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To objectively quantify changes in steroid hormones in organisms caused by adverse environmental loads, we developed a simple and sensitive UPLC-MS/MS (ultra-performance liquid chromatography triple quadrupole mass spectrometry) method for the simultaneous determination of 18 steroid hormones on the HPG axis. This analytical method was based on liquid extraction and a multimode electrospray and atmospheric pressure chemical ionization (ESCi) source, which was optimized by mass spectrometry, liquid phase and pretreatment for the quantification of cholesterol (CH), aldosterone (A), cortisone (E), hydrocortisone (F), 21-deoxycortisol (21-DF), corticosterone (B), 11-deoxycortisol (11-DF), androstenedione (A2), estradiol (E2), estrone (E1), 2-methoxyestradiol (2-MeE2), 21-hydroxyprogesterone (21-OHP), 17-α hydroxyprogesterone (17α-OHP), testosterone (T), dehydroepiandrosterone (DHEA), progesterone (P4), dihydrotestosterone (DHT), and pregnenolone (P5). The method exhibits linearity in the analyte-concentration range 0.03–1000 μg mL−1 (r2 > 0.99), the spiked recoveries for the concentration range tested are 76.22–113.66%, and the relevant parameters of precision are 7.52–1.14%. Compared to other methods, this new method not only uses a small amount of serum (only 100 μL), but also permits the analysis of the challenging steroid, cholesterol. Furthermore, the method was successfully applied to the determination of steroids in Mus musculus, Carassius auratus, Rana catesbeiana Shaw, and Rana nigromaculata serum samples from randomly selected individuals. Therefore, this method is efficient and a very useful tool for assessing changes in steroid hormones. [ABSTRACT FROM AUTHOR]

Carbocyclic steroids D-annulated at 16α and 17α positions with a 5-membered ring E are easily accessible via the interrupted Nazarov cyclization. Three steroid series have been structurally studied: chlorine-containing D-annulated pentacyclic steroids 2, their reduced derivatives 3 and their synthetic precursors – benzylidines of tetracyclic steroids 1. The presence of two conformers for pentacyclic steroids 2 and 3 have been found in crystals, where formation is evidently caused by the conformational flexibility of the ring E. Geometries obtained from X-ray diffraction experiments have been used as the initial ones for semi-empirical and ab initio quantum chemical calculations to confirm the reproducibility of the steroids' geometries by these methods and further used for docking studies against the human androgen receptor ligand-binding domain. The docking results have confirmed that the geometries of the studied D-annulated pentacyclic steroids perfectly match the human androgen receptor active site. Geometry analysis of docking results allowed us to explain the influence of subtle structural differences in series 2 and 3 on the ligand binding energy. Compounds with the highest binding energies have been studied in experiments on AR-positive 22Rv1 prostate cancer cells. Selected steroids revealed antiproliferative potency and inhibited AR pathways in 22Rv1 prostate cancer cells, including downregulation in NKX3.1 and PSA expression. A combination of pentacyclic steroid 2d with non-steroidal antiandrogen bicalutamide exhibited significant antiproliferative effects in 22Rv1 cells. The obtained results for this family of pentacyclic steroids provides insights for further structure–activity relationship studies towards novel drugs targeting nuclear receptors in cancer cells. Pentacyclic steroids are of great interest for the development of innovative therapeutic approaches in the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

The 13C NMR shifts of a variety of organic compounds including pinanes and steroids were predicted using combined molecular mechanics (Pcmod 9.1/MMFF94) and ab initio GIAO (B3LYP/DFT,6-31G(d)) calculations. The accuracy of the ab initio calculations was no different when using the 6-31G(d) basis set than more complex basis sets but the calculations were much faster. The MM calculations for steroids took a few seconds and the Gaussian calculations ca. 45 minutes on a desktop computer. For the data set of alkanes, alkenes and alkynes the 13C shifts were obtained from the GIAO isotropic shieldings (Ciso) by (a) empirically scaled shieldings (δc = A−B·Ciso) and (b) separate references for sp3, sp2 and sp carbons (δc = δref−Ciso). The second method gave better agreement and was used henceforth. For the data set of 34 alkanes, alkenes and aromatics with 96 shifts the mean absolute error (MAE) was excellent (0.90) ppm but the alkyne data set (7 compounds with 27 shifts) did not give as good agreement (MAE 1.7 ppm). For the more complex substituted compounds, pinanes and steroids the MAE ＜ 2 ppm but with large errors mainly from the C–X carbons (X = F, Cl, Br, O, =O). The agreement for fluorocyclohexane (axial and equatorial) was, apart from C1 so good that the fluorine substituent effect is completely explained by this model. The calculations for the pinanes gave good agreement with two exceptions, C6 in the cyclobutane ring and the C=C–CO carbon fragment. In both cases the use of different geometries (MM3 and 6-31G(d) res.) gave good results (apart from the CO carbon) suggesting that the errors are due to the molecular geometries in these strained molecules. In contrast for the seven steroids examined the MAE was 1.4 ppm and the assignments of the calculated and experimental shifts were almost identical. Thus this method could be very useful in assigning the 13C spectra and determining the conformations of relatively large molecules. [ABSTRACT FROM AUTHOR]

A solid support-mediated p-TsOH-catalyzed milder transformative protocol was developed to furnish diverse ring-A and/or ring-B oxo-functionalized steroids. To furnish interesting isomers involving the A/B-ring of biomolecules in a one-pot approach, only solid supports (and not solution!) were found to be effective. p-TsOH/SiO2-oxidation of 4β-hydroxycholesterol, the major oxysterol in human circulation, into a mixture of cholest-4-en-3-one, cholest-4-ene-3,6-dione, and 5α-cholestane-3,6-dione was the starting point for the investigations herein. The reaction protocol was optimized in detail, and efforts were carried out toward gaining an understanding of the mechanistic aspects favoring the solid support, and a possible synergetic catalytic system involving p-TsOH and SiO2 was expected to be a key part. Application of the novel methodology to 4β,7α-dihydroxy steroids resulted in the desired diverse ketosteroids through oxidation/oxidative dehydration, which generalized the process as a facile multi-oxo-functionalization steroidal transformation. [ABSTRACT FROM AUTHOR]

The stratum corneum is the outermost layer of the skin; its barrier function is highly dependent on the composition and the structure as well as the organization of lipids in its extracellular matrix. Ceramides, free fatty acids and cholesterol represent the major lipid classes present in this matrix. They play an important role in maintaining the normal hydration levels required for the normal physiological function. Despite the advancement in the understanding of the structure, composition and the function of the stratum corneum (SC), the concern of “dry skin” remains important in dermatology and care research. Most studies focus on the quantification of water in the skin using different techniques including Raman spectroscopy, while the studies that investigate the effect of hydration on the quality of the barrier function of the skin are limited. Raman spectroscopy provides structural, conformational and organizational information that could help elucidate the effect of hydration on the barrier function of the skin. In order to assess the effect of relative humidity on the lipid barrier function; we used Raman spectroscopy to follow-up the evolution of the conformation and the organization of three synthetic ceramides (CER) differing from each other by the nature of their polar heads (sphingosine, phytosphingosine and α hydroxyl sphingosine), CER 2, III and 5 respectively. CER III and 5 showed a more compact and ordered organization with stronger polar interactions at intermediate relative humidity values, while CER 2 showed opposite tendencies to those observed with CER III and 5. [ABSTRACT FROM AUTHOR]

Rotational strengths were calculated, by the TD-DFT (time-dependent density functional theory) method, for a series of 5,7-diene steroids, ergosterol, lumisterol, pirocarciferol, isopirocarciferol, as well as structurally related compounds such as 2,4-cholestadiene and levopimaric acid. The results were discussed in the context of the CH/π hydrogen bond; we suggest that the CH/π hydrogen bond plays an important role in the enhancement of CD amplitude of cisoid dienes. [ABSTRACT FROM AUTHOR]

Within the combined DFG research project “Sulfated Steroids in Reproduction” an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, 4A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL−1. Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL−1. Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy – determined in a two-level spike experiment – showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices. [ABSTRACT FROM AUTHOR]

Several crystal modifications of two steroids (androsterone and beclomethasone dipropionate) have been characterised by solid-state NMR methods, and the chemical shifts between the different forms compared. The gradual loss of water from androsterone hemihydrate has been monitored by XRPD. The crystal structures are discussed in relation to the NMR data. That of the beclomethasone dipropionate ethyl acetate solvate has been determined for the first time. It is found that there are clear channels containing the ethyl acetate molecules, which are apparently not incorporated by hydrogen bonding. Carbon-13 CP MAS spectra with long contact times and MAS proton spectra are used to show the mobility of ethyl acetate molecules in the BDP solvate. The second-order effect of chlorine on the 13C spectrum of beclomethasone dipropionate monohydrate has been explored by varying the applied magnetic field. [ABSTRACT FROM AUTHOR]