HPLC Determination of Diphacinone and Chlorophacinone in Urine with Solid Phase Extraction

Diphacinone (DPCN) and/or chlorophacinone (CPCN) in samples of urine were separated,enriched and purified by solid phase extraction (SPE) on Oasis HLB C18 column.The SPE procedure micro column was rinsed successively with mixtures of HOAc-CH3OH and NH3(aq.)-CH3OH to remove impurities,and eluted with mixture of CH3OH-H2O (90+10) to transfer DPCN and CPCN into the eluate from the column.The eluate was evaporated to dryness by N2-blowing at 40 ℃,and the residue was taken up with 1.0 mL of CH3OH.An aliquot of 10.0 μL was used for HPLC determination.Zorbax Extend C18 column was used as the stationary phase and a mixture of 0.02 mol·L-1 NH4OAc-HOAc buffer solution (pH 5.5) and methanol (30+70) was used as the mobile phase.UV detection at 285 nm was adopted.Linear relationship was found in the range of 0.02-5.0 mg·L-1 for both compounds.Test for recovery was made,giving values of recovery in the range of 96.0%-100.0% (for DPCN) and in the range of 92.0%-96.0% (for CPCN).Values of intraday RSD′s (n=6) found were less than 6.2% and 4.9%,interday RSD′s (n=18) were less than 9.8% and 8.9% for DPCN and CPCN respectively.