Introduction

Fusarium is widely distributed in nature, mainly causing mildew contamination in corn and other cereal crops, such as soybean, sorghum, barley, and wheat [1]. Moniliformin (MON) is a toxic secondary metabolite produced by various Fusarium species and exists in nature in the form of sodium and potassium salts. It cannot be destroyed after heating for 60 min in a solution with pH 4 at 100 °C. Freeze drying also does not affect the stability of the beading bacteriocin [2]. MON, chemically named 1, 2-cyclocyclobutene-1, 2-dione [C4HO3R (R = Na or K)], is harmful to many tissues and organs, such as the cardiovascular and immune system of animals. It mainly acts on actively proliferating cells, such as myocardium, liver, spleen and bone cells [3]. Some researchers had measured the different pollution levels of corn and grain in Poland [4], Peru [5], Morocco [6] and Keshan disease area of China [7]. One of the most polluted was the Polish corn, and the pollution level reached 530 mg/kg. Due to its widespread distribution and pollution, processed products such as soybean oil and corn oil are likely to be contaminated by MON. However, there are limited sources reporting the content and solubility of MON in vegetable oils. As a highly water-soluble toxin, MON will not only exist in about 1% water content in vegetable oils but also depend on the degree of contamination. This article established a method for the determination of MON in vegetable oils to census a considerable amount of vegetable oils on the market to provide a reference for related scientific researchers. Similar to MON, fumonisin and deoxysuccinol are also toxic secondary metabolites produced by Fusarium. These two mycotoxins have been detected in vegetable oils. Therefore, MON may contaminate vegetable oils during plant growth, harvesting, processing, transportation, or sales [8].

The previous common detection methods mainly include ion chromatography (IC) [9], capillary electrophoresis (CE) [10], thin-layer (TLC) and high-performance liquid chromatography (HPLC) [11]. Nevertheless, it takes a long time to analyze samples by IC and CE. Although TLC could detect the sensitivity and reproducibility of the sample, it can only achieve semi-quantitative data compared with HPLC, which has a higher accuracy and lower detection limit. Due to the complex matrix in the research object, it is necessary to establish an advanced extraction and purification method to reduce the negative impact on vegetable oils. At the same time, due to the limited information on MON in vegetable oils in previous work, there is a critical need for a noval and accurate method for the analysis and detection. In this experiment, we established a hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS) rapid analysis method for MON in vegetable oil using an Oasis MAX solid-phase extraction cartridge column in solid-phase extraction purification technology. Therefore, an accurate and appropriate measurement method was established according to these advantages.

Materials and Methods

Apparatus and Reagents

LC-30A ultra-high-performance liquid chromatography was from Shimadzu (Kyoto, Japan). AB SCIEX QTRAP®4500 LC/MS mass spectrometry was from AB SCIEX (Massachusetts, USA). HILIC column and Oasis MAX solid-phase extraction column (3 mL, 60 mg) were from Waters Corporation (Milford, Massachusetts, USA). Nitrogen cyclone concentrator and vortex mixer were from Biotage (Uppsala, Sweden). Moniliformin standard (sodium salt form, purity ≧ 98.5%) was purchased from Merck & Co Inc. (Darmstadt, Germany). Methanol, formic acid, acetonitrile and ammonium acetate are chromatographically pure and purchased from Dikma Technologies (Beijing, China).

Sample Extraction

The vegetable oil sample 1.00 g (accurate to 0.01 g) was poured into a 10 mL plastic centrifuge tube, along with 5 mL of methanol. The solution was vortexed, then mixed vigorously for 10 min with a shaker and centrifuged at high speed (10,000 r/min, 5 min). Then, 4 mL of supernatant was transferred to another centrifuge tube, and 5 mL of methanol was added to the residue for secondary extraction. After repeating the above steps, 5 mL was taken, and the supernatant was combined and mixed for purification.

Extract Purification

The Oasis MAX solid-phase extraction column was activated with 3 mL of methanol followed by water before injecting the extract to pass through the activated solid-phase extraction column. After passing, it was rinsed with 5 mL of water followed by methanol and drained. The target compound adsorbed on the filler was eluted with 10 mL of 40% formic acid/methanol, and the eluate was purged to near dryness at 70 °C. At last, reconstituted with 1 mL of 80% acetonitrile water, and passed through a 0.22-μm filter to be tested.

Chromatography and Mass Spectrometry Conditions

For chromatography, we used a Waters UPLC BEH HILIC column (2.1 mm × 100 mm, 1.7 μm). For the mobile phase, we used 5-mM ammonium acetate solution and acetonitrile, injecting 10 μL of sample into the column at 30 ℃. From 0 to 1 min, the mobile phase B is 100%, then decreased to 50% at 2 min, and reached to 100% from 2.5 to 12 min.

For mass spectrometry experiments, we used an electrospray ion source (ESI) with a negative ionization mode to detect multiple-reaction monitoring (MRM). The parameters of the source were used with the following settings: electrospray voltage (IS) 4000 V, atomization gas (NEB) pressure 0.5 MPa, air curtain gas pressure (CUR) pressure 0.35 MPa, auxiliary gas (AUX) pressure 0.7 MPa, auxiliary gas temperature (TEM) 550 ℃, collision cell outlet voltage (CXP) 13 V, and focusing voltage (FP) 150 V. Both the monitored ion pair and the quantified ion pair were 97/41, and the deconvolution voltage (DP) and collision energy (CE) were − 5 V and − 25 eV, respectively.

Results and Discussion

Determination of Mass Spectrometry Conditions

MON was optimized by a 10 μL/min needle pump for primary mass spectrometry of MON in negative ion mode. The parent ion m/z 97 was formed; then the secondary ion mass spectrometry was performed on the parent ion to obtain a characteristic fragment ion peak m/z 41 with a higher response value. Finally, m/z 97 > 41 was selected as the quantitative ion pair.

Selection of Chromatographic Conditions

The column commonly used in the laboratory is a C18 column, but we found that the C18 column did not retain MON. Therefore, according to the polar molecular characteristics of MON, we selected the Waters UPLC BEH HILIC column (100 mm × 1.7 mm, 3 μm). In our experiments, we investigated the separation effect with acetonitrile–water, acetonitrile − 0.1% formic acid water, and acetonitrile with 5 mM ammonium acetate as the mobile phase. We found that acetonitrile with 5 mM ammonium acetate made the peak shape sharp, symmetrical and sensitive. In the negative ion mode, the addition of formic acid in most cases inhibits the ionization of the compound, and the addition of ammonium acetate acts as a buffer salt while increasing the ionization efficiency of the compound. The chromatograms with different mobile phases are shown in Fig. 1.

Fig. 1
figure 1

Effect of different mobile phases on peak (a: acetonitrile/water; b: acetonitrile/0.1% formic acid water; c: acetonitrile with 5 mM ammonium acetate)

Full size image

Selection of Pretreatment Conditions

Selection of Extract

Jörg Barthel et al. used different proportions of methanol/water and acetonitrile aqueous solution to extract MON from corn, finding that acetonitrile/water (50/50%) was the best test condition [12]. Because water and vegetable oil are not mutually soluble, and their mutual permeability is poor, water was first excluded from extracting MON. Since the main component of vegetable oil is glycerol of unsaturated fatty acid, it is necessary to remove oil and fat while purifying the sample, and to ensure the extraction rate of MON simultaneously. Therefore, the extraction efficiencies of methanol and acetonitrile were compared here. We found that methanol extraction produced a high yield when the concentration of MON is 1.0 μg/kg. Afterwards, the extraction efficiencies of different proportions of methanol/water and acetonitrile/water (90%, 80%, 70%, 60%, 50%) were compared. We found that the extraction efficiency of 100% methanol was the best (Fig. 2).

Fig. 2
figure 2

Efficiency of different extracts

Full size image

Purified Extract

The vegetable oil sample matrix is complex and is susceptible to matrix effects when analyzed by mass spectrometry. It is essential to determine a proper purification method to reduce matrix effects. In our experiments, we compared the purification effects and extraction efficiency of Oasis MAX, Oasis MCX, Prime HLB, Oasis WAX, and Oasis HLB solid-phase extraction columns by first performing a recovery test using the rinsing and elution conditions recommended by the manufacturer of the solid-phase extraction column. When the concentration of MON added is 1.0 μg/kg, the results showed that Oasis HLB and Prime HLB solid-phase extraction columns did not purify the vegetable oil sample because the eluent still contained oil, meaning that the sample just passed through the column. The Oasis MCX and Oasis WAX solid-phase extraction columns only partially retained the target compound, with a recovery rate of less than 60%. The Oasis MAX solid-phase extraction column, an ion-exchange column, is packed with a mixed anion-exchange reversed-phase sorbent with high selectivity and sensitivity to acidic compounds. In theory, it can completely absorb MON, but the average recovery was 87%, which needs improvement. This recovery rate may be due to the incomplete elution; therefore, further optimization is necessary.

Leaching and Elution Conditions

Because the rinse was water and methanol, to prevent loss of the target compound after rinsing, we measured the solution after rinsing and found that MON was not present. This indicated that MON was adsorbed in the solid-phase extraction column and was not washed off from the column, allowing us to use methanol and water as the rinse.

The target compound was eluted using different ratios of formic acid/methanol solution, and the recovery rates were compared to select the appropriate eluent (Fig. 3). It was sufficient to add 10 mL of 40% formic acid/methanol elution for best results, with a recovery rate of up to 99.4%. The results showed that the Oasis MAX solid-phase extraction column was suitable.

Fig. 3
figure 3

Effect of different elution systems on recovery

Full size image

Method Validation

Matrix Effect

Matrix effect can affect the accuracy of mass spectrometry. To evaluate the matrix effect, we observed the ratio of the matrix slope by matching the standard curve to the solvent standard curve [13]. If their ratio is closed to 1.0, the matrix effect is weaker. When the ratio is between 0.8 and 1.2, the matrix effect is considered negligible. Thus, a standard working solution prepared with a blank matrix solution was compared with a standard working solution prepared from 80% acetonitrile/water to examine the matrix effect of MON in vegetable oil. The experiment (repeated three times) showed that the matrix effect value of this experimental method was between 0.7 and 0.8, indicating the presence of a matrix effect.

Standard Curve and Sensitivity

To further reduce the influence of the matrix effect, a blank sample matrix solution was used to prepare standard solutions of different concentrations. The standard working curve was plotted with a peak area (Y) as the ordinate and the mass concentration (X, μg/L) as the abscissa. We found that the mass concentration and peak area showed good linearity in the range of 0.1–10 μg/L. The regression equation was ‘y = 355,000x + 5770’ with a correlation coefficient (r) of 0.9997. The standard deviation of the slope was 19.794, and the intercept of the calibration curve was 5710. After calculating the limit of detection (LOD) by the signal/noise ratio method S/N = 3 and the limit of quantitation (LOQ) by the method of signal/noise ratio S/N = 10, they were 0.03 and 0.1 μg/L, respectively.

Recovery and Precision

The external standard method was used for quantification, using negative vegetable oil samples for the recovery and precision tests. After adding three different concentrations of MON (0.5, 1, and 2 μg/kg) to vegetable oil samples, the spiked samples were treated according to the pretreatment method. The measurement was repeated six times, with results shown in Table 1.

Table 1 Spiked recovery and precision of MON in vegetable oil samples (n = 6)
Full size table

Real Samples Analysis

Real sample analysis was applied to evaluate a total of 224 vegetable oil samples purchased at a local supermarket including imported vegetable oils. These samples were divided into four types: soybean oil, corn oil, peanut oil, and rapeseed oil. No MON was found in any of these samples. Although MON was not found in the samples this time, there was still a risk of contamination. In the European Union (EU), the maximum limit of fumonisin in corn products for direct human consumption is 1000 µg/kg [14]. We advocated that the maximum limit for MON in vegetable oils is also defined as 1000 µg/kg, because MON and fumonisin are secondary metabolites of fusarium.

Conclusion

In this paper, we established an analytical method to measure fusarium in vegetable oils by solid-phase extraction–hydrophilic interaction chromatography–tandem mass spectrometry. This method, with its simple pretreatment steps, is highly precise, quantitative and accurate. It is suitable for the determination of MON in various vegetable oils. Our method may provide favorable technical support for the relevant law enforcement agencies to regulate and control the grain and oil market.