Mycotoxins are mainly produced by filamentous fungi in a complex matrix [1
]. Mycotoxins can contaminate different agricultural commodities and they are mainly detected in cereals, such as barley, wheat, maize, and even fruit and related products [3
]. Considering the severe toxicity, the presence of mycotoxins in foods could induce a high potential risk to human health, such as endocrine disorders, immunosuppression, teratogenic, carcinogenic and mutagenic effects, and so on [7
]. In recent decades, due to the high frequency of contamination and widespread occurrence, mycotoxins have increasingly attracted attention worldwide.
It is well known the kelps are major keystone species which remain deep rooted in the marine environment [9
]. Also, there are ample minerals and nutrients in kelps, which make them highly bioactive for human beings. Kelps usually grow on the bottom of the sea. They contain fiber, protein, beta carotene, amino acids, enzymes and chlorophyll, leading to the high quality in foods. In addition, there are also phosphorus, iron, sodium, potassium, calcium, magnesium, and other minerals in kelp [10
]. Considering the similar components as cereals with protein and polysaccharose, fungi may also grow in kelps during the storage stage. Therefore, mycotoxins might also occur in kelps and related food and feeds. To the best of our knowledge, there is scarcely any information regarding the presence of mycotoxins in kelps. Reports about the transformation and generation of mycotoxins in kelps have also not been presented. In order to control mycotoxins in foods and feeds, the first and most important step is to develop sensitive and reliable methods for mycotoxin monitoring.
In the last decades, there have been numerous studies on mycotoxin detection with different chromatographic equipment, such as High Performance Liquid Chromatography (HPLC) [11
], Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) [14
]), Gas Chromatography (GC) [17
], Gas Chromatography Tandem Mass Spectrometry (GC-MS) [19
], and so on. Antibody-based immunoassays were also applied for mycotoxin detection with advantages of simplicity, low–cost and high throughput. These immunoassays mainly include enzyme linked immunosorbent assay (ELISA) [23
], fluorescence polarization immunoassay (FPIA) [26
], surface plasmon resonance (SPR) [29
], flow cytometric microsphere immunoassay [33
], and rapid strip tests [36
]. However, these methods mainly focused on cereal matrix and related products. As far as we know, there are very few reports on the detection of these targets in marine-derived products, especially kelps. Considering the high frequency of contamination of mycotoxins in cereals, exposure to kelps with similar components as cereals should be taken seriously. For the exposure investigation, the first and most important step is to develop a reliable detection method for mycotoxins in kelps.
In this work, a rapid, reliable, and sensitive LC-MS/MS method was developed for mycotoxin exposure detection in kelp. In order to obtain a satisfactory recovery for each analyte, sonication and an acidulated extraction pretreatment were investigated and optimized in this work. In order to minimize the matrix effect, each sample was further purified by a PLEXA cartridge. Based on this method, in 43 of 50 kelp samples in Shandong Province, 3AcDON/15AcDON was detected with a positive rate of 86%. In China, Shandong Province is one of the major kelp production and consumption areas, and the contamination of mycotoxins will lead to high dietary exposure risk to human beings.
In conclusion, a sonication based quantitative and confirmatory LC-MS/MS procedure was developed for the determination of 7 major mycotoxins (3AcDON, 15AcDON, DON, F-X, NIV, T-2, and ZEA). Specifically, target analytes were extracted with acidulated methanol/acetonitrile/formic acid (49.5/49.5/1, v/v/v). After the extraction, each sample was further purified by a PLEXA cartridge to minimize the matrix effect. The validation of this developed procedure proved the suitability of the method for the confirmatory analysis of mycotoxins with mean recoveries from 72.59~107.34%, intra-day RSD < 9.21%, inter-day RSD < 9.09%, LOD < 5.55 μg kg−1, and LOQ < 18.5 μg kg−1. With respect to real samples, T-2, F-X, DON, ZEA and NIV were not detected in any sample, while all samples had 3AcDON/15AcDON that ranged from 57.5 to 162.5 μg kg−1 with a positive rate of 86%. Considering that Shandong Province is one of the major kelp production and consumption areas in China (over 40%), the contamination of mycotoxins will lead to high dietary exposure risk to human beings.
4. Materials and Methods
4.1. Chemicals and Reagents
3-Acetyldeoxynivalenol (3AcDON), 15-Acetyldeoxynivalenol (15AcDON), Deoxynivalenol (DON), Nivalenol (NIV), T-2 toxin (T-2), and Zearalenone (ZEA) (Figure 4
) were obtained from Fermentek Biotechnology (Jerusalem, Israel).
Acetonitrile and methanol (HPLC) were adopted in this work (Dima Technology Inc.) (Muskegon, MI, USA). Formic acid (HPLC) was purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). Milli–Q Synthesis system (Millipore, Bedford, MA, USA) was used for water purification. Bond Elut PLEXA cartridges (500 mg, 6 cc) (Agilent Technologies, CA, USA) were used in this work. Other reagents were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China).
The LC system adopted in this research was obtained from AB SCIEX (Redwood City, CA, USA) with a Venusil ASB C18 column (100 mm × 2.1 mm i.d., 3 μm particle size). The quadrupole mass spectrometer used in this work was a AB4000 triple from AB SCIEX (Redwood, CA, USA). The vortex mixer was from North TZ–Biotech Develop Co., Ltd. (Beijing, China). The N-EVAP 112 nitrogen evaporator was from Organomation Associates (Berlin, MA, USA).
4.3. Sample Preparation
Two grams of dried kelp sample was weighed into a 50-mL polypropylene centrifuge tube. Two experiment groups were fortified with 50 and 100 µg kg−1 of each analyte. One unfortified group was set as the negative control. Twenty milliliters of methanol/ethyl acetate/formic acid (49.5/49.5/1, v/v/v) was added and ultrasound was performed for 2 min followed by vortexing for 3 min for extraction. Each sample was centrifuged at 9000 rpm for 10 min at 4 °C. The supernatant was transferred and dried using a nitrogen evaporator at 60 °C. The residues were re-dissolved with 10 mL of water by vortexing for 3 min. Each sample was loaded onto a PLEXA cartridge with 5 mL of methanol and 5 mL of water in turn. After rinsing with 5 mL of water, analytes were eluted with 5 mL of methanol. After drying using a nitrogen evaporator at 60 °C, target analytes were re-dissolved with 1 mL of water/acetonitrile(9/1, v/v). Samples were filtered through a 0.22-μm filter and 10 μL was injected for LC-MS/MS analysis.
4.4. Instrumental Conditions
Target analytes were separated via LC system with Venusil ASB C18 column. The mobile phase was as follows: solvent A (water containing 50 μM of ammonium acetate) and solvent B (acetonitrile). The column temperature was set to 25 °C, and the flow rate was 0.5 mL min−1 with injection volume of 10 μL. The gradient elution program was performed for chromatography separation as follows: 0–1 min, 98% A; 1 to 3 min, 98–60% A; 3.0–4.0 min, 60% A; 4.0–5.0 min, 60–10% A; 5.0–6.1 min 10% A; 6.1–6.2 min 10–98% A; 6.2–8.0 min 98% A.
For detection, the LC system was coupled to an AB4000 triple quadrupole mass spectrometer (Redwood, CA, USA) with an electrospray ionization source (ESI). For maximum intensity detection, the mass conditions were optimized as follows: Capillary voltage at 5.0 kV; Source temperature at 550 °C, IonSpray voltage at 5500 V. Ion Source Gas 1 was 55 Psi, and Ion Source Gas 2 was 55 Psi. The MS instrument was operated in integrate ESI positive (ESI+) and negative (ESI−) multiple reaction monitoring (MRM) mode (Table 1