2.1. Optimization of Extraction Process
Multi-mycotoxin extractions were prepared by using three different sample preparation techniques in corn. Immunoaffinity (method A) [23
], solid-phase extraction (method B) [24
] and QuEChERS (quick, easy, cheap, effective, rugged and safe) (method C) [25
] methods applied for this study are shown in Table 1
. They were evaluated based on analyte recovery (Rec.) and relative standard deviation (RSD).
The number of mycotoxins giving acceptable recoveries is seven in method A, eight in method B and nine in method C with significant improvement for DAS, FB1 and OTA in respect to the total number of 14 analyzed mycotoxins.
As shown in Table 1
, extraction efficiency using acetonitrile-water in method C was increased for FB1 while there was no change for FB2 and FB3 due to their chemical structure. FB1 with two hydroxyl groups has more solubility than FB2 and FB3 with one hydroxyl group in acetonitrile-water as an extraction solution. Aqueous acetonitrile in method C in comparison with aqueous methanol and acetonitrile:methanol in methods A and B provided better recovery for FB1, DAS and OTA. In method C, cleaning of the sample extract was carried out by the EMR lipid removal in order to minimize ion source contamination. Thus, the QuEChERS method was further modified and used as a reference method. In method D, the extraction efficiency is affected by changing the pH because the addition of 0.3% formic acid in the extraction mixture changes the state of ionization for FB1, FB2, FB3, DAS and OTA as acidic and NIV as high polar compounds. It promotes the extraction of the neutral form of acidic mycotoxins into the organic phase due to their ionization constant. Moreover, this degree of acidity prevents the retention of acidic mycotoxins such as OTA in the cleanup process. Acetonitrile and methanol cannot extract NIV lonely, and the presence of low amounts of acid is necessary during extraction due to the incomplete partitioning. However, nivalenol as the most polar mycotoxin cannot meet the satisfactory level of recovery, 55% is a remarkable change in this report. The reconstitution step at the end of the process plays an important role in improving the signal responses and better peak shapes.
shows extracted ion chromatogram (XIC) and total ion chromatogram (TIC) for all mycotoxins with 50 ng/mL AFB1, AFB2, AFG1 and AFG2; 1000 ng/mL FB1, FB2, FB3, T-2, HT-2, DON and ZON; 2000 ng/mL NIV; 500 ng/mL DAS and OTA in corn matrix blank. XIC is a chromatogram created by taking intensity values at a single, discrete mass value, or a mass range, from a series of mass spectral scans. TIC is a chromatogram created by summing up intensities of all mass spectral peaks belonging to the same scan. The co-elution of some mycotoxin compounds was acceptable because these related compounds illustrate different MRM transitions in LC-MS/MS.
Solvent Mixture and Mobile Phase
Among different solvents, acetonitrile provides better recovery for a wide range of mycotoxins and matrices. However, there are reports of using methanol as a suitable co-extraction solvent in this field [27
]. Different proportions of acetonitrile, methanol and water have been studied to discover an efficient extraction procedure with high recoveries for all analytes. In addition, to obtain the best chromatogram with the lowest noise signal, different mobile phases were used.
To optimize the extraction step, different proportions of acetonitrile and water were applied. Three volume ratios, acetonitrile:water (80:20, v:v), (60:40, v;v) and (50:50, v:v) were employed and acetonitrile:water (50:50, v:v) was confirmed as the most appropriate volume ratio. Various proportions of acidic water (0.1%, 0.2%, 0.3%, 0.5% and 1%) were tested and the extraction with equal proportion of acetonitrile and water containing 0.3% formic acid was suitable for all acidic and neutral mycotoxins.
Different various mobile phases and additives were evaluated, including acetonitrile and methanol (mobile phase) and formic acid, ammonium formate, ammonium fluoride, acetic acid and ammonium acetate (additive). Methanol containing formic acid, ammonium formate, and ammonium fluoride was found to give superior peak shape compared to acetonitrile. The reconstitution mixture was added at the end of the sample preparation step because the compatibility of mobile phases with final extraction solvent leads to improved signal response and prevents peak fronting and unstable retention time compared with no reconstitution. Since the nature of polar compounds makes them prone to dissolve in more polar solutions, reconstitution of samples with polar mobile phases is strongly recommended.
In this research, the combination of acetonitrile and acidic water as an extraction solvent at the beginning of the sample preparation and reconstitution with methanol:water (50:50, v:v) provides higher recoveries and better resolution for all 14 mycotoxins.
Animal feeds contain a mixture of crop ingredients, which makes them complex for mycotoxin analysis. Among the feedstuffs, corn has attracted the most attention in mycotoxin analysis due to its high production, consumption and contamination. This protein rich grain expedites the animal growth to prepare them for market weight quickly at a low cost. Corn is a good source of proteins, carbohydrates, vitamins, unsaturated lipids and minerals. It has a high level of matrix effect that makes it a great candidate for research. Therefore, the proposed extraction method was initially developed and validated in corn and further evaluated using different feed matrices.
As a part of validation study, this method was used in 5 different feed matrices (sheep food, dried distiller grain, dairy food, fish food and goat starter) provided from Association of American Feed Control Officials. AAFCO program materials were prepared from different commercial feedstuffs purchased from US marketplaces in order to monitor the use and performance of methods in analytical laboratories. Participating in these proficiency tests is a valid source of method evaluation because several laboratories take part simultaneously and determine 12 mycotoxins in various animal feeds with different level of contaminations. These samples are delivered to the laboratories as powdered samples, and there is not usually enough information about their ingredients and components. They are mixtures of various crops and agricultural products. The matrices considered in this validation were selected among 2019–2010 AAFCO proficiency tests. Sheep food, dried distiller grain, dairy food, fish food and goat starter represent a wide range of feedstuffs with diverse physicochemical properties. As shown in Table 2
, the applicability of the method was confirmed by satisfactory results for all mycotoxins (z ≤ ±2), although it is worth noting that sheep food, dried distillers’ grain and fish food with lower z-scores were affected by the presence of co-eluting matrix interferences leading to signal suppression. The Chromatogram of the dried distiller grain is presented in Figure 2
. It depicts the separation of DON, AFB2, AFB1, HT-2, FB1, T-2, FB3, OTA, ZON and FB2 at different retention times.
In addition, a FERN comparison exercise was conducted for Aflatoxin B1 in 12 dog foods, which was successfully reported. All these matrices were spiked and analyzed for quality control purpose.
Researches indicate that matrix effect is the most important factor in the extraction process for mycotoxin analysis in feed. To eliminate matrix effects, matrix-matched calibration must be in the same or similar matrices that are being studied. Therefore, it is possible that the matrix-matched calibration cannot completely account for different degrees of incurred mycotoxins, and matrix interferences lead to signal suppression and enhancement. As shown in the accuracy and precision section, the recoveries for the in-house validation were obtained using the same matrices for calibration standard and matrix spike samples while there was no such condition for samples in proficiency tests. Recoveries higher than 70% were obtained for DON, DAS, FB1, FB2, FB3, HT-2, T-2, OTA, ZON, AFG1, AFG2, AFB1 and AFB2 and 55% for NIV with relative standard deviation lesser than 12%.
The presented analytical procedure was used for the analysis of more than 50 routine feed samples. The applicability of this method was confirmed by comparison with our old laboratory methods using water and acetonitrile/0.5% acetic acid for extraction followed by salting out reagent and hexane. Additionally, the method validation was performed with three different levels of spike and five replicates.
Chromatograms of all analyzed mycotoxins in the lowest matrix-matched calibration standard are presented in Figure 3
. Fragmentation reactions were carried out in Multiple Reaction Monitoring mode and two product ions, a quantifier ion and a qualifier ion, were measured for 14 mycotoxins. During the evaluation, it was proved that the sensitivity of the MRM transitions is related to the quality and freshness of the used solvents.