Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection

Copyright

PII: s0041-0101(%)00069-4

To.+m, Vol. 35, No 2, pp 271 XI. IVY7 d> 1997 Elsev~er Science Ltd. All rights rcwned Printed m Great Rntm 004-0101197 51.50 - 000

DETERMINATION OF OKADAIC ACID BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION NOUREDDINE

BOUA’I’CHA,’ MARIE-CLAIRE

HENNION’

and

PAT SANDRA2 ‘CEMATMA, Laboratoire de Chimie Analytique, ESPCI, 10 Rue Vauquelin, 75231, Paris. France; and *Department of Organic Chemistry, University of Ghent, Krijgslaan 281 S4, 9000. Ghent, Belgium (Received 18 January 1996; accepted 20 March 1996)

N. Boua’icha, M.-C. Hennion and P. Sandra. Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection. Toxicon 35, 273-281, 1997.-Micellar electrokinetic chromatography (MEKC) with ultraviolet (UV) detection was applied for the determination of non-derivatized phycotoxins associated with diarrhoetic shellfish poisoning. A detection limit for 40 pg of okadaic acid (OA) was achieved. The UV intensities of this toxin measured at 200 nm showed good linearity in the range 40-640 pg. OA was detected in mussels spiked with 10 rig/g whole tissue. The presence of OA and dinophysistoxin-2 was observed in the crude extract of the dinoflagellate Prorocentrum lima. 0 1997 Elsevier Science Ltd. All rights reserved

INTRODUCTION

Diarrhoetic shellfish poisoning (DSP) is a severe gastrointestinal illness caused by the consumption of shellfish contaminated with toxic dinoflagellates such as certain Dinophysis and Prorocentrum species (Yasumoto et al., 1984). The toxins responsible for DSP comprise a group of lipid-soluble polyether compounds that include okadaic acid (OA). dinophysistoxin-1 (DTX-l), dinophysistoxin-2 (DTX-2), and dinophysistoxin-3 (DTX-3). the latter being a complex mixture of 7-0-acyl derivatives of the first three toxins (Fig. 1). These compounds are potent inhibitors of type 1 and 2A protein phosphatases (Bialojan and Takai, 1988), and have recently been shown to be powerful tumour promoters (Yasumoto and Murata, 1990; Suganuma et al., 1988; Haystead et al., 1989). The most commonly used method for DSP toxin determination in shellfish tissue is the one based on a mouse bioassay (Association of Official Analytical Chemistry, 1980: Vernoux, 1991). This approach suffers from poor reproducibility, low sensitivity, and interference matrices. Sensitive immunologically based assays such as enzyme-linked immunosorbent assay (ELISA) have been developed for OA (Usagawa et al.. 1989; Shestowsky et al., 1992), but these methods cannot be used for precise quantitative analysis of toxin mixtures because of differences in the cross-reactivities of individual toxins with antibodies. Different approaches for the determination of DSP toxins by high-performance 273

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liquid chromatography (HPLC) have been evaluated. They mostly involve derivatization of the carboxylic acid moiety to form highly fluorescent derivatives which are then separated by reverse-phase HPLC (Lee et al., 1987; Dickey et al., 1993; Allenmark et al., 1990; Pleasance et al., 1992a; Shen et al., 1991; Luckas, 1992). To the best of our knowledge, the only reports to date on the direct determination of OA in shellfish involve the use of HPLC combined with ion-spray mass spectrometry (MS) (Pleasance et al., 1990, 1992a), and capillary electrophoresis combined with ion-spray MS (Pleasance et al., 1992b). However, the equipment needed for these methods is too expensive for most routine analytical laboratories. Capillary electrophoresis is a relatively recent addition to the arsenal of analytical techniques utilized in the analyses of biological samples (Gorbon et al., 1988; Karger, 1989). A variety of separation principles has arisen using capillary electrophoresis. Micellar electrokinetic chromatography (MEKC) is one such principle and is an interface between electrophoresis and chromatography. It is characterized by two distinct phases, an aqueous and a micellar phase or pseudostationary phase (Terabe et al., 1985). These two phases are established by employing buffers containing surfactants (e.g. sodium dodecyl sulphate, SDS), which are added above their critical micellar concentration. The separation is based on the differential distribution of the solute molecule between the electroosmotically pumped aqueous mobile phase and the slower moving, electrophoretitally retarded micellar phase. This technique has been used to separate neutral and charged species. In this paper we describe the development and application of MEKC for the rapid and highly sensitive determination of OA in phytoplankton and shellfish extracts. Detection of non-derivatized toxin was performed by ultraviolet (UV) absorption at 200 nm.

MATERIALS AND METHODS Equipment

An HP 3D-CE system (Hewlett Packard, Waldbronn, Germany) equipped with diode array detection was used for the MEKC experiments. An untreated fused-silica capillary column (Composite Metal Services Ltd, U.K.) of 50 pm internal diameter and 64.5 cm length (56 cm to the detector) was used in all separations. The new column was preconditioned by flushing it with several column volumes (c. IO ~1) of borate buffer (12.5 mM, pH 9.2) and then with the running buffer. The column was exclusively used for the analyses described here. Before storage, it was purged with borate buffer (12.5 mM, pH 9.2) and water, and then dried in an air stream. Sample

Okadaic acid (OA) : R’ = R2 = H, R3 = Me Dinophysistoxin-1 Dinophysistoxin-2 Dinophysistoxin

(DTX-1) : R’ = H, R2 = R3 = Me (DTX-2) : R’ = R3 = H, R2 = Me -3 (DTX-3) : R’ = Acyl, R2 = H or Me, R3 = H or Me

Fig. 1. Structures of phycotoxins associated with diarrhoetic shellfish poisoning.

Okadaic Acid Determination

215

introduction was performed using hydrodynamic injection at 5 kPa for 7 sec. The analysis was done at ambient temperature (20 + 1°C). The micellar phase was composed of 12.5 mM borate buffer at pH 9.2 and 20 mM SDS. The voltage applied was 30 kV with positive polarity and UV detection was performed at 200 nm.

Materials

Okadaic acid of the highest purity ( + 95% by HPLC) was obtained from Sigma (Bornem. Belgium). Methanol, diethyl ether and n-hexane were HPLC grade. HPLC-grade water was generated using a Millipore Milh-Q system (Millipore Corp., Bedford, MA, U.S.A.). Borate buffer (pH 9.2) was prepared by the addition of 477 mg sodium borate to 100 ml water.

Mussel sample preparation

One-hundred grams of mussels was spiked with OA at 10 rig/g whole tissue and then sonicated in 200 ml cooled aqueous 80% methanol for 10 min. After centrifuging this homogenate for 5 min at 3200 g, the supernatant was extracted twice with n-hexane (v/v). After discarding the hexane layers, 50 ml of water was added and the aqueous methanol layer was extracted twice with diethyl ether (v/v). The pooled diethyl ether extracts were evaporated to dryness at reduced pressure. The residue was redissolved in 50 ~1 methanol for analysis. In a similar way, 100 g of non-spiked mussels was extracted and used as a blank.

Algae sample preparation

Six grams (wet weight) of cultured Prorocentrum lima strain SBOI (Diogine et al., 1995) was sonicated in 100 ml cooled aqueous 80% methanol for IO min. After centrifuging the homogenate for 5 min at 3200 g, 50 ml of water was added and the aqueous methanol layer was extracted twice with diethyl ether (v/v). The pooled diethyl ether extracts were evaporated to dryness at reduced pressure. The residue was then dissolved in methanol at a concentration of 20 mg/ml for analysis.

RESULTS AND DISCUSSlON

Initial experiments for the OA analysis were performed under pure electrophoretic conditions using 12.5 mM borate buffer at pH 9.2 [Fig. 2(A)]. The negatively charged solute migrated towards the cathode and eluted at 4.40 min (peak 1). At 3.6 min, corresponding to the electroosmotic flow, several solutes, i.e. the neutrals, eluted. indicating that the standard okadaic acid was far from pure. The analysis in Fig. 2(A) corresponds to a sample size of 400 pg. The detection wavelength was set at 200 nm, providing an acceptable molar absorptivity. For the real samples, capillary zone electrophoresis (CZE) could hardly be applied. owing to coelution of OA with matrix solutes. Therefore MEKC was carried out by adding 20 mM SDS to the buffer. The MEKC analysis is shown in Fig. 2(B). The migration time of OA was very similar to that in the CZE analysis in Fig. 2(A) (4.17 min), indicating that OA was moving mainly by electrophoretic mobility, whereas the neutrals were shifted to 7.18 min, emphasizing their hydrophobic nature. The MEKC separation provided good linearity for OA over the concentration range lo-160 pg/ml (the injection volume was in the order of 4 nl). A calibration graph for OA is presented in Fig. 3. The graph shows a linear response over at least two orders of magnitude, with a correlation coefficient of 0.998. The detection limit, as defined by a signal-to-noise ratio of 2, was 40 pg, and this is confirmed in the insert of Fig. 3. The sensitivity could be increased by a factor 2 by setting the UV detector to 190 nm instead of 200 nm. However, in view of the stability of the baseline noise over a day-long operation. analysis at 200 nm was preferred. Reproducibility of the migration time was adequate to ensure proper peak assignment; extremes of migration time varied by no more than 5% on a daily basis. To maintain reproducible migration times and electrophomtic performance, it is necessary to flush the column with several column volumes (c. 5 ~1) of

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Fig. 2. Electropherograms of okadaic acid. (A) CZE: UV detection at 200 nm, 7 set hydrodynamic injection at 5 kPa, + 30 kV, 12.5 mM borate buffer, pH 9.2. (B) MEKC: UV detection at 200 nm, 7 set hydrodynamic injection at 5 kPa, + 30 kV, 12.5 mM borate buffer, pH 9.2, 20 mM SDS. The electropherograms represent an injection of 400 pg of okadaic acid.

running buffer after each analysis. In addition, it is also common practice after three analyses to wash the column with borate buffer (12.5 mM, pH 9.2) to remove adsorbed material from the walls of the capillary. To evaluate the MEKC method for the rapid and sensitive determination of OA in shellfish extracts, 100 g mussels was spiked with OA at 10 rig/g whole tissue and extracted as described in Materials and Methods. Figure 4(A) shows the electropherogram of the spiked extract. Peak 1 in this electropherogram corresponds with OA, as can be deduced from Fig. 4(B) showing a standard injection under the same conditions, and from Fig. 4(C)

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in which the standard was added to the spiked sample after extraction. The extract prepared from the unspiked mussels gave no OA peak in the electropherogram [Fig. 4(D)]. The detection limit of OA in mussels by HPLC-fluorescence detection after derivatization was reported to be about 40 rig/g digestive glands (digestive glands = 10% of whole tissue) (Lawrence and Scott, 1993). The major problem with this method, besides its low sensitivity, is that OA has a very similar polarity to many naturally occurring fatty acids, and it is necessary to remove these interfering carboxylic acids as much as possible to obtain high derivatization signals. In the MEKC analysis the detection limit is lower than 10 rig/g whole tissue of mussels, which is good enough to detect OA levels well within the maximum allowance level currently employed for shellfish monitoring. Japanese law sets the maximum level for DSP in shellfish at 2 pg/g hepatopancreas (Lee et al., 1987), and in Norway the maximum tolerable level was set at 20 pg OA per 100 g shellfish meat (Stabell et al., 1991; Underdal et al., 1985). The determination of OA in shellfish by HPLC combined with ion-spray MS (Pleasance et al., 1990, 1992a) gave a detection limit of about 10 rig/g for OA toxin in the digestive glands of mussels. Capillary electrophoresis combined with ion-spray MS (Pleasance et al., 1992b) was also applied for the determination of OA, and a 16 ng sample size was required to generate a good spectrum. The MEKC method was also applied for the determination of toxins, especially OA and DTX-2, in the crude extract of the dinoflagellate P. lima. Figure 5(A) shows the electropherogram of the ether extract from this microorganism and Fig. 5(B) the analysis of the extract spiked with OA (peak 1) and DTX-2 (peak 2). The broad peak (Y) in the electropherogram corresponds to the peak of the neutrals in the okadaic acid standard. This is not surprising as the commercially available standard is extracted from cultured P. lima.

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Fig. 3. Calibration curve for okadaic acid in the concentration range l&l60 pg/ml. For conditions see Fig. 2(B). The insert shows the MEKC analysis achieved at the detection hmlt of 40 pg for okadaic acid with a signal-to-noise ratio of 2. Bars represent SE. (n = 3).

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Acid Determination

Many other peaks (X), presenting electrophoretic mobilities in the range of that of OA, were observed in the crude extract of P. lima [Fig. 5(A)]. Work is in progress to determine the identification of these compounds. Thus, as the commercially available okadaic acid that was used as standard was not 100% pure, the values obtained do not reflect the exact concentrations of okadaic acid in our samples. A pure standard will lead to a higher detection limit of okadaic acid in the mussel tissues.

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N. BOUA’iCHA et al. CONCLUSION

MEKC with UV detection at 200 nm allows the determination of okadaic acid in mussels at the 10 rig/g whole tissue level. This detection limit is below the maximum allowance level currently employed for shell~sh monitoring, In comparison with existing techniques such as the mouse bioassay and HPLC using fluorescence or mass spectrometric detection, the determination of DSP toxins by MEKC-UV holds promise for routine screening of these compounds in natural extracts. Particularly attractive features are its ease of operation, low cost, small sample consumption, speed of analysis, separation efficiency and the potential for automation.

Ac~n~~fedgements-We

thank V. Fessard for providing the algae sample and M. Ammar for donation of the DTX-2 standard. This work has been carried out with financial support from the European Commission (DG XII, Science and Technology, Division XII-H-l}, in the framework of the Human Capital and Mobility Programme, as part of the network project ‘Hyphenated Analytical Chemistry for Environmental and Public Health Research in the European Union’ (grant no. CHRXCT930274).

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