4.3.2. Mycotoxin Solutions

The following mixed mycotoxin solutions were prepared in acetonitrile, according to concentrations specified in the following:



**Table 2.** Preparation of the calibration solutions.

Aliquots of 0.5 mL of spiking solutions A, B, and C were dispensed in 2 mL amber vials. The spiking solutions were labeled as Vial #1, Vial #2, and Vial #3; mycotoxin concentrations were blind. Approximately 2 mL of mixed standard solution (labeled as Vial #4) and approximately 4 mL of mixed ISTD solution (labeled as Vial #5) were dispensed in 4 mL amber vials; the mycotoxin concentrations were specified.

To prepare the calibration solutions, different volumes of the mixed standard solution and the mixed ISTD solution were added to six autosampler vials as listed in Table 2 to obtain six calibration levels across the calibration range. After evaporation to dryness under a stream of air or nitrogen at approximately 40 ◦C, the dried residue was re-dissolved by adding 400 μL of HPLC injection solvent.

#### 4.3.3. Sample Preparation

The test samples (10 g) were extracted with 50 mL (V3) acetonitrile/water 84/16 (*v*/*v*) for 60 min on an orbital shaker. The extract was filtered through filter paper (Whatman No. 4), and 5 mL (V4) of filtrate (equivalent to 1 g sample) were evaporated to dryness at 40 ◦C under a stream of air. The residue was reconstituted with 100 μL of methanol and then 900 μL water were added (to obtain a methanol: water ratio of 10:90, *v*/*v*). The Oasis® HLB column was activated and conditioned prior to use as follows. The column was attached to a vacuum manifold, conditioned with 2 mL methanol, and equilibrated with 2 mL methanol/water 10/90 (*v*/*v*). The reconstituted sample extract was then passed through the column at a flow rate of about one drop per second. The column was washed with 1 mL methanol/water 20/80 (*v*/*v*) and dried. Afterwards, the toxins were eluted with 1 mL methanol. To prepare the sample test solution, 100 μL of the mixed ISTD solution were added to the SPE eluate. Then the SPE eluate was evaporated to dryness under a stream of air or nitrogen at 40 ◦C. The dried residue was re-dissolved by adding 400 μL (V1) of HPLC injection solvent and filtered through 0.20 μm regenerated cellulose filter.

For the determination of the recoveries, spiking was performed with the mixed stock solutions A, B, and C for wheat, wheat flour, and wheat crackers, respectively, with an evaporation time of approximately one hour.
