3.1 Solid-phase extraction

During solid-phase extraction, a solution of the analyte is passed through an extraction column containing the adsorbent. The analytes are absorbed by the particle bed, and the column is washed to remove the impurities. In the next step, the absorbed analyte is washed out with a suitable solvent. Numerous examples of solid-phase extraction with different sorbents (Table 1), including silica gel modified by the octadecyl hydrocarbon chain (C18) [19,20,34], Florisil [19] and Oasis^® HLB polymers [24], as well as extract purification tools such as immunosorbents (IAC) [20,35,36] can be found in the literature.

Jodlbauer et al. [34] used the octadecyl sorbent to isolate ZEN and its derivatives from urine and animal tissue samples. The recovery of ZEN and its metabolites from urine and tissues ranged from 86% to 102% in the evaluated method. Andres et al. [19] used C18 and Florisil as sorbents in the process of ZEN isolation and purification. Both sorbents were tested on urine samples, and Florisil was found to be less selective. The octadecyl sorbent was selected for further analyses, where ZEN recovery ranged from 92% to 108%.

In other studies, ZEN and its metabolites were isolated from urine, plasma and equine feces, and were purified by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC) [20]. An ammonium acetate buffer with pH 4.8 and D₂-ZAN was initially added to the urine sample. The solution was incubated with β-glucuronidase/ arylsulfhatase solution and brought to pH 4. The solution was applied to a sorbent filled with ISOLUTE^® C18, which was previously conditioned with methanol and H₂O. The sorbent was washed with 30% MeOH (2 mL) and dried under vacuum. The analytes were eluted with methanol (1.25 mL), and the eluate was diluted with a phosphate buffer solution at pH 7.4. The extract was purified with an immunosorbent (Easi-Extract^®Zearalenone) which was previously conditioned with phosphate buffered saline solution (PBS). The cited study demonstrated that effective sample preparation and sensitivity of the method are determined by the complexity of the analyzed matrix. Single-stage purification before IAC was sufficient for plasma samples, whereas urine samples had to be purified by both SPE and IAC. Urine recovery ranged from 56% to 100%.

Zearalenone and its metabolites can also be isolated with molecularly imprinted polymers (MIP). In the work of Mausia et al. [21], the zearalenone template was synthesized by precipitation polymerisation and applied as a sorbent in molecularly imprinted solid-phase extraction (MISPE). The sorbent was characterized by high stability and affinity for the ZEN molecule. The sorbent was previously conditioned with acetonitrile; the sample was applied in an acetonitrile/ water mixture (60/40% v/v) and washed with an ACN/ H₂O mixture (30/70% v/v). The analytes were eluted with a 95/5% (v/v) mixture of methanol and acetic acid. The obtained recovery for ZEN ranged from 90.8% to 99.6%.

Comments: Since the introduction of solid phase extraction as a sample preparation method (especially for liquid samples), this technique has been widely used to isolate and enrich various compounds and has often been used to purify extracts from interfering compounds. The rapid development of SPE is due to progress aimed at the synthesis of new sorbents that are characterized by specific interactions with an isolated compound in order to achieve high recovery. Future research should focus on reducing the amount of sorbent used for extraction, thus minimizing or eliminating the use of toxic solvents. An alternative to traditional sorbents can be molecularly imprinted polymers (MIPs), which enable selective and specific interaction with the analyte. This ensures not only selective retention of the isolated compound, but also purification of the extract from interfering compounds. Undoubtedly, an important issue is the repeatability and reproducibility of the obtained sorbents.

3.2 QuEChERS

The quick, easy, cheap, effective, rugged and safe (QuEChERS) method is suitable for preparing biological samples in line with green chemistry rules. In the QuEChERS approach, the isolated compound is extracted with organic solvents with the addition of salt, and analyte and matrix components are split between the organic phase and water. A purified extract is obtained by dispersion and solid phase extraction. The QuEChERS method is simple, it relies on non-chlorinated solvents, involves fewer sample preparation steps, and can be used to analyze a larger number of samples. Pajewska at el. [17] and Woźniak at el. [37] used the QuEChERS technique to isolate ZEN and its metabolites from muscle tissues, and obtained a recovery rate of 56-115%. This technique was also used by Cunha and Fernandes [38] to analyze breakfast cereals and contaminated flour. The procedure was modified to produce a recovery rate of 67-101% for breakfast cereals and 52-103% for flour.

3.3 Novel methods of sample preparation for the isolation of ZEN and its metabolites

Zearalenone levels should be monitored in food and feed to protect humans and animals from consuming products contaminated with this mycotoxin. Therefore, innovative methods are being sought to minimize the health problems associated with toxins.

Kim et al. [39] developed an innovative system for removing and monitoring ZEN levels in feed, which offers an alternative to IAC. The proposed approach relies on the use of monoclonal antibodies (mAbs) and magnetic nanoparticles (MNP). In the cited study, ground and enriched samples were shaken and extracted in 70% methanol. The collected samples were passed through paper filters, and MNP-mAb conjugates were used to remove the mycotoxin. Zearalenone was removed magnetically from the supernatant, and its concentration was measured. The mycotoxin was also extracted by IAC for comparative purposes. The recovery rate was significantly higher in the proposed approach (92-100%) than in IAC (81-88%).

Amoli-Diva et al. [40] used dispersive liquid-liquid micro-extraction (DLLME) combined with microsolid phase extraction (μ-SPE) to determine ZEN levels in wheat samples. This simple and low-cost extraction method was characterized by satisfactory repeatability and reproducibility as well as high recovery in the range of 91.6% to 99.1%. Antep and Merdivan [41] investigated the effectiveness of the DLLME method for quantitative determination of ZEN in beer, with the aim of developing a simple, fast and sensitive extraction method. The obtained recoveries ranged from 71% to 108%. The simplicity, accuracy and low cost of the DLLME-μ-SPE method were confirmed by Hashemi et al. [42] who extracted ZEN from an acetonitrile mixture with the use of hydrophobic magnetic nanoparticles. The magnetic absorber was collected with an external magnet, and water from the supernatant was decanted. The recovery rate for maize samples was high in the range of 93.2% to 102.1%. The study demonstrated that ZEN levels in samples could be effectively determined with the use of the two-stage micro-extraction procedure based on DLLME and μ-SPE with hydrophobic magnetic nanoparticles.

Porto-Figueira et al. [43] combined μ-QuEChERS with ultrasound-assisted extraction (USAE) to improve the original QuEChERS method. The described approach involved a reduced amount of separating salts, smaller samples and fewer solvents. The cited authors demonstrated the potential of the proposed method.

Magnetic molecularly imprinted polymers (mMIP) were used for selective extraction of ZEN from various cereal flours [44]. Quercetin was the inert template, and it was also used to synthesize mMIP for ZEN. Flour extraction mixtures were prepared by centrifugation and sonication with acetonitrile and water (80:20, v/v) and 0.2% HCOOH. The extracts were centrifuged, separated and placed in centrifuge tubes, and ultra-pure water and 100 mg of mMIP were added. The solution was decanted, and the supernatant was removed with an external magnet. The analyte with mMIP was transferred to a water test tube, and the supernatant was centrifuged and removed. Zearalenone was eluted three times with methanol and twice with acetonitrile. The sample was centrifuged each time, and mMIP was magnetically decanted. The ZEN recovery rate was above 95%, which indicates that this method is useful for isolating trace amounts of the mycotoxin in food analyses.

Another unconventional method for ZEN detection was presented by Fang et al. [45]. The authors synthesized a new molecularly imprinted optosensing material (MIOM) by anchoring the MIP layer on ionic liquid-modified CdSe/ ZnS quantum dots. The MIOM was used to detect ZEN in maize, rice and wheat flours. The recovery rates ranged from 84% to 107%.

Comments: The novel approaches to sample preparation differ from conventional methods. The separation of the extracts from other co-extracted compounds in the matrix is also an important issue during sample preparation. Effective sample preparation and high sensitivity of the method are determined by the complexity of the matrix, therefore each step is an inherent element to obtaining the best possible recovery results. It is preferable to replace the expensive sorbents (e.g., antibodies) by magnetic particle imprinted polymers with structural analogs of the analytes. The developed methodologies for isolating ZEN and its metabolites with the use of mMIP allow obtaining high and not always repeatable recoveries, but they allow high efficiency of cleaning the samples from compounds that coexist in them as contaminants. Future research should focus on the search for a template for the molecular imprint and the development of a repeatable synthesis of the prepared polymers. In addition, new applications such as integrated artificial intelligence systems to predict appropriate extraction conditions are expected to ensure the future viability of this technique.

3.4 Solvent extraction

Liquid extraction is a popular method of extracting analytes from the matrix, including samples of food and infected animal tissues, in analyses of ZEN and its metabolites. Additional ultrasonic (UE) or microwave (MEA) energy can be used in this extraction technique, which can successfully increase the efficiency of extraction and shorten the time of its operation.

Pallaroni et al. [22] described the pressurized liquid extraction (PLE) method that relies on environmentally acceptable and less harmful organic solvents. Zearalenone was extracted from maize with a 50/50% (v/v) mixture of isopropanol and an aqueous solution of triethanolamine (1%) at 80°C. The obtained recovery was 85%.

Uracca et al. [4] determined the content of ZEN and α-zearalenone in cereal samples (corn, wheat, rice, barley, rye) and swine feed by ASE. Optimal extraction conditions were obtained by using a solvent mixture MeOH/ACN (50:50 v/v) at the temperature of 50°C, a pressure of 1500 psi, a static time of 5 min and flush volume 60%. The obtained recovery exceeded 96% for ZEN (RSD < 4.0%) and reached 98% for α-ZAL (RSD < 4.6%) [4].

Comments: One of the advantages of ASE method is that the process is automated. The solvent thoroughly penetrates the sample, which reduces extraction time and decreases cost associated with slow sample preparation.

4.1 High-performance liquid chromatography

The presence of ZEN and its metabolites can be determined by high-performance liquid chromatography (HPLC) with various detectors. Zearalenone is a naturally fluorescent compound, and it is identified by fluorimetric detection.

Ok et al. [36] isolated ZEN by HPLC with fluorimetric detection, where the Synergi^TM Hydro-RP 80Å column (Phenomenex, 4.6 × 250 mm; 4 μm) was maintained at 40°C. The excitation and emission wavelengths were 275 nm and 450 nm, respectively. A H₂O/ACN/MeOH (35/10/55% v/v/v) mixture was the mobile phase, and the flow rate was 1.0 mL min^-1. The LOD value was 4.0 μg kg^-1, and LOQ was determined at 10.0 μg kg^-1.

Marczuk et al. [35] analyzed clinical cases of dairy cattle infected by Fusarium fungi, including ZEN. Blood samples were collected and subjected to IAC extraction with a Zearala-Test^® column to determine the plasma concentration of ZEN. The extracts were analyzed by HPLC with fluorescent detection. An ODS Hypersil chromatographic column (4 × 250 mm; 5 μm) was used. The mobile phase was an ACN/MeOH/H₂O mixture (46/8/46 or 8/10/82% v/v/v) with a flow rate of 1.8 mL min^-1 or 1.5 mL min^-1 at 30°C. The excitation and emission wavelengths were 218 and 438 nm, respectively.

Hewitt et al. [33] identified ZEN in corn and maize products with the use of an H₂O/MeOH/ACN mixture as the mobile phase at an excitation wavelength of 274 nm and an emission wavelength of 440 nm. Pajewska et al. [17] relied on the QuEChERS technique to isolate ZEN and its metabolites from human tissues. The compounds were separated by HPLC with fluorimetric detection at the excitation and emission wavelengths of 270 and 440 nm, respectively. Zearalenone and α-ZEL were detected as compounds with the highest fluorescent intensity. High-performance liquid chromatography with fluorescence detection is characterized by high selectivity and sensitivity, and it is one of the most popular methods of ZEN detection.

Liquid chromatography coupled with mass spectrometry is becoming an increasingly popular detection technique despite its high cost. Liquid chromatography with one mass analyzer (LC-MS) and tandem mass spectrometry (LC-MS/MS) ensure unambiguous identification of the analyte. This method requires an ion source to determine the mass-to-charge ratio (m/z). Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most widely used ionization techniques [46,47].

Jodlbauer et al. [34] relied on the LC-MS/MS technique with an APCI type ionization source to determine the concentrations of ZEN and its metabolites in urine and tissue samples collected from cows and pigs. The analysis was performed on the Supersphere^®RP-18 Merck column (125 × 3.0 mm; 4 μm) at 35°C. The mobile phase was an ACN/MeOH/ H₂O (10/45/45% v/v/v) mixture with 15 mM ammonium acetate at a flow rate of 0.5 mL min^-1. The LOD values ranged from 0.1 to 0.5 μg L^-1, and LOQ – from 0.5 to 1 μg L^-1.

Soleimanes et al. [47] determined ZEN levels in cereal matrices by the UPLC-MS/MS technique with ESI in positive and negative ion mode. The LOD values in the tested matrices were determined at 0.1-0.4 ng g^-1, and LOQ values – at 0.2-1 ng g^-1. Liquid chromatography coupled with mass spectrometry is increasingly used in analyses, but other analytical methods are often selected due to the high cost of mass spectrometry.

4.2 Gas chromatography

Gas chromatography-mass spectrometry (GC-MS) is also used to analyze ZEN and its metabolites. A GC-MS method for detecting ZEN and its metabolites in samples of bovine urine, bile, meat and kidneys was described by the Standard Operating Procedure ARO/458 [48]. The GC-MS/MS method was also used by Rodríguez-Carrasco et al. [49] to detect ZEN in grain-based products. Dudziak [50] relied on GC-MS to detect ZEN in aqueous environments. Zearalenone concentrations were determined at 0.3 to 0.5 ng/L in water samples, and recovery rates ranged from 62% to 80%.

4.3 Thin-layer chromatography

Classical thin-layer chromatography (TLC) is a less accurate technique than GS-MS. Quantitative results are compared with the standard, and the purity, quality and concentration of the standards affect the accuracy of the analysis. This method has been used to detect ZEN in maize. The fluorescence of ZEN was compared against the applied standards. Samples that tested positive for ZEN in TLC were extracted again to validate the results [51].

Schaafsma et al. [57] relied on TLC to analyze Fusarium toxins in maize and wheat. A comparison of the costs associated with different analytical methods revealed that TLC was the cheapest technique. However, toxin concentrations were less accurately determined by TLC than HPLC.