Development of a single chiral HPLC method for the separation of brodifacoum enantiomers

AGRO 55

Randal S. Stahl, Randal.S.Stahl@aphis.usda.gov and John J. Johnston. National Wildlife Research Center, USDA/APHIS/WS, 4101 LaPorte Avenue, Fort Collins, CO 80521
Brodifacoum(3-[3-(4'-Bromo[1,1'-biphenyl]-4-yl)1,2,3,4-tetrahydro-1- naphtalenyl]-4-hydroxy-2H-1-benzo-pyran-2-one), a 2nd generation anti coagulant rodenticide, has two chiral centers, and thus four enantiomers. It is common practice to isolate the enantiomer pairs from each chiral center and then optimize their individual separation using a second chromatography method. We evaluated the effect of varying mobile phase composition on the separation of the four enantiomers in a single chromatography method on Chiracel OD-H, Chiralpak AD-H, Chiracel OJ-H and Chiralpak AS-H, 250 x 4.6 mm columns. The mobile phase combinations and solvent volume percent ranges we evaluated were: 1. hexane (60-95%)/isopropyl alcohol (5-40%). 2. hexane (80-95%)/ethanol (5-20%). 3.hexane (60-95%)/methanol:ethanol (1:1) (5-40%). The hexane/isopropyl alcohol mobile phase provided baseline resolution between the enantiomer pairs associated with each chiral center on the Chiralpak AD-H column under the conditions investigated. This approach will be used for the isolation of brodifacoum enantiomers to permit improved risk characterization for wildlife which feed on brodifacoum poisoned target species.