Elsevier

Analytica Chimica Acta

Volume 914, 31 March 2016, Pages 117-126
Analytica Chimica Acta

Simultaneous screening for lipophilic and hydrophilic toxins in marine harmful algae using a serially coupled reversed-phase and hydrophilic interaction liquid chromatography separation system with high-resolution mass spectrometry

https://doi.org/10.1016/j.aca.2016.01.062Get rights and content

Highlights

  • Simultaneous separation of lipophilic and hydrophilic toxins in a single injection is presented.

  • Serially coupled RPLC and HILIC-TOF/MS method is an efficient tool for screening of toxins with different polarities.

  • Synchronous extraction conditions for lipophilic and hydrophilic toxins in algae were optimized.

  • Recoveries between 89.4 and 105.8% and repeatability between 4.18 and 14.87% were obtained.

Abstract

The presence of toxins in harmful algal blooms (HABs) poses considerable concerns because of their potential adverse effects on ecological environments and human health. When marine HABs occur, efficient screening and identification of toxins in different kinds of HAB algae remains a challenge. In this study, the applicability of serial coupling of reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) combined with high resolution mass spectrometry (HR-MS) for the simultaneous screening and identification of various kinds of known lipophilic and hydrophilic toxins in HAB algae was investigated for the first time. Ultrasound-assisted extraction (UAE) was explored to extract both lipophilic and hydrophilic toxins in algae simultaneously. As in most cases, toxin standards were not available; therefore, an identification procedure based on accurate mass data and chromatographic behavior was proposed. According to this procedure, eight known lipophilic toxins and 11 hydrophilic toxins were successfully detected in a single injection, and the proposed method was validated. Satisfactory sensitivity, repeatability (RSD <14.87%) and recovery (89.4–105.8%) of the method were achieved. A major advantage of the proposed method is that it can almost detect members of all eight groups of marine algal toxins in a single run. Using this method, several known toxins in different marine toxigenic algae including Alexandrium tamarense, Alexandrium minutum and Prorocentrum lima were successfully observed and identified. This work demonstrates that RPLC/HILIC-HR-MS combined with an accurate mass list of known marine algal toxins may be used as a powerful tool for screening of different classes of known toxins in marine harmful algae.

Introduction

In recent decades, harmful algal blooms (HABs) caused by seawater eutrophication and climate change have seriously threatened the balance among marine fishery resources, marine breeding industries, and marine ecosystems [1]. A number of marine HAB algae species generate various toxins that can be bioaccumulated in shellfish. Consumption of shellfish contaminated with algal toxins can act on the human digestive, nervous or cardiovascular systems, resulting in human poisoning or even death. Thus, in China, when HABs occur, the Department of Marine Environment Management need to rapidly confirm whether the HAB algae generates toxins and what types of toxins are produced. Currently, the morphology-based classification method is often used to determine whether a HAB algae is toxic via the identification of its constituent species. However, numerous types of HAB algae can produce toxins, and even for the same toxigenic algae, the toxin amount and category can also vary significantly depending on the sea area and growth conditions [2]. In addition, a HAB caused by more than one species of algae often appear. It becomes more difficult to identify the various toxins given the existence of multiple toxigenic algae. Therefore, a highly efficient detection method for the synchronous, efficient identification of different classes of toxins in marine harmful algae is urgently needed in China.

To date, over 200 marine algal toxins and derivatives have been discovered [3]. They can be classified as lipophilic and hydrophilic toxins. The joint FAO/IOC/WHO workshop on toxins in bivalve molluscs classify the toxins into eight groups based on chemical structure, namely, the azaspiracid (AZA) group, brevetoxin (PbTx) group, cyclic imine (CI) group, domoic acid (DA) group, okadaic acid (OA) group, pectenotoxin (PTX) group, saxitoxin (STX) group and yessotoxin (YTX) group [4]. AZA, CI, OA, PbTx, PTX and YTX are lipophilic toxins while STX and DA are hydrophilic toxins. The difference in chemical structure and polarity of these toxins in HAB algae make them extremely difficult to be simultaneously screened and identified.

Biological assays with white mice [5], [6], high performance liquid chromatography (HPLC) [7], [8], [9], [10] and liquid chromatography-mass spectrometry (LC-MS) [11], [12], [13], [14], [15], [16], [17], [18], [19], [20] are usually applied to determine the marine algae toxins. Biological assays with white mice have some drawbacks with respect to sensitivity, false positive results and in relation to animal welfare and ethics [21], [22]. Recently, LC-MS technology has been the most advanced method to detect various algae toxins in algae and seafood products. LC-MS technology mostly divides into two categories. One category is the combination of LC and low resolution MS to detect a target toxin in the sample [11], [12], [13], [14], [15], [16], [17]; the other is the combination of LC and HR-MS, which is a powerful tool to conduct synchronous target and nontarget analyses of various algae toxins [3], [18], [19], [20], [23], [24], [25]. Gerssen et al. [3] applied a combination of RPLC and Orbitrap HR-MS with a molecular weight database of over 200 lipophilic algae toxins to establish a simultaneous screening method for multiple lipophilic toxins in shellfish and toxigenic algae. Orellana et al. [26] also developed a method for profiling lipophilic marine algae toxins based on RPLC and Orbitrap HR-MS. HILIC is a chromatographic technique that uses a hydrophilic stationary, and it is well suited for the separation of polar and hydrophilic compounds [27]. Aqueous mobile phases (usually acetonitrile/water) are used, making this separation technique compatible with ESI-MS [28]. HILIC also has the advantage of not requiring the use of ion pairing reagents [29]. HILIC coupled with ESI-MS detection has been widely used in the paralytic shellfish toxins (PST) analysis [21], [29], [30]. In our previous research [2], we applied a combination of HILIC and time of flight mass spectrometry (TOF-MS) with an accurate molecular weight database of over 20 STX group toxins to develop a simultaneous screening and identification method for multiple hydrophilic toxins in marine harmful algae. Currently, no studies have been reported about the combination of HR-MS and chromatographic separations for simultaneous screening and identification of multiple classes of lipophilic and hydrophilic toxins in marine harmful algae.

Given the high separation orthogonality of the two-dimensional LC separation system constituted by RPLC and HILIC, this technique has been used to analyze complex mixtures containing a wide range of compound polarities [31], [32], [33], [34]. However, its development has not reached maturity and the method is too complicated, thus, it is not applicable for routine testing work. In 2008, Louw et al. [35] proposed a simple chromatographic separation technology combining RPLC and HILIC to broaden the elution window for the analysis of hydrophilic and hydrophobic compounds. In recent years, this simple and reliable technology has been successfully applied to the simultaneous separations of multiple hydrophilic and hydrophobic compounds [36], [37], [38], [39], [40], [41]. Rajab et al. [41] used serial coupling of RPLC and zwitterionic hydrophilic interaction LC with TOF-MS to screen and identify suspected transformation products of diclofenac with different polarities. As far as we know, there have been no reports of combined RPLC and HILIC to simultaneously separate lipophilic and hydrophilic algae toxins. This research uses RPLC combined with HILIC and HR-MS for a systematic optimization of the chromatographic conditions and extraction methods for the samples and establishes a new method for the simultaneous screening and identification of different classes of lipophilic and hydrophilic toxins in marine harmful algae to provide technical support in the case of a toxigenic HAB disaster.

Section snippets

Reagents and materials

Formic acid and ammonium acetate were purchased from Fluka (Buchs, Switzerland). HPLC-grade acetonitrile (ACN) and methanol were purchased from Merck (Darmstadt, Germany). Water was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Reference standards of gonyautoxins (GTX1, GTX2, GTX3, and GTX4), decarbamoyl gonyautoxins (dcGTX2 and dcGTX3), saxitoxin (STX), domoic acid (DA), neosaxitoxin (NEO), N-sulfocarbamoyltoxins (C1 and C2), gymnodimine (GYM), spirolide 1

Optimization of RPLC/HILIC separation conditions

Retention and separation of both polar and non-polar solutes in complex samples is a challenge that frequently requires a combination of methods, e.g., RPLC and NPLC [35]. In the present study, serial coupling of analytical RPLC and HILIC was developed to simultaneously separate multiple classes of lipophilic and hydrophilic toxins in a single LC-TOF/MS run. The first column, which has an I.D. of 2.1 mm, exhibits optimal flow between 0.2 and 0.4 mL min−1. Using the T-piece and mixer, the flow

Conclusions

In the present study, we demonstrate that serial coupling of RPLC and HILIC separation systems may be easily used in conjunction with HR-MS and that the resultant method is suitable for the simultaneous screening of different classes of known hydrophilic and hydrophobic toxins in marine toxigenic algae. This study describes the successful development of a generic simultaneous extraction procedure for hydrophilic and hydrophobic toxins in toxigenic algae. Hydrophilic and hydrophobic toxins in

Acknowledgments

The researchers gratefully acknowledge the financial support from the National Natural Science Foundation of China-Shandong Joint Funded Project (U1406403), the National Science & Technology Pillar Program of China (2013BAK12B00), the Basic Scientific Fund for National Public Research Institutes of China (2014T05), the Science & Technology Program of Lianyungang (SH1516),the Shandong Provincial Natural Science Foundation (ZR2015PD003), the Open Research Fund Program of Key Laboratory of Marine

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