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Measurement by isotope dilution mass spectrometry of equiline and oestrone in serum of women taking tablets of equine oestrogens
Author(s) -
Siekmann Lothar,
Siekmann Anita,
Breuer Heinz,
Dehennin Louis
Publication year - 1983
Publication title -
biomedical mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 0306-042X
DOI - 10.1002/bms.1200100311
Subject(s) - chemistry , chromatography , isotope dilution , sephadex , mass spectrometry , ether , dilution , organic chemistry , enzyme , physics , thermodynamics
The technique of isotope dilution mass spectrometry was used for the measurement of equiline and oestrone as well as the sulphate esters of the two phenolic steroids in serum. Serum samples were obtained from an ovariectomized woman who received a tablet of 1.25 mg of various conjugated equine oestrogens. For the measurement of the non‐conjugated oestrogens 20 pg (2,4,16,16‐ 2 H 4 )equiline and 100 pg (6,7‐ 3 H 2 )oestrone were added to serum samples of 0.5–4.0 ml. The steroids were extracted with 15 ml ether and separated from each other by column chromatography on Sephadex LH‐20. This was followed by derivative formation with heptafluorobutyric anhydride. The ester derivatives of the two steroids were injected into a SE‐52 capillary column which was coupled to a mass spectrometer. For the recording of equiline and the corresponding isotopically labelled equiline the instrument was adjusted to m / z 464 and 468, respectively. For the measurement of oestrone and (6,7‐ 3 H 2 )oestrone the m / z values 466 and 470 were monitored continuously. The amounts of equiline and oestrone in the serum samples were calculated from the isotope ratios measured by selected ion monitoring. For the determination of the sulphate esters of the oestrogenic steroids the serum samples (0.5–4.0 ml) from which the non‐conjugated steroids had been removed by ether extraction were treated with 15 ml methanol. The serum proteins were sedimented by centrifugation and the methanolic supernatant was evaporated to dryness. The sulphate esters of the phenolic steroids were then hydrolysed by enzymatic cleavage. After an incubation period of 48 h, 50 pg (2,4,16,16‐ 2 H 4 )equiline and 150 pg (6,7‐ 3 H 2 )oestrone were added to the mixture. The oestrogens were then extracted with cyclohexane and determined as described for the measurement of the nonconjugated phenolic steroids. The precision of the method for the measurement of equiline in the range from 1.5–50 pg was at 7.3% (CV) and for oestrone in the range from 30–150 pg at 3.6% (CV). The accuracy of the procedure was achieved by the use of the isotope dilution principle in combination with the highly specific technique of selected ion monitoring.