HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine.
A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(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 (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).
Gatti
R
R
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Franchina
M
M
Gioia
M G
MG
Cavrini
V
V
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004855:Equilenin / Q000652:urine*; D004857:Equilin / Q000652:urine*; D005260:Female; D006801:Humans; D017698:Postmenopause; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D004857:Equilin; D004855:Equilenin