Transient isotachophoresis-capillary zone electrophoresis with contactless conductivity and ultraviolet detection for the analysis of paralytic shellfish toxins in mussel samples
Introduction
Paralytic shellfish toxins (PSTs) are potent natural biotoxins produced mainly by marine dinoflagellates (e.g. Alexandrium catenella, Alexandrium tamarense, Alexandrium minutum, Gymnodinium catenatum) [1], [2], [3], [4] and freshwater cyanobacteria (e.g. Anabaena circinalis) [5], [6]. PSTs are of particular concern in shellfish-producing areas because they accumulate in the digestive gland of filter-feeding shellfish such as mussels, scallops and oysters. While they do not harm the shellfish, consumption of PSTs-contaminated shellfish can, in serious cases, result in the death of seafood consumers from respiratory arrest and cardiovascular shock due to blockage of voltage-gated sodium channels [7], [8], [9], [10]. The allowable level of PSTs in shellfish is 800 μg/kg [11], [12], which corresponds with 800 ng/mL of PSTs in shellfish extract following the procedure described in the AOAC methods [12], [13].
Rapid, quantitative on-site PSTs detection and quantification using portable analytical instrumentation would improve biotoxin management by providing timely biotoxin information to shellfish producers to manage stock harvests around biotoxin closure periods, and reduce losses due to harvested/packed seafood product loss or recall, and the risk of the sale of seafood exceeding limits for safe human consumption. Various analytical techniques have been developed and employed for the assay of PSTs, including mouse assay [13], enzyme-linked immunosorbent assay (ELISA) [14], [15], [16], [17], receptor binding assay (RBA) [18], [19], saxiphilin-based assay [20], [21] and high performance liquid chromatography (HPLC) [12], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]. Among these methods, ELISA is the only to have been commercially developed in a test kit for PSTs detection in contaminated shellfish or water samples (i.e. www.r-biopharm.com, www.biosense.com, www.abraxiskits.com, www.anconbiotech.com and www.beaconkits.com). While these commercial kits are considered portable and can be used to detect PSTs on-site, Campbell et al. [34] pointed out they have the potential to produce subjective interpretation of results. The Association of Official Analytical Chemists (AOAC)-approved HPLC methods with pre- or post-column oxidation for fluorescence detection (HPLC-FLD) [11], [12], [30], and more recently, HPLC-MS, are the most widely used analytical methods for PSTs determination [31], [32], [33], [35], but are not easily miniaturized for on-site PSTs detection.
Capillary electrophoresis (CE) has been developed as an alternative separation method for PSTs [36], [37], [38], [39], [40], [41], [42], [43], [44] as it provides rapid and efficient separation with minimal consumption of sample and reagents. Significantly, it is inherently more miniaturizable than HPLC and a number of portable capillary and microchip electrophoresis devices have been developed [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], including the Mars Organic Analyser [45], [51], [53], hand-held portable isotachophoresis (ITP) system [49] and the Medimate Multireader® [50], [56]. CE methods developed for the analysis of PSTs used either UV [36], [40], [41], [42], [43], [44] or/and MS [36], [39], [40], [44] detection. MS is currently not readily amenable to being portable, while for UV, photometric detector based on light-emitting diodes (LEDs) has to be used for portability option [57], [58]. We previously compared these two detection modes with another two that have been realized in portable instrumentation, namely, C4D and FLD for the analysis of PSTs in mussel sample [44]. Whilst micellar electrokinetic chromatography (MEKC) coupled with FLD had good sensitivity (60.9–1040 ng/mL), selectivity was compromised by the AOAC pre-column oxidation method required to render the PSTs fluorescent. C4D achieved similar limit of detections (LODs) to FLD without the need for this reaction, but failed when applied to mussel extracts due to sample matrix interference.
In this work, capillary zone electrophoresis (CZE) with counter-flow transient isotachophoresis (tITP) is used for the pre-concentration of the PSTs and reduction of matrix interferences by using the sodium ion from the saline sample matrix as the leading ion. The method was successfully used to determine concentration of PSTs in a contaminated mussel sample and the outcomes validated against data obtained using the AOAC-approved HPLC-FLD method [12].
Section snippets
Standards and reagents
Standards of PSTs preserved in acid medium (saxitoxin (STX), decarbamoylSTX (dcSTX), neosaxitoxin (NEO), decarbamoylNEO (dcNEO), gonyautoxin1 to gonyautoxin4 (GTX1-4) and decarbamoylgonyautoxin2-3 (dcGTX2-3)) were purchased from National Research Council of Canada, Institute for Marine Biosciences (Halifax, Canada). All standards, each having different concentrations, were diluted with Milli-Q water (Millipore, Bedford, MA, USA) to the desired concentrations. A PST mixture for method
Results and discussion
In ITP, target analytes are focused into discrete zones between leading electrolyte (co-ion with electrophoretic mobility, μep higher than the target analytes) and TE (co-ion with μep lower than target analytes). For the PSTs, sodium ion is an attractive leading ion because its μep (51.9 × 10−9 m2 V−1 s−1 [59]) is greater than many organic cations. Conveniently, there is a high abundance of sodium in seawater and marine or freshwater organism extracts which allows establishment of an ITP system
Conclusions
The current study presents a new method for the analysis of PSTs in shellfish usingCZE-C4D in combination with tITP for analyte concentration and reduction of matrix effects. The method and the quantitation results of PSTs in mussel sample were validated using the AOAC-approved HPLC-FLD method. CZE-C4D was selected because of its potential for miniaturization and portability, enabling its deployment for on-site screening to inform the shellfish producers of potential PSTs contamination so they
Acknowledgements
The authors would like to thank the Australian Research Council, (ARC), for a QEII Fellowship (DP0984745) and Future Fellowship (FT130100101) and the University of Tasmania for funding support. We also acknowledge scholarships from the Ministry of Education Malaysia (MOE) and Universiti Teknologi Malaysia for A.S.A.K. We would like to thank Department of Primary Industries, Parks, Water and Environment of Tasmania (DPIPWE) and Advanced Analytical Australia Sydney for the contribution of
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