Quantification of Trichothecene Mycotoxins

The DON stock solution was prepared at 2 mg/mL in ACN/H₂O (84 + 16, v/v) and the concentration of dilutions in ACN was established using the extinction coefficient of 6,805 L/mole*cm [44 (link)]. Stock solutions of NX-2, NX-3, 7-hydroxy-isotrichodermol, 7-hydroxy-isotrichodermin, 7-hydroxy-diacetoxyscirpenol, and 7-hydroxy diacetoxyscirpenol were prepared gravimetrically in ACN or ACN/H₂O (84 + 16). Intermediate dilutions were prepared at 50 μg/mL in H₂O/ACN (4 + 1 v/v). For most experiments, the working dilutions of each toxin were prepared daily in a 1 + 9 (v/v) mixture of MeOH and PBS (10% MeOH/PBS). For NX-2, the calibration standards were generally prepared over the range of concentrations from 0.1 to 50 ng/mL. For NX-3, the range was generally 0.5 to 100 ng/mL. To study the effects of MeOH concentration, NX-2 standards were also prepared in MeOH/PBS at MeOH concentrations of 0% (PBS alone), 20% (1 + 4 v/v), and 30% (3 + 7 v/v). Because of the effects of MeOH, for high MeOH concentrations, the upper limit for the calibration standards was increased, to a maximum of 500 ng/mL (in 30% MeOH). Cross-reactivity studies were conducted with trichothecene analogs prepared in 10% MeOH/PBS at concentrations up to 5000 ng/mL.