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The new natural products reported in 2018 in peer-reviewed articles in journals with good reputations were reviewed and analyzed. The advances made by Asian research groups in the field of natural products chemistry in 2018 were summarized. Compounds with unique structural features and/or promising bioactivities originating from Asian natural sources were discussed based on their structural classification. [ABSTRACT FROM AUTHOR]

Background: Asphodelus tenuifolius Cav. (Asphodelaceae) has traditional reputability in treatment of diarrhea and constipation but no scientific study has been reported for its gastrointestinal effects. Present study was conducted to evaluate antidiarrheal and laxative activities of the plant. Methods: Aqueous-ethanol crude extract of Asphodelus tenuifolius (At.Cr) was subjected to phytochemical screening and liquid-liquid fractionation. In vivo studies of charcoal meal intestinal transit test, antidiarrheal activity against castor oil induced diarrhea and laxative activity were performed in mice. In vitro experiments were conducted upon rabbit jejunum preparations using standard tissue bath techniques. Results: Phytochemical screening indicated presence of alkaloids, anthraquinones, flavonoids, saponins, steroids, tannins and phenols in At.Cr. In charcoal meal intestinal transit test, At.Cr increased (p < 0.001) intestinal motility at 100 mg/kg dose, but decreased (p < 0.001) it at 500 mg/kg dose, when compared to the control group. At.Cr (300–700 mg/kg) provided protection from castor oil induced diarrhea in mice, which was significant (p < 0.001) at 500 and 700 mg/kg doses, as compared to the saline treated control group. At.Cr (50 and 100 mg/kg) enhanced total and wet feces counts in normal mice, as compared to saline treated control. In jejunum preparations, At.Cr inhibited spontaneous, K+ (80 mM) and K+ (25 mM) mediated contractions, similar to verapamil. Pre-incubation of jejunum preparations with At.Cr resulted in rightward nonparallel shift in Ca+ 2 concentration response curves, similar to verapamil. The spasmolytic activity was concentrated in ethylacetate fraction. Aqueous fraction exhibited spasmogenicity upon spontaneous contractions, which was blocked in presence of verapamil, but remained unaffected by other tested antagonists. Conclusion: The Asphodelus tenuifolius crude extract possesses gut modulatory activity, which may normalize gut functions in diarrhea and constipation. The spasmolytic activity of the extract was found to be mediated through Ca+ 2 channel blocking action. The spasmogenic activity, found partitioned in aqueous fraction, possibly involves Ca+ 2 influx through voltage gated Ca+ 2 channels. The study supports ethnic uses of the plant in diarrhea and constipation. [ABSTRACT FROM AUTHOR]

• New capillary photoionization (CPI) ion source design for coupling of GC to MS. • CPI produces intensive molecular ions of trimethylsilylated steroids. • GC-CPI-MS/MS method validated for the analysis of 18 endogenous steroids in human urine. • CPI applied for the first time for quantitative analysis of biological samples. A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF 2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC–MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL−1 and limits of quantitation (LOQ) were between 0.05 to 2 ng mL−1. In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples. [ABSTRACT FROM AUTHOR]

Ganodermasides E–H (1 – 4), four new ergosterol derivatives and two known ones (5 and 6) were isolated from the fermentation of the endophytic fungus Epicoccum poae DJ-F in the stems of Euphorbia royleana Boiss. Their structures were elucidated by spectroscopic analysis, including extensive 1D NMR, 2D NMR, and HRESIMS techniques. All the isolated compounds were tested for their vitro antibacterial activity. Compounds 1 – 6 showed weak inhibitory effects on Staphylococcus epidermidis , Pseudomonas syringae , and Ralstonia solanacearum with MIC values ranging from 0.4 to 3.6 mM. [Display omitted] • Four new ergosterol derivatives were isolated from Epicoccum poae DJ-F. • Their structures were elucidated by spectroscopic analysis. • Compounds 1 – 6 were evaluated for in vitro antibacterial activity. [ABSTRACT FROM AUTHOR]

Eight previously undescribed lanostane triterpenoids and nine known ones were identified from the fruiting bodies of Ganoderma lingzhi S.H. Wu, Y. Cao & Y.C. Dai. Their structures were determined based on spectroscopic data and quantum chemical calculations. Structurally, ganoderane GL-1, featuring a hydrogenated tetramethyls-phenanthraquinone, represents the first example in lanostane nor-triterpenoid group. Biologically, ganoderanes GL-2 and GL-3, distinguished by the presence of a rare "1,11-epoxy" moiety, exhibited significant inhibition against nitric oxide production induced by lipopolysaccharide in RAW264.7 macrophage cells, while ganoderanes GL-4 and GL-8 exhibited bifunctional activities of anti-proliferation and anti-inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Anabolic steroids are of high biological interest due to their involvement in human development and disease progression. Additionally, they are banned in sport due to their performance-enhancing characteristics. Analytical challenges associated with their measurement stem from structural heterogeneity, poor ionization efficiency, and low natural abundance. Their importance in a variety of clinically relevant assays has prompted the consideration of integrating ion mobility spectrometry (IMS) into existing LC-MS assays, due primarily to its speed and structure-based separation capability. Herein we have optimized a rapid (2 min) targeted LC-IM-MS method for the detection and quantification of 40 anabolic steroids and their metabolites. First, a steroid-specific calibrant mixture was developed to cover the full range of retention time, mobility, and accurate mass. Importantly, this use of this calibrant mixture provided robust and reproducible measurements based on collision cross section (CCS) with interday reproducibility of <0.5%. Furthermore, the combined separation power of LC coupled to IM provided comprehensive differentiation of isomers/isobars within 6 different isobaric groups. Multiplexed IM acquisition also provided improved limits of detection, which were well below 1 ng/mL in almost all compounds measured. This method was also capable of steroid profiling, providing quantitative ratios (e.g., testosterone/epitestosterone, androsterone/etiocholanolone, etc.). Lastly, phase II steroid metabolites were probed in lieu of hydrolysis to demonstrate the ability to separate those analytes and provide information beyond total steroid concentration. This method has tremendous potential for rapid analysis of steroid profiles in human urine spanning a variety of applications from developmental disorders to doping in sport.

Steroids are tetracyclic aliphatic compounds, and most of them contain carbonyl groups. The disordered homeostasis of steroids is closely related to the occurrence and progression of various diseases. Due to high structural similarity, low concentrations in vivo, poor ionization efficiency, and interference from endogenous substances, it is very challenging to comprehensively and unambiguously identify endogenous steroids in biological matrix. Herein, an integrated strategy was developed for the characterization of endogenous steroids in serum based on chemical derivatization, ultra-performance liquid chromatography quadrupole Exactive mass spectrometry (UPLC-Q-Exactive-MS/MS), hydrogen/deuterium (H/D) exchange, and a quantitative structure-retention relationship (QSRR) model. To enhance the mass spectrometry (MS) response of carbonyl steroids, the ketonic carbonyl group was derivatized by Girard T (GT). Firstly, the fragmentation rules of derivatized carbonyl steroid standards by GT were summarized. Then, carbonyl steroids in serum were derivatized by GT and identified based on the fragmentation rules or by comparing retention time and MS/MS spectra with those of standards. H/D exchange MS was utilized to distinguish derivatized steroid isomers for the first time. Finally, a QSRR model was constructed to predict the retention time of the unknown steroid derivatives. With this strategy, 93 carbonyl steroids were identified from human serum, and 30 of them were determined to be dicarbonyl steroids by the charge number of characteristic ions and the number of exchangeable hrdrogen or comparing with standards. The QSRR model built by the machine learning algorithms has an excellent regression correlation, thus the accurate structures of 14 carbonyl steroids were determined, among which three steroids were reported for the first time in human serum. This study provides a new analytical method for the comprehensive and reliable identification of carbonyl steroids in biological matrix., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A new oxygenated lanostane-type triterpenoid, 20 S ,24 S -epoxy-lanosta-7,9(11)-dien-3 β ,15 α ,25 R ,26-tetraol ( 1 ), together with three known compounds ( 2 - 4 ) were isolated from the fruiting bodies of Ganoderma weberianum . Extensive NMR spectrometry and HRESIMS analysis, as well as NMR and ECD calculations elucidated the structure of the new compound. 27-nor-3 β -hydroxylanosta-7,9(11),23 E -trien-25-one ( 2 ) showed superior α -glucosidase inhibitory activity with IC 50 value of 122.1 μM to that of positive control acarbose (304.6 μM).

Iridoid-monoterpenoid indole alkaloid hybrids (IMIAHs) represent a rare class of natural products reported from only several plants of Rubiaceae and Dipsacaceae families, while their structural assignments remain a very challenging work due to complexity and flexibility. In the current study, a new IMIAH (1) was isolated from the roots of Dipsacus asper and its structure with absolute configuration was unambiguously established by a combination of spectroscopic analyses, chemical degradation and ECD calculation. A new oleanane-type triterpenoid saponin (2) and 15 known co-metabolites were also obtained and structurally characterized. Our biological evaluations showed that compound 2 exhibited moderate inhibition against acetylcholine esterase (AChE) with an IC 50 value of 15.8 ± 0.56 μM, and compound 15 displayed potent cytotoxicity selectively against human A549 and H157 lung cancer cells with IC 50 values of 6.94 ± 0.24 and 9.06 ± 0.12 μM, respectively. Unlabelled Image [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Ackee plant (Blighia sapida K. D. Koenig) (Sapindaceae) is used in Sub-Saharan Africa (where it has its origin) and in different parts of the world (The Caribbean, North and South America, Europe). Traditionally it is used to manage numerous ailments like backache, constipation, cancer, fever in young children, gonorrhea, dysentery, psychosis, hernia, stomach ache, malaria, rheumatism, typhoid etc. It is also used as a cosmetic and food source. The fruit aril is the main constituent of the Jamaican National Dish. This review summarizes its phytochemistry, nutritional properties, ethnobotany and pharmacology. The need to gather up to date information on this plant, to facilitate in vivo and in vitro investigations for verification of some of the local claims necessitated this review. Materials and methods A literature search was performed on Ackee using ethno botanical text books and scientific databases such as PubMed, Scopus, EBSCO, Science-direct, Google, Google Scholar and other web sources like records from PROTA, PROSEA, JSTOR and Botanical Dermatology Database. Results and discussion This review highlights the traditional uses of parts of the Ackee plant: the bark, the leaves, capsules, roots and seeds. They are used in the management of diverse disease conditions such as diarrhoea, conjunctivitis, fever, internal hemorrhage, dysentery, cutaneous skin infections, and bacterial infections amongst others. Only nineteen compounds have been reportedly isolated from the parts of B. sapida ; Alkaloids, quinines, polyphenols, and steroids, their glycosides, sesquiterpenes and triterpenes. Some of the plant extracts and its isolated compounds showed anticancer, antimicrobial, antidiarrheal, antioxidant and hypoglycemic activities both in vitro and in vivo. The seed/leaves have also been used as insect repellants and the leaves have been reported to have lethal effects on larvae of various mosquitoes' species. The oil contains a lot of nutrients and may be considered for edible consumption after safety has been confirmed. Hypoglycin A and the less malignant hypoglycin B are found in the unripe aril of Ackee and consumption results in hypoglycemia, vomiting, gluconeogenesis disruption which can result in coma and death. The untapped economic potential of its fruits is glaring in West Africa countries. Conclusion Though B.sapida has been put to enormous traditional use, the pharmacological studies conducted are not sufficient, most studies are either in-vivo or in-vitro. More work is required (well-designed pharmacological tests, randomized clinical trials) to evaluate these medicinal claims. This review provides a basis for future research. The isolation of more compounds,detailed pharmacological investigations, exploration of food use and detoxification techniques are key areas to investigate. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Abstract A new, relatively simple sample processing and detection workflow has been developed for the quantification and confirmation of banned growth-promoting substances in a wide variety of animal-based food products. The method covers all required compounds (belonging to the so-called A1, A3, A4 and B2f groups as termed by the relevant EU legislation) which are currently monitored by the official European community surveillance programs. The sample processing includes a thermal sample denaturation step, intended to prevent undesirable side-reactions during the following enzymatic deconjugation of covalently bound analytes. A pH-adjusted dual liquid/liquid-extraction produces sufficient clean extracts for a wide range of matrices (urine, muscle, liver, serum, full blood). The method has been validated using two hybrid quadrupole high-resolution mass spectrometers (Orbitrap and time-of-flight technology–based instruments). Full-scan data acquisition, interlaced with targeted modes (unit mass isolation of the precursors, followed by collision-induced fragmentation), produces sufficiently sensitive and selective detection of the analytes within all the validated matrices. The proposed method is an alternative to currently used methods that are restricted to a limited set of analytes and matrices. Graphical abstract Image 1 Highlights • A comprehensive steroid multi-residue method is proposed. • Q-Orbitrap and Q-TOF based detection was compared. • HRMS provides reliable quantitative validation data. • Q-HRMS provides sensitive and selective confirmation. [ABSTRACT FROM AUTHOR]

Toad venom (venenum bufonis, also called Chan'su) has been widely used for centuries in China to treat different diseases, especially for cancer. Bufadienolides are mainly responsible for the anti-cancer effects of toad venom. However, systematic chemical composition and cytotoxicity as well as key pharmacophores of these bufadienolides from toad venom have not yet been defined clearly. To enrich the understanding of the diversity of bufadienolides and to find bufadienolides with better activities from toad venom. This study was carried out to isolate chemical constituents, research their anti-tumor effects and mechanisms by MTT assay, flow cytometry and Western blotting, and develop a CoMFA and CoMSIA quantitative structure-activity relationship (QSAR) model for illustrating the vital relationship between the chemical structures and cytotoxicities. Among 47 natural bufadienolides, most of bufadienolides (21 compounds isolated in this study and 26 compounds isolated previously) could significantly inhibit the proliferation of cancer cells, and compounds 1 , 8 , 12 , 18 and 19 showed the most potent inhibitory activity against four types of human tumor cells. Compound 18 induced G 2 /M cell cycle arrest and apoptosis. Moreover, 3D contour maps generated from CoMFA and CoMSIA identified several pharmacophores of bufadienolides responsible for the anti-tumor activities. Our study might provide reliable information for future structure modification and rational drug design of bufadienolides with anticancer activities in medical chemistry. Unlabelled Image [ABSTRACT FROM AUTHOR]

Rationale: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers. Methods: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)‐IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11‐keto‐etiocholanolone and 11β‐hydroxyandrosterone. The obtained 13C delta values were statistically analyzed to evaluate their inter‐ and intra‐individual distribution. Results: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra‐individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway. Conclusions: For the selected ERCs of non‐sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts. [ABSTRACT FROM AUTHOR]

Abstract Roylea cinerea (D.Don) Baillon Roylea cinerea (D.Don) Baillon family Lamiaceae is a shrub of the monotypic genus. Aerial parts of the plant are used traditionally in Indian sub-Himalayas and Nepal for the treatment of jaundice, skin diseases, malaria, diabetes, febrifuge and contusions. Method This article reviews botanical description, phytochemistry, ethnomedicinal uses and pharmacological activities of R. cinerea to evaluate if the scientifically evaluated pharmacological profile of the plant can corroborate ethnomedicinal uses. A survey was conducted to document ethnomedicinal and folklore uses of the plant in five districts of Himachal Pradesh, India. Results Phytochemical studies of R. cinerea reveal the presence of glycosides, diterpenes, flavonoids, tannins, steroids, saponins and phenols. R. cinerea extracts. The compounds showed anticancer, antifungal, hepatoprotective, antiperiodic, antiprotozoal, antidiabetic and antioxidant activities on scientific evaluation. A diterpenoid from the plant, precalyone, exhibited antiproliferative activity against P-388 lymphocytic leukemia cell line. Cinereanoid D, a labdane diterpenoid that inhibits ATP binding of heat shock protein Hsp90, is a potential anticancer lead. Two compounds from aerial parts of the plant, 4-methoxybenzo[ b ]azet-2(1H)-one and 3 β -hydroxy-35-(cyclohexyl-5′-propan-7′-one)-33-ethyl-34-methylbacteriohop-16-ene, showed antidiabetic activity. Thus, the scientific reports confirm the ethnomedicinal use of this plant in diabetes, malaria and liver diseases. Conclusion Roylea cinerea is a traditionally used medicinal plant from Western Himalayas. The pharmacological evaluation confirmed the ethnomedically claimed antidiabetic activity using scientifically accepted protocols and controls, although some of the studies require reconfirmation. The bioactivity-guided fractionation attributes the activity to 4-methoxybenzo[ b ]azet-2(1H)-one and 3 β -hydroxy-35-(cyclohexyl-5′-propan-7′-one)-33-ethyl-34-methylbacteriohop-16-ene. Further, cinereanoid D is a potential lead for targeting Hsp90 and its medicinal chemistry studies can lead to a potent anticancer compound. The plant extract also showed antimalarial and hepatoprotective activities. Some of the studies discussed in this review require reconfirmation, as the protocols lacked proper positive and negative controls. Thus, the review of the scientific reports on Roylea cinerea supports ethnomedicinal use as antidiabetic, antimalarial and hepatoprotective. Further studies to prove scientific basis for use in leucorrhea, skin diseases, inflammation and strengthening of claims for liver tonic are required. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Four new steroidal saponins, 3-O-β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl -(25R)-spirost-5-en-3β,17α-diol (1), 3-O-β-D-glucopyranosyl(1→4)-β-D- fucopyranosyl-(25S)-spirost-5-en-3β,17α-diol (2), 3-O-β-D-glucopyranosyl(1→2) -β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl-(25R)-spirost-5-en-3β,17α-diol (3), 3-O-β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl-(25R/S)-spirost-5-en-3β,12β-diol (4), together with five known steroidal saponins were isolated from the ethanolic extract of the rhizome of Polygonatum sibiricum. Chemical structures of these compounds were elucidated by spectroscopic techniques. Anti-inflammatory activities of these new compounds were evaluated. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A new LC–MS/MS method for the determination of steroids was developed and validated. • Multiple sulfated steroid have been analyzed for the first time in AF. • LC–MS/MS based reference data on steroids in AF of mid-gestation were provided. • Classical steroid and sulfated steroid pathway were confirmed in feto-placental unit. Abstract Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC–MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ± SD, 8.6 ± 3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ± 2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ± 3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ± 10.7 ng/mL), androsterone sulfate (AnS: 9.2 ± 7.4 ng/mL), estrone sulfate (E1S: 3.0 ± 3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ± 4.0 ng/mL) and estriol (E3: 1.2 ± 0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway. [ABSTRACT FROM AUTHOR]

Background: Graft-versus-Host Disease (GvHD) causes significant morbidity and mortality in patients after allogeneic stem cell transplantation. Donor T-cells cause inflammation and tissue damage in GvHD target organs such as liver, gut and skin. Cytokine receptor associated kinases JAK1 and JAK2 are critical for inflammatory cytokine response in GvHD. Ruxolitinib is a small molecule inhibitor of JAK1 and JAK2. Preliminary data indicated substantial clinical activity in patients with steroid-refractory (SR) acute and chronic GvHD.Methods: The RIG-study is an investigator-initiated open-label, multicenter, prospective randomized controlled two-arm phase 2 study, comparing the efficacy of ruxolitinib and best available treatment (BAT) versus BAT in steroid-refractory acute GvHD (SR-aGvHD). Patients with acute skin, intestinal or liver GvHD > grade 1 and failure of previous treatment are eligible. The trial aims to include 160 patients who will be randomized in a 1:1 ratio and stratified by GvHD grade (≤ grade 3 versus grade 4) and number of previous immunosuppressive treatments (≤ 3 versus ≥4). The primary endpoint is the overall response rate at day 28, defined as: Improvement of at least one stage in the severity of acute GvHD in one organ without deterioration in any other organ, or disappearance of any GvHD signs from all organs without requirement for new systemic immunosuppressive treatment. Secondary objectives include time to response, overall survival, event-free survival, non-relapse mortality (NRM), failure-free survival, graft failure rates, quality of life and changes in serum levels of pro-inflammatory cytokines and GvHD-related biomarkers.Discussion: This randomized prospective trial will provide further evidence if the retrospectively collected data demonstrating activity of ruxolitinib for SR-aGvHD can be reproduced. A major advantage of ruxolitinib might be the limited and predictable toxicity profile compared to other immunosuppressive therapies that mainly includes viral reactivation and cytopenias. This trial will establish candidate biomarkers to predict and monitor responses to ruxolitinib. As a next step ruxolitinib might be tested upfront against steroids or in a preemptive manner to prevent GvHD to occur.Trial Registration: NCT02396628 (registration date 17.07.2015); DRKS00007939 (registration date 26.03.2015). [ABSTRACT FROM AUTHOR]

Abstract Saline and freshwater lacustrine oils from the Dongpu Depression (Bohai Bay Basin) were characterized by negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). ESI-FT-ICR MS detected a large range of nitrogen and oxygen compounds in the oils, mostly comprising N 1 , O 1 , O 2 and N 1 O 1 species. The low mature oils are dominated by O 1 species with double bond equivalent of 4 (DBE = 4; mainly alkylphenols), O 2 species with DBE = 5–6 (i.e., hopanoid, secohopanoic and steroid acids) and DBE = 1 (i.e., fatty acids), indicating a genetic relationship between oxygen compounds and their precursors. The nitrogen and oxygen compounds were significantly influenced by maturity, with the lower maturity oils analyzed enriched in O 2 species, particularly fatty acids and 4–5 ring naphthenes with biologically sourced hydrocarbon skeletons: ratios of O 2 /N 1 > 0.7; C 20–30 /C 15–45 -DBE 1 -O 2 > 0.4; and DBE 5–6 /DBE 12–22 -O 2 > 6.0 may be useful indicators of low maturity. With increasing maturity, the condensation degree of the compounds increased and the carbon number decreased. The relative abundances of N 1 and O 1 species with the following specific DBE carbon number ranges were particularly sensitive to maturity: DBE 9–12 /DBE 15–18 -N 1 , C 20–28 /C 29–40 -DBE 12 -N 1 , C 20–30 /C 31–50 -DBE 15 -N 1 , DBE 9–12 /DBE 4–20 -O 1 and C 20–30 /C 31–50 -DBE 8 -O 1. In some instances the FT-ICR MS proxies provided a more reliable indication of high maturity levels than traditional sterane based maturity parameters. However, several of these species were also responsive to other influences, namely C 20–28 /C 29–40 -DBE 12 -N 1 and C 20–30 /C 31–50 -DBE 15 -N 1 ratios reflected an organofacies control and DBE 9–12 /DBE 4–20 -O 1 and C 20–30 /C 31–50 -DBE 8 -O 1 ratios changed with thermochemical sulfate reduction (TSR)– confirmed by δ34S variances of OSCs. In light of the multiple controls on the nitrogen and oxygen compounds, DBE 9–12 /DBE 15–18 -N 1 ratio seems unaffected by organofacies or TSR and was considered the most reliable maturity proxy displaying a similar or better maturity relationship than Ts/(Ts + Tm). Aside from secondary alteration effects, the freshwater samples showed several compositional differences to the saline samples, albeit from a limited data set, including generally higher O 2 and N 1 (particularly low MW) product abundances, lower abundances and quite different distribution of O 1 products. These distinctions suggest ESI FT-ICR MS analysis has potential for distinguishing these sample types. Highlights • Heteroatom compounds in saline lacustrine oils were detected. • Abundant O and N compounds were identified in low mature oils. • Parameters of N and O compounds indicating maturity were proposed. • NSO compounds in several saline lacustrine oils were probably impacted by TSR. [ABSTRACT FROM AUTHOR]

Abstract Phytochemical studies of the seeds of the Cameroonian medicinal plant, Salacia staudtiana , resulted in the isolation and identification of five new cardenolides (1 – 5) as well as a new dihydro-β-agarofuran (9), along with eight known compounds. The structures of all compounds were elucidated by 1D/2D NMR, ESI-HRMS data and comparison with literature data. The relative configurations of the new compounds were defined by X-ray crystallography analysis, NOESY correlations and coupling constants. We evaluated their antibacterial efficacy against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis , and two pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa , compared to the standard antibiotics, streptomycin and gentamicin. Moreover, we assessed the antibacterial activity of the crude extract of the seeds in parallel to evaluate the plausible synergistic effects of the compounds in chemical defense of the seeds during germination and plant reproduction. The isolated compounds showed moderate antibacterial activities against the tested organisms. Compounds 1 and 3 and the crude extract exhibited distinct antibacterial activities against B. subtilis and S. aureus. The isolated compounds showed weak DPPH radical scavenging properties compared to the reference standard (Trolox). Our study lends evidence to the antibacterial chemical defense of S. staudtiana seeds by seed-borne compounds. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Abstract Two new 19-norbufadienolides (1 and 2) and one new 14,15-epoxy bufadienolide (3) alongside 16 known bufadienolides (4 – 19) were isolated from Bufonis Venenum that originated from the skin and parotid venom glands of an Asiatic toad (Bufo bufo gargarizans Cantor). The structures of these bufadienolides were elucidated based on the interpretation of their HRESIMS and NMR data. Compound 1 , which had a unique peroxide, was established through extensive single-crystal X-ray diffraction. The two 19-norbufadienolides exhibited more potent cardiotonic activity in the isolated toad heart model and lower cytotoxicity against U87, U251, and LN-18 cell lines than other bufadienolides, such as bufalin and bufotalin. The results suggested that 19-norbufadienolides might be more suitable for developing cardiotonic agents with low cytotoxicity. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Objective: To study the role of the prenatal environment in regulating reproductive development by measuring the prospective association between umbilical cord concentrations of sex hormone binding globulin (SHBG; principal regulator of sex steroid activity), bioavailable sex steroids, and age at menarche.Design: Prospective population-based cohort.Setting: Not applicable.Patient(s): In 286 female members of the Western Australian Pregnancy (Raine) cohort, concentrations of SHBG and steroids (estrogens: estrone, estradiol, estriol and estetrol [E4]; androgens: total testosterone, Δ4-androstenedione, androstenedione and dehydroepiandrosterone) were measured by liquid chromatography-tandem mass spectrometry from archived umbilical cord blood samples collected at birth. Bioavailable concentrations of testosterone and estradiol were calculated along with total composite measures of androgen and estrogen bioactivity. SHBG was measured by ELISA.Intervention(s): None.Main Outcome Measure(s): Age of menarche was calculated from date of menarche, collected prospectively by questionnaire sent home with participants at the year 10 follow-up.Result(s): Higher maternal education, higher body mass index, and the presence of antepartum hemorrhage were all significantly associated with earlier age at menarche. The bioavailable sex steroid measures accounted for 8.3% of the variance in age at menarche. Further, both SHBG and E4 concentrations accounted for a significant proportion of unique variance in age at menarche.Conclusion(s): Lower SHBG and higher E4 concentrations in umbilical cord blood were associated with earlier age at menarche. These results suggest that the prenatal sex steroid environment contributes toward pubertal development and age at menarche. [ABSTRACT FROM AUTHOR]

Steroids are key players in a high variety of physiological processes and are typically analyzed for the diagnosis of hormonal disorders. Due to their chemical and structural similarity many of these metabolites cannot be separated by conventional techniques such as liquid chromatography. Herein, we present an analysis strategy based on two dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOF MS) which demonstrates superior separation power and enables comprehensive screening of steroids. We show absolute quantitation of 40 steroids in human urine over three orders of magnitude with limits of detection ≤50 nM and the tentative identification of additional 30 steroids based on accurate mass, isotopic pattern analysis and spectral similarity matching to known steroids. The method displays excellent inter- and intra-day stability, repeatability and recovery and was validated for clinical routine analysis. Additionally, we demonstrate the potential of the approach for untargeted analysis of urinary steroids in mouse and rat. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the efficiency and clinical safety of postnatal administration of a GnRH agonist on canine puberty postponement. Sexual steroids and histological gonadal changes were also described. Twenty-four littermate puppies were randomly assigned to: Deslorelin acetate 18.8 mg sc (DESLO; n = 12) or Placebo: sc (PLACE; n = 12) postnatally. The dogs were clinically and endocrinologically followed up until puberty when they were gonadectomized and their gonads histomorphometrically studied. Deslorelin postponed the age of puberty (72.7 ± 4.8 vs. 35.8 ± 1.9 weeks; P < 0.01) in these dogs. At the time of this submission, 3 DESLO dogs (108 weeks old) remain non-pubertal. All dogs concluded growing at a similar age (29.75 ± 2.44 vs. 29.25 ± 0.90 weeks; P > 0.1) independently of their group and pubertal status. None of the females had side effects while the 2 non pubertal DESLO males presented bilateral cryptorchydism. All the bitches ovulated at puberty (P > 0.1) and the 2 DESLO that were mated became pregnant. Deslorelin postponed basal serum sexual steroids up to puberty in both genders (P < 0.01). The histomorphometrical study of the testes revealed that the tubular diameter (P < 0.05), germinal epithelium height and composition (P < 0.01) were decreased in DESLO group. Ovarian structures did not differ between treatments (P > 0.05). It was concluded that postnatal deslorelin decreased sexual steroids reversibly postponing puberty in both genders without side effects in bitches and causing 2/6 of cryptorchydism and impairment of testicular histomorphometry in male dogs. [ABSTRACT FROM AUTHOR]

Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors’ laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS 2 mode was developed and validated to improve the coverage to 65 AAS and/or their esters in a single injection. In addition, a GC/MS/MS method in selected reaction monitoring (SRM) mode was developed to screen for another seven AAS and/or their esters not adequately covered by the UHPLC/HRMS method using the same sample extract after derivatisation with pentafluoropropionic anhydride. The UHPLC/HRMS and GC/MS/MS methods in combination allowed the detection of 72 AAS and/or their esters with estimated limits of detection down to sub to low ppb levels with good interday precision. Method applicability was demonstrated by the detection of boldione and 4-androstenedione in two out-of-competition hair samples and testosterone propionate in a referee hair sample. [ABSTRACT FROM AUTHOR]

The yield and fatty oil components of the seed kernels of Annona squamosa L. (Family: Annonaceae) were determined by solvent extraction method and gas chromatography-mass spectrometry (GC-MS). Seeds were extracted with ethanol and further fractionated with n-hexane. The free radical-scavenging activities of both ethanolic and n-hexane fraction against 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were determined by UV spectrophotometer at 517 nm. Phytochemical screening revealed the presence of numerous bioactive compounds including steroids, flavonoids, terpenoids, fatty acids, and different types of ester compounds. The prevailing compounds found in ethanolic fraction were n-hexadecanoic acid (10.08%), heptadecene-(8)-carbonic acid-(1) (29.68%), octadecanoic acid (3.61%), 9-octadecenoic acid (Z)-2,3-dihydroxypropyl ester (5.14%), ergost-5-en-3-ol (3.68%), stigmasta-5,22-dien-3-ol (5.93%), and y-sitosterol (8.25%). Compounds found in n-hexane fraction were mainly n-hexadecanoic acid (14.42%), 9,12-octadecadienoic acid (2.87%), cis-vaccenic acid (10.39%), 9-octadecenoic acid (7.03%), hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (4%), 9-octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester (13.33%), ergost-5-en-3-ol (4.04%), stigmasta-5,22-dien-3-ol, (3.beta.,22e) (6.07%), and y-sitosterol (10.87%). The crude fatty oil was converted into methyl esters and analyzed by GC-MS. Eleven compounds constituting 99.9% of the oil were identified. The presence of saturated and unsaturated fatty acids in ethanolic and n-hexane fraction of A. squamosa seed extract justify the use of this plant to treat many ailments in folk and herbal medicine. Both the fractions have shown significant antioxidant activity. The presence of phenolic compounds and unsaturated fatty acids are reported as possible contributors for antioxidant activity of seed extract. [ABSTRACT FROM AUTHOR]

Two new lanostane triterpenoids (1 and 2), two new ergostane-type steroids (3 and 4) together with two known lanostane triterpenoids (5 and 6) and one known steroid (7) were isolated from the cultured mycelia of Ganoderma capense (CGMCC 5.71). Their structures were determined on the basis of extensive spectroscopic (HRESIMS, 1D NMR, 2D NMR) data analyses. Compound 1 exhibited moderate cytotoxic activity against the human cancer cell line NCI-H1650 with an IC50 value of 22.3 μM, and 7 displayed cytotoxic activity against the human cancer cell line HCT116 with an IC50 value of 17.4 μM. In addition, compounds 2, 3, 5, and 6 displayed weak anti-HIV activity with IC50 values of 23.5, 46.7, 21.6, and 30.1 μM, respectively. [ABSTRACT FROM AUTHOR]

This brief review questions the belief that just because it is possible to measure vertebrate steroids (such as estradiol-17β, testosterone and progesterone) in the tissues of invertebrates, this necessarily means that they are endogenously derived or are hormones. There is a surprisingly large number of studies, mainly on mollusks, showing that they can readily absorb vertebrate steroids from the environment. They are also able to conjugate these steroids to fatty acids with great efficiency, and subsequently retain them for very long periods (with half-lives measured in weeks rather than days). This, plus the fact that key enzymes that are required for the biosynthesis of vertebrate steroids (e.g. aromatase) do not appear to be present in invertebrates, calls into doubt the claims in many studies on invertebrates that steroid concentrations are functionally linked to reproductive cycles or that invertebrates can be used as biomarker for vertebrate-type endocrine disrupters. [ABSTRACT FROM AUTHOR]

The steroid 20-hydroxyecdysone (20E) is a major component of phytoecdysteroid in plants and may play a defensive role against insect pests in higher plants. In spinach, the biosynthesis and accumulation of 20E have been investigated during the vegetative stage; however, these processes have not been clearly studied during the reproductive stage, particularly in male and female individuals. In this study, we analyzed the level and distribution of 20E in individual male and female spinach plants during the reproductive stage via high performance liquid chromatography (HPLC). We found that 20E biosynthesis and accumulation were markedly different between male and female spinach during the late flowering stage. Compared with the male plant, biosynthesis of 20E in the leaves was more active and its accumulation in the floral parts was higher in female plants during the late flowering stage. These results indicate that the female reproductive organs at least in PE-positive plants could be effectively protected against harmful insects via active biosynthesis and accumulation of PE during the late flowering stage to protect floral parts from harmful insects for seed formation and store the available 20E in seeds for the next generation. [ABSTRACT FROM AUTHOR]

In this study we describe an approach to enhance the sensitivity of an online comprehensive two-dimensional liquid chromatography (LC × LC) high-resolution mass spectrometry method for the separation and detection of trace levels of anabolic-steroid residues in complex urine matrices. Compared to one-dimensional liquid chromatography (1D-LC), LC × LC methods offer higher separation power, thanks to the combined effect of two different selectivities and a higher peak capacity. However, when using state-of-the-art LC × LC instrumentation, the price paid for the increase in separation power is a decrease in sensitivity and detectability of trace-level analytes. This can be ascribed to the sample dilution that takes place during each of the two chromatographic steps. The way in which fractions are collected and transferred from the first to the second column is also of paramount importance, especially the volume and the solvent composition of the fractions injected in the second column. To overcome the detection limitation, we present an active-modulation strategy, based on concentrating the fractions of the first-dimension effluent using a modulation interface that employs trap columns. We obtained a signal enhancement for anabolic-steroid compounds in a bovine-urine sample by a factor of 2.4–7.6 and an increase in the signal-to-noise ratio up to a factor of 7 in comparison with a standard loop-based modulation interface. In addition, thanks to the increased sensitivity of our method, a substantially larger number of peaks were detected (76 vs. 36). Moreover, we could reduce the solvent consumption by a factor of three (160 mL vs. 500 mL per run). [ABSTRACT FROM AUTHOR]

Clinacanthus nutans (Burm.f.) Lindau (Acanthaceae), commonly known as Sabah snake grass, is a vegetable and a well-known herb that is considered an alternative medicine for insect bites, skin rashes, herpes infection, inflammation, and cancer and for health benefits. Current review aims to provide a well-tabulated repository of the phytochemical screening, identification and quantification, and the pharmacological information of C. nutans according to the experimental design and the plant preparation methods which make it outstanding compared to existing reviews. This review has documented valuable data obtained from all accessible library databases and electronic searches. For the first time we analyzed the presence of flavonoids, triterpenoids, steroids, phytosterols, and glycosides in C. nutans based on the results from phytochemical screening which are then further confirmed by conventional phytochemical isolation methods and advanced spectroscopic techniques. Phytochemical quantification further illustrated that C. nutans is a good source of phenolics and flavonoids. Pharmacological studies on C. nutans revealed that its polar extract could be a promising anti-inflammation, antiviral, anticancer, immune and neuromodulating, and plasmid DNA protective agent; that its semipolar extract could be a promising antiviral, anticancer, and wound healing agent; and that its nonpolar extract could be an excellent anticancer agent. [ABSTRACT FROM AUTHOR]

One new ergostane steroid, ergosta-4,6,8,22E-tetraen-11β-ol (1), together with five known analogues (2-6), was isolated from the fruiting bodies of Coprinus setulosus for the first time. Their structures were elucidated by analysis of 1D and 2D spectroscopic data as well as MS. The inhibitory activity of these isolates against acetylcholinesterase (AChE) showed that compound 1 exhibited weak inhibitory activity. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study was to investigate the sterol profiling and predict the pharmacological potential of marine gastropod Telescopium telescopium (T. telescopium), collected from mangrove ecosystem in the South west coast of India. Methods: Sterol fractions were separated from the crude lipids using 15% ethyl acetate. Ethyl acetate fractions were dried under ultrahigh purity N2 and analyzed using GC-MS. The biological activity was predicted using the software CLC-Pred; In silico predictions of cytotoxicity for tumor and non-tumor cell lines and PASS. Results: This study proved the existence of four sterols, of which cholesterol was abundant. It was found that most of the steroids profiled from T. telescopium displayed activity against reproductive system as well as skin related diseases. Conclusion: The predicted anti infertility and skin related activity of the steroids identified from the marine gastropod T. telescopium is useful to attract industrial interest towards this species which will be helpful in rising new combinations with added therapeutic and nutritional worth. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to investigate the gas chromatographic properties and mass spectrometric fragmentations of anabolic androgenic steroids (AASs) after trimethylsilylated derivatization. Materials & methods: A total of 113 AASs were analyzed through gas chromatography-mass spectrometry in the full-scan mode. Results: New fragmentation pathways yielding m/z 129, 143 and 169 ions were analyzed. Based on the characteristics of the A-ring, seven classes of drugs were identified and analyzed. Conclusion: The fragmentation pathway of a new classification of 4-en-3-hydroxyl was reported for the first time. The relationship between the chemical structures of AASs and their retention time, along with their molecular ion peak abundance, was also reported herein for the first time.

In this study, we assessed 80 autopsy samples to investigate the relationships between cause of death and the concentrations of multiple steroids in serum and cerebrospinal fluid (CSF). First, we developed and validated analytical methods to quantify seven steroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycortiocosterone, progesterone, and testosterone) by using liquid chromatography coupled with electrospray ionization-tandem mass spectrometry. Next, we statistically evaluated the levels of each steroid for six causes of death: hypothermia, traumatic injury, fire fatality, asphyxia, intoxication, and internal disease. We observed that cortisol concentrations in serum and CSF obtained from cadavers who died from hypothermia were significantly higher than those in samples obtained from cadavers who died from the remaining causes of death (P < 0.05). Similarly, corticosterone concentrations obtained from cadavers who died from hypothermia were significantly higher than those in samples from several other causes of death. However, concentrations of the remaining steroids analyzed did not differ significantly among the causes of death. We further elucidated the correlations between steroid concentrations in serum and CSF. Except for 11-deoxycorticosterone and progesterone, steroid concentrations were significantly positively correlated in serum and CSF. Although data on cadaveric steroid concentrations are limited-especially in CSF-values obtained were in the approximate range of the living human data reported to date., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Hormones in biological media reveal endocrine activity related to development, reproduction, disease and stress on different timescales 1 . Serum provides immediate circulating concentrations 2 , whereas various tissues record steroid hormones accumulated over time 3,4 . Hormones have been studied in keratin, bones and teeth in modern 5-8 and ancient contexts 9-12 ; however, the biological significance of such records is subject to ongoing debate 10,13-16 , and the utility of tooth-associated hormones has not previously been demonstrated. Here we use liquid chromatography with tandem mass spectrometry paired with fine-scale serial sampling to measure steroid hormone concentrations in modern and fossil tusk dentin. An adult male African elephant (Loxodonta africana) tusk shows periodic increases in testosterone that reveal episodes of musth 17-19 , an annually recurring period of behavioural and physiological changes that enhance mating success 20-23 . Parallel assessments of a male woolly mammoth (Mammuthus primigenius) tusk show that mammoths also experienced musth. These results set the stage for wide-ranging studies using steroids preserved in dentin to investigate development, reproduction and stress in modern and extinct mammals. Because dentin grows by apposition, resists degradation, and often contains growth lines, teeth have advantages over other tissues that are used as records of endocrine data. Given the low mass of dentin powder required for analytical precision, we anticipate dentin-hormone studies to extend to smaller animals. Thus, in addition to broad applications in zoology and palaeontology, tooth hormone records could support medical, forensic, veterinary and archaeological studies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Doping in elite or amateur athletes with testosterone, androgens and anabolic steroids (A/AS) has as a main objective to develop muscle strength and mass to improve sports performance. Massive doping is a worldwide public health issue insufficiently known by physicians in general and by endocrinologists in particular. Yet, its prevalence, probably underestimated, would be between 1 and 5% at the international level. Numerous deleterious effects associated with A/AS abuse have been identified: inhibition of the gonadotropic axis responsible for hypogonadotropic hypogonadism and infertility in men, and masculinization (defeminization), hirsutism and anovulation in women. Metabolic (very low HDL cholesterol), hematological (polycythemia), psychiatric, cardiovascular and hepatic complications have also been documented. As a result, anti-doping agencies have developed increasingly effective techniques for detecting A/AS, both to identify and punish cheating athletes and to protect the health of the greatest number of them. These techniques use a combination of liquid and gas chromatographic methods coupled with mass spectrometry, known respectively by the acronyms LC-MS and GC-MS. These detection tools have a remarkable sensitivity and specificity to detect natural steroids and synthetic A/AS of known structure. Furthermore, by distinguishing isotopes, it is also possible to distinguish natural endogenous hormones, testosterone and androgenic precursors from those administered for doping purposes. For elite athletes, a "biological passport" has also been introduced. It consists of monitoring the evolution of steroids and their metabolites, as well as other biological parameters in the blood and urine over time after having established a basal state athlete signature, established, a priori, without doping. Better training of health professionals, general practitioners and specialists should be a priority for academic institutions and medical societies. It would provide them with better knowledge of the populations at risk and the clinical and biological semiology of male and female doping, including withdrawal syndrome associated with anxiety and depression following cessation of chronic A/AS use. The ultimate goal is to provide these physicians with the keys to treating these patients while combining medical rigor and empathy. These points will be addressed in this short manuscript., (Copyright © 2023. Published by Elsevier Masson SAS.)

Chemical signals deposited in feces play an important role in intraspecific and interspecific communication of many mammals. We collected fresh feces of adult wolves from wild breeding groups. All samples visually identified as belonging to wolves were subsequently identified to species level by sequencing a small fragment of mtDNA and sexed typing DBX6 and DBY7 sex markers. Using gas chromatography-mass spectrometry (GC-MS), we identified 56 lipophilic compounds in the feces, mainly heterocyclic aromatic organic compounds, such as indole or phenol, but also steroids, such as cholesterol, carboxylic acids and their esters between n-C 4 and n-C 18 , aldehydes, alcohols and significant quantities of squalene and α-tocopherol, which would increase the chemical stability of feces on humid substrates. There was variability in the number and proportions of compounds between sexes, which could be indicative of their function as chemical signals. We also found variability in different reproductive states, especially in odorous compounds, steroids and α-tocopherol. Feces with a presumed marking function had higher proportions of α-tocopherol and steroids than feces with non-marking function. These compounds could be involved in intragroup and intergroup communication of wolves and their levels in feces could be directly related with the wolf's sex and physiological and reproductive status., (© 2023. The Author(s).)

Context: Determination of steroid levels in the amniotic fluid gives some insight on fetal adrenal and gonadal functions., Objective: Our objectives were to establish reference ranges of 12 steroid levels throughout pregnancy and to compare them with steroid levels from pregnancies with fetuses presenting with 21-hydroxylase deficiency (21OHD)., Methods: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was applied to 145 "control" amniotic fluid samples from gynecology activity (12 + 6 to 32 + 4 gestational weeks, GW). The following steroids were analyzed according to gestational age and compared to 23 amniotic fluid samples from fetuses with classic 21OHD confirmed by molecular studies: delta-4-androstenedione (D4), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (11OH), 21-deoxycortisol (21OH), corticosterone, deoxycorticosterone (DOC), testosterone, pregnenolone, 17-hydroxypregnenolone (17Pregn), cortisol, and cortisone. Chromosomal sex was determined by karyotype and gestational age by biometric measurements., Results: Analysis of control samples showed a statistically significant difference for D4 and testosterone levels according to fetal sex. Cortisol, corticosterone, and DOC had lower concentrations before 20 GW than after 20 GW, whereas 17Pregn and pregnenolone had higher concentrations before 20 GW. This allowed us to establish age- and sex-dependent reference values. We observed higher 21OH, 17Pregn, D4, and testosterone levels in females with 21OHD than female controls. The ratios 17OHP/17Pregn, D4/DHEA, and 11OH/17OHP appeared discriminant for the diagnosis of 21OHD., Conclusion: Our study provides information on fetal steroidogenesis and suggests reference values for 12 steroids during pregnancy. This allows a prenatal diagnosis of 21OHD within 24 hours and might be useful in the diagnosis of other variations of sex development., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An analytical method for multiclass determination of steroid hormones in compost has been developed to fill the lack of methods for steroid residuals monitoring in this waste-derived product, increasingly produced and recycled in the circular-economy approach. The procedure simply entails an ultrasound-assisted extraction (UAE) on 300 mg compost by 3 × 2.5 mL methanol × 5 min sonication steps followed by a quick clean-up by solid-phase extraction (SPE) on the silica-based Supelclean™ LC-NH 2 that avoids use of organic solvents. The clean extract is analysed by HPLC-MS/MS achieving firm identification and quantitation of the 16 steroids, i.e., glucocorticoids, progestins, androgens, oestrogens. The analytical figures of merits were assessed, viz. selectivity, sensitivity, linearity, matrix effect, trueness, precision, carry-over and robustness, in line with updated guidelines. Recovery was investigated in the concentration range 15-800 ng g -1 , and at the quality control levels (15, 50, 200 and 400 ng g -1 ) was in the range 60-120%, with inter-day precision RSDs < 20% (n = 3). The experimental quantification limit was 15 ng g -1 for all the hormones. The method was applied to analysis of different compost samples proving to be functional to environmental monitoring., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The present review describes the distribution and biological activities of natural mono-, di-, and triaromatic steroids. It is shown that the producers of aromatic steroids are microorganisms, fungi, and marine invertebrates, and also they were found in plants, animals, marine sediments, and karst deposits. Eighty biologically active aromatic steroids likely have an anti-tumor, anti-inflammatory, and neuroprotection activity with a confidence of 78 to 92%. The structures and predicted biological activities of aromatic steroids are available. This review emphasizes the role of aromatic steroids as an important source and potential leads for drug discovery and they are of great interest to chemists, physicians, biologists, pharmacologists, and the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Background The inconsistency in results of cortisol alterations after childhood maltreatment (CM) might arise due to the fact that no study so far considered the effects of environmental factors such as maltreatment load and genetic factors such as the influence of FKBP5 genotype on stress hormone regulation. This study analyzed the interaction between the single nucleotide polymorphism rs1360780 within the FKBP5 gene and the severity of maternal CM experiences (maltreatment load) on hair steroid levels of mother-infant-dyads. Methods Hair samples of N  = 474 mothers and N  = 331 newborns were collected < 1 week after parturition enabling a retrospective assessment of cortisol, cortisone, and dehydroepiandrosterone (DHEA) using mass spectrometry. The sum score of the Childhood Trauma Questionnaire operationalized the maternal maltreatment load. DNA from whole blood or buccal cells was used for FKBP5 genotyping. Results The higher the maltreatment load, the higher maternal hair cortisol and cortisone levels in T allele carriers of FKBP5 rs1360780 were observed. Hair cortisol and DHEA levels of newborns with the T allele were reduced with an increasing maternal maltreatment load, while there was an increase of hair cortisol and DHEA in newborns homozygous for the C allele. Conclusions This study is the very first uncovering a gene ( FKBP5 ) × environment (maltreatment load) interaction on hair steroids in mothers and their offspring, indicating an intergenerational transmission of hypothalamic-pituitary-adrenal axis alterations. These results may help to explain the inconsistency in previous findings on steroid hormone alterations after chronic and traumatic stress and should be considered in future studies. [ABSTRACT FROM AUTHOR]

Obesity is increasing worldwide, and since obesity is associated with dyslipidemia, the consumption of cholesterol-lowering pharmaceuticals has increased. The aim of this study was therefore to study potential endocrine disrupting effects of one of the world’s most frequently prescribed drugs, the cholesterol-lowering drug, atorvastatin (ATO) in vitro using the H295R steroidogenesis assay and in vivo using male Sprague-Dawley rats. We analyzed all major steroids in the mammalian steroidogenesis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vitro, ATO significantly decreased all steroids in the H295R steroidogenesis at concentrations close to human plasma Cmax values, with an IC50 value for testosterone of 0.093 ± 0.033 µM. Additionally, we determined steroid hormone levels in testis, adrenals, brain and plasma from rats after 14 days of exposure to three therapeutically relevant doses of ATO and observed pronounced decreasing steroid levels in particular in testis and adrenals but also in brain and plasma. In testis, all major steroidogenic enzymes were up-regulated, indicating autocrine and/or paracrine compensation for the decrease in steroid production by this tissue. In adrenals, StAR and CYP11A1 gene expression were decreased, whereas little effects were observed in the brain. Furthermore, we analyzed plasma LH and ACTH levels to investigate feedback via the PT and HPA axes. No effects were observed on LH levels, indicating little compensation via the PT axis. In contrast, ACTH levels increased during ATO exposure, indicating that the HPA axis to some extend compensated for the decrease in adrenal steroid production. Overall, ATO exerted pronounced effects on steroid production both in vitro and in vivo at therapeutically relevant doses. This clearly demonstrates the high potency of ATO to affect steroid homeostasis during therapeutic treatment. Further clinical and epidemiological studies should be conducted to investigate the relevance of these observations in patients treated with cholesterol-lowering pharmaceuticals. [ABSTRACT FROM AUTHOR]

An LC–MS/MS method was developed and validated to analyze simultaneously estrogens (estradiol, E2; estrone, E1), androgens (testosterone, T; androstenedione, A4; dehydroepiandrosterone, DHEA), progestagens (17a-hydroxypregnenolone, 17OHP5; 17a-hydroxyprogesterone, 17OHP4; progesterone, P4), glucocorticoids (cortisol, F; cortisone E; corticosterone, B; 11-deoxycortisol, S; 21-hydroxyprogesterone, 21OHP4), and mineralocorticoids (aldosterone, A) from 150 μl of human serum, plasma, or endometrium and endometriotic tissue homogenates. Samples spiked with isotope-labeled steroids as internal standards were extracted with toluene prior to LC–MS/MS analysis. The chromatographic separation of underivatized steroids was achieved on a biphenyl column with 0.2 mM NH 4 F as the eluent additive and a water-methanol gradient to improve E2 and E1 ionization. Method validation was performed with human plasma samples, and analysis of certified E2, T, F, and P4 reference serums (BCR-576, ERM-DA346, ERM-DA192, ERM-DA347), as well as homogenates of endometrium and endometriotic tissue. A total of 27 steroids were included in the method development to ensure the specificity of the method. After validation, the method was found suitable for quantitative analysis of 11 steroids: E2 (6.7 pM-13 nM), E1 (1.3 pM-6.6 nM), T (3.3 pM-13 nM), A4 (13 pM-33 nM), 17OHP5 (32 pM-65 nM), 17OHP4 (33 pM-13 nM), F (33 pM-133 nM), E (13 pM-130 nM), B (33 pM-134 nM), S (13 pM-129 nM), and A (32 pM-32 nM). In addition, DHEA (333 pM-32 nM), P4 (13 pM-13 nM) and 21OHP4 (13 pM-13 nM) can be analyzed semiquantitatively. [ABSTRACT FROM AUTHOR]

Background: Serum steroids are crucial molecules altered in prostate cancer (PCa). Mass spectrometry (MS) is currently the elected technology for the analysis of steroids in diverse biological samples. Steroids have complex biological pathways and stoichiometry and it is important to evaluate their quantitative ratio. MS applications to patient hormone profiling could lead to a diagnostic approach. Methods: Here, we employed the Surface Activated Chemical Ionization-Electrospray-NIST (SANIST) developed in our laboratories, to obtain quantitative serum steroid ratio relationship profiles with a machine learning Bayesian model to discriminate patients with PCa. The approach is focused on steroid relationship profiles and disease association. Results: A pilot study on patients affected by PCa, benign prostate hypertrophy (BPH), and control subjects [prostate-specific antigen (PSA) lower than 2.5 ng/mL] was done in order to investigate the classification performance of the SANIST platform. The steroid profiles of 71 serum samples (31 controls, 20 patients with PCa and 20 subjects with benign prostate hyperplasia) were evaluated. The levels of 10 steroids were quantitated on the SANIST platform: Aldosterone, Corticosterone, Cortisol, 11-deoxycortisol, Androstenedione, Testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), 17-OH-Progesterone and Progesterone. We performed both traditional and a machine learning analysis. Conclusion: We show that the machine learning approach based on the steroid relationships developed here was much more accurate than the PSA, DHEAS, and direct absolute value match method in separating the PCa, BPH and control subjects, increasing the sensitivity to 90% and specificity to 84%. This technology, if applied in the future to a larger number of samples will be able to detect the individual enzymatic disequilibrium associated with the steroid ratio and correlate it with the disease. This learning machine approach could be valid in a personalized medicine setting. [ABSTRACT FROM AUTHOR]

Ion mobility spectrometry enhances the performance characteristics of liquid chromatography-mass spectrometry workflows intended to steroid profiling by providing a new separation dimension and a novel characterization parameter, the so-called collision cross section (CCS). This work proposes the first CCS database for 300 steroids (i.e., endogenous, including phase I and phase II metabolites, and exogenous synthetic compounds), which involves 1080 ions and covers the CCS of 127 androgens, 84 estrogens, 50 corticosteroids, and 39 progestagens. This large database provides information related to all the ionized species identified for each steroid in positive electrospray ionization mode as well as for estrogens in negative ionization mode. CCS values have been measured using nitrogen as drift gas in the ion mobility cell. Generally, direct correlation exists between mass-to-charge ratio (m/z) and CCS because both are related parameters. However, several steroids mainly steroid glucuronides and steroid esters have been characterized as more compact or elongated molecules than expected. In such cases, CCS results in additional relevant information to retention time and mass spectral data for the identification of steroids. Moreover, several isomeric steroid pairs (e.g., 5β-androstane-3,17-dione and 5α-androstane-3,17-dione) have been separated based on their CCS differences. These results indicate that adding the CCS to databases in analytical workflows increases selectivity, thus improving the confidence in steroids analysis. Consequences in terms of identification and quantification are discussed. Quality criteria and a construction of an interlaboratory reproducibility approach are also reported for the obtained CCS values. The CCS database described here is made publicly available. [ABSTRACT FROM AUTHOR]

Sex steroids in clinical endocrinology have been mainly investigated with peripheral blood and urine samples, while there is limited information regarding the local levels within tissues. To improve analytical properties of sex steroids from trace amounts of tissue samples, two-phase extractive ethoxycarbonlyation and subsequent pentafluoropropionyl derivatization coupled to gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed. The optimized analytical conditions led to excellent chromatographic separation of 15 estrogens, 6 androgens, and 2 progestins. The quantitative results were calculated based on in-house control samples as the steroid-free tissues, and the precision and accuracy were 4.2%–26.8% and 90.8%–116.4%, respectively. The on-column limit of quantification was from 180 fg to 0.5 pg for androgens and estrogens, and 1.25 pg for progestins, which were found to be linear ( r 2  > 0.990). The validated method was then applied to quantify 7 sex steroids from three 100-μm-thick frozen breast tissue slices from postmenopausal patients with breast cancer. This is the first report on the improved GC–MS/MS method for the detection of androgens and pregnenolone from breast cancer tissues, and it can be a useful technique to measure the local levels of sex steroids, thus, enhancing our understanding of the pathophysiological significances of steroidogenesis. [ABSTRACT FROM AUTHOR]

Atrazine, a widely use herbicide, has been classified as a potential endocrine disruptor, especially for freshwater species. In this study, we tested the hypothesis that atrazine can affect reproduction in crayfish through dysregulation of vitellogenin expression and hormone synthesis. Adult female crayfish ( Procambarus clarkii ) were exposed during one month to atrazine at concentrations of either 1 or 5 mg/L. At the end of the exposure, ovaries, hepatopancreas, and hemolymph samples were harvested for analysis of vitellogenin expression and steroid hormone levels. Ovarian tissue was also sampled for both biochemical and histological analyses. Our results show that atrazine-exposed crayfish had a lower expression of vitellogenin in the ovary and hepatopancreas, as well as smaller oocytes, and reduced vitellogenin content in the ovary. Despite these effects, circulating levels of estradiol increased in females exposed to 5 mg/L of atrazine, showing that the inhibiting effect of atrazine on vitellogenin production was not related to a lower secretion of sexual steroids. Instead, some early stimulating effects of estradiol on vitellogenesis could have occurred, particularly in the hepatopancreas. On the other hand, atrazine caused a higher metabolic effort, in terms of lactate production, presumably triggered to provide the energy needed to face the unspecific stress produced by the herbicide. Lipid peroxidation was not affected by atrazine, but glutathione levels were significantly increased. [ABSTRACT FROM AUTHOR]

Objective: The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples.Study Design: We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5).Results: Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC.Conclusions: We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum.Implications: This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing. [ABSTRACT FROM AUTHOR]

The present study evaluated changes of fecal sexual steroids in two female cheetahs (Geijsha and Duchessa) in Northern Italy throughout one year. Wet feces were collected daily from two sibling animals of the same age, housed with conspecific males and managed in the same conditions, and estrogens and progestogens concentrations were analyzed by radioimmunoassay (RIA). Evidence of ovarian activity based on regular fluctuation in estrogen excretion was demonstrated in both females. None of the animals was continuously cycling, as follicular activity was interrupted by anestrous periods, during the spring and early winter. No significant increases of progestogens were recorded after the estrogen peaks, indicating that induced or spontaneous ovulations did not occur during the observation period. The wavelet decomposition evidenced the temporal pattern of ovarian activity in the two females, underlying throughout the year a more pronounced rhythmical ovarian estrogenic activity in Geijsha than in Duchessa. However, this statistical approach had a smoothing effect in depicting the hormonal patterns and the number of follicular phases might be lower than that revealed by the iterative method. In this study, RIA on wet feces performed very well to determine sexual steroid concentrations, and an ovarian activity interrupted by anestrous periods along the year in captive cheetahs co-housed in a small group was demonstrated. More information on estrous behavior of captive cheetahs were obtained in this study, but the effects of husbandry and management conditions on natural reproductive physiology of this species remain to elucidate. [ABSTRACT FROM AUTHOR]