Isotope dilutionmass spectrometry

Unlike protein hormones, which are heterogeneous, steroids can be quantified very accurately in biological samples by means of isotope dilution-mass spectrometry. This procedure allows the absolute identification and quantification oftestosterone inblood and other specimens. The procedure is based on the highly specific recognition of the steroid by mass spectrometry coupled to an exact estimation of recovery by addition of labeled testosterone (isotope dilution). According to a well validated method, 14C-labelled testosterone is added to the serum sample and the steroid fraction is extracted with an organic solvent and purified by gel chromatogra-phy on Sephadex LH-20. The testosterone-containing fraction is then chemically reacted with heptafluorobutyric anhydride to produce the 3-enol, 17^-diester of testosterone, a step which improves the sensitivity and the specificity of mass frag-mentography, since it increases the molecular mass of the steroid and minimizes the probability of spuriously detecting small molecular impurities contained in the sample. The derivative is then injected into the mass spectrometer and multiple ion detection is performed by recording m/e 680 and 682. The quantity of testosterone in the sample is then calculated from the ratio of the peak heights or peak areas and from the known amount of 14C-testosterone added (Siekmann 1979). The method is highly precise (1% variation in duplicate determinations) and sensitive (limit of detection: 5 pg) and is the reference method for testosterone determination currently used for measurement of testosterone concentrations in serum pools to be distributed in quality control programs (Thienpont etal. 1996).

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