title: Determination of 11-deoxycortisol (Reichstein's compound S) in human plasma by clinical isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection.
abstract: 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.
authors.affiliation: Steroid Laboratory, Children's Hospital, Justus Liebig University of Giessen, Giessen, Germany. stefan.wudy@paediat.med.uni-giessen.de
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Wudy
authors.forename: Michaela
authors.initials: M
authors.lastname: Hartmann
authors.forename: Janos
authors.initials: J
authors.lastname: Homoki
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000175:diagnosis; D000728:Androgens / Q000097:blood; D002138:Calibration; D003350:Cortodoxone / Q000097:blood* / Q000138:chemical synthesis; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D013256:Steroids / Q000097:blood
chemical_list: D000728:Androgens; D013256:Steroids; D003903:Deuterium; D003350:Cortodoxone