Biochar-based polymeric film as sustainable and efficient sorptive phase for preconcentration of steroid hormones in environmental waters and wastewaters.

BACKGROUND: The environmental impact of sample preparation should be minimized through simplification of the procedures and the use of natural, renewable and/or reusable materials. In such scenario, thin-film microextraction fulfils the former criteria, as it enables few steps and miniaturization, thus small amount of extraction phase. At the same time, the use of sorbents such as biochars obtained from biomass waste is even more promoted due to their availability at low cost and increased life-cycle in a circular economy vision. However, it is not always easy to combine these criteria in sample preparation. RESULTS: A thin film microextraction was developed for the determination of steroids in aqueous samples, entailing a membrane made of cellulose triacetate and a wood-derived biochar (Nuchar®) as carbon precursor. Different characterization techniques showed the successful preparation, whereas the sorption kinetics experiments demonstrated that biochar is responsible for the extraction with the polymer acting as a smart support. After a study about membranes' composition in terms of biochar amounts (4 %, 10 %, 16 % wt) and type of synthesis set up, the ceramic 3D-mold was selected, achieving reproducible and ready-to-use membranes with composition fixed as 10 %. Different elution conditions, viz. type and time of agitation, type, composition and volume of eluent, were evaluated. The final microextraction followed by HPLC-MS/MS quantification was successfully validated in river and wastewater treatment plant effluent samples in terms of accuracy (R% 64-123 %, RSD<19 % in river; R% 61-118 %, RSD <18 % in effluent, n = 4), sensitivity (MQLs 0.2-8.5 ng L-1) and robustness. SIGNIFICANCE: This novel biochar-based polymeric film proved to be a valid and sustainable sorbent, in terms of extraction capability, ease of preparation and greenness. By comparison with literature and the greenness evaluation with the most recent metric tools, this method expands the potential applicability of the thin-film microextraction and opens up innovative scenarios for sustainable procedures entailing the use of biochars entrapped in bio-polymers.

Occurrence and spatiotemporal distribution of natural and synthetic steroid hormones in soil, water, and sediment systems in suburban agricultural area of Guangzhou City, China.

Steroid hormones are highly potent compounds that can disrupt the endocrine systems of aquatic organisms. This study explored the spatiotemporal distribution of 49 steroid hormones in agricultural soils, ditch water, and sediment from suburban areas of Guangzhou City, China. The average concentrations of Σsteroid hormones in the water, soils, and sediment were 97.7 ng/L, 4460 ng/kg, and 9140 ng/kg, respectively. Elevated hormone concentrations were notable in water during the flood season compared to the dry season, whereas an inverse trend was observed in soils and sediment. These observations were attributed to illegal wastewater discharge during the flood season, and sediment partitioning of hormones and manure fertilization during the dry season. Correlation analysis further showed that population, precipitation, and number of slaughtered animals significantly influenced the spatial distribution of steroid hormones across various districts. Moreover, there was substantial mass transfer among the three media, with steroid hormones predominantly distributed in the sediment (60.8 %) and soils (34.4 %). Risk quotients, calculated as the measured concentration and predicted no-effect concentration, exceeded 1 at certain sites for some hormones, indicating high risks. This study reveals that the risk assessment of steroid hormones requires consideration of their spatiotemporal variability and inter-media mass transfer dynamics in agroecosystems.

Anabolic steroids in livestock production: Background and implications for chemical food safety.

The use of steroids in livestock animals is a source of concern for consumers because of the risks associated with the presence of their residues in foodstuffs of animal origin. Technological advances such as mass spectrometry have made it possible to play a fundamental role in controlling such practices, firstly for the discovery of marker metabolites but also for the monitoring of these compounds under the regulatory framework. Current control strategies rely on the monitoring of either the parent drug or its metabolites in various matrices of interest. As some of these steroids also have an endogenous status specific strategies have to be applied for control purposes. This review aims to provide a comprehensive and up-to-date knowledge of analytical strategies, whether targeted or non-targeted, and whether they focus on markers of exposure or effect in the specific context of chemical food safety regarding the use of anabolic steroids in livestock. The role of new approaches in data acquisition (e.g. ion mobility), processing and analysis, (e.g. molecular networking), is also discussed.

Simultaneous Determination of Selected Steroids with Neuroactive Effects in Human Serum by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Neuroactive steroids are a group of steroid molecules that are involved in the regulation of functions of the nervous system. The nervous system is not only the site of their action, but their biosynthesis can also occur there. Neuroactive steroid levels depend not only on the physiological state of an individual (person's sex, age, diurnal variation, etc.), but they are also affected by various pathological processes in the nervous system (some neurological and psychiatric diseases or injuries), and new knowledge can be gained by monitoring these processes. The aim of our research was to develop and validate a comprehensive method for the simultaneous determination of selected steroids with neuroactive effects in human serum. The developed method enables high throughput and a sensitive quantitative analysis of nine neuroactive steroid substances (pregnenolone, progesterone, 5α-dihydroprogesterone, allopregnanolone, testosterone, 5α-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and epiandrosterone) in 150 µL of human serum by ultrahigh-performance liquid chromatography with tandem mass spectrometry. The correlation coefficients above 0.999 indicated that the developed analytical procedure was linear in the range of 0.90 nmol/L to 28.46 µmol/L in human serum. The accuracy and precision of the method for all analytes ranged from 83 to 118% and from 0.9 to 14.1%, respectively. This described method could contribute to a deeper understanding of the pathophysiology of various diseases. Similarly, it can also be helpful in the search for new biomarkers and diagnostic options or therapeutic approaches.

Enantiopure Corral[4]BINOLs as Ultrastrong Receptors for Recognition and Differential Sensing of Steroids.

The precise recognition and sensing of steroids, a type of vital biomolecules, hold immense practical value across various domains. In this study, we introduced corral[4]BINOLs (C[4]BINOLs), a pair of enantiomeric conjugated deep-cavity hosts, as novel synthetic receptors for binding steroids. Due to the strong hydrophobic effect of their deep nonpolar, chiral cavities, the two enantiomers of C[4]BINOLs demonstrated exceptionally high recognition affinities (up to 1012 M-1) for 16 important steroidal compounds as well as good enantioselectiviy (up to 15.5) in aqueous solutions, establishing them as the most potent known steroid receptors. Harnessing their ultrahigh affinity, remarkable enantioselectivity, and fluorescence emission properties, the two C[4]BINOL enantiomers were employed to compose a fluorescent sensor array which achieved discrimination and sensing of 16 structurally similar steroids at low concentrations.

Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring.

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng µL-1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80-120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

Exploring the potential of using ion mobility-mass spectrometry to separate matrix interferences from analytes in food control.

During residue analysis in complex matrices for food safety purposes, interfering signals can sometimes overlap with those of the analyte of interest. Access to an additional separation dimension besides chromatographic and mass separation, such as ion mobility, can aid in removing interfering signals, allowing for correct analyte identification in these cases. In our laboratory, during routine LC-MS/MS analysis of liver samples for growth promoter residues, an interfering signal was found that matches the retention time and m/z values for stanozolol, a synthetic anabolic steroid. In the present work, the performance of a liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS) method has been evaluated to study whether this LC-MS/MS false positive in liver samples could be eliminated by LC-IM-MS analysis. A cyclic ion mobility system already allowed the separation of stanozolol from the interfering peak after only one pass, showing a significant improvement compared to the conventional LC-MS/MS method. Additionally, collisional cross section (CCS) values were calculated and successfully compared with those from literature for identification purposes, eventually allowing both the identification and quantification of stanozolol in this complex matrix.

Exploration of the profiles of steroidal saponins from Rhizoma Paridis and their metabolites in rats by UPLC-Q-TOF-MS/MS.

INTRODUCTION: Steroidal saponins characterised by intricate chemical structures are the main active components of a well-known traditional Chinese medicine (TCM) Rhizoma Paridis. The metabolic profiles of steroidal saponins in vivo remain largely unexplored, despite their renowned antitumor, immunostimulating, and haemostatic activity. OBJECTIVE: To perform a comprehensive analysis of the chemical constituents of Rhizoma Paridis total saponins (RPTS) and their metabolites in rats after oral administration. METHOD: The chemical constituents of RPTS and their metabolites were analysed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). RESULTS: A reliable UPLC-Q-TOF-MS/MS method was established, and a total of 142 compounds were identified in RPTS. Specifically, diosgenin-type saponins showed the diagnostic ions at m/z 415.32, 397.31, 283.25, 271.21, and 253.20, whereas pennogenin-type saponins exhibited the diagnostic ions at m/z 413.31, 395.30, and 251.20. Based on the characteristic fragments and standard substances, 15 specific metabolites were further identified in the faeces, urine, plasma, and bile of rats. The metabolic pathways of RPTS, including phase I reactions (de-glycosylation and oxidation) and phase II reactions (glucuronidation), were explored and summarised, and the enrichment of metabolites was characterised by multivariate statistical analysis. CONCLUSION: The intricate RPTS could be transformed into relatively simple metabolites in rats through de-glycosylation, which provides a reference for further metabolic studies and screening of active ingredients for TCM.

Testosterone synthesis was inhibited in the testis metabolomics of a depression mouse model.

INTRODUCTION: Depression is a common emotional disorder. Previous studies have suggested that depression is associated with the central nervous system. Recent studies have suggested that reduced testosterone level is the core inducement of depression. Testis is the vital organ for the synthesis of testosterone. How does testis mediate depression is still unknown. OBJECTIVES: We adopted a classical depression model of mouse caused through chronic mild stress (CMS). The metabolomics liquid chromatography-mass spectrometry was adopted to analyse the influence of CMS on testis metabolism. Then we confirmed the possible abnormal metabolism of the testis in depression mice by pathway analysis and molecular biological technique. RESULTS: Compared with control mice, 16 differential metabolites were found in CMS mice by multivariate statistical analysis. In comparison with control mice, CMS mice showed higher levels for campesterol, ribitol, citric acid, platelet activating factor, guanosine, cytosine and xanthine and lower levels for docosahexaenoic acid, hippuric acid, creatine, testosterone, dehydroepiandrosterone, progesterone, l-carnitine, acetyl carnitine and propionyl carnitine. The pathway analysis indicated that these differential metabolites are associated with steroid hormone synthesis, purine metabolism and phenylalanine metabolism. In addition, we also first discovered that testicular morphology in depression mice was damaged and steroid hormone synthetases (including steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage) were inhibited. CONCLUSION: These findings may be helpful to parse molecular mechanisms of pathophysiology of depression. It also pointed out the direction to search for potential therapy schedules for male depression and provide novel insights into exploring the pathogenesis of male depression.

Differentiation of steroid isomers by steroid analogues adducted trapped ion mobility spectrometry-mass spectrometry.

Steroids are one of the important indicators of health and disease. However, due to the high similarity of steroid structures, there are several potential obstacles in the differentiation of steroids, especially for their isomers. Herein, we described a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) approach based on the steroid analogue adduction for isomer-specific identification of steroids. The application of dexamethasone (DEX) to form heterodimers with steroids enhanced the separation of their isomers in TIMS. Two isomer pairs including 17-hydroxyprogesterone/11-deoxycorticosterone and androsterone/epiandrosterone were successfully separated as the heterodimers with DEX by TIMS. The stability of DEX-adducted heterodimers is comparable with steroid dimers. Owing to the high separation efficiency and stability, the relative quantification of steroid isomers was demonstrated with the proposed method.

Lanostane triterpenoids from the fruiting bodies of Ganoderma amboinense.

Lanostane-type triterpenoids are the main characteristic constituents in Ganoderma mushrooms. Phytochemical analysis on the ethanol extract of the fruiting bodies of Ganoderma amboinense led to isolation and identification of twelve previously undescribed lanostane triterpenoids (1-12). Their chemical structures were determined by HR-ESI-MS, IR, and NMR spectroscopic analysis, NMR calculation, as well as X-ray crystallography. All isolates were evaluated for the α-glucosidase inhibitory and anti-inflammatory activities. Compounds 1, 5, 6, and 11 showed significant α-glucosidase inhibitory activity with IC50 values ranging from 33.5 µM to 96.0 µM. Moreover, compound 12 showed anti-inflammatory activity with IC50 value of 21.7 ± 2.1 µM.

Steroidal Alkaloids from Food Waste of Tomato Processing Inhibit Neuroblastoma Cell Viability.

Nowadays, there is considerable attention toward the use of food waste from food processing as possible sources of compounds with health properties, such as anticancer activity. An example is tomato processing, which is responsible for generating a remarkable amount of waste (leaves, peel, seeds). Therefore, our goal was to evaluate the potential anticancer property of tomato extracts, in particular "Datterino" tomato (DT) and "Piccadilly" tomato (PT), and to study their phytochemical composition. Liquid chromatography with tandem mass spectrometry (LC/MS-MS) results showed that these extracts are rich in alkaloids, flavonoids, fatty acids, lipids, and terpenes. Furthermore, their potential anticancer activity was evaluated in vitro by MTT assay. In particular, the percentage of cell viability was assessed in olfactory ensheathing cells (OECs), a particular glial cell type of the olfactory system, and in SH-SY5Y, a neuroblastoma cell line. All extracts (aqueous and ethanolic) did not lead to any significant change in the percentage of cell viability on OECs when compared with the control. Instead, in SH-SY5Y we observed a significant decrease in the percentage of cell viability, confirming their potential anticancer activity; this was more evident for the ethanolic extracts. In conclusion, tomato leaves extracts could be regarded as a valuable source of bioactive compounds, suitable for various applications in the food, nutraceutical, and pharmaceutical fields.

Revealing Structure and Localization of Steroid Regioisomers through Predictive Fragmentation Patterns in Mass Spectrometry Imaging.

Identifying and mapping steroids in tissues can provide opportunities for biomarker discovery, the interrogation of disease progression, and new therapeutics. Although separation coupled to mass spectrometry (MS) has emerged as a powerful tool for studying steroids, imaging and annotating steroid isomers remains challenging. Herein, we present a new method based on the fragmentation of silver-cationized steroids in tandem MS, which produces distinctive and consistent fragmentation patterns conferring confidence in steroid annotation at the regioisomeric level without using prior derivatization, separation, or instrumental modification. In addition to predicting the structure of the steroid with isomeric specificity, the method is simple, flexible, and inexpensive, suggesting that the wider community will easily adapt to it. We demonstrate the utility of our approach by visualizing steroids and steroid isomer distributions in mouse brain tissue using silver-doped pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging.

Development and validation of a deep eutectic solvent-assisted liquid-liquid extraction method for simultaneous quantification of six steroid hormones in serum by liquid chromatography-tandem mass spectrometry.

Steroid hormones have been reported to be associated with endocrine system diseases. This paper proposes a novel procedure of deep eutectic solvent (DES)-assisted liquid-liquid extraction (LLE) to extract six steroid hormones (including cortisone, cortisol, androstenedione, testosterone, 17-hydroxyprogesterone, and progesterone) from serum coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of five types of L-proline, choline chloride, and citric acid-based DESs were tailored; the DES from L-proline and ethylene glycol at a molar ratio of 1:4 with 20 % acetonitrile was selected as the best-fit assisted solvent for the six steroid hormones compared with other DESs. The parameters for extraction by selected DES were optimized using Box-Behnken design (BBD), and the optimal extraction conditions are 200 µL of acetonitrile, 100 µL of the sample, and 80 µL of DES. Under optimum conditions, the method has good linear calibration ranges (between 0.07 ng mL-1 and 600 ng mL-1), correlation coefficients of determination (r2>0.99), and low limits of quantification (between 0.02 and 0.60 ng mL-1). The extraction recoveries were in the range of 81.84-114.43 %, and the intra-day and inter-day relative standard deviations (RSDs) were less than 10 %.In general, the DES-LC-MS/MS method is a simple and environmentally-friendly method, which can be complementary to the presently available methods for determining steroid hormones in serum.

Lanostane triterpenoids with anti-proliferative and PTP1B/α-glucosidase inhibitory activities from the fruiting bodies of Ganoderma calidophilum.

Twelve previously undescribed and four known lanostane triterpenoids were isolated from the fruiting bodies of Ganoderma calidophilum. The structures of undescribed compounds, ganodecalones H-S (1-12), were elucidated by extensive spectroscopic analysis as well as ECD and NMR calculations. Compound 4 showed significant inhibitory activity against human leukaemia cell line K562, gastric cancer cell line SGC-7901, and cervical cancer cell line HeLa with IC50 values of 13.10 ± 0.19, 17.26 ± 4.75, and 4.36 ± 0.58 µM, respectively. Compound 16 exhibited inhibitory potency against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase with IC50 values of 30.2 ± 0.13 µM and 120.6 ± 0.14 µM, respectively. The binding sites and interactions of 16 with PTP1B and α-glucosidase were revealed using molecular docking simulations.

Gas chromatography properties and mass spectrometry fragmentation of anabolic androgenic steroids in doping control.

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.

Lanostane triterpenoids with anti-proliferative and anti-inflammatory activities from medicinal mushroom Ganoderma lingzhi.

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.

Development of a Rapid, Targeted LC-IM-MS Method for Anabolic Steroids.

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.

Identification of endogenous carbonyl steroids in human serum by chemical derivatization, hydrogen/deuterium exchange mass spectrometry and the quantitative structure-retention relationship.

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.

Testosterone histories from tusks reveal woolly mammoth musth episodes.

Hormones in biological media reveal endocrine activity related to development, reproduction, disease and stress on different timescales1. Serum provides immediate circulating concentrations2, whereas various tissues record steroid hormones accumulated over time3,4. Hormones have been studied in keratin, bones and teeth in modern5-8 and ancient contexts9-12; however, the biological significance of such records is subject to ongoing debate10,13-16, 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 musth17-19, an annually recurring period of behavioural and physiological changes that enhance mating success20-23. 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.