Anal. Chem. 1999, 71, 247-250
HPLC/Tandem Electrospray Mass Spectrometry for the Determination of Sub-ppb Levels of Pacific and Caribbean Ciguatoxins in Crude Extracts of Fish Richard J. Lewis,*,†,‡ Alun Jones,‡ and Jean-Paul Vernoux§
Queensland Agricultural Biotechnology Centre (QDPI), The University of Queensland, Qld 4072, Australia, Centre for Drug Design and Development, The University of Queensland, Qld 4072, Australia, and Laboratoire de Microbiologie alimentaire, Universite´ de Caen, Esplanade de la Paix, Caen Cedex, France
Ciguatera is a significant food-borne disease caused by potent polyether toxins (ciguatoxins) which accumulate in the flesh of ciguateric reef fish at risk levels >0.1 ppb for Pacific ciguatoxins. Research on ciguatera has been severely hindered by the lack of analytical methods that detect and characterize low levels of ciguatoxin in crude extracts of fish. Here we report a new procedure for ciguatoxin analysis based on gradient reversed-phase HPLC/tandem mass spectrometry (HPLC/MS/MS). The method gave a linear response to pure Pacific and Caribbean ciguatoxins (P-CTX-1 and C-CTX-1) and the structurally related brevetoxin (PbTx-2) spiked into crude extracts of fish. Levels equivalent to 40 ppt P-CTX-1, 100 ppt C-CTX-1, and 200 ppt PbTx-2 in fish flesh could be detected by HPLC/MS/MS. Using P-CTX-1 as an internal standard, the analysis of extracts of 30 ciguateric fish from the Caribbean Sea (8 toxic, 12 borderline, and 10 nontoxic by mouse bioassay) confirmed the reliability of the method and allowed an estimated risk level of >0.25 ppb C-CTX-1 to be established. HPLC/MS/MS provides a sensitive analytical approach, not previously available, for the determination of Pacific and Caribbean ciguatoxins at sub-ppb levels in fish flesh. Ciguatera (fish poisoning) is a major economic and social problem throughout tropical and subtropical waters, with an estimated 25 000 persons poisoned annually. The disease is characterized by neurological and gastrointestinal disorders which typically appear from 1 to 24 h following the consumption of contaminated fish.1,2 The toxins involved are potent sodium channel activator toxins known as ciguatoxins that are produced by the benthic dinoflagellate Gambierdiscus toxicus.3 The ciguatoxins and structurally related brevetoxins (e.g., PbTx-24) compete at site 5 on the voltage-sensitive sodium channel (Figure 1). Two related families of Pacific ciguatoxins (P-CTX) have been identified * Corresponding author: (phone) 617 3365 1924; (fax) 617 3365 1990; (email)
[email protected]. † Queensland Agricultural Biotechnology Centre. ‡ Centre for Drug Design and Development. § Universite ´ de Caen. (1) Gillespie, N. C.; Lewis, R. J.; Pearn, J.; Bourke, A. T. C.; Holmes, M. J.; Bourke, J. B.; Shields, W. J. Med. J. Aust. 1986, 145, 584-590. (2) Glaziou, P.; Legrand, A. M. Toxicon 1994, 32, 863-873. (3) Lewis, R. J.; Holmes, M. J. Comp. Biochem. Physiol. 1993, 106C, 615628. 10.1021/ac980598h CCC: $18.00 Published on Web 11/26/1998
© 1998 American Chemical Society
in Pacific Ocean fish.5-10 A third family of Caribbean ciguatoxins (C-CTX) has been identified in fish of the Caribbean Sea.11-14 The ciguatoxins are heat-stable polyether toxins of 1023-1157 Da. P-CTX-1 is the major toxin in the flesh of carnivorous fish of the Pacific, contributing to ∼90% of total lethality and posing a health risk at levels above 0.1 ppb.5,6,15,16 The minimum risk level for C-CTX-1 has not been determined.13 The traditional method of detecting the presence of ciguatoxins in fish involves testing lipid extracts by the mouse bioassay.17-20 More recently, cytotoxicity,21 radioligand binding,7,22 and antibody(4) Lin, Y.; Risk, M.; Ray, S. M.; Van Engen, D.; Clardy, J.; Golik, J.; James, J. C.; Nakanishi, K. J. Am. Chem. Soc. 1981, 103, 6773-6774. (5) Murata, M.; Legrand, A. M.; Ishibashi, Y.; Fukui, M.; Yasumoto, T. J. Am. Chem. Soc. 1990, 112, 4380-4386. (6) Lewis, R. J.; Sellin, M.; Poli, M. A.; Norton, R. S.; MacLeod, J. K.; Sheil, M. M. Toxicon 1991, 29, 1115-1127. (7) Lewis, R. J.; Norton, R. S.; Brereton, I. M.; Eccles, C. D. Toxicon 1993, 31, 637-643. (8) Satake, M.; Murata, M.; Yasumoto, T. Tetrahedron Lett. 1993, 34, 19751978. (9) Satake, M.; Ishibashi, Y.; Legrand, A.-M.; Yasumoto, T. Biosci. Biochem. Biotechnol. 1996, 60, 2103-2105. (10) Legrand, A.-M.; Teai, T.; Cruchet, P.; Satake, M.; Murata, K.; Yasumoto, T. In Harmful Algae; Reguera, B., Blanco, J., Fernan´dez, M. L., Wyatt, T., Eds.; Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Santiagode Compostela, Spain, 1998; pp 473-475. (11) Vernoux, J. P. Oceanol. Acta 1988 11, 37-46. (12) (a) Crouch, R. C.; Martin, G. E.; Musser, S. M.; Granade, H. R.; Dickey, R. W. Tetrahedron Lett. 1995, 36, 6827-6830. (b) Poli, M. A.; Lewis, R. J.; Dickey, R. W.; Musser, S. M.; Buckner, C. A.; Carpenter, L. G. Toxicon 1997, 35, 733-741. (13) Vernoux, J.-P.; Lewis, R. J. Toxicon 1997, 35, 889-900. (14) Lewis, R. J.; Vernoux, J.-P.; Brereton, I. M. J. Am. Chem. Soc. 1998 120, 5914-5920. (15) Lewis, R. J.; Sellin, M. Toxicon 1992 30, 915-919. (16) Legrand, A. M.; Fukui, M.; Cruchet, P.; Ishibashi, Y.; Yasumoto, T. In Proceedings Third International Conference on Ciguatera Fish Poisoning, Puerto Rico; Tosteson, T. R., Ed.; Polyscience Publications: Quebec, 1992; pp 25-32. (17) Hoffman, P. A.; Granade H. R.; MacMillan, J. P. Toxicon 1983, 21, 363369. (18) Lewis, R. J.; Sellin, M. Toxicon 1993, 31, 1333-1336. (19) Vernoux, J. P. Mem. Qld. Museum 1994, 34, 625-629. (20) Lewis, R. J. In Manual on Harmful Marine Microalgae; Hallegraeff, G. M., Anderson, D. M., Cembella, A. D., Eds.; IOC Manuals and Guides 33; UNESCO: Paris, France, 1995; pp 135-161. (21) Manger, R. L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Hokama, Y.; Dickey, R. W.; Granade, H. R.; Lewis, R.; Yasumoto, T.; Wekell, M. M. J. Assoc. Off. Anal. Chem. 1995, 78, 521-527. (22) Pauillac, S.; Ble´chaut, J.; Cruchet, P.; Lotte, C.; Legrand, A.-M. In Harmful Marine Algal Blooms; Lassus, P., Arzul, G., Erad, E., Gentien, P., Marcaillou, C., Eds.; Lavoisier: Paris, France, and Intercept Ltd.: Andover, UK, 1995; pp 801-808.
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Figure 1. Structures of the marine polyether toxins P-CTX-1,5 C-CTX-1,14 and PbTx-2.4 P-CTX-1, C-CTX-1, and PbTx-2 are potent sodium channel activator toxins ([M + H]+ m/z 1111.6, 1141.6, and 895.5, respectively).
based23 methods have been reported that have potential to be developed into cost-effective screens for ciguateric fish. Analytical HPLC/MS methods have also been developed for determining ciguatoxins24,25 and brevetoxins.26 However, HPLC/MS is not sufficiently sensitive to detect clinically relevant levels of ciguatoxins in crude extracts of fish.27 In this report, we describe an HPLC/MS/MS method for the simultaneous determination of Pacific and Caribbean ciguatoxins in crude extracts of fish flesh and assess the potential of PbTx-2, which is commercially available, as an internal standard. A brief account of parts of this work have been published.28 MATERIALS AND METHODS Extraction and Bioassay of Crude Extracts of Fish. Portions of the flesh of 30 fish captured from the Caribbean Sea were extracted and partially purified on a Florisil column to yield an (23) (a) Pauillac, S.; Sasaki, M.; Naar, J.; Inoue, M.; Branaa, P.; Cruchet, P.; Chinain, M.; Legrand, A.-M. Eur. J. Ichthyol., in press. (b) Pauillac, S.; Inoue, M.; Sasaki, M.; Naar, J.; Murata, M.; Tachibana, K.; Chinain, M.; Legrand, A. M. In Harmful Algae; Reguera, B., Blanco, J., Fernan´dez, M. L., Wyatt, T., Eds.; Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Santiagode de Compostela, Spain, 1998; pp 563-566. (24) Lewis, R. J.; Holmes, M. J.; Alewood, P. F.; Jones A. Nat. Toxins 1994, 2, 56-63. (25) Lewis, R. J.; Jones, A. Toxicon 1997, 35, 159-168. (26) Hua, Y.; Lu, W.; Henry, M. S.; Pierce, R. H.; Cole, R. B. Anal. Chem. 1995, 67, 1815-1823. (27) Lewis, R. J., unpublished results. (28) Lewis, R. J.; Jones, A. J.; Vernoux, J.-P.; Marquais, M. In Harmful Algae; Reguera, B., Blanco, J., Fernan´dez, M. L., Wyatt, T., Eds.; Xunta de Galicia, Intergovernmental Oceanographic Commission of UNESCO, Santiagode de Compostela, Spain, 1998; pp 523-524.
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acetone/methanol 9:1 fraction as described by Vernoux and Lewis.13 An estimate of the potency of these extracts was determined by ip injection of extracts into mice,19 where 72 ng of C-CTX-1 is taken as the minimum lethal dose for a 20-g mouse (C-CTX-1 LD50 ) 3.6 µg/kg).13 The relationship between mouse potency, expressed in C-CTX-1 equivalents, and human risk from consumption of fish flesh is obtained from Vernoux,11 where toxic (inedible) fish contain an equivalent of g1.8 ppb C-CTX-1, borderline toxic fish (edible in small quantities) contain the equivalent of 0.9 to 0.25 ppb, 2.5 times higher than that for P-CTX-1 which is estimated to (29) (30) (31) (32)
Vernoux, J. P.; Talha, F. Comp. Biochem. Physiol. 1989 94B, 499-504. Vernoux, J. P.; Abbad El Andaloussi, S. Biochimie 1986, 68, 287-291. Vernoux, J. P., unpublished results. Marquais, M.; Vernoux, J.-P.; Molgo. J.; Sauviat, M. P.; Lewis, R. J. In Harmful Algae; Reguera, B., Blanco, J., Fernan´dez, M. L., Wyatt, T., Eds.; Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Santiagode de Compostela, Spain, 1998; pp 476-477.
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affect humans at 0.1 ppb. This difference in human risk level could stem from differences in potency of the two ciguatoxins, with C-CTX-1 being 10-fold less potent than P-CTX-1 when tested ip in mice. With the maximum safe level of C-CTX-1 set at >0.25 ppb, 5 of 12 borderline toxic fish and all fish determined toxic (inedible) by mouse bioassay contained levels of ciguatoxin C-CTX-1 that are likely to affect humans (see Figure 4). Conclusions. HPLC/MS/MS is the first analytical method capable of detecting, in crude extracts of fish, sub-ppb levels of the major ciguatoxins contributing to human ciguatera poisoning in the Pacific Ocean and Caribbean Sea. The method provides a significantly improvement in sensitivity over results obtained by fluorometric HPLC analysis.16,22 An appropriate internal standard and faster extraction and HPLC methods that take advantage of the sensitivity and specificity of HPLC/MS/MS would further improve the speed and accuracy of this analysis. Rapid advances in MS technology, such as ion trap MS, should soon bring the cost of MS/MS instrumentation within reach of most analytical laboratories.
ACKNOWLEDGMENT This work was supported by the Centre for Drug Design and Development and the Queensland Agricultural Biotechnology Centre (to R.J.L.), and the French Ministry of Overseas Territories (DOM-TOM; CORDET) and IFREMER (to J.-P.V.). We thank M. Poli (UAMRIID) for the gift of PbTx-2.
Received for review June 1, 1998. Accepted October 15, 1998. AC980598H
