Simultaneous determination of luteolin and apigenin in dog plasma by RP-HPLC

A specific and accurate high-performance liquid chromatographic method has been developed and validated for the simultaneous determination of luteolin and apigenin in the plasma of dog. The sample was treated with 6.0% perchloric acid to precipitate the protein. Luteolin and apigenin were extracted with ethyl acetate. The organic layer separated was dried and reconstituted in the mobile phase. The HPLC separation was performed on C18 column and the UV detector was set at 350 nm. The standard curve for luteolin and apigenin in plasma were linear over the range of 38.5-4350 and 16.5-1860 ng/ml, with the correlation coefficients 0.9996 and 0.9999, respectively. The assay recoveries for luteolin and apigenin ranged from 102.7 to 104.5% and 93.8-101.8%, respectively. The intra- and inter-day precisions (R.S.D.) for luteolin and apigenin were all less than 7.9%. The sample was stable within 24 h at 4 degrees C storage, 30 days at -20 degrees C storage, and undergoing four freeze-thaw-assay cycles. The limits of detection (LOD) of luteolin and apigenin were 1.82 and 1.94 ng/ml, while the limits of quantification (LOQ) were 7.84 and 6.29 ng/ml, respectively. The method developed was applied successfully to study pharmacokinetics of the effective composition (luteolin) of Chrysanthemum morifolium extract in dogs after single dose of oral administration.