Broad-specific monoclonal antibody based IACs purification coupled UPLC-MS/MS method for T-2 and HT-2 toxin determination in maize and cherry samples

ABSTRACT

In this work, broad-specific monoclonal antibody based immunoaffinity columns (IACs) coupled to ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for purification and determination of T-2 and HT-2 toxin in maize and cherry samples. Based on broad-specific anti-T-2 monoclonal antibody, the prepared IACs could be used for both T-2 and HT-2 purification with cross reactivity values at 100% for T-2 and 108% for HT-2. Column capacity was 3.14 μg mL⁻¹ gel for T-2 toxin, and 3.20 μg mL⁻¹ gel for HT-2 toxin. After 10 cycles of usage at intervals of 2 days, column capacity decreased to 0.85 μg mL⁻¹ gel for T-2 and 1.22 μg mL⁻¹ gel for HT-2 toxin. The developed method exhibited high sensitive results with limit of detection (LOD) lower than 0.35 μg kg⁻¹ and 1.42 μg kg⁻¹.

KEYWORDS:

• Broad-specific immune-affinity columns
• T-2 toxin
• HT-2 toxin
• UPLC-MS/MS

1. Introduction

Naturally occurring mycotoxins contaminated in cereals and fruits bring sever health risks to animals and even human beings. T-2 toxin and it major metabolite HT-2 toxin (structure shown in Figure 1), belong type A trichothecenes, are mainly produced by various species including F. soprotrichioides, F. poae, and F. acuinatum (Brezina et al., 2014; McCormick et al., 2015). It is reported that humid and cool conditions were associated with increased fusarium mycotoxin accumulation in harvested cereals, fruits and even seafoods (Hjelkrem et al., 2018; Li, Wu, et al., 2017; Lu et al., 2016). T-2 toxin and HT-2 toxin are most important fusarium mycotoxins with severe acutely toxic mycotoxins. T-2 toxin was reported to exhibit protein synthesis and mitochondrial function, and they were also identified to show immunosuppression and general cytotoxicity toxic effects both in vivo and in vitro(Li, Zou, et al., 2017; Lin et al., 2019; Zhang, Li, Xu, Pan, & Sun, 2019). As the major metabolite, the toxic effects of HT-2 toxin were comparable as T-2 toxin itself (Maruniakova, Kadasi, Sirotkin, Bulla, & Kolesarova, 2014, 2015; Zhang et al., 2019). It seems that the toxicity of T-2 toxin might be induced by the sum contamination of HT-2 toxin as well. Considering their high toxic effects, T-2 toxin and HT-2 toxin received lots of concerns in recent years (Wei et al., 2019; Wu et al., 2019; Zhang, Zhang, Liu, Wu, & Zhang, 2018). In 2011, the Panel on Contaminants in the Food Chain (CONTAM) from the European Food Safety Authority (EFSA) has established a group tolerable daily intake (TDI) for the sum of T-2 and HT-2 as low as 0.1 μg kg⁻¹·B·W per day.

Figure 1. Chemical structures of T-2 toxin and major metabolites HT-2 toxin.

Considering the severe toxic effects of T-2 and HT-2 toxin, reliable and sensitive analytical methods are needed for the determination of these mycotoxins to control their contamination in foods. Immunoassays, such as enzyme-linked immuno sorbent assay (ELISA) (Arola, Tullila, Nathanail, & Nevanen, 2017) and immobilized activated beads (Edupuganti, Edupuganti, O'Kennedy, Defrancq, & Boullanger, 2013), can allow specific recognition without complicated sample preparation. These methods are fast and high-throughput, and can be applied for the screening of T-2 and HT-2. Compared to the immunoassays, instrument methods are much more reliable for the application of confirmation methods. Literature about analysis of T-2 and HT-2 as well as other mycotoxins has been reported in the recent years. In general, gas chromatography (GC) coupled with electron capture detector (ECD) (Majerus, Hain, & Scheer, 2008) or mass spectrometric detection (MS) (Rodriguez-Carrasco, Molto, Manes, & Berrada, 2014) has been applied. Moreover, high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) coupled with UV were the most frequently applied techniques rencently (Grajewski, Kosicki, Twaruzek, & Blajet-Kosicka, 2019). Based on the structures, T-2 and HT-2 are less sensitive via ultraviolet (UV) or FLD detector. Therefore, extra derivatization procedure is necessary before UV or FLD detector is applied (Visconti, Lattanzio, Pascale, & Haidukowski, 2005). In recent years, high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) is the most popular technique than others because of its sensitivity and the simplified sample preparation without derivatization. Apart from this, LC-MS/MS is also applicable for simultaneous detection of multiple mycotoxins in a single analysis.

However, as most previous reports, food matrices are complex and contain substances which might inhibit ionization in LC-MS/MS results. Therefore, sample preparation is the most important step prior to the instrument analysis steps. During the past decades, solid phase extraction (SPE) procedures including reverse-phase C18, Oasis HLB (Gentili et al., 2007; Stecher et al., 2007), and MycoSep 227 cartridges (Haubl et al., 2007) were mostly reported for T-2 and HT-2 clean-up. These purification protocols were mainly based on non-specific, hydrophobic interactions between SPE adsorbents and analytes, which might lead to the co-extraction of interferences as well as target analytes and result in matrix effects in the following LC-MS/MS analysis.

Recently, immunoaffinity columns (IACs) based on high-specific antigen and antibody recognization were proved to be an efficient tool with high selectivity and affinity characters for isolation, purification, and concentration of target analytes from complex matrix (Li et al., 2008). Compared to traditional SPE methods, IACs showed higher efficiency for targets purification in diverse sample matrices (Li, Wang, et al. 2012). However, reports about T-2 toxin immune-affinity purification were either focused on T-2 toxin itself (Pascale, Haidukowski, & Visconti, 2003) or coupled with fluorescence or ECD detector with lower sensitivity than LC-MS/MS (Kong, Zhang, Shen, Ou-Yang, & Yang, 2012; Visconti et al., 2005). What’s more, most of these literature mainly focused on cereals samples, neglecting other exposure pathway in fruit foods. In this research, new broad-specificity anti-T-2 monoclonal antibody (mAb) was used to develop IACs for the T-2 toxin and HT-2 toxin purification coupled an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for high sensitive determination of T-2 and HT-2 toxin in maize and cherry samples.

2. Materials and methods

2.1. Chemicals and reagents

T-2 and HT-2 standard were obtained from Pribolab (Qingdao, China). Anti-T-2 mAb (NO. 021T-0101) was obtained from Zeyang Biotechnology (Beijing, China). CNBr-activated Sephrose 4B gel was purchased from Amersham Biotech (Uppsala, Sweden). HPLC grade acetonitrile and methanol were purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). Other chemicals and solvents were analytical grade and obtained from Beijing Chemical Reagent Co. (Beijing, China). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.2. Apparatus

Ultraviolet–visible detector was from Shanghai Analytical Instrument (Shanghai, China). Acquity UPLC system with Acquity BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm particle size), and Micromass Quattro Premier XE triple quadrupole mass spectrometer were from Waters (MA, USA). Vortex mixer (model HQ-60) was obtained from North TZ-Biotech Develop Co. Ltd. (Beijing, China). Nitrogen evaporator (N-EVAP 112) was from Organomation Associates (MA, USA).

2.3. Standard solutions and buffers

T-2 and HT-2 standard solutions were prepared by dissolving 1 mg of each standard to a final volume of 1 mL with methanol, respectively (1 mg/mL for each). Posphate buffered saline (PBS, pH 7.4), HCl solution (1 mM), NaHCO3 solution (0.1M, pH 8.4), Tris-HCl buffer (0.1 M, pH 8.0), and Acetate buffer (0.1 M, pH 4.0) were prepared in this work as follows.

PBS (0.01M, pH 7.4) was prepared with 0.2 g of KH2PO4, 0.2 g of KCl, 2.9 g of Na2HPO4·12 H2O, and 8.0 g of NaCl dissolved to a final volume of 1 L with deionized water. HCl solution (1 mM) was prepared by diluting 84 μL of HCl (37%) to a final volume of 1 L with deionized water. NaHCO3 solution (0.1M, pH 8.4) was prepared with 8.4 g of NaHCO3 and 29.3 g of NaCl dissolved to a final volume of 1 L with deionized water. Tris-HCl buffer (0.1 M, pH 8.0) was prepared with 12.1 g of Tris, 29.3 g of NaCl, and 2.4 mL of HCl (37%) dissolved to a final volume of 1 L with deionized water. Acetate buffer (0.1 M, pH 4.0) was prepared with 2.5 g of CH3COONa·3H2O, 29.3 g of NaCl, and 4.7 mL of glacial acetic acid dissolved to a final volume of 1 L with deionized water.

2.4. Preparation of immunoaffinity columns

For immunoaffinity columns (IACs) preparation, the first and most important step is the immunosorbents preparation. In this work, immunosorbents were prepared according to the previous protocol with some minor revisions (Yao et al., 2020). Details were as follows: 1 g of CNBr Sepharose 4B was dissolved to a final volume of 5 mL with HCl for activation. The activated gel was transferred to a sintered-glass funnel and washed with 200 mL of 1 mM HCl. After that, the active gel was washed with 600 mL of NaHCO3 solution and then mixed with 30 mg of anti-T-2 mAb (NO. 021T-0101). The coupling was processed by stirring at 4°C for 20 h. Then, unbound mAb was removed by adding 50 mL of PBS for washing. The concentration of mAb in elute was tested via a UV-Vis spectrometry to determine the coupling efficiency. Excess binding sites of activated gel were blocked by adding 10 mL of Tris-HCl buffer and stirred at 4°C for 2 h. After washing with 20 mL of acetate buffer (0.1 M, pH 4.0) and 20 mL of Tris-HCl buffer in turn for 3 cycles, 1 mL of prepared gel was transferred to a glass column (3 cc) with a filter plate (10 mm × 0.8 mm, i.d.). The prepared IACs were stored in PBS at 4°C until use.

2.5. Column capacity determination for T-2 toxin and HT-2 toxin

Column capacities for T-2 toxin and HT-2 toxin were tested with 10 μg of each standard dissolved in 10 mL of methanol/PBS (10/90, v/v). First, IACs were conditioned with 10 mL of PBS, and then the standard analyte was loaded onto each column by gravity. Each column was washed with 10 mL of methanol/water (10/90, v/v) and 10 mL of water. Target analyte was eluted with 3 mL of methanol and dried under a stream of nitrogen at 65°C. The residues were redissolved with 1 mL of acetonitrile/water (50/50, v/v) and then filtered through a 0.22 μm filter for analysis. For regeneration of the IACs after using, each column was washed with 50 mL of PBS and then stored in PBS at 4°C for the next use.

2.6. Sample preparation

Before analysis, maize samples were crushed and sift through 50 mesh sieve and cherry samples were kernel removed. An amount of 1 g (±0.01 g) of negative cereal or fruit samples (maize and cherry) were weight and transferred to a 15 mL of polypropylene centrifuge tube. Samples were spiked at three groups at concentration of 10, 40, and 100 μg kg⁻¹ with five replicates each (n = 5). One unfortified sample was set as negative control. Spiked group samples were vortexed and stay at room temperature for 15 min for incubation. Then, 10 mL of methanol/water (60/40, v/v) were added and vortexed for 3 min for extraction. Each sample was centrifuged at 10,000 × g for 15 min, and 5 mL of each supernatant was transferred to another polypropylene tube. Then, 25 mL of PBS was added to each tube for sample dilution to decrease the percentage of methanol as low as 10% before purification. Immunoaffinity purification protocol was the same as described in column capacity determination.

2.7. UPLC-MS/MS method

A Waters UPLC system was selected in this research for separation using an Acquity BEH C18 column with column temperature at 30°C. Mobile phase was consisted of 5 μM ammonia water (A) and 5 μM ammonia acetonitrile (B). Flow rate was settled at 0.3 mL min⁻¹ with injection volume at 10 μL. Gradient elute programme was adopted as follows: 0∼1 min 100%∼90% A, 1∼6 min 90%∼20% A, 6∼6.5 min 20∼100% A. Detection of T-2 and HT-2 was performed in positive ionization mode with desolvation temperature at 300°C, source temperature at 80°C, and capillary voltage at 2.8 kV. Nitrogen was performed as desolvation gas at a flow rate of 650 L/h, with a cone gas flow rate of 30 L/h. Argon was performed as collision gas at a pressure of 2.5 × 10⁻³ mbar in the collision cell. Multiple reaction mode (MRM) was adopted for qualitative and quantitative analysis (Parameters were shown in Table 1).

Table 1. MRM parameters of T-2 toxin and HT-2 toxin.

Analyte	Scan mode	Precursor ion (m/z)	Product ion (m/z)	Cone voltage (V)	Collision energy (eV)
T-2	ESI+	484.8 [M + NH4]^+	215.1^a 245.2	15	20 15
HT-2	ESI+	442.8[M + NH4]^+	215.2^a 263.3	12	15 12

^aIon for quantification.

3. Results and discussion

3.1. Performance and optimization of IAC conditions

CNBr-actived Sepharose 4B is suitable for immunosorbent preparation due to its easy derivation charicater (Li, Wang, et al. 2012). For conjugation, 5∼10 mg of mAb are recommended for each millilitre gel. CNBr-actived Sepharose 4B is adopted in this experiment and 6 mg mAb were employed for each millilitre of gel in this research. Coupling efficiency is monitored over 95% with 5.5 mg of mAb per millilitre gel.

After that, IACs purification steps for T-2 and HT-2 toxin were optimized in this work. On the basis of sample preparation procedure, loading solvent containing no more than 10% of methanol led to satisfactory results for both T-2 toxin and HT-2 toxin. Then, 10 mL of deionized water was used to remove the ions in PBS to minimize ionization effects and 10 mL of 10% methanol was used to remove non-specific binding to reduce matrix interferences. Finally, target analytes were eluted with 3 mL of methanol for antibody–antigen dissociation. Methanol is a mild elution medium without causing irreversible denaturation in order to allow the re-usage of these prepared IACs. What’s more, methanol performed as the elution solvent is easy to concentrate without extra salt ions effect in MS/MS analysis, which is superior to acid or other buffer solutions (Vera-Avila, Vazquez-Lira, Garcia, & Covarrubia, 2005).

Specificity of this mAb was tested by cross reactivity (CR) value according to previous report (Li, Zhang, et al. 2012). This mAb showed broad-specific character and can recognize both T-2 and HT-2 with negligible CR value (<0.01) for other analogues, including structure related DON and DOM-1 (Table 2). Therefore, the prepared IACs could be used for T-2 and HT-2 purification. In order to test the column capacity, 10 μg of T-2 toxin and 10 μg of HT-2 toxins were passed through the prepared IACs, respectively. After loading, washing, and eluting procedure of IAC treatment, concentrations of T-2 toxin and HT-2 toxin were detected with LC-MS/MS method. The column capacity was 3.14 μg mL⁻¹ gel for T-2 toxin, and 3.20 μg mL⁻¹ gel for HT-2 toxin.

Table 2. Performances of IC50, cross-reactivity (%CR) of T-2 toxin analogues (DON, DOM-1, HT-2, NIV, T-2 tetrael, T-2 toxin, T-2 triol).

Analogue	IC50 (ng mL^−1)	CR (%)
DON	>10,000	<0.01
DOM-1	>10,000	<0.01
HT-2	26.78	108
NIV	>10,000	<0.01
T-2 tetrael	>10,000	<0.01
T-2 toxin	28.90	100
T-2 triol	>10,000	<0.01

Reusability depends on activity of the immobilized antibodies and the chemical stability of the support is an important IACs parameter. The column capacity of repeat usage was evaluated in 20 days at 2 days intervals (shown in Figure 2). The column capacity decreased following the increasing cycles of usage. During 10 times of usages, the column capacity was 0.85∼3.14 μg mL⁻¹ gel for T-2 toxin and 1.22∼3.2 μg mL⁻¹ gel for HT-2 toxin. Considering the determination of mycotoxins is trace analysis, it is suitable for T-2 and HT-2 toxin purification with these prepared IACs for at least ten cycles.

Figure 2. IAC capacity for T-2 toxin and HT-2 toxin for 20 days at intervals of every other day.

3.2. UPLC-MS/MS method

In order to minimize potential interruption and obtain satisfactory sensitivity and peak shape, gradient UPLC elution programme was settled and optimized for isolation within 5 min (Figure 3). MRM mode was selected for T-2 toxin and HT-2 toxin in ESI positive mode by injecting standard solution with the tandem mass spectrometer directly. The most abundant precursor ion for both T-2 and HT-2 are [M+NH4]+. MS/MS product ions were tested at different collision energy and two abundant product ions were used for qualitative and quantitative ions (Table 1). According to the identification criteria of Commission Decision 2002/657/EC, this development UPLC-MS/MS method is suitable for qualitative and quantitative of T-2 and HT-2 toxin.

Figure 3. MRM chromatograms of matrix standard (A), spiked samples (B), and blank control samples (C) for T-2 toxin (484.4 > 215.1) and HT-2 toxin (442.8 > 215.2).

3.3. Method validation

3.3.1. Linearity

Linearity was evaluated based on matrix-spiked calibration curves at 7 spiked levels with concentrations ranged from 1 to 500 μg kg⁻¹ for T-2 toxin and 5 to 500 μg kg⁻¹ for HT-2 toxin. Calibration curves for maize and cherry samples were shown in Table 3. From the table, the liner range obtained is wide to cover most nature contaminated concentrations. Samples of especially high concentrations of T-2 or HT-2 could be diluted before detection.

Table 3. Calibration curve, LOD, and LOQ of T-2 and HT-2.

Matrix	Analyte	Liner range (μg kg^−1)	Regression equitation	R^2	LOD (μg kg^−1)	LOQ (μg kg^−1)
Maize	T-2	1–500	y = 28.50x + 18.74	0.9871	0.28	0.93
	HT-2	5–500	y = 19.85x + 27.96	0.9892	1.25	4.16
Cherry	T-2	1–500	y = 20.85x + 28.32	0.9925	0.35	1.17
	HT-2	5–500	y = 19.68x + 17.75	0.9941	1.42	4.73

3.3.2. LOD and LOD

LOD (limit of detection) and LOQ (Limit of Quantification) are determined on the basis of S/N (Signal to Noise) ratio. LOD in different samples depend on S/N ratio over 3 was determined lower than 0.35 μg kg⁻¹ and 1.42 μg kg⁻¹ for T-2 toxin and HT-2 toxin, respectively. LOQ depend on S/N ratio over 10 were lower than 1.17 μg kg⁻¹ and 4.73 μg kg⁻¹, respectively (Table 3).

3.3.3. Accuracy and precision

Accuracy and precision were determined based on recoveries of spiked samples at three different spiked concentrations. Recoveries of samples were determined by calculated and spiked concentrations. Intra–day and inter–day RSD (Relative Standard Deviation) were determined on the basis of 5 replicates at different spiked levels of 3 consecutive days (results shown in Table 4). From the table, mean recoveries ranged from 89.6∼96.6% for T-2 toxin and 89.2∼96.2% for HT-2 with intra-day and inter-day RSD less than 7.33% and 6.49% in maize samples. In cherry samples, mean recoveries ranged from 89.9∼98.8% for T-2 toxin and 88.9∼98.7% for HT-2 with intra-day and inter-day RSD less than 7.88% and 6.95%. From the results, it can be concluded that the developed method could be applied for T-2 and HT-2 toxin determination in maize and cherry samples.

Table 4. Accuracy and precision of mycotoxins in maize and cherry.

Matrix	Analyte	Spiked level (µg kg-1)	Day 1		Day 2		Day 3		Inter-day RSD (%)
			Recovery (%)	Intra-day RSD (%) (n = 5)	Recovery (%)	Intra-day RSD (%) (n = 5)	Recovery (%)	Intra-day RSD (%) (n = 5)
Maize	HT-2	10	89.8 ± 6.25	7.02	93.4 ± 4.20	6.23	92.3 ± 7.18	6.98	5.77
		40	86.9 ± 5.33	6.29	93.0 ± 4.31	5.38	96.2 ± 6.88	6.75	6.21
		100	94.5 ± 3.91	3.87	95.6 ± 4.36	5.18	89.2 ± 3.26	3.86	3.58
	T-2	10	89.6 ± 4.25	4.66	93.5 ± 6.93	6.05	95.2 ± 6.78	7.33	6.49
		40	95.6 ± 6.05	6.22	94.3 ± 4.38	5.84	96.6 ± 7.52	6.78	6.29
		100	94.3 ± 5.98	5.27	92.9 ± 5.23	4.99	95.7 ± 4.31	6.87	5.81
Cherry	HT-2	10	91.8 ± 5.38	4.89	94.3 ± 5.28	5.19	98.7 ± 6.18	4.29	3.97
		40	88.9 ± 5.40	5.64	95.9 ± 5.67	5.84	94.8 ± 6.27	6.49	5.46
		100	92.7 ± 6.71	6.48	92.8 ± 5.44	6.97	95.1 ± 5.74	5.11	6.29
	T-2	10	91.8 ± 4.88	5.27	95.3 ± 7.21	7.85	91.2 ± 6.84	6.91	6.95
		40	94.8 ± 5.65	5.08	91.7 ± 6.82	6.19	98.8 ± 5.39	5.47	5.66
		100	89.9 ± 6.29	7.88	91.7 ± 5.84	6.47	94.6 ± 5.28	5.80	6.11

The result showed this developed IACs purification coupled UPLC-MS/MS protocol exhibited higher sensitivity results with lower LODs than published ELISA (Kawamura et al., 1990; Kierek Jaszczuk et al., 1995) or UPLC-MSMS methods (Kafouris, Christofidou, Christodoulou, Christou, & Ioannou-Kakouri, 2017). What’s more, this developed protocol is the first report for T-2 and HT-2 toxin detection in cherry samples.

3.4. Contamxination exposure of T-2 toxin and HT-2 toxin in commercial samples

Totally 30 commercial samples (15 maize samples and 15 cherry samples) were obtained from local farmer's market in Shandong Province to investigate the exposure of T-2 toxin and HT-2 toxin. Samples were processed according to the developed IAC-UPLC-MS/MS methods, and concentrations of targets in each sample were determined on the basis of each calibration curves. Finally, 12 samples were detected T-2 toxin positive, in which 5 samples were also detected HT-2 toxin positive in maize samples with positive rate at 80% in maize. In cherry samples, only 2 samples were detected T-2 toxin positive, and none HT-2 positive samples detected (positive rate 13.3%) (Table 5).

Table 5. T-2 and HT-2 contamination exposure in commercial maize and cherry samples in Shandong Province.

Maize No.	T-2 (μg kg^−1)	HT-2 (μg kg^−1)	Cherry No.	T-2 (μg kg^−1)	HT-2 (μg kg^−1)
1	325	96.1	16	ND	ND
2	65.4	ND	17	ND	ND
3	ND	ND	18	ND	ND
4	75.8	ND	19	28.8	ND
5	257	149	20	ND	ND
6	178.5	77.9	21	ND	ND
7	35.9	ND	22	ND	ND ND
8	66.7	ND	23	19.7
9	159	107	24	ND	ND
10	107	ND	25	ND	ND
11	118	68.7	26	ND	ND
12	ND	ND	27	ND	ND
13	ND	ND	28	ND	ND
14	65.7	ND	29	ND	ND
15	49.3	ND	30	ND	ND

4. Conclusions

In this work, a broad-specific mAb based IACs were prepared and coupled to UPLC-MS/MS detection method was developed for T-2 and HT-2 toxin purification and determination in maize and cherry samples. The mAb used in this work showed broad-specific CR values to both T-2 and HT-2 toxin. Negligible CR values were obtained with structure related mycotoxins in this work. Under the optimized conditions, this IAC-UPLC-MS/MS protocol could be used for T-2 and HT-2 determination in both maize and cherry samples. With high sensitive UPLC-MS/MS conditions, lower LOD of T-2 and HT-2 were adopted at 0.28 μg kg⁻¹ and 1.25 μg kg⁻¹ for maize samples and 0.35 μg kg⁻¹ and 1.42 μg kg⁻¹ for cherry samples. Applying to commercial samples, 12 samples were detected T-2 toxin positive with 5 detected HT-2 toxin positive in maize samples (positive rate 80.0%). In cherry samples, only 2 samples were detected T-2 positive (positive rate 13.3%). The proposed protocol meets the requirement for sample preparation and highly sensitive identification of multiple T-2 and HT-2 in agricultural product and food safety.

Disclosure statement

Shaoxia Lin and Yanshen Li are equally contributors. No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was funded by China Postdoctoral Science Foundation Funded Project (No. 2019M650023), National Natural Science Foundation of China (No.31871718), and Natural science foundation of Shandong Province (No. ZR2017LC022).