C−H Acetoxylation‐Based Chemical Synthesis of 17 β‐Hydroxymethyl‐17 α‐methyl‐18‐norandrost‐13‐ene Steroids

Palladium‐catalyzed C?H acetoxylation has been proposed as a key transformation in the first chemical synthesis of steroids bearing a unique 17β‐hydroxymethyl‐17α‐methyl‐18‐nor‐13‐ene D ‐fragment. This C?H functionalization step was crucial for inverting the configuration at the quaternary stereocenter of a readily available synthetic intermediate. The developed approach was applied to prepare the metandienone metabolite needed as a reference substance in anti‐doping analysis to control the abuse of this androgenic anabolic steroid.     

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC‐CI‐MS/MS: a methandienone case study

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC‐CI‐MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long‐term metabolite (18‐nor‐17β‐hydroxymethyl,17α‐methyl‐androst‐1,4,13‐trien‐3‐one) could be detected up to 26 days by using GC‐CI‐MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long‐term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

Development and validation of a qualitative screening method for the detection of exogenous anabolic steroids in urine by liquid chromatography-tandem mass spectrometry

A screening method for the urinary detection of 34 exogenous anabolic steroids has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography-tandem mass spectrometry. The use of some adducts such as [M+NH₄]⁺, [M+CH₃COO]⁻ and [M+H+MeOH]⁺ was necessary in order to detect some analytes at the required level (lower than 10 ng/ml). Two transitions were selected for each analyte. Different concentration factors have been studied in order to increase the sensitivity. A concentration factor of 50 was selected for the screening method although the high ion suppression observed under these conditions can hamper its application as a quantitative method. The method was validated and limits of detection were obtained by spiking ten different blank urine samples at five different concentration levels. Up to 29 analytes were detected in all spiked urines at the required level. Limits of detection between 1 and 10 ng/ml were obtained for most analytes which fulfil current requirements. The applicability of the method was shown by analysing positive samples.     

25‐Hydroxyvitamin D3 Synthesis by Enzymatic Steroid Side‐Chain Hydroxylation with Water

The hydroxylation of vitamin D₃ (VD₃, cholecalciferol) side chains to give 25‐hydroxyvitamin D₃ (25OHVD₃) is a crucial reaction in the formation of the circulating and biologically active forms of VD₃. It is usually catalyzed by cytochrome P450 monooxygenases that depend on complex electron donor systems. Cell‐free extracts and a purified Mo enzyme from a bacterium anaerobically grown with cholesterol were employed for the regioselective, ferricyanide‐dependent hydroxylation of VD₃ and proVD₃ (7‐dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD₃ strictly depends on a cyclodextrin‐assisted isomerization of VD₃ into preVD₃, the actual enzymatic substrate. This facile and robust method developed for 25OHVD₃ synthesis is a novel example for the concept of substrate‐engineered catalysis and offers an attractive alternative to chemical or O₂ /electron‐donor‐dependent enzymatic procedures.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

Transferred multipolar atom model for 10β,17β‐dihydroxy‐17α‐methylestr‐4‐en‐3‐one dihydrate obtained from the biotransformation of methyloestrenolone

Biotransformation is the structural modification of compounds using enzymes as the catalysts and it plays a key role in the synthesis of pharmaceutically important compounds. 10β,17β‐Dihydroxy‐17α‐methylestr‐4‐en‐3‐one dihydrate, C₁₉H₂₈O₃·2H₂O, was obtained from the fungal biotransformation of methyloestrenolone. The structure was refined using the classical independent atom model (IAM) and a transferred multipolar atom model using the ELMAM2 database. The results from the two refinements have been compared. The ELMAM2 refinement has been found to be superior in terms of the refinement statistics. It has been shown that certain electron‐density‐derived properties can be calculated on the basis of the transferred parameters for crystals which diffract to ordinary resolution.     

Catalytic Transformation of Oxygenated Organic Compounds into Pure Hydrogen

The continual growth in transportation fuels and more strict environmental legislations have led to immense interest in developing green biomass energy. In this work, a proposed catalytic transformation of oxygenated organic compounds (related to bio-oil) into pure hydrogen was desighed, involving the catalytic reforming of oxygenated organic compounds to hydrogen-rich mixture gas followed by the conversion of CO to CO₂ via the water gas reaction and the removal of CO₂. The optimization of the different reforming catalyst, the reaction conditions as well as various sources of oxygenated organic compounds were investigated in detail. The production of pure hydrogen, with the H₂ content up to 99.96% and the conversion of 97.1%, was achieved by the integrated catalytic transformation. The reaction pathways were addressed based on the investigation of decomposition, catalytic reforming, and the water gas reaction.     

Selective hydrogenations of steroids catalyzed by heterogenized Ru complexes

Summary [RuCl₂(PPh₃)₃], [{RuCl₂(TPPMS)₂}₂] and their heterogenized analogs were applied in the selective hydrogenation of 17-keto- and α,β-unsaturated ketosteroids. In basic conditions these complexes selectively hydrogenated the C=O bonds, similarly to the results obtained in the case of α,β-unsaturated aldehydes. A new method was developed for the synthesis of an expensive steroid alcohol, which can be prepared traditionally in a more complicated way.     

Determination of estrogenic steroids in surface water and wastewater by liquid chromatography-electrospray tandem mass spectrometry

An analytical method is presented which permits trace level determination of 17alpha-ethynylestradiol (EE2), 17beta-estradiol (E2), and estrone (E1). Using this method, the estrogenic steroids were analyzed in drinking water, surface water, and wastewater (sewage influents and effluents) at concentrations down to 0.1 ng/L. Sample volumes between 100 and 500 mL are concentrated using automated solid-phase extraction. Analysis is performed by liquid chromatography with detection by tandem mass spectrometry. Applying simple clean-up procedures and internal standard calibration, recovery losses resulting from matrix-dependent ion suppression during electrospray ionization could be compensated for all of the investigated compounds. Recoveries around 100% were obtained for all analytes after correction using the internal standards. Limits of quantification (LOQ) were between 0.1 and 0.4 ng/L for purified sewage, surface, ground, and drinking water and between 1 and 2 ng/L in the case of raw sewage. Water treatment by wastewater treatment plants (WWTPs) or by a surface water treatment plant affected the removal of all estrogenic steroids. Thus, E1, E2, and EE2 were removed in the municipal WWTPs to the extent of 93%, 93%, and 80%, respectively. In the effluents of the WWTP in Ruhleben (Berlin, Germany), E1, E2, and EE2 were detected at the low ng/L level. E2 and EE2 were, however, not present in the Berlin surface water above the LOQ (0.2 ng/L). E1 was the only compound that could be detected in surface water samples. After additional surface water treatment it was still detectable but only at trace-level concentrations with a mean value of 0.16 ng/L.