High pressure liquid chromatography of corticosteroids :
I. Preliminary report of the determinations of plasma corticosterone by HPLC combined with radioimmunoassay
1977 Volume 53 Issue 6 Pages 765-775
Details
1977 Volume 53 Issue 6 Pages 765-775
The analysis of corticosteroids (CS) was preliminarily investigated using a DU PONT 830 High Pressure Liquid Chromatography (HPLC) equipped with a UV detector and a Permaphase column ETH.
1) Firstly, the best condition for the separation of mixed CS was investigated. In a linear gradient elution, ten authentic steroids, varying greatly in polarity (including native CS such as DOC, Corticosterone and Cortisol) were sharply separated in about 20 minutes. Recovery of the steroid injected into the column was 96-100%.
2) Standard CS added to the chloroform extracts from plasma or urine were also clearly separated in the same condition as above. In the experiment using predonisolone and estrone-propionate in different doses, the synthetic steroids were quantitatively separated, giving a linear calibration curve.
3) Sensitivity of the steroid determination was ng order photochemically in a plot study, but the method, using a UV detector, was not sufficient to apply to small amount of samples extracted with chloroform. To solve this problem, radioimmunoassay was applied to the eluate containing biological steroid, which was roughly separated using the synthetic steroid marker.
As for the determination of plasma corticosterone using this method of HPLC combined with radioimmunoassay, the results were satisfactory for practical use; the mean based values and SD of 10 normal controls were 0.4±0.2 μg/100ml for corticosterone, and they showed 4 to 5-fold increases to 250 μg of synthetic ACTH injection.
