*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) [25,26] 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.

Figure 1 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.


**Table 1.** Recovery (Rec.) and Relative Standard Deviation (RSD) values (*n* = 5) of 14 mycotoxins using different extraction techniques.

Method A (Vicam), Method B (solid-phase extraction (SPE)), Method C (QuEChERS (quick, easy, cheap, effective, rugged and safe)) and Method D (Modified QuEChERS).
