β Scribed by Rita Gatti; Michele Franchina; Maria Grazia Gioia; Vanni Cavrini
No coin nor oath required. For personal study only.
A high-performance liquid chromatographic (HPLC) method with fluorescence detection (! ex = 280 nm; ! em = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (! ex = 280 nm; ! em = 312 nm, after photoderivatization) and 10 fmol for equilenin (! ex = 280 nm; ! em = 410 nm).
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