Determination of 11-deoxycortisol (Reichstein's compound S) in human plasma by clinical isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection.

A first assay based on stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS) has been developed for plasma 11-deoxycortisol (Reichstein's compound S), the leading hormonal marker of 11beta-hydroxylase deficiency. A suitable internal standard being unavailable, we synthesized dideuterated 11-deoxycortisol according to a newly devised synthetic procedure. 17,21-Dihydroxy-pregna-1,4-diene-3,20-dione underwent selective deuteration using Wilkinson's catalyst. Our product [1alpha,2alpha-2H2]11-deoxycortisol was obtained in good yield (35.6%) and high isotopic purity (0.1% 2H0, 99.9% 2H2). Structural confirmation was done by MS and NMR. Our plasma work up consisted of equilibration of plasma with internal standard ([1alpha,2alpha-2H2]11-deoxycortisol), solid phase extraction with Extrelut NT columns, a clean up step using Sephadex LH-20 mini columns and preparation of heptafluorobutyrates as derivatives. Quantification was achieved by selected ion monitoring of m/z 465.40 (analyte) and m/z 467.40 (internal standard). One hundred twenty picograms of 11-deoxycortisol gave a signal to noise ratio of 10. Calibration plot was linear. Spiking experiments showed good accuracy with relative errors <3.0%. Intraassay precision CV was 4.78% and interassay precision CV was 4.56%. We succeeded in integrating our new analyte into our already existing multisteroid ID/GC-MS plasma assay, which now, in its expanded version, is capable of determining all major diagnostic steroids of androgen related disorders in a single profile: 11-deoxycortisol, 17alpha-hydroxyprogesterone, testosterone, 4-androstenedione, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, androstanediol and 5alpha-dihydrotestosterone. The diagnostic potential of our multisteroid ID/GC-MS assay, the small amounts of plasma (0.5 ml) required, the rapid and convenient sample work up, the application of benchtop GC-MS instrumentation, and highest specificity offered by mass spectrometric detection prove our assay suitable for routine clinical use, especially in pediatric endocrinology.

The scarlet letter: Reichstein's Substance S. A comparison of the angiostatic properties of 5 alpha-tetrahydro S and 5 beta-tetrahydro S.

5 beta-Tetrahydro-Reichstein's Substance S (3 alpha, 5 beta-THS) from different sources yielded variable bioassay activity in the chick chorio-allantoic membrane assay system. Physical characterization showed impure products. Synthesis of this compound by two different routes yielded active and inactive 3 alpha, 5 beta-THS. Of the other two epimers, 3 beta, 5 beta-THS (epi-THS) and 3 alpha, 5 alpha-THS (allo-THS), only the latter was active. These results suggest that the impurities present in 3 alpha, 5 beta-THS synthesized by reduction of the alpha, beta-unsaturated ketone of Substance S might be either or both the epi-/allo-epimers (3 beta, 5 beta-THS and 3 alpha, 5 alpha-THS, respectively), with only the latter contributing the positive angiostatic activity to the mixture. Of the two synthetically derived compounds, only the latter was shown to maintain the activity, whereas 3 alpha, 5 beta-THS was not antiangiogenic.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol. Synthesis and immunogenic properties.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol have been synthesized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. No major difference in the steroid specificity of the antisera was observed when either 7 alpha- or 7 beta-epimers were used for immunization. In both cases, highly specific antisera were obtained which may possibly be used to assay human plasma 17-hydroxyprogesterone and 11-deoxycortisol without chromatographic purification.