James A. Demetriou

Analysis of urinary metabolites of estradiol-17β, -4-14C. A rapid paper chromatograph 1c method for obtaining a profile of urinary metabolites ☆ ☆☆

Abstract A method is described for the analysis of numerous urinary radiometabolites of estradiol-17β,-4- 14 C. The method is very useful in any study of the metabolism of estrogens where a rapid survey or profile of radiometabolites is desired. The method consists of: 1) enzymatic hydrolysis, 2) extraction, 3) paper chromatography, 4) radiochromatogram scanning, 5) triangulation of the scans and 6) calculation of the percentage composition of the extract. The validity of the designation and quantitation of estrone, estradiol and estriol has been established by radioisotope dilution and recrystallization to constant specific activity. Metabolites designated exclusively on the basis of paper chromatographic mobilities are: 2-methoxyestrone, 16-ketoestrone, ketols and epiestriols. The method of triangulation for the quantitation of metabolites has been shown to be reproducible and reliable.

An automatic continuous-flow spotting/striping machine

Abstract This paper presents the principle, design and application of an automatic continuous-flow spotting/striping machine. The apparatus was specifically designed to apply multiple organic solvent extracts automatically and simultaneously as spots or stripes to chromatography paper. The machine applied twelve 300-μl volumes of a methanol—dichloromethane (4:1) solution to chromatography paper in 30 min as uniform and compact stripes. The dynamic action of a unique mechanical “heart-shaped” cam was responsible for obtaining uniform and compact stripes. A recovery range of 93–98% for [3H]aldosterone (in methanol—dichloromethane) was obtained after pumping the solution through Acidflex® and polyethylene tubing. The remaining [3H]aldosterone was removed by two 100-μl methanol—dichloromethane washes. Urine aldosterone values showed no significant differences when extracts were applied by the machine or manually. This machine reduces the man-hours required for sample applications three-fold and reduces operator tension built up by long hours of tedious manual applications.