ABSTRACT
A method for the chemical determination of 16-epi-oestriol in the urine of nonpregnant women with a qualitative sensitivity of less than 0.5 μg/24 h is described. The separation of 16-epi-oestriol and oestriol is accomplished by converting 16-epi-oestriol into its acetonide, a reaction which is stereoselective for cis-glycols and therefore not undergone by oestriol as a trans-glycol. Following partition between chloroform and aqueous alkali, the acetonide of 16-epi-oestriol is completely separated with the organic layer whereas oestriol as a strong phenol remains in the alkaline phase. 16-epi-oestriol is chromatographed on alumina as the acetonide and determined as a Kober chromogen. This procedure can easily be incorporated into the method of Brown et al. (1957 b) thus making possible the simultaneous routine assay of oestradiol-17β, oestrone, oestriol and 16-epi-oestriol from one sample of urine.
The specificity of the method was established by separation of 16-epi-oestriol from nonpregnancy urine as the acetonide, hydrolysis of the acetonide by phosphoric acid, isolation of the free compound by microsublimation and identification by micro melting point, colour reactions and chromatography. The accuracy of the method is given by a mean recovery of 64% for pure crystalline 16-epi-oestriol when added to hydrolysed urine in 5–10 μg amounts. The precision is given by s = 0.24 μg/24 h. For the duplicate determination of 16-epi-oestriol the qualitative sensitivity is 0.44 μg/24 h, the maximum percentage error being ± 100% The quantitative sensitivity (±25% error) is 1.7 μg/24 h.