Determination of microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin in water and fish tissue using isotope dilution liquid chromatography tandem mass spectrometry
Introduction
Cyanobacteria are prokaryotic microorganisms found globally in both inland waters and coastal and marine systems [1,2]. Across varying environmental conditions, cyanobacteria can produce harmful secondary metabolites called cyanotoxins, which possess various physiochemical properties, structures, and toxicological mechanisms of action [2,3]. Microcystins (MCs) and nodularin (NOD) are hepatotoxic cyclic peptides [1,2]. Cylindrospermopsin (CLD) is a cytotoxic, dermotoxic, and hepatotoxic cyclic guanidinic alkaloid [4]. Anatoxin-a (ANA) is a neurotoxic bicyclic secondary amine [1,2]. Saxitoxin (SAX) is a neurotoxic guanadinium derivative with two amine functional groups [1,2]. Occurrence of these cyanotoxins in the environment, resulting from cyanobacterial Harmful Algal Blooms (HABs), have been reported globally in surface waters [2].
Cyanobacterial HABs result in various water quality problems and severe economic damage by impairing water supplies, recreational activities and fisheries [1,5]. Further, elevated exposure to cyanotoxins through food and water can be fatal to both humans and wildlife [[1], [2], [3]]. Despite the complexity of environmental exposures and economic losses caused by these contaminants of emerging concern, few water quality criteria and regulations for exposure to cyanotoxins exist, especially in the developing world [1,6]. Many countries have regulatory values for exposure to microcystin-LR that are in agreement with the recommended exposure level (1.0 μg L−1) provided by the World Health Organization [1,7]. In the United States, the Environmental Protection Agency (EPA) has revised the Unregulated Contaminant Monitoring Rule (UCMR 4) for Public Water Systems to add 10 cyanotoxins [8], has added several toxins to the Contaminant Candidate List 3 and 4 (CCL3&4) [9], and has proposed the Draft Human Health Recreational Ambient Water Quality Criteria or Swimming Advisories for MCs (4 μg L−1) and CLD (8 μg L−1) [10]. However, a lesser studied pathway to exposure in humans is through the consumption of contaminated food, such as dietary supplements, invertebrates, and fish [2]. Thus, a multi-toxin screening method for cyanotoxins in water and fish tissue is necessary to support ecological and public health studies of cyanotoxins in surface waters and organisms consumed by human populations.
Recent developments in reverse phase chromatography (RPLC) stationary phases have resulted in separation methods that allow for the simultaneous detection of ANA, CLD, MCs, and NOD in water and fish tissue [[11], [12], [13], [14], [15]]. However, resolving all toxin classes on a single RPLC column is challenging due to the diverse range of physiochemical properties, charge states, and structures exhibited by cyanotoxins [16]. For example, coelution of ANA and d-phenylalanine (DPA), which are isobaric and produce similar fragment ions [17,18], can lead to misidentification of ANA [13] on RPLC columns. Additionally, the high water solubility of some cyanotoxins requires use of ion-pairing agents to achieve sufficient retention on an RPLC column, which increases background noise, decreases ionization efficiency, and results in higher detection limits [16]. Alternatively, other multi-toxin screening methods obtain successful retention and separation of the polar cyanotoxins, ANA, CLD, and SAX using hydrophilic interaction liquid chromatography (HILIC) [16,[19], [20], [21], [22]]. The advantage of HILIC includes functionality similar to traditional normal phase chromatography with the compatibility of solvents suitable for RPLC, allowing the same mobile phases to be used for both separation techniques [23]. Thus, use of HILIC separation in addition to RPLC separation could allow for the regular incorporation of SAX in analytical screening methods.
Another challenge for the development of cyanotoxin analytical methods is finding reliable strategies that account and correct for the influence of matrix effect on ionization efficiency, a frequent problem when using electrospray ionization (ESI), and during extraction recovery. Robust methods to correct for matrix effect and recovery involve use of a surrogate/internal standards that shares physiochemical and structural properties with that of the target compound, resulting in similar column retention, ionization efficiency, and extraction recovery [24,25]. To date, several compounds have been used as internal standards for cyanotoxin method development [11,15,17,19,26]. Unfortunately, no compounds have been generally accepted as suitable internal standards because robust evaluation in terms of observed variation in relative response and recoveries compared to the target compounds has not been performed [15]. Ideally, an isotopically labeled version of all target analytes could be used in an isotope dilution method to correct for recovery bias and matrix effects because variations in retention, ionization efficiency, and recovery would be rendered negligible [24,25]. However, commercially available isotopically labeled internal standards for cyanotoxins were not available until recently.
Herein, we report an initial multi-toxin screening method to incorporate commercially available isotopically labeled internal standards for anatoxin-a, cylindrospermopsin, microcystin-LA, LR, RR, and YR to correct for matrix effect and extraction bias of MCs. This approach further evaluates a zwitterionic HILIC analytical column to separate ANA, CLD, and SAX simultaneously, apparently for the first time. Use of optimized HILIC and RPLC separation methods allows for rapid detection of ANA, CLD, SAX, MCs, and NOD in water and fish tissue. Method development of solid phase extraction (SPE) for water and liquid-liquid extraction (LLE) for fish tissue involved the optimization of extraction techniques where highly polar cyanotoxins (ANA, CLD, and SAX) were isolated separately from moderately polar cyanotoxins (MCs and NOD). SPE extraction methods in the present study were built from previously existing methods to incorporate SAX. Multiple solvent systems were evaluated for optimized extraction from fish tissue, followed by cleanup of extracted tissue samples using the SPE methods developed for water. This method was subsequently used to examine target analytes in water and fish from a caged fish study staged in a hypereutrophic impoundment located in Waco, TX, USA.
Section snippets
Chemicals and reagents
All chemicals were reagent grade or better, obtained from various commercial vendors, and used as received. The cyanotoxin standards microcystin-LA (M-LA), microcystin-LR (M-LR), microcystin-LY (M-LY), microcystin-RR (M-RR), microcystin-YR (M-YR), nodularin, anatoxin-a, anatoxin-a-13C4 (ANA-13C), cylindrospermopsin, cylindrospermopsin-15N5 (CLD-15N) and saxitoxin (Fig. S4) were purchased from Abraxis (Warminster, PA, USA). Isotopically labeled 15N internal standards (IS) microcystin-LA-15N7
LC–MS/MS methodology
Compound specific mass spectrometry parameters were automatically determined using MassHunter Optimizer (Agilent Technologies, Santa Clara, CA, USA) by flow injection analysis. Optimized MS/MS transitions and instrument parameters are provided in Table 1. Typically, MCs containing a single arginine residue will form [M+H]+ precursor ions at the arginine moiety [31]. Similarly, M-RR will form [M+2H]2+ precursor ions due to the presence of two arginine residues [32]. In contrast, MCs without an
Acknowledgements
Funding for this work was provided by the United States Department of Agriculture (USDA), National Institute of Food and Agriculture (NIFA) (#20166900725093) to JLC and BWB. Research reported in this publication was also supported by the National Institute Of Environmental Health Sciences of the National Institutes of Health under award number 1P01ES028942 to BWB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National
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