Synthesis of multiply deuterated 3- and 21-monosulfates of allo-tetrahydrocorticosteroids as internal standards for mass spectrometry

► We synthesized the 3- and 21-monosulfates of multiply deuterated 5α-tetrahydrocorticosteroids. ► Exchange reaction of the 5α-3- or -11-ketosteroids with NaOD in MeOD followed by NaBD4 was employed. ► 3β-Hydroxy group was epimerized into 3α configuration by Walden inversion. ► Sulfation of C-3 and C-21 hydroxy groups was attained by treatment with SO3-TMA complex. ► The isotopic purity of labeled compound was enough high to use them as a internal standard in LC/MS. The accurate analysis of trace components in complex biological matrices requires the use of reliable internal standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled analogues of the analyte molecules are the most appropriate internal standards. In this paper the synthesis of the 3- and 21-monosulfates of allo-tetrahydrocorticosteroids labeled with four or five deuterium atoms is described. The principal reactions used were (1) hydrogen–deuterium exchange reaction of active methylene groups adjacent to 3- and 11-oxo group of 17,20;20,21-bismethylenedioxy derivatives of 5α-3-ketosteroids and/or 5α-11-ketosteroids with NaOD in CH3OD followed by reduction with NaBD4, (2) epimerization of the 3β-hydroxy group into a 3α configuration, (3) sulfation of hydroxy groups at C-3 or C-21 in the resulting substrates with sulfur trioxide–trimethylamine complex, and (4) removal of 17,20;20,21-bismethylenedioxy groups with hydrogen fluoride in ethanol. Isotopic purity was found to be satisfactory by MS, and NMR properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.