The mobile phase plays an important role in the ionization efficiency when the analytes enter the MS/MS system. Acetonitrile, methanol, water, formic acid, and buffers such as ammonium acetate are commonly used as the mobile phase in UPLC. We have selected formic acid and ammonium acetate because of their high ionization efficiency and solubility in the presence of methanol. Therefore, we investigated mixtures of methanol/water and acetonitrile/water, separately, as the mobile phase. The results showed that the response of fumonisins was 15 times higher when using methanol/water than when using acetonitrile/water, with an acceptable peak shape. Moreover, the use of methanol/water produced better separation of the mycotoxins compared with acetonitrile/water. Therefore, we selected methanol as the elution mobile phase.

Adding ammonium acetate (10 mM) and formic acid (0.1%, v/v) to the mobile phase of methanol improved the responses of all mycotoxins. Adding formic acid greatly enhanced the response especially of positively charged ENNs, BEA, and FBs. Thereby, the formic acid in water was selected to improve the mobile phase. The possible reason for the superior performance is that ionic strength can increase after adding a suitable amount of formic acid solution to the mobile phase; the change in ionic strength can affect the behavior of the separated material, which is advantageous to the positive ion protons.

Varying concentrations of formic acid solution (0.05%, 0.1%, 0.5%, 1.0%, and 5.0%, v/v) showed minimal effect, but the responses of zearalenone (ZEN), ENN A1, ENN B1, BEA, fusarenon-X (FUS-X), gliotoxin (GLT), and neosolaniol (NEO) were relatively higher with 0.5% formic acid compared to the other proportions (Figure 1). Considering the efficiency of all mycotoxins, 0.5% (v/v) formic acid in water was selected for the mobile phase.

The gradient elution program of mobile phase had effects on the retention time and peak shape of target compounds, and also influenced the ionization efficiency and sensitivity of the target compounds. The gradient elution supports an efficient separation of the multiple mycotoxins, shortens the retention time, and enhances the peak shape and sensitivity.

The initial ratio of 0.5% (v/v) formic acid water solution/methanol was set to 80:20 (v/v), resulting in a suitable response and peak shape for the ENNs, while there was no response for FBs, ochratoxin A (OTA), DON, 3-AcDON, and 15-AcDON. In this condition, the results showed that when increasing the proportion of methanol, the ENNs and BEA mycotoxins presented two peaks with varying retention times (Figure 2), which can seriously affect the accuracy of qualitative and quantitative results of the compounds. When the initial ratio of 0.5% (v/v) formic acid water solution/methanol was set to 95:5 (v/v), we could observe better-resolved peaks. The optimized gradient elution of eluent A (0.5% formic acid water solution) was as follows: starting with 5% and rapidly increasing the proportion to 85% in 5.5 min, slowly increasing to 100% within 5.8 min, then linearly reducing to 5% in 10.0 min. Under this condition, the concentration of DON, 3-AcDON, 15-AcDON, FUS-X, 4,5-diacetoxyscirpenol (DAS), GLT, FB1, FB2, and FB3 is 200 µg·L⁻¹; the concentration of ZEN, OTA, verruculogen (VCG), sterigmatocystin (SMC), T-2 toxin (T-2), HT-2 toxin (HT-2), NEO, ENNA, ENNA1, ENNB, ENNB1, and BEA is 50 µg·L⁻¹; and the concentrations of AF B1, AF B2, AF G1, and AF G2 are 10, 3, 10, and 3 µg·L⁻¹, respectively. The 25 mycotoxins could be monitored as shown in Figure 3. A good peak shape and resolution were achieved for all the target mycotoxins within 10 min except for the conjugations 3-AcDON and 15-AcDON, which were selected as different MS/MS daughter ions for quantification.

The extraction solvent was selected according to the method described in a previous work [15]. The extraction yield greatly improved when the acetonitrile/water content gradually increased until reaching the maximum yield at the proportions of 80% and 84%, respectively [33]. In this study, we have made a slight modification. When using pure acetonitrile as the extraction solvent, the yields of ENNs, BEA, and FBs were less than 60% in all matrices. However, when extracting with a solution containing a ratio of 99:1 (v/v) of 0.1% formic acid water solution/acetonitrile, we observed more prominent intensities of the peaks, especially for the FBs.

Sorbents C18, PSA, GCB, SPE C18, and the amino column (NH₂) were selected for the purification of mycotoxins. The sorbent C18 can be used to remove the fatty and nonpolar components of the matrix, whereas PSA is mainly used for the removal of organic acids, pigments, and phenol, among others, whereas GCB mainly removes pigments of the crude sample.

We have optimized the purification by separately testing 100 mg of C18, 150 mg of PSA, and 150 mg GCB. The results are summarized in Table 1. The mycotoxins used with PSA and GCB were recovered with less than 50% yield, and the extraction yield with GCB was less than 20%. The low yield can be explained by the high affinity of the mycotoxins to the sorbents, except for DON mycotoxin. Published results show that GCB is suitable for application with biscuits when using an amount smaller than 500 mg with dichloromethane/methanol (80:20, v/v) [26]. Nevertheless, dichloromethane is toxic and is not recommended. The sorbent GCB was not suitable for the detection of mycotoxins in pomegranate juices [17] and beer-based drinks [2] due to the sorption of the mycotoxins, so we may not choose GCB as an alternative cleaning sorbent.

Using 0.4 g of PSA produced significant improvements in the recovery, reaching yields superior to 80% for DON, AF B1, AF B2, AF G1, AF G2, T-2 toxin, HT-2 toxin, OTA, ZEN, FB1, and FB2 in rice [28], but the yields decreased when using PSA in sesame, with recoveries less than 10% for T-2, HT-2, OTA, ZEN, FB1, FB2, and FB3, and lower than 60% for AF B1. When the amount of PSA increased, the recovery of AF B2 decreased from 70.4% to 36.4%, and did not affect the recoveries of AF G1 and AF G2 [21].

Our results showed that C18 obtained satisfactory yields of about 60.0%~101.7% for most of the mycotoxins, excepted for T-2, HT-2, SMC, NEO, and DAS, which is consistent with the results obtained for sesame butter [21]. However, using a C18 sorbent concentration of 50 mg/mL did not improve the purification of human breast milk [20].

Using a combination of C18 with PSA in the range of 10, 20, 50, and 100 mg showed that when the amount of PSA increased, the recovery of most mycotoxins decreased. However, the mycotoxins VCG, FUS-X, and GLT were not affected, with extraction yields inferior to 30%. Our results demonstrated that the type and quantity of sorbents greatly influence the extraction yield.

Evaluation of the commonly used purification column MycoSep 226, 227, and 400 (Romer Labs Inc., Union, MO, USA) showed that the three columns were all suitable for DON, 3-AcDON, 15-AcDON, T-2, HT-2, VCG, FUS-X, and DAS, with recoveries from 61.3% to 125.1%, with the MycoSep 227 superior to the 226 and 400, although the three MycoSep columns were not suitable for OTA, ENNs, BEA, NEO, and FBs. Our results are consistent with the observations of Sun et al., who reported on the extraction of mycotoxins from wheat, rice, and corn with MycoSep 226 [15], and the observations reported by other researchers extracting other feedstuffs with MycoSep 400 [34].

The comparison of column C18 with amino column NH₂ showed that FBs and OTA were not properly recovered from the column NH₂, as reported in the case of multiple mycotoxins determination of sorghum [35].

Satisfactory recoveries were obtained when using column C18 for FBs, compared with a MultiSep 211 FUM purification cartridge [36], with recoveries of 80.9%–97.0%. In addition, the cost of a C18 column is much lower than an immunoaffinity column. The method thus provides a simple and cheap purification. The results showed that the amino column NH₂ is also suitable for the extraction of ENNs and BEA, although the yields ranged from 61.3% to 83.6%. This work presents the first simple and cost-effective purification of ENNs and BEA.

It can be clearly observed that by optimization of the extraction parameters, satisfactory results could be obtained for most of the mycotoxins, although a few mycotoxins presented low yields. No cleanup was applied for the following experiments of 25 mycotoxins.

Matrix effects (MEs) are unavoidable, and the removal of the matrix interference is challenging. At present, reports from scientific literature offer solutions such as the internal standard method using the internal standards zearalenone (ZEN) and deepoxy-deoxynivalenol (DOM) [35] or isotopically labeled standards [37]. Although using a standard curve with an acceptable linear relation presents the advantage of high precision, the choice of an appropriate internal standard for a multicomponent analysis is often difficult and expensive. The response of the target mycotoxins can be suppressed or enhanced on account of the interfering matrix components. The ME was calculated by the Equation (1) for different blanks of wheat, corn, and rice samples as shown in Figure 4. (1) ME = 100 × ( 1 − area of mycotoxin seandard in blank matrix area of mycotoxin standard in solent )

It can be observed that the signal enhancement was prominent for the FBs, with an ME ranging from 28.4% to 132.1%. The effect is emphasized in the case of corn, which exhibited an ME of about twice that of wheat or rice. The signal enhancement of the FBs was also reported in spices (enhancement of about 20%) [38] and sesame butter [21], but the was suppressed in white rice (ME between 13.2% and 17.5%) [9]. The MEs of OTA and DAS were slightly enhanced, presenting values ranging from 24.3% to 50.9% and 6.8% to 31.4%, respectively.

Most of the mycotoxins had a signal suppression effect and were close to the tolerable range between +20% and −20% [23]. However, the signals of HT-2, T-2, and VCG were significantly suppressed by 48.8%–81.3%, as reported for cereal syrups [38].

It is worth mentioning that AF B1 and B2 in corn presented a strong signal suppression effect, as reported in white rice, edible nuts, and seeds by Arroyo-Manzanares et al. [18]. Compared to literature results, we obtained acceptable ME percentages except for AF B1 and B2. The mycotoxins NEO and DAS in wheat showed a strong signal enhancement. The signal suppression for the ENNs, especially for ENNB in rice, was more important than that for wheat and corn.

In conclusion, the matrix effects caused by different matrices were significant for most of the mycotoxins. The calibration curves help to reduce the MEs and, therefore, increase the extraction yields to improve the accuracy of the analysis.

The developed and optimized method was applied for the analysis of 65 samples, comprising 26 wheat samples from different areas of China, 14 corn samples collected from two different years (2014 and 2015), and 25 various brands of rice obtained from the local supermarket. As shown in Figure 5, 90% of the samples were contaminated with mycotoxins. The detected mycotoxins were DON, 3-AcDON, 15-AcDON, ENNA, ENNA1, ENNB, ENNB1, FB1, FB2, FB3, T-2, OTA, BEA, ZEN, and SMC. The other mycotoxins (AF B1, AF B2, AF G1, AF G2, HT-2, VCG, NEO, FUS-X, and DAS) were not detected.

In contaminated samples, all the wheat samples contained DON within concentrations ranging from 42.1 to 718.0 µg·kg⁻¹, but the values are still below the state limit standards. Nevertheless, we should pay attention to the addition of the concentrations of 3-AcDON and 15-AcDON reaching values ranging from 6.1 to 32.8 µg·kg⁻¹. It is worth mentioning that 19%, 27%, 69%, and 35% of the total wheat samples were contaminated with ENNA, ENNA1, ENNB, and ENNB1, respectively. The detected concentrations ranged from 0.1 to 17.2 µg·kg⁻¹. The concentrations of ENNB were similar to that of ENNA in the wheat sample, as reported in the case of wheat grains from Poland [30] and Morocco [32]. Moreover, the values of FBs (FB1+, FB2+, and FB3), T-2, and OTA ranged from 0.1 to 5.9 µg·kg⁻¹, 0.2 to 0.7 µg·kg⁻¹, and 0.2 to 3.7 µg·kg⁻¹, respectively. These values are all far below the maximum residual limits authorized by the European Union regulations [40].

The main mycotoxins detected in corn (12 samples out of 14 were contaminated) were FBs with concentrations ranging from 0.1 to 1190.7 µg·kg⁻¹. Samples from 2014 were more contaminated than the ones from 2015, indicating that storage conditions should be better controlled. The quantity of detected mycotoxins in rice was lower than that of wheat and corn. The concentrations of mycotoxins FBs, OTA, BEA, and SMC ranged from 0.1 to 11.0 µg·kg⁻¹, and are below the maximum residual limits, as well as the mycotoxins that are not mentioned in the regulations, such as ENNs and BEA. We should strengthen supervision and try to identify the potential risks and the effect of toxin combinations.

The reagents methanol, acetonitrile, and formic acid were of HPLC grade and were purchased from Thermo Fisher Scientific Inc. (Shanghai, China). The extraction kits QuEChERS (Part No: 5982-0650, Agilent Technologies Inc., Santa Clara, CA, USA), sorbent C18, GCB, and PSA were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The solid-phase extraction column C18 and the amino column NH₂ were purchased from Agilent Technologies Inc. (Santa Clara, CA, USA). Ultra-pure water was produced by a Mill-Q system (Millipore, Billerica, MA, USA). All other reagents were of analytical grade.

Solid mycotoxin standards of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), fusarenon-X (FUS-X), neosolaniol (NEO), 4,5-diacetoxyscirpenol (DAS), aflatoxin B1 (AF B1), aflatoxin B2 (AF B2), aflatoxin G1 (AF G1), aflatoxin G2 (AF G2), T-2 toxin, HT-2 toxin, fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), sterigmatocystin (SMC), verruculogen (VCG), ochratoxin A (OTA), zearalenone (ZEN), gliotoxin (GLT), enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1), and beauvericin (BEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The standards AF B1, AF B2, AF G1, and AF G2 were dissolved in methanol at concentrations of 200, 60, 200, and 60 µg·L⁻¹, respectively. Stock solutions of the other standards were prepared in methanol at a concentration of 100 mg/L. All the standard solutions were stored at −18 °C in darkness. The working standard solutions were freshly prepared before use by diluting the stock solution in order to obtain a 50:50 v/v ratio of methanol/water. The concentrations of the working mixed solutions were set accordingly to the response values of the mycotoxins by MS. The concentration of the working mixed standard solutions for DON, 3-AcDON, 15-AcDON, FUS-X, DAS, GLT, FB1, FB2, and FB3 were 200 µg·L⁻¹. In the case of ZEN, OTA, VCG, SMC, T-2, HT-2, NEO, ENNA, ENNA1, ENNB, ENNB1, and BEA, the concentration of the working mixed solutions was 50 µg·L⁻¹. Finally, the concentrations of the working mixed solutions for AF B1, AF B2, AF G1, and AF G2 were 10, 3, 10, and 3 µg·L⁻¹, respectively.

A blank substrate was measured with the matrix of a standard solution according to Section 2.3, with the same concentration as the working mixed standard solutions.

The sample (2 ± 0.05 g) was finely milled in a 50 mL centrifuge tube before extraction. In the case of spiked sample, the required volume of spiking standard solution was added prior to the extraction step. A mixture of acetonitrile/water (20 mL, 80:20, v /v) containing 0.1% (v/v) formic acid was added to the solution. The mixture was then vortexed for 30 s and shaken on an automatic thermostatic cultivation shaker (Yiheng Technology Co., Ltd., Shanghai, China) for 30 min. The QuEChERS extraction kit, containing sodium chloride (1.0 g), anhydrous magnesium sulfate (4.0 g), sodium citrate (1.0 g), and sodium hydrogen citrate sesquihydrate (0.5 g), was added to the mixture and the tube was vigorously shaken by hand for 2 min. The sample was then centrifuged (3-18K, Sigma, St. Louis, MO, USA) at 8000 revolutions per minute (relative centrifugal force, RCF, 3500× g) for 5 min at 10 °C. The supernatant (2 mL) containing the extracts was transferred to a centrifuge tube, and submitted to a stream of N₂ at 55 °C until complete dryness. The residue was sequentially dissolved in a mixture of methanol/water (0.5 mL, 1:1, v/v), vortexed for 1 min, then filtered through nylon filter (0.22 µm, Membrana GmbH, Wuppertal, Germany), and the extracts were finally recovered in a 200 µL microtube for UPLC–MS/MS analysis.

The extracted mycotoxins were analyzed by ultrahigh-performance liquid chromatography coupled to tandem quadrupole mass spectrometry (UPLC–MS/MS, XEVO-TQ, Waters Corp., Milford, MA, USA). The data processing was performed using the MassLynx 4.0 software (Waters Corp., Milford, MA, USA).

The separation of the mycotoxins was performed using a CORTECS C18 column (100 × 2.1 mm, 1.6 µm, Waters Corp., Manchester, NH, USA) under a flow rate of 200 µL·min⁻¹. The volumes of the strong wash (90% methanol) and weak wash (10% methanol) solvents were 100 µL and 600 µL, respectively. The proportion of the mobile phase was set as described in Section 2.2. Mobile phase A was methanol and mobile phase B was a water solution containing 0.5% (v/v) formic acid. The mobile phase A was gradually eluted from 5% during 5.5 min, 850% during 5.8 min, 100% during 9.0 min, 5% during 10.0 min. The column was maintained at room temperature and the sample temperature was 20 °C. The injection volume was 5 µL.

Tandem mass spectrometry (MS/MS) detection was performed in a positive electrospray ionization mode (ESI+). For the infusion experiments, the mycotoxins standards (0.1 mg/L) were dissolved in methanol and a flow rate of 25 µL·min⁻¹ was applied. The parameters were optimized by using the IntelliStart program supplied by Waters Corp. (Milford, MA, USA). The capillary voltage was set at 2.5 kV. Gaseous nitrogen was used as the cone, nebulizing, and desolvation gas. The source and desolvation temperature was 110 °C and 500 °C, respectively. The cone and desolvation gas flows were maintained at 20 L·h⁻¹ and 800 L·h⁻¹, respectively. The collision gas flow was 0.17 mL·min⁻¹. The analysis of the mycotoxins was performed in a multiple reaction monitoring (MRM) mode. For each mycotoxin, at least one precursor ion and two fragment ions were monitored. The most abundant product ion was selected for quantification while the second-most intense ion was used for the qualitative analysis. The shape of the peaks was optimized by waiting 1 min before and after the retention time. The acquisition parameters for the 25 analyzed mycotoxins are summarized in Table 4.