Determination of steroid hormones by the use of isotope dilution-mass spectrometry: A definitive method in clinical chemistry

Isotope dilution-mass spectrometry (ID-MS) is a reliable analytical tool for the highly accurate measurement of volatile compounds in biological fluids. In the present investigation the technique of ID-MS has been applied to the specific and accurate determination of oestradiol-17β, oestriol, testosterone, progesterone, aldosterone and cortisol in human serum. The analytical procedure for the measurement of steroid hormones comprises the following steps: (1) addition of a 14C-labelled steroid to the serum sample; (2) extraction of the 14C-labelled and of the non-labelled steroid by the use of an organic solvent; (3) purification of the steroid fraction by column chromatography on Sephadex LH-20 or on Lipidex-5000; (4) formation of a derivative; (5) combined gas chromatography-mass spectrometry. During gas chromatography the mass spectrometer continuously records two m/e values corresponding to the labelled and the non-labelled hormone. Quantitative determination is performed by comparing the peak heights of the hormone to be determined and of the labelled internal standard. The accuracy of the methods is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. Therefore, the analytical procedures described here may be recommended as definitive methods in clinical chemistry.