Development of a liquid chromatography-mass spectrometry method to quantitate deoxynivalenol in Harlan Sprague-Dawley (HSD) rat plasma, amniotic fluid and fetal homogenate

Abstract

Deoxynivalenol (vomitoxin or DON) is a mycotoxin primarily produced by fungi of the Fusarium genus. It may be found in Fusarium-infected wheat, corn, and barley and hence there is potential for human exposure. Although some toxicity data exists for DON, data following perinatal exposure is generally lacking. The objective of this work was to develop a method to quantitate DON in HSD rats to assess fetal and lactational transfer of DON in support of a National Toxicology Program (NTP) toxicology study. Standards were prepared by spiking 100 μL of matrix with 20 μL of DON spiking solution and 20 μL of 13C15-DON (internal standard, IS) in acetonitrile. Samples were extracted by adding 360 μL of acetonitrile followed by vortex mixing and centrifugation. The supernatants were dried, the residue was reconstituted in 150 μL of 20% aqueous acetonitrile and analyzed by liquid chromatography-tandem mass spectrometry. An Acquity UPLC BEH C18 column was used with mobile phases consisting of water and methanol. The electrospray ionization source was operated in negative ion mode. The MRM transitions were 295/265 and 310/279, for DON and 13C15-DON, respectively. The method was successfully qualified over a calibration range ~2 to 1000 ng/mL in male HSD rat plasma. The matrix standard curve was linear (r > 0.99). The lower limit of quantitation was 2.06 ng/mL and the limit of detection (LOD) was 0.354 ng/mL. The accuracy (determined as % relative error, RE) and precision (determined as % relative standard deviation, RSD) for the lowest matrix standard was ≤± 12% and 5.5%, respectively. The method was qualified for GD18 HSD dam rat plasma and fetal homogenate (in water) at ~ 5 and 500 ng/mL. DON was stable in HSD GD18 maternal rat plasma and fetal homogenate when stored at ~-70 oC for at least 29 and 43 days, respectively. The method was applied to samples from a dose range finding study where HSD dams were dosed via gavage from GD 6 to PND 28 with either 0 (control) or 1 mg/kg/day DON. DON was quantified in GD 18 dam plasma (17.7 to 21.1 ng/mL), amniotic fluid (9.65 to 14.8 ng/mL), and fetal homogenate (21.5 to 24.7 ng/g) demonstrating fetal transfer. Although DON was found in PND 4 dam plasma (16.2 to 18.6 ng/mL), the levels in pup plasma were below the LOD suggesting absence of lactational transfer.