Measurement by isotope dilution mass spectrometry of 17 α‐ethynyloestradiol‐17 β and norethisterone in serum of women taking oral contraceptives

The highly specific and accurate technique of isotope dilution mass spectrometry has been used for the measurement of 17α‐ethynyloestradiol‐17β and norethisterone in serum. Serum samples were obtained from female volunteers who received 2.5 mg lynestrenol and 50 μg 17α‐ethynyloestradiol‐17β in two different galenical preparations. The determination of total 17α‐ethynyloestradiol‐17β (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‐ 3 H 2 ] 17α‐ethynyloestradiol‐17β 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‐ 3 H]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‐17β‐trimethylsilyl ether of norethisterone and [ 3 H]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‐ 3 H]norethisterone. Accuracy was achieved by the use of the 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 17α‐ethynyloestradiol‐17β and 2.5% (CV) for norethisterone. The lower limit of detection was at 20 pg ml −1 for both methods.