Derivative ratio analysis: A new method for measurement of steroids and other compounds with specific functional groups using radioassay by gas-liquid chromatography
✍ Scribed by Arthur Karmen; Irmgarde McCaffrey; Bernard Kliman
- Publisher
- Elsevier Science
- Year
- 1963
- Tongue
- English
- Weight
- 495 KB
- Volume
- 6
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A general method has been developed for sensitive, specific assay of chemical compounds with certain functional groups. A known amount of a subst'ance possessing a similar group is added, as an internal standard, to the solution to be assayed. Radioactive derivatives of both the test material and the internal standard are prepared by reacting the mixture with a carbon-14-labeled reagent such as acetic anhydride-C'". An aliquot of the solution is then assayed by gas chromatography using a detector highly sensitive to radioactivity. The ratio of the radioactivity in the test material to that in the internal standard is the same as the molar ratios of the two substances in the test solution.
This procedure was evaluated by assaying for testosterone (4-androstene-17-ol-3-one) using methyl ricinoleate (12-hydroxy-9-octadecenoic acid methyl ester) as imernal standard.
Methods
Testosterone USP, was obtained from Calbiochcm, Inc. Testosterone was acetylated to the 17-monoacetatc derivative with acetic anhydride and pyridine, 0.1 ml of each, at 37"C, for 14 hr.
Methyl ricinoleate and I-methyl-12-acetoxyricinoleate were obtained from the Baker Castor Oil Company (120 Broadway, New York 5, N. Y.).
Solutions of each of the above in benzene were used to determine the retention times on the gas-liquid chromatographic columns used.
Three samples, prepared by the addition of 0.1 mg of methyl ricinoleate to 0.1, 0.5, and 1.0 mg of testosterone, respectively, were acetylated with acetic-l-W anhydride, specific activity 3.7 mc/mmole (New Eng-