins, GTX4 (1), GTX1 (2), GTX3 (3), GTX2 (4), NEO (5), STX (6), B1 and dcGTX3 (7), and dcGTX2 (8). (A) Separation of a standard solution of the carbamates using mobile phase 1 (see text). (B) Sample clean-up with the HLB solid phase extraction. Spiked and blank mussel extract in the upper and lower trace, respectively. (C) Separation of a standard solution of the gonyautoxins using mobile phase 2 (see text).
the linear correlation is 0.98. The amount of STX eq./MU for each toxicity level is also presented graphically (Fig. 3C), revealing that this was not constant throughout the toxicity scale. It increased from 0.13 mg STX eq./MU at 150 MU and up to 0.30 mg STX eq./MU at 400 MU and then levelled off with the exception of a lower value at 500 MU.
The toxicity scale for the test kit was given as either positive or negative. This was compared with different cutoff levels on the continuous scale for the mouse bioassay method (Fig. 4). The best agreement between the test kit and the mouse bioassay was expressed by the kappa values detected for 200– 210 MU/100 g. The two methods differed
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Table 1 Limits of detection for the carbamates with the post-column HPLC method expressed as nmol/g shellfish meat and mg STX equivalents/100 g shellfish meat Toxin
cLOD (nmol/g)
cLOD (mg STX eq./100 g)
STX NEO GTX 1 GTX 2 GTX 3 GTX 4
0.13 0.20 0.05 0.04 0.03 0.15
4 6 2 ,1 ,1 3
in sensitivities with 10 samples positive on the test kit below 200 MU/100 g and three samples negative on the test kit above this toxicity level (but still below the regulatory limit of 400 MU/100 g).
Fig. 3. (A) Toxicity estimates from the mouse bioassay and the HPLC-method plotted against each other. The second-degree relation with its 95% confidence lines are included. (B) Graphic presentation of the pairs of toxicities showing the best agreement. (C) STX equivalents per MU throughout the MU toxicity scale.
4. Discussion One obvious objection against basing an analytical method for determination of PSP toxicity on only the carbamates (STX, NEO, GTX 1 – 4) is the possibility of underestimation of total toxicity when some of the other PSP toxins are present in the shellfish. However, the AOAC extraction procedure used in the sample preparation cause at least partial hydrolysis of sulfocarbamoyl toxins (C1 – 4, B1, B2) present into their corresponding carbamates (Hall et al., 1990; Laycock et al., 1995; Anderson et al., 1996). The carbamates are considerably more toxic than the corresponding sulfocarbamoyls, and consequently, this will increase the toxicity of the shellfish extract (Kao, 1993). In addition, unknown peaks in the same retention region as the GTX’s, originating from sulfocarbamoyls present in the extract would result in reanalysis of the sample utilizing mobile phase 2 in order to get a better separation of the early eluting toxins. However, only a few samples showed peaks that could be attributed to these toxins and even then in very small amounts. The decarbamoyl toxins (dcSTX, dcNEO, dcGTX 1– 4), on the other hand, will not be included in the toxicity estimate based on the carbamates. If any of the toxins dcSTX, dcGTX 2 and 3 are present in the extracts, they will appear in the chromatograms. Peaks with coinciding retention times with these toxins were very rare. Thus, it appeared that the dominating toxins in Norwegian PSP contaminated shellfish are the carbamates and possibly sulfocarbamoyls that hydrolyse to their respective carbamates during extraction with boiling 0.1 M HCl. When comparing the HPLC method with the mouse bioassay, the linear correlation was 0.84 when results lower than 800 MU/100 g were plotted in an XY-diagram. This can be considered as a fairly good linear correlation between the methods. However, the linear correlation dropped to 0.76 if only the results between 150 and 500 MU/100 g were included, leaving out the lowest and highest values with the largest error in the estimated MU/100 g. This suggests that the correlation between the two toxicity scales was actually non-linear, at least for toxicity levels around the regulatory limit and lower. The agreement analysis support this assumption by showing that there seemed to be an increasing amount STX eq./MU with increasing toxicities up to 400 MU/100 g. At the toxicity level of 200 MU/100 g, 0.20 mg STX equals 1 MU, and this increase to 0.30 mg STX eq./MU at 400 MU. In the literature, 0.20 mg STX diHCl equals 1 MU (equals 0.16 mg STX) is most often used. This figure is based on results showing that 400 MU/100 g shellfish equals the regulatory limit of 80 mg STX diHCl/100 g. The reason why the HPLC method used in this study gave consistently higher toxicity estimates than the mouse bioassay compared with the average figures from the literature is unclear. The toxicity values of the different toxins present will, however, greatly influence the toxicity estimates achieved from the chromatographic
Table 2 Cross-tables with kappa values (k) and their 95% confidence intervals from the agreement analysis between the mouse bioassay and the HPLC method
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Kappa-values in bold print show the best agreements and values below 0.50 are not included in the table.
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from the Norwegian Research Council is gratefully acknowledged.
References
Fig. 4. Comparison between the MIST Alerte test kit and the mouse bioassay by kappa values with 95% confidence limits for different toxicity levels.
method. In this study, the toxicity factors reported by Oshima were used. In the literature, there are also several references stating that the mouse bioassay tend to underestimate the amount of PSP toxicity present in low-toxicity shellfish compared with liquid chromatographic methods (Lawrence et al., 1995; Oshima, 1995b; Salter et al., 1989). Agreement analysis between the commercial test kit (MIST Alerte) and the mouse bioassay gave a cut-off value of 200– 210 MU/100 g for the test kit, and this is in agreement with the cut point of 200 MU/100 g given by the manufacturer. The test kit gave no false negative results compared with the regulatory limit determined by the mouse bioassay, but 30% of the samples with PSP levels below the cut point were also determined as positive. This is also demonstrated by the agreement analysis by the difference in sensitivities of the two methods.
5. Conclusions Based on the results with PSP positive mussel samples through several years, an HPLC method determining all six carbamates STX, NEO and GTX 1 – 4, gives good estimates of total PSP toxicity in shellfish. The method can safely substitute the biological assay. However, the mouse bioassay should be used at intervals to safeguard against major changes in the PSP toxin profile. The MIST Alerte test kit shows promising results in identifying clearly PSP positive samples and used in the field it could greatly reduce the number of samples in need for a more comprehensive analysis of PSP toxins.
Acknowledgements The authors are grateful to Brit Heidenreich for technical assistance with the mouse bioassay. Financial support
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