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Modified Procedure for Estimating Estrogens in Urine¹

Department of Animal Sciences, Purdue University, Lafayette, Indiana

ABSTRACT

Mellin et al. (4) published detailed procedures for extraction, purification, separation, and quantification of the predominant estrogens in bovine urine. The purpose of this note is to describe a technique for using gas-liquid chromatography (GLC) to combine the separation and quantitative steps outlined earlier (4).

Experimental Procedure

A comparative study was made on the GLC properties of 13 estrogen standards known to occur as urinary metabolites in mammals. These compounds were made up to a concentration of 2 µg/microliter, and 1 µl was injected. The procedure of VandenHeuvel and Horning (7) was used to calculate steroid numbers and T values for purposes of interlaboratory comparison.

The chromatographic analyses were performed on a Model 600-D Aerograph HY-FI, equipped with a flame ionization detector, coupled to a Model 328 Isothermal Temperature Control (Wilkens Instrument & Research, Inc.). A 1-mv Leeds and Northrup recorder was used. Operating conditions were as follows: oven 250±0.2 C; injector 295±5 C; hydrogen and nitrogen flow rates 30 ml/minute each.

¹ Journal Paper no. 3004, Purdue University Agricultural Experiment Station. This work was supported in part by Grant no. 4291 from the Purdue Research Foundation, Lafayette, Indiana.