Natural toxins are chemical substances that are not toxic to the organisms that produce them, but which can be a potential risk to human health when ingested through food. Thus, it is of high interest to develop advanced analytical methodologies to control the occurrence of these compounds in food products. Current trends in sample preparation involve moving towards “greener” approaches by scaling down analytical operations, miniaturizing the instruments and integrating new advanced materials as sorbents. The combination of these new materials with sorbent-based microextraction technologies enables the development of high-throughput sample preparation methods, which improve conventional extraction and clean-up procedures.
1. Introduction
Natural toxins are chemical substances naturally produced by living organisms (animal, plants or microorganisms) that are not toxic to them, but which can be potential health hazards to humans when ingested through food. These substances may naturally occur in food endogenously (toxic compounds that are implicit constituents of food resulting from the metabolism of a genus, species or strain, e.g., glycoalkaloids in potato or tetradotoxin in pufferfish) or exogenously (toxic compounds resulting from the metabolism of living organisms that occur in food as contaminants as they are not intentionally added, e.g., mycotoxins produced by molds grown in different products and toxins produced by algae that may be accumulated in edible marine organisms)
[1][2]. The World Health Organization (WHO) encourages national authorities to monitor the most relevant natural toxins in the food supply. In this context, natural toxins of exogenous origin have received the most attention because of their potential harmful health risks and their involvement as natural contaminants. With respect to international organisms, these natural toxins of exogenous origin can be grouped in mycotoxins, phycotoxins (or marine toxins) and plant alkaloids
[1][3][4]. Mycotoxins are toxic metabolites produced by certain types of molds, which can grow on a large number of foodstuffs such as cereals, dried fruits, nuts and spices. Most of these mycotoxins are chemically stable and survive food processing. The most common are aflatoxins (B1, B2, G1, G2 and M1), ochratoxins (A, B and C), patulin and fusarium toxins (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, zearalenone and fumonisins)
[5]. On the other hand, marine toxins are produced during blooms of particular naturally occurring microalgae species in the ocean and fresh water. Thus, these toxins can be retained and bioaccumulated in shellfish and fish or contaminate drinking water. Their intake can be a potential hazard to consumers, since they are not eliminated by cooking or freezing, and might cause several adverse effects
[6]. Conversely, in recent years, awareness about alkaloids of plant origin, such as pyrrolizidine, tropane and opioid alkaloids, has raised because of their occurrence as contaminants in different food products and the lack of data and knowledge about their exposure through food. These alkaloids are secondary metabolites of some plants, which can grow in fields as weeds and contaminate food crops appearing throughout the production of plant-derived products and finally be ingested, being toxic to humans
[4][7][8][9][10][11][12]. The control of all these exogenous natural toxins in food is of high importance since they can cause from mild disorders (headache, vomiting, diarrhea, etc.) to serious situations (neurological disorders, carcinogenic, teratogenic or/and mutagenic effects, hepatic and renal damage, etc.) and can even be lethal. Moreover, they may cause the appearance of chronic diseases due to their harmful effects after a long-term exposure at high levels
[1][2][3][4][5][6][7][8][9][10][11][12]. Therefore, food safety plays an essential role in reducing the risks related to the presence of harmful substances in food in order to protect consumers. In fact, the WHO in collaboration with the European Food Safety Authority (EFSA), the Food and Agriculture Organization (FAO) and the Codex Alimentarius Commission have established a legislation for mycotoxins and marine toxins
[13][14], whereas pyrrolizidine, tropane and opioid alkaloids are in the process of being legislated, and at the moment only recommendations have been established for them
[15][16][17]. In this sense, maximum residue limits (MRLs) for many of these natural toxins have been established in these guidelines to control the occurrence of these compounds in food
[13][18].
Nonetheless, to achieve these limits and ensure the health of consumers it is important to develop high-throughput, sensitive and selective analytical methods to determine in a feasible way the presence of these natural toxins in foodstuffs
[19]. However, the analysis of these compounds in food samples constitutes a challenging task because of the extreme complexity of these matrices, which considerably hinders the selective extraction of the target analytes and decreases the sensitivity of the method
[20]. Despite significant advances in analytical instrumentation, particularly with respect to the combination of mass spectrometry and chromatographic separation, these techniques are not sensitive enough for direct analysis of complex matrices. Therefore, sample preparation is still a crucial step in food analysis in order to achieve an effective isolation and/or preconcentration of the analytes and provide an adequate clean-up of matrix interferences prior to instrumental analysis
[21].
For many years, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) have been the most extensively used sample preparation techniques. Due to the inherent drawbacks of LLE (such as: time-consumption, limited ability to extract polar compounds, requirement of large volumes of solvents, etc.), SPE has become more popular, as it provides more efficient recoveries and lower solvent consumption than LLE
[22]. Nevertheless, current trends in sample preparation involve moving towards “greener” approaches by scaling down analytical operations and miniaturizing the instruments
[23][24]. This has led in recent years to the development of different microextraction techniques for sample preparation procedures. In this sense, the SPE technique has been the axis of improving and creating even better and greener sorbent-based sample preparation techniques, which require less time and labor than SPE, such as: miniaturized solid-phase extraction (m-SPE), micro-dispersive solid-phase extraction (µ-dSPE), microextraction by packed sorbents (MEPS), pipette-tip solid-phase extraction (PT-SPE), solid-phase microextraction (SPME), stir-bar sorptive extraction (SBSE), and micro-solid-phase extraction (µ-SPE). These sorbent-based microextraction techniques have been proposed in recent years as an alternative to conventional sample preparation techniques to meet the Green Analytical Chemistry (GAC) requirements, as they involve advantages such as minimal solvent and sample consumption, fewer treatment steps, and reduction of waste generation
[25]. Thus, they enable the development of cheaper, more cost-effective, and more environmentally friendly extraction and purification procedures.
On the other hand, the synthesis of new advanced materials for their application as sorbents in sample preparation has achieved considerable progress in the last decade, since these materials can play an important role in preconcentration processes and, in some cases, provide selective extraction of the target compounds
[20][21][23][26][27]. Magnetic nanoparticles (MNPs), silica-based nanomaterials, metal-organic frameworks (MOFs), multiwalled carbon nanotubes (MWCNTs) and graphene oxide (GO) are currently the most used materials for the extraction of natural toxins from food samples, as they present large surface area and advanced physicochemical properties that enhance the efficiency, selectivity and sensitivity of the analytical procedures
[21][27][28][29]. Additionally, the combination of these new materials with microextraction technologies enables the development of high-throughput sample preparation methods, which provide the advantages of both strategies leading to meet the GAC requirements and improving conventional extraction and clean-up technologies
[23][30].
Some works in the literature have previously reviewed the determination of several natural toxins, such as phytotoxins
[27] or mycotoxins
[31], in food samples and other matrices. However, these works have just focused on one type of compounds but have not considered other natural toxins. On the other hand, other published reviews have addressed the development of new materials for their application to extract or detect chemical contaminants in order to ensure food safety
[27][32][33].
The miniaturization of conventional sample preparation procedures has been proposed as an alternative for developing analytical methods with improved analytical characteristics (accuracy, precision, sensitivity, etc.) along with a decrease in sample and solvent consumption, reduction of hazardous reagents and wastes, and saving energy and time. As a result, new formats and configurations have arisen to carry out microextraction procedures, which overcome drawbacks of conventional techniques.
Table 1 collects the most relevant works published in the last decade dealing with microextraction techniques based on sorbent-adsorption, which have been applied for the isolation of natural toxins from different food samples. In this sense, Solid-Phase Microextraction (SPME) has been the most popular
[34][35][36][37][38]. However, procedures based on the dispersion of the sorbent material, such as micro-dispersive solid-phase extraction (µ-dSPE) and micro-solid-phase extraction (µ-SPE) have also been used
[39][40][41]. All the works reviewed were performed for the analysis of mycotoxins (ochratoxins, aflatoxins, zearalenone, fumonisins and patulin) in different food matrices (mainly, wine, cereals and nuts). Only three of the methodologies developed in these articles perform the simultaneous determination of different types of mycotoxins
[37][39][40], while the other works only described the individual determination of a specific analyte
[34][35][36][38][41][42]. Concerning detection mode, mass spectrometry (MS) and fluorescence detection (FLD) were the techniques employed to detect these natural toxins (
Table 1). Most of these works used MS detection, which is the most suitable technique to detect the presence of contaminants in food at trace levels thanks to its high sensitivity and to its structural elucidation capability, which enables the unequivocal identification and confirmation of the target analytes. In contrast, the FLD also provides high sensitivity and selectivity, but if the analytes do not show fluorescence it is necessary to carry out a derivatization process (pre-column or post-column derivatization) for their detection, which can sometimes be time consuming.
Table 1. Application of sorbent-based microextraction techniques for isolation of natural toxins in food samples (2009–2019).
* Elution performed with mobile phase (online system); ACN: Acetonitrile; AF: Aflatoxin; F: Fumonisin; HPLC-FLD: High performance liquid chromatography coupled to fluorescence; HPLC-MS/MS: High performance liquid chromatography coupled to tandem mass spectrometry; HPLC-MS: High performance liquid chromatography coupled to mass spectrometry; MeOH: Methanol; MEPS: Microextraction by packed sorbent; MWCNTs: Multiwalled carbon nanotubes; OTA: Ochratoxin A; OTB: Ochratoxins B; PAT: Patulin; SPME: Solid-phase microextraction; T-2: T-2 toxin; UHPLC-FLD: Ultra High performance liquid chromatography coupled to fluorescence; UHPLC-MS: Ultra High performance liquid chromatography coupled to tandem mass spectrometry; ZAL: Zearalanol; ZAN: Zearalanone; ZEL: Zearalenol; ZEN: Zearalenone; µ-dSPE: Micro-dispersive solid-phase extraction; µ-SPE: Micro-solid-phase extraction.
Sometimes, the commercially available microextraction techniques and sorbent materials used limit the development of the analytical methodologies. One of the crucial parameters that determine success of sample preparation is the choice of the sorbent material. Depending on the analytes to be extracted, the sorbent material must have specific characteristics that allow obtaining the highest extraction efficiency. In addition, using minimal amounts of sorbents is one of the requirements of the Green Analytical Chemistry (GAC) when developing an analytical procedure
[29]. Thus, the sorbent must have advanced functional properties to be able to potentially interact with the target analytes to achieve high extraction efficiency by using minimal amounts of it. In this sense, current trends in the development of analytical methods are focused on the synthesis of new advanced materials to apply them as sorbents in sample preparation procedures. Among these materials, magnetic nanoparticles (MNPs), silica-based nanomaterials, metal-organic frameworks (MOFs), multiwalled carbon nanotubes (MWCNTs) and graphene oxide (GO) have been the most employed for the extraction of natural toxins from food products (
Table 2). The advanced properties of these materials, such as their large surface area, low resistance to diffusion, fast sorption kinetics and large adsorptive capability make them very suitable for sample preparation, as they improve the efficiency, selectivity and sensitivity of the analytical procedures. Moreover, the integration of these new materials in microextraction technologies enables developing high-throughput analytical methods with the advantages of both strategies. Thanks to this integration, conventional and commercially available procedures can be improved and GAC requirements can be accomplished. In this sense, in the last decade, different new materials have been used to extract natural toxins from food products by their combination with different microextraction techniques, such as m-SPE, in-syringe SPE, PT-SPE, µ-dSPE, µ-MSPE, µ-SPE, SPME and SBSE (
Table 2). They have proved their efficiency in the extraction of several mycotoxins (mainly aflatoxins, ochratoxins, patulin and zearalenone) and marine toxins, which have been mainly extracted from cereals, drinks, dairy products and seafood (
Table 2). Sometimes, these sorbent materials lack or have little selectivity during the extraction procedure, leading to the extraction of matrix interferences along with the analyte that may hinder its detection. To overcome this problem, MIPs can be synthesized as sorbents by polymerization processes
[30]. In this sense, different MIPs have been applied in m-SPE, µ-MSPE and SBSE for the extraction of patulin, T-2 toxin, fumonisin, aflatoxins and ochratoxins from food samples (
Table 2). Nevertheless, when developing multicomponent methods to simultaneously extract different compounds belonging to different chemical families in a single run, the lack of selectivity of the materials is desirable, since in this case the sorbent must be able to extract a wide range of compounds. Therefore, in these cases specificity is not required. On the other hand, the analytical procedures published in the last decade, which integrate new materials in microextraction techniques for the extraction of natural toxins from food, have been mainly combined with the detection of analytes by HPLC coupled to MS or FLD, and to a lesser extent with ultraviolet detection (UV), such as the diode array detection (DAD) (
Table 2). In contrast, there are no works using GC as a separation technique instead of HPLC. Indeed, for the analysis of these natural toxins, it is more suitable to use HPLC, since they are not very volatile compounds. Therefore, sometimes, to achieve their analysis by GC it is necessary to perform a derivatization process, which is more complex and time consuming than the determination by HPLC.
Table 2. Application of new advanced materials on sorbent-based microextraction techniques to isolate natural toxins from food samples (2009–2019).
* Elution performed with mobile phase (online system); ACN: Acetonitrile; AF: Aflatoxin; AMNPs: Aptamer-functionalized magnetic nanoparticles; AZA: Azaspiracid; CD: Cyclodextrin; CNT: Carbon nanotube; DA: Domoic acid; DAD: Diode array detector; DCM: Dichloromethane; DON: Deoxynivalenol; DTX: Dinophysistoxin; F: Fumonisin; Fe3O4 SPs@ZIF8/Zn2+: Modified magnetic zeolite imidazolate framework-8; Fe3O4@PDA MIPs: Magnetic polydopamine-based molecularly imprinted polymer; Fe3O4@pDA NPs: Core–shell polydopamine magnetic nanoparticles; Fe3O4@SiO2@UiO-6: Magnetite@silica core-shell magnetic microspheres; FLD: Fluorescence; GO: Graphene oxide; GYM: Gymnodimine; HPLC: High performance liquid chromatography; LTL: Zeolites linde type; M/ZIF-8: Magnetic zeolite imidazolate framework-8; MAA-co-DVB: Methacrylic acid-co-divinyl-benzene; MeOH: Methanol; MEPS: Microextraction by packed sorbent; MGNP: Magnetic-graphene nanoparticles; MGO: Magnetic graphene oxide; MIP: Molecular imprinted polymer; MMIP: Magnetic molecularly imprinted polymer; MMIP-SB: Magnetic molecularly imprinted stir-bars; MMM: Magnetic mesoporous microspheres; MNM: Magnetic nanostructured materials; MNPC: Magnetic nanoporous carbon; MNPs: Magnetic nanoparticles; MS: Mass spectrometry; MS/MS: Tandem mass spectrometry; m-SPE: Miniaturized solid phase extraction; MWCNTs: Multiwalled carbon nanotubes; n.p.: Not provide; OA: Okadaic acid; OTA: Ochratoxin A; OTB: Ochratoxin B; OTC: Ochratoxin C; PAT: Patulin; PBS: Phosphate buffer saline; PDA: Photodiode array; PD-MNPs: Polydopamine magnetic nanoparticles; PT-SPE: Pipette-tip solid phase extraction; PTX2: Pectenotoxin-2; PU: Polyurethane; rGO: Reduced Graphene oxide; SBSE: Stir-bar sorptive extraction; SPE: Solid-phase extraction; SPME: Solid-phase microextraction; SPX1: Spirolides-1; STEH: Sterigmatocystin; TFA: Trifluoroacetic acid; T-2: T-2 toxin; UHPLC: Ultra high performance liquid chromatography; UV/vis: Ultraviolet/visible; YTX: Yessotoxins; ZAL: Zearalanol; ZAN: Zearalanone; ZEL: Zearalenol; ZEN: Zearalenone; β-CDPG: β-cyclodextrin supported on porous graphene nanohybrid; µ-dSPE: Micro-dispersive solid-phase extraction; µ-MSPE: Micro-magnetic solid-phase extraction; µ-SPE: Micro-solid-phase extraction; 3DG@Fe3O4: Magnetic three-dimensional graphene sorbent.