Siekmann L, Siekmann A, Breuer H
Biomed Mass Spectrom. 1980 Nov;7(11-12):511-4. doi: 10.1002/bms.1200071111.
The highly specific and accurate technique of isotope dilution mass spectrometry has been used for the measurement of 17 alpha-ethynyloestradiol-17 beta and norethisterone in serum. Serum samples were obtained from female volunteers who received 2.5 mg lynestrenol and 50 micrograms 17 alpha-ethynyloestradiol-17 beta in two different galenical preparations. The determination of total 17 alpha-ethynyloestradiol-17 beta (conjugated and non-conjugated) was carried out by the following procedure: (1) adsorption of the steroids from 1 ml serum to Amberlite XAD-2; (2) enzyme hydrolysis of the conjugated steroid; (3) addition of 1 ng [6,7-3H2] 17 alpha-ethynyloestradiol-17 beta as internal standard; (4) column chromatography on Sephadex LH 20; (5) derivative formation with heptafluorobutyric anhydride; (6) isotope dilution mass spectrometry at m/z 474 and 478 using a glass capillary gas-liquid chromatography column. For the measurement of norethisterone, which is the major metabolite of lynestrenol, 1 ng [7-3H]norethisterone was added to 0.5 ml serum. The labelled and the non-labelled steroids were extracted and purified by column chromatography on Sephadex LH 20. The norethisterone was reacted to form the 3-enol-17 beta-trimethylsilyl ether of norethisterone and [3H]norethisterone. For isotope dilution mass spectrometry the derivative was injected into the glass capillary column which was coupled to the mass spectrometer. The instrument was adjusted to m/z 442 and 444, corresponding to the molecular ions of the ether derivatives of norethisterone and [7-3H]norethisterone. Accuracy was achieved by the use of highly specific technique of mass spectrometry and the exact control of recovery using the isotope dilution principle. The precision was 4.5% (CV) for the determination of 17 alpha-ethynyloestradiol-17 beta and 2.5% (CV) for norethisterone. The lower limit of detection was at 20 pg ml-1 for both methods.
