Analysis Of Steroids Research Articles

Hair and Saliva Endocannabinoid and Steroid Hormone Analysis by Liquid Chromatography Paired with Tandem Mass Spectrometry.

Endocannabinoids are lipid neurotransmitters that play an important part in human health. Recent methods have found that quantification of endocannabinoids in hair and saliva samples is possible using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). This chapter describes two simple sample preparation methods that can be used to prepare hair and saliva samples for analysis using LC-MS/MS. Our LC-MS/MS method can be applied to both hair and saliva samples and is sufficiently sensitive for endocannabinoid, as well as steroid hormone, quantification in both of these sample matrices. This chapter provides a comprehensive description of how this can be achieved and provides tips and tricks for troubleshooting problems users may experience.

An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS).

The crucial role of steroid hormones in health and diseases merits their high-throughput, accurate and affordable measurements in biological specimens. Despite advances in analytical methods, sensing and quantifying steroid hormones remains challenging. Immunoassays offer excellent sensitivity but are inherently labour-intensive, costly, and prone to false positives. Mass spectrometry (MS) has been increasingly utilised, with the main hurdle being the isobaric tendencies of similar analytes, which complicates their separation and accurate quantification. This study compares ultrahigh-performance supercritical fluid chromatography separation (UHPSFC) and ultra-high-performance liquid chromatography (UHPLC) for MS detection. It optimises the column chemistry, temperature, and pressure to provide an operational protocol for the resolution and quantification of analytes. It presents the systematic characterisation of UHPSFC-MS performance by investigating spiked blood samples using Solid-Phase Extraction (SPE) and describes the matrix effects associated with MS measurements. Although both separation methods showed adequate resolution, specificity, and retention time, UHPSFC-MS was superior for five out of seven columns tested. With added high-throughput capacities, UHPSFC-MS, thus, offers an optimal solution for the analysis of steroid hormones for research, medical chemistry, and clinical diagnostics.

Triple-template surface imprinted magnetic polymers for wide-coverage extraction of steroid hormones from human serum.

In this study, novel triple-template magnetic nanospheres by surface imprinting, called triple-template magnetic molecularly imprinted polymers (tri-MMIPs), were prepared for the detection of steroid hormones in human serum. The polymers were constructed by Fe3O4 as support, estrone (E1), progesterone (PROG), and estradiol (E2) as triple templates, acrylic acid (AA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The resulting tri-MMIPs were further characterized and applied as sorbents in human serum pretreatment. Coupled with ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) profiling after derivatization, 15 steroids (E1, 6-ketoE1, 4-OHE1, 16α-OHE1, DHEA, 4-MeOE1, PROG, PREG, 17-OHPROG, 7-DHE2, E2, 2-OHE2, 16-epiE3, E3, 2-MeOE2) were successfully quantified. The developed method exhibited a good recovery (78.9-114%) and low limit of quantitation (LLOQ, 5-10pg/mL), which indicates a great potential for application in the analysis of multiple steroid hormones in complex biological samples.

Comprehensive targeted profiling of multiple steroid classes in rodent plasma using liquid chromatography-mass spectrometry

BackgroundReliable quantification of multiple steroid classes in biological fluids within a single method remains an analytical challenge despite many previously published methods. Crosstalk of positional isomers, overlap of stereoisomer fragmentation patterns, differing proton affinities, in-source fragmentation, varying stability of protonated ions in the gas phase across steroid classes, and non-existence of steroid-free matrix are the main challenges limiting the number of simultaneously profiled steroids. ResultsIn this study, we focused on the development of a derivatization-free, achiral, high-throughput, and cost-effective UHPLC-MS/MS approach that allows simultaneous profiling of a spectrum of 38 steroids covering progestogens, androgens, corticosteroids, and estrogens, while properly addressing the hurdles of steroid analysis. Within a 20-min method, 16 stereoisomers and 15 positional isomers were fully resolved within a single run while separated from 7 additional non-interfering steroids and matrix interferences in rodent plasma. Protein precipitation (PP) and supported liquid extraction (SLE) methods using only 40 μL of sample were developed to achieve the lowest possible limits of quantification. Nevertheless, 5α-dihydroprogesterone and 3α,5α-THDOC could be only qualitatively assessed when using PP. In contrast, DHEA-S could not be quantified or identified when using SLE. A novel surrogate matrix-background subtraction approach, using rat plasma after the animal's adrenalectomy, has been implemented into the optimized PP-UHPLC-MS/MS workflow, successfully validated according to the unified ICH/EMA M10 guidelines, and compared to the traditional quantification strategies. Moreover, the validity of the newly adopted approach has been verified by the targeted profiling of multiple biologically active endogenous steroids in more than 500 samples of mouse plasma in total. SignificanceUnderestimation of hurdles associated with steroid analysis often compromises the accurate steroid quantification. Our comprehensive, fully validated UHPLC-MS/MS method targeting a wide spectrum of endogenous steroids, mitigating steroid crosstalk and using a minimal sample volume together with a novel surrogate matrix-background subtraction approach significantly advances steroid analysis for research and clinical applications covering multiple biological scopes.

RESEARCH OF STEROID AND VOLATILE COMPOUNDS IN VERBENA OFFICINALIS L. HERB

Biologically active compounds of medicinal plants are able to affect various links of processes in the living organism, namely, they regulate the processes of metabolism, growth and development of organisms. The therapeutic potency of medicinal plant raw materials is derived by a complex of biologically active compounds that influence various biologically processes, including metabolism, growth, and development. Numerous steroid-like remedies of plant origin exhibit remarkable activity. Phytosterols, chemical compounds similar to cholesterol, are synthesized in plant organisms and have been found to have anti-tumor effects and lower blood cholesterol. Additionally, steroid and volatile compounds demonstrate antimicrobial and anti-inflammatory activity. The genus Verbena includes 250 species. 3 species grow on the territory of Ukraine: Verbena officinalis L., Verbena supinа L., Verbena hybrida Hort. Verbena officinalis L. is globally recognized for its antioxidant and antimicrobial properties, showcasing anti-inflammatory, antiseptic, antispasmodic, and anti-allergic effects. However, comprehensive chemical studies of Verbena officinalis L. are limited, necessitating a detailed analysis of phitosterol and volatile compound content using modern precise methods. The aim of the research was to qualitatively and quantitatively determine the content of steroid and volatile compounds in Verbena officinalis L. herb using gas chromatography/mass spectrometry. We chose the herb Verbena officinalis L. as the object of the study. Materials and methods. The content of phytosterols and volatile compounds in Verbena officinalis L. herb was studied using the GC/MS method. We chose the herb Verbena officinalis L. as the object of the study. An Agilent Technologies 6890 chromatograph with a 5973 mass spectrometric detector with an HP-5 ms capillary column were used. The mass spectra library in conjunction with AMDIS and NIST identification programs was employed for components were identification. Research results. The study of phytosterols in Verbena officinalis L. identified campesterol (9.32%), stigmasterol (7.13%), γ-sitosterol (72.21%) and androst-5,15-dien-3-ol-acetate were identified (11.35%). In the volatile fraction 8 compounds were identified, with benzophenone (33.96%) and hexahydro farnesyl acetone (22.57%) being dominant. Conclusions. The GC/MS method allowed for the qualitative and quantitative analysis of steroid and volatile compounds in Verbena officinalis L, identifying 4 phytosterols and 8 volatile compounds. The identified phytosterols and volatile compounds in the herb have significant implications for the production of phytoremedies and the development of raw material standardization parameters. We identified campesterol, stigmasterol, γ-sitosterol and androst-5,15-dien-3-ol-acetate in the herb Verbena officinalis L. Eight volatile compounds were identified in the herb Verbena officinalis L. by the chromato-mass spectrometric method. Benzophenone and hexahydro farnesyl acetone are dominant. Steroid compounds (mg/kg) were identified and quantified: campesterol, stigmasterol, γ-sitosterol and androst-5,15-dien-3-ol-acetate. The obtained research results will be taken into account in the further production of phytoremedies based on the medicinal Verbena herb, as well as in the development of raw material standardization parameters.

B-159 Fast Cleanup for 19 Steroid Analytes in Serum Using Supported Liquid Extraction (SLE)

Abstract Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool that holds promise in designing the future of steroid analysis in the clinical laboratory. It is already widely used in steroid analysis in endocrinology and is being utilized increasingly in newer applications for individual steroids and panels relevant in clinical research. LC-MS/MS is gaining user base owing to greater sensitivity and specificity and gradually even replacing some of the legacy test methods. Improved sample preparation, modern chromatography methods, and sensitive, faster scanning mass spectrometers have all played a role in improving LC-MS/MS. Steroid analysis for clinical research can require very low limits of detection which demand high recoveries and very clean extracted samples. Supported Liquid Extraction (SLE) can provide very clean extracted samples with less method development and a streamlined workflow compared to solid phase extraction (SPE). Laboratories desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Chromatographic separation of steroid isomers with the same m/z is necessary for accurate quantitation by LCMS/MS. Meeting these criteria can be challenging in a single LC-MS/MS method. Methods Each 200 µL serum sample was pretreated with 200 µL of pH 6 Ammonium Acetate buffer (aq) before being loaded onto a Novum™ PRO SLE, MAX 96-well plate, following the standard SLE protocol. Samples were eluted using 2 × 900 µL of Dichloromethane or Ethyl Acetate. After evaporation of the elution solvent, samples were reconstituted in 100 µL of 0.5 mM Ammonium Fluoride / Methanol (60:40, v/v). A Kinetex™ 2.6 μm C18, 50 x 3.0 mm column using a gradient of 0.5 mM Ammonium Fluoride (aq) and Methanol on an Agilent® 1290 Infinity UHPLC and SCIEX® 7500 Triple Quad™ were used for quantitation. Results Here, we present an effective sample extraction method and LC-MS/MS analysis method for the quantitation of a 19-analyte steroid panel from serum. The Kinetex 2.6 μm C18 column provided a fast 8-minute chromatographic separation and good selectivity for steroid analytes in both positive ion mode and negative ion mode. Calibration curves with a linear fit and 1/x weighting, showed good linearity with R2 of >0.99 for all analytes except 17-OH-Pregnenolone and DHEAS when using Dichloromethane as the elution solvent, and R2 of >0.980 for all analytes when using Ethyl Acetate as the elution the solvent. Accuracy of samples using Dichloromethane as the elution solvent were 80-112 %, except for 17-OH-Pregnenolone and DHEAS. Accuracy of all samples using the Ethyl Acetate elution solvent were 91-118 %. Conclusions The-96 well plate extraction method was robust, reproducible, and can be automated. SLE using Novum Pro MAX combined with a fast LC method provides a simple, rapid, high-throughput workflow for identification and quantitation of 19 steroids from serum.

B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis

Abstract Background Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities. Methods The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (>0.06ng/g Creatinine). Results Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney. Conclusions The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.

Retrospective Analysis of Systemic Steroid and Combined Systemic-Intratympanic Steroid Treatment Results and Prognostic Factors in Sudden Idiopathic Hearing Loss

Retrospective Analysis of Systemic Steroid and Combined Systemic-Intratympanic Steroid Treatment Results and Prognostic Factors in Sudden Idiopathic Hearing Loss

Transcriptome analysis of four types of gonadal tissues in largemouth bass (Micropterus salmoides) to reveal its sex-related genes.

The sex determination system of largemouth bass (Micropterus salmoides, LMB) is XX/XY; however, the underlying molecular mechanisms involved in early sex differentiation, gonadal development, and exogenous hormone-induced sex reversal remain unknown. In this study, LMB at 15days post-hatching (dph) were fed diets containing 20mg/kg of 17α-methyltestosterone (17α-MT) or 30mg/kg of 17β-estradiol (17β-E2) for 60days, respectively. Serum steroid levels, histological observations of the gonads, and identification of sex-specific markers were employed to screen the gonads of 60-day-old normal female fish (XX-F), normal male fish (XY-M), 17β-E2 induced pseudo-female fish (XY-F), and 17α-MT-induced pseudo-male fish (XX-M) for transcriptome sequencing in order to uncover genes and pathway involved in the process of sexual reversal. The results from histology and serum sex steroid hormone analysis showed that both 17α-MT and 17β-E2 were capable of inducing sex reversal of LMB at 15dph. Transcriptome results revealed a total of 2,753 genes exhibiting differential expression, and the expression pattern of these genes in the gonads of XX-M or XY-F resembled that of normal females or males. The male sex-biased genes that are upregulated in XX-M and downregulated in XY-F are referred to as key genes for male reversal, while the female sex-biased genes that are upregulated in XY-F and downregulated in XX-M are referred to as key genes for female reversal. Finally, 12 differentially expressed genes (DEGs) related to male sex reversal were screened, including star2, cyp17a, cyp11b1, dmrt1, amh, sox9a, katnal1, spata4, spata6l, spata7, spata18 and foxl3. 2 DEGs (foxl2a and cyp19a1b) were found to be associated with female sex reversal. The changes in these genes collectively influence the direction of sex differentiation of LMB. Among them, star2, dmrt1 and cyp19a1b with significantly altered expression levels may play potentially crucial role in the process of gender reversal. The expression patterns of 21 randomly selected genes were verified using qRT-PCR which confirmed the reliability and accuracy of the RNA-seq results. These findings not only enhance our understanding of the molecular basis underlying sex reversal but also provide crucial data support for future breeding research on unisexual LMB.

Synthesis of steroid tracers with a oxyme ligation method and use thereof in fluorescent polarisation immunoassay

A method for the synthesis of ketosteroid oxime conjugates with a fluorescein (6-FAM) derivative connected via a short bifunctional aminooxypropyl linker is proposed. The conjugates were used as tracers for fluorescence polarization immunoassay (FPIA) with monoclonal antibodies. Stereoisomers by the oxime group were separated by preparative RP TLC and HPLC and tested in the FPIA method. Binding parameters of of tracers with monoclonal antibodies (mAbs) to progesterone were studied. Tracers showed high efficiency in the analysis of steroid hormones, the detection limit of progesterone being lower than that for a previously described analog. The Z-isomer was found to be more sensitive in the FPIA analysis compared to the E-isomer.

Towards Improved Steroid Hormone Analysis: The Shift from Immunoassays to Mass Spectrometry

Towards Improved Steroid Hormone Analysis: The Shift from Immunoassays to Mass Spectrometry

Substantial benefits of an inert biphenyl column for the analysis of steroids and their phase II metabolites in biological samples.

Steroids can be used as biomarkers in clinical metabolomics and other fields related to human toxicology. This chemical group is known for its complexity, considering its number of isobaric compounds and the wide variety of phases I and II metabolic pathways that parent compounds can undergo. For a successful analysis of steroids in biological samples, liquid chromatography separation must be finely tuned. It is especially challenging for glucuronidated and sulfated steroids derivatives that bear polar heads and can be affected by non-specific adsorption. The benefits of a biphenyl stationary phase chemistry for the selectivity of the separation of steroids and their phase II metabolites and the extent to which nonspecific adsorption phenomena could degrade chromatographic performance were investigated. Replacing a conventional hardware by a passivated hardware allowed to considerably reduce peaks width and asymmetry of sulfated species. The addition of weak ion pairing agents in the mobile phase could also help to reduce non-specific adsorption but are detrimental to mass spectrometry detection. As confirmed by the successful detection of 52 steroids in plasma, the use of a biphenyl stationary phase complemented by a passivated column hardware is of great help for a successful biomedical analysis of steroids and their phase II metabolites.

Steroidal 21-imidazolium salt derivatives: Synthesis and anticancer activity

Nitrogen-containing steroids are known as prostate cancer therapeutics. In this work, a series of pregnane derivatives bearing an imidazolium moiety were synthesized using Δ16-20-ketones as starting material. An improved approach for the construction of the 20-keto-21-heterocycle-substituted fragment involved the rearrangement of 16,17-epoxides with HCl, followed by reaction of the formed α-chloroketone with 1-substituted imidazoles. Binding affinity analysis of the imidazolium steroids and their synthetic intermediates to human CYP17A1 showed only type I (substrate-like) interactions. The strongest affinity was observed for 16α,17α-epoxy-5α-pregnan-20-on-3β-ol (Kd = 0.66 ± 0.05 µM). The steroid derivatives have been evaluated for antitumor activity against a range of prostate cancer cells as well as against various other solid tumor and hematologic cancer cell lines. All 21-imidazolium salts were active against the hormone-dependent prostate cancer line LNCaP. The most pronounced cytotoxicity in solid tumor and hematologic cancer cell lines was observed for intermediate product, 21-chloro-5α-pregn-16-en-20-on-3β-ol. Among the imidazolium salts, the derivatives with a single bond were more cytotoxic than their unsaturated congeners.

Innovative molecular networking analysis of steroids and characterisation of the urinary steroidome

Steroids are cholesterol-derived biomolecules that play an essential role in biological processes. These substances used as growth promoters in animals are strictly regulated worldwide. Targeted assays are the conventional methods of monitoring steroid abuse, with limitations: only detect known metabolites. Metabolism leads to many potential compounds (isomers), which complicates the analysis. Thus, to overcome these limitations, non-targeted analysis offers new opportunities for a deeper understanding of metabolites related to steroid metabolism. Molecular networking (MN) appears to be an innovative strategy combining high-resolution mass spectrometry and specific data processing to study metabolic pathways. In the present study, two databases and networks of steroids were constructed to lay the foundations for the implementation of the GNPS-MN approach. Steroids of the same family were grouped together, nandrolone and testosterone were linked to other analogues. This network and associated database were then applied to a few urine samples in order to demonstrate the annotation capacity in steroidome study. The results show that MN strategy could be used to study steroid metabolism and highlight biomarkers.

Circulating adrenal and gonadal steroid hormones heterogeneity in active young males and the contribution of 11-oxy androgens

The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency.

Mitochondrial dysfunction results in enhanced adrenal androgen production in H295R cells

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2 h) and slower accumulation of androstenedione and testosterone (24 h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17β-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

Cyanoacetohydrazide as a Novel Derivatization Agent for the Determination of UHPLC-HRMS Steroids in Urine.

The possibility of cyanoacetohydrazide usage as a novel derivatizing agent is demonstrated in the presented article, and a comparison with hydroxylamine as the most commonly used reagent is provided. Optimal conditions for steroid derivatization with cyanoacetohydrazide are provided. According to the collected data, the maximum yield of derivatives was observed at pH 2.8 within 70 min at 40 °C with 5 ng/mL limit of detection for all investigated analytes. It was shown that cyanoacetohydrazide derivatives produces both syn- and anti-forms as well as hydroxylamine, and their ratios were evaluated and shown in presented work. An efficiency enchantment from two to up to five times was achieved with a novel derivatization reagent. Its applicability for qualitative analysis of steroids in urine was presented at real samples. Additionally, the reproducible fragmentation of the derivatizing agent in collision-induced dissociation offers opportunities for simplified non-targeted steroidomic screening. Furthermore, cyanoacetohydrazide increases ionization efficiency in positive mode, which can eliminate the need for redundant high-resolution instrument runs required for both positive and negative mode analyses.

Sex and disease severity-based analysis of steroid hormones in ME/CFS

PurposeMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by persistent fatigue and decreased daily activity following physical and/or cognitive exertion. While ME/CFS affects both sexes, there is a higher prevalence in women. However, studies evaluating this sex-related bias are limited.MethodsCirculating steroid hormones, including mineralocorticoids (aldosterone), glucocorticoids (cortisol, corticosterone, 11-deoxycortisol, cortisone), androgens (androstenedione, testosterone), and progestins (progesterone, 17α-hydroxyprogesterone), were measured in plasma samples using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Samples were obtained from mild/moderate (ME/CFSmm; females, n=20; males, n=8), severely affected patients (ME/CFSsa; females, n=24; males, n=6), and healthy controls (HC, females, n=12; males, n=17).ResultsAfter correction for multiple testing, we observed that circulating levels of 11-deoxycortisol, 17α-hydroxyprogesterone in females, and progesterone in males were significantly different between HC, ME/CFSmm, and ME/CFSsa. Comparing two independent groups, we found that female ME/CFSsa had higher levels of 11-deoxycortisol (vs. HC and ME/CFSmm) and 17α-hydroxyprogesterone (vs. HC). In addition, female ME/CFSmm showed a significant increase in progesterone levels compared to HC. In contrast, our study found that male ME/CFSmm had lower circulating levels of cortisol and corticosterone, while progesterone levels were elevated compared to HC. In addition to these univariate analyses, our correlational and multivariate approaches identified differential associations between our study groups. Also, using two-component partial least squares discriminant analysis (PLS-DA), we were able to discriminate ME/CFS from HC with an accuracy of 0.712 and 0.846 for females and males, respectively.ConclusionOur findings suggest the potential value of including steroid hormones in future studies aimed at improving stratification in ME/CFS. Additionally, our results provide new perspectives to explore the clinical relevance of these differences within specific patient subgroups.Graphical abstract

Electric field-assisted dried blood spot sample preparation for analysis of steroids using LC–MS/MS

Despite being a minimally invasive sample source, dried blood spots (DBS) generally pose the challenge of efficient sample extraction for broad metabolomics applications. This is particularly true for the quantification of steroids using non-derivatized liquid chromatography tandem mass spectrometry assays. To address these limitations, we have demonstrated the use of electric field as a driver for sample preparation from DBS samples to simultaneously quantify testosterone (T), 17α-hydroxyprogesterone (17-OHP), progesterone (P), and cortisol (C), using both standard electroporation cuvettes, as well as a novel custom vertical electric field setup. Our findings, backed by computational modeling, show that a 10V DC application for 180 s can draw out twice the amount of the aforementioned steroids from both Whatman-903 and DMPK-C sample collection cards using our novel device when compared to standard solvent-based collection methods. This study not only introduces the use of electric field for sample preparation for metabolomics, but additionally introduces a novel device that eliminates the electric double layer effect in the process.