Total urinary estriol determined by "on-line" liquid chromatography with ultraviolet detection

We report a fully automated method for the specific assessment of total estriol in urine. Acid-hydrolyzed urine is concentrated on a short reversed-phase column and prepurified with methanolic Tris buffer, the pH of the buffer increasing from 7 to 11. The anionic form of the estriol molecule is eluted with methanolic NaOH. By acidification in a mixing chamber, estriol in its neutral form is focused on the top of a second short reversed-phase column, effectively chromatographed on an analytical column, and quantified by ultraviolet absorbance at 278 nm. Losses of estriol throughout the total procedure are negligible and thus external calibration is feasible for quantification. Analytical recoveries for estriol-supplemented urines ranged from 98.3 to 105%. Replicate analyses of a urine containing 68 mumol of estriol per liter gave a CV of 2.76%. As little estriol as 2 mumol/L can be detected. Results from pregnancy urines correlated well with those of radioimmunoassay. The method is especially suited for clinical emergencies in a routine laboratory.