Highly sensitive determination of amanita toxins in biological samples using β-cyclodextrin collaborated molecularly imprinted polymers coupled with ultra-high performance liquid chromatography tandem mass spectrometry

Highlights

• Determination of five amanita toxins in biological samples using MIPs coupled with UPLC-MS/MS

• A β-cyclodextrin functional vinyl monomer was synthesized and the common moiety of toxins was used as the template for preparing MIPs.

• A higher level of analytical performance was achieved as compared to previous works.

• The method was applied to the determination of the amanita toxins in suspected samples and showed great potential in the diagnosis of mushroom poisoning.

Abstract

In this work, a β-cyclodextrin functional vinyl monomer was synthesized and the common moiety of five amanita toxins was used as the template for preparing molecularly imprinted polymers (MIPs). Chemical calculation was used to evaluate and describe the binding interactions between the template and the functional monomer. The preparation conditions were optimized and the resultant MIPs were characterized and employed as solid-phase extraction (SPE) sorbents. The SPE conditions including the amount of sorbent, extraction solution, and eluting solution were also optimized for the enrichment of the five toxins. Using an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), detection limits ranging from 0.34–0.42 µg/L, 0.16–0.33 µg/L, and 0.035–0.056 µg/kg were achieved for the five toxins in serum, urine and liver samples, respectively. The proposed method was further applied to the determination of the amanita toxins in suspected samples and showed great potential in the diagnosis of mushroom poisoning.

Introduction

Mushrooms are rich in vegetable proteins, amino acids and vitamins, and their growing popularity as part of the diet is due to their nutritive properties and good taste [1], [2]. Some of them are also great sources of bioactive secondary metabolites with antioxidant, antitumor and antimicrobial properties [3], [4], [5]. However, mushroom poisonings caused by ingestion of wild poisonous mushroom remain a public health problem in many countries [6], [7]. The main mushroom toxins can be divided into three categories: slow-acting but highly toxic amatoxins, quick-acting phallotoxins, and virtoxins [8], [9]. Amanitins are the principal toxins of the amatoxins family that are produced by death-cap mushrooms and are one of the deadliest toxins known to humans with a LD₅₀ of 50–100 µg/kg [10]. They specifically inhibit DNA-dependent RNA polymerase II enzyme activity leading to block protein synthesis and then cell necrosis [11]. Moreover, they are thermally stable and their toxicity won't decrease during conventional processing and storage. It is estimated that the acute liver failure induced by amanita toxins accounts for over 90% of mushroom poisoning related fatalities worldwide [12], [13].

Accurate detection of the toxins is critical for precise diagnosis and rehabilitation monitoring in the clinical treatment of mushroom poisoning [14]. Leite et al. reported the determination of α- and β-amanitins in urine and liver samples using UHPLC-MS/MS coupled with protein precipitation and SPE sample preparations [15]. Helfer et al. reported an on-line turbulent flow chromatography coupled with HPLC-MS/MS for the direct analysis of α- and β-amanitins in human urine [16]. Tomková et al. described the simultaneous determination of muscarine, α- and β-amanitins in human urine by using weak cation-exchange polymers as SPE sorbents and UHPLC-TOF MS [17]. Despite the high sensitivity and selectivity of mass spectrometry, efficient sample preparation is usually still required to reduce matrix effect and avoid injecting an extremely dirty sample [18]. Moreover, most of the above reports were focused on the determination of amatoxins and few works have been done for determining phallotoxins in biological samples [19].

SPE has been widely used for separating and enriching target analytes ranging from a simple matrix removal to solving highly selective enrichment challenges [20]. The most commonly used SPE sorbents are C8/C18 chemically bonded silica and hydrophilic–lipophilic balance polymers. However, their specificities are not high enough for toxins, and the C8/C18 chemically bonded silica have narrow pH stability and the residual silanol groups have a negative effect on the retention of target analytes [21]. Thus, it is of great interest to develop more specific sorbents for separating and enriching trace toxins in biological samples. Molecular imprinting technology enables the fabrication of polymers with selective recognition ability through polymerizing self-assembled functional monomers and cross-linkers around a template molecule [22], [23]. Because of their advantages such as ease preparation, tailor-made selectivity and good chemical stability, molecularly imprinted polymers (MIPs) have attracted great research attention and have been widely used in many fields [24]. Unfortunately, the recognition ability of MIPs in aqueous environments remains a challenge. This could be related with the fact that the most commonly used hydrogen bonding in non-covalent molecular imprinting is disturbed in aqueous environment which is detrimental to the recognition performance of the MIPs [25]. One solution is to take advantages of hydrophobic interactions in aqueous solutions. Cyclodextrins are amphiphilic macromolecules consisting of hydrophilic outer surface and hydrophobic inner cavities, which can form inclusion complexes with non-polar molecules or their moieties [26], [27]. Particularly, the combination of the size inclusion effect of cyclodextrins and the selectivity of molecular imprinting has attracted burgeoning interest. The exploration of cyclodextrins-based functional monomers for molecular imprinting could significantly improve the recognition performance of MIPs in aqueous solutions, and this aspect has been reviewed recently [28].

Qiu and coworkers reported the determinantion of α-amanitin based on molecularly imprinted photonic crystals by using synthesized moiety of α-amanitin as template and SiO₂ colloidal photonic crystal as solid support [29]. In our previous works, a target recognition determinant imprinting was proposed to prepare MIPs for the selective capture, removal and fluorescent sensing of α-amanitin, but the resulting MIPs showed an unsatisfactory adsorption capacity in aqueous solutions [30], [31], [32]. In this research, a β-cyclodextrin functionalized vinyl monomer was synthesized and used for the preparation of the MIPs that can recognize and capture the three amatoxins and two phallotoxins. The obtained MIPs were employed as SPE sorbents for the separation and enrichment of α-amanitin, β–amanitin, γ–amanitin, phallacidin, and phalloidin in biological samples, and the main factors affecting the recovery were carefully optimized. The analytical performance of the proposed method was further investigated and compared with previous works. Finally, the method was applied to the determination of the amanita toxins in mushroom poisoning clinical samples.