Determination of microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin in water and fish tissue using isotope dilution liquid chromatography tandem mass spectrometry

Highlights

• Optimized extraction and separation of cyanotoxins for water and fish tissue.

• Novel use of a zwitterionic hydrophilic interaction liquid chromatography column.

• Novel characterization of matrix effects using isotopically labeled standards.

• Targeted and nontargeted analyses using the same extraction and separation methods.

• Novel data mining techniques for use with nontargeted screening.

Abstract

Cyanobacteria can form dense blooms under specific environmental conditions, and some species produce secondary metabolites known as cyanotoxins, which present significant risks to public health and the environment. Identifying toxins produced by cyanobacteria present in surface water and fish is critical to ensuring high quality food and water for consumption, and protectionn of recreational uses. Current analytical screening methods typically focus on one class of cyanotoxins in a single matrix and rarely include saxitoxin. Thus, a cross-class screening method for microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin was developed to examine target analytes in environmental water and fish tissue. This was done, due to the broad range of cyanotoxin physicochemical properties, by pairing two extraction and separation techniques to improve isolation and detection. For the first time a zwitterionic hydrophilic interaction liquid chromatography column was evaluated to separate anatoxin-a, cylindrospermopsin, and saxitoxin, demonstrating greater sensitivity for all three compounds over previous techniques. Further, the method for microcystins, nodularin, anatoxin-a, and cylindrospermopsin were validated using isotopically labeled internal standards, again for the first time, resulting in improved compensation for recovery bias and matrix suppression. Optimized extractions for water and fish tissue can be extended to other congeners in the future. These improved separation and isotope dilution techniques are a launching point for more complex, non-targeted analyses, with preliminary targeted screening.

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.