Abstract
A liquid chromatographic (LC) method is described for the determination of the plant estrogens daidzein, formononetin, and coumestrol and the estrogenically active metabolite equol in bovine blood plasma and urine. The blood and urine samples are incubated overnight with and without β3-glucuronidase/sulfatase for analysis of both free and conjugated forms of estrogens. Samples are applied to Extrelut ® columns, extracted with ethyl acetate, and evaporated to dryness. Residues from urine samples are dissolved in methanol, diluted with water, acidified with HCl, and purified by injection through a Sep-Pak® C,8 cartridge. This eluate is used for LC analysis. Residues from blood samples are dissolved in benzene-petroleum ether (1 + 1), extracted with ammonium hydroxide, acidified with glacial acetic acid, and extracted with ethyl acetate. The ethyl acetate extract is evaporated, dissolved in 80% methanol, injected onto a LC reversephase column, and separated in a linear gradient system between 40 and 80% methanol in phosphate buffer. Quantitation is performed by means of UV and fluorescence responses. The method was sensitive enough to determine 0.4 μg/mL of daidzein and formononetin and 0.1 and 13 ng/mL of coumestrol and equol, respectively, in blood, and 130,80, and 7 ®g/mL of daidzein, formononetin, and coumestrol, respectively, and 4 Mg/mL of equol in urine. The applicability of the method was checked by the determination of total and free plant estrogens in blood samples from a dairy cow fed a normal diet.
Babesia canis vogeli infection in dogs and ticks in the semiarid region of Pernambuco, Brazil