Ready-Solv IX,“ and counted in a liquid scintillation spectrometer.” Another 
4 ml aliquot of each plasma sample was extracted as before, the steroids 
chromatographically separated, and iodine-stained bands with Rm values corre- 
sponding to authentic testosterone or ®-estradiol cut off the columns. After 
oxidation the radioactivity in these bands, and that in the remaining portion 
of each column was measured. 
RESULTS 
Steroid Hormone Standards 
Chromatographic separation and sensitivity of detection 
Figure 1 illustrates the mobility of testosterone and $-estradiol when 
added to separate columns, and that of testosterone, f-estradiol, and choles-— 
terol on the same column. Hormones were made up at a concentration of 1 ug/ul 
in chloroform:methanol (97:3) and 10 ug of each was normally added to the 
colums. In addition serial dilutions of testosterone and 8-estradiol were 
added to the columns to check sensitivity; it was possible to detect the pres- 
ence of 50 nanograms of these hormones with the iodine stain. 
The Rm values of the steroid hormone bands stained with iodine were suffi- 
ciently distinct to easily separate testosterone (Rm of 27 mm) and 8-estradiol 
(Rm of 34 mm) from each other, and both from cholesterol (Rm of 38 mm). In 
this solvent system, the Rp values of other steroids in the androstane series 
clustered around that of testosterone (R's of androstendione and dehydroiso- 
androsterone were 30 mm), and those in the estrane series clustered around B- 
estradiol (Rm's of progesterone and estrone were 32 and 36 mm). Charring with 
sulfuric acid resulted in a red color for the 8-estradiol band, a blue color 
for the testosterone band, and a rust color for the cholesterol band. 
Scanning of absorption peaks 
The absorption peaks in the sulfuric acid chromogen spectra for testoster- 
one and 8-estradiol are shown in Figures 2 and 3, and were respectively, 285 - 
290 nm, and 355 - 365 nm. These absorption peaks were found to be directly 
proportional to steroid hormone concentration up to 10 ug (fig. 4). 
Figures 5 and 6 depict typical sulfuric acid chromogen spectra of eluates 
of standards of testosterone and B-estradiol, or after extraction of 4 ml of 
plasma from male and female, mature Japanese quail. The absorption maxima of 
the extracts were almost identical to those obtained when authentic steroids 
were added directly to the sulfuric acid and scanned, but peak heights were 
much less in magnitude. 
2 Beckman Instruments, Inc., Silver Spring, Md. 
° Packard Instrument Co., Inc., LaGrange, T1l. 
