Analysis Of Steroids Research Articles

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma

Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

Natural Luffa cylindrica sponge sorbent for the solid phase extraction of estrone, 17-β-estradiol, and testosterone in aquaculture water

The bare natural fibrous skeleton of Luffa cylindrica (LC) sponge gourd was simply prepared and used as a sorbent in the vortex-assisted solid phase extraction (VA-SPE) of three steroid hormones, estrone (E1), 17-β-estradiol (E2), and testosterone (T) from aquaculture water samples. The extracted compounds were analysed by high performance liquid chromatography with a diode array detector (HPLC-DAD). Extraction with the prepared sorbent was highly efficient, completing within 10 min. The developed method showed good linearity in a concentration range of 15–1000 μg L−1 for E1, E2, and T. The limits of detection (LODs) were 9.6 ± 0.1 μg L−1 for E1, 9.8 ± 0.1 μg L−1 for E2, and 9.7 ± 0.1 μgL−1 for T. The respective limits of quantitation (LOQs) were 32.1 ± 0.3 μg L−1, 32.7 ± 0.4 μg L−1, and 32.2 ± 0.5 μg L−1. Good reproducibility (RSD < 3.5%, n = 6), good precision (RSD < 6.8%, n = 6), and excellent reusability (n = 22, RSD < 8%) were achieved. Recoveries from real spiked samples ranged from 70.2 ± 0.1% to 98.9 ± 4.7% with RSDs lower than 6.0%. The proposed method is simple, fast, inexpensive, and eco-friendly, with potential applications in the trace analysis of steroid hormones. Novelty statementOnly a few studies have focused on sample preparation for the simultaneous determination of steroid hormones in aquaculture water. The authors believe that the use of unmodified naturally available Luffa cylindrica as a solid phase extraction (SPE) sorbent has not yet been reported. This work successfully developed, for the first time from the bare fibrous skeleton of Luffa cylindrica, a very simple and low-cost SPE sorbent to extract E1, E2, and T for simultaneous determination by HPLC.

Flowers and Inflorescences of Selected Medicinal Plants as a Source of Triterpenoids and Phytosterols.

Steroids and triterpenoids are compounds valued for their various biological and pharmacological properties; however, their content in medicinal and edible plants is often understudied. Flowers have been consumed since the ancient times as a part of traditional cuisine and as alternative medicines. Currently, the interest in medicinal and edible flowers is growing since contemporary consumers are incessantly seeking innovative natural sources of bioactive compounds. The aim of this report was the GC-MS (gas-chromatography-mass spectrometry) analysis of steroid and triterpenoid content in flowers, inflorescences and leaves of several plants (Berberis vulgaris L., Crataegus laevigata (Poir.) DC., Pulsatilla vulgaris Mill., Rosa rugosa Thunb., Sambucus nigra L. and Vinca minor L.), applied in herbal medicine in various forms, including isolated flowers (Flos), inflorescences (Inflorescentia) or aerial parts (Herba, i.e., combined flowers, leaves and stems). The most abundant source of triterpenoids was V. minor flowers (6.3 mg/g d.w.), whereas the steroids were prevailing in P. vulgaris flowers (1.8 and 1.1 mg/g). The profiles of triterpenoid acids and neutral triterpenoids in C. laevigata and S. nigra inflorescences were particularly diverse, involving compounds belonging to lupane-, oleanane- and ursane-type skeletons. The obtained results revealed that some flowers can constitute an abundant source of phytosterols and bioactive triterpenoids, valuable for utilization in functional foods, dietary supplements and cosmetic products.

Prior to versus after Metformin Treatment-Effects on Steroid Enzymatic Activities.

Background: We recently reported that metformin administration has substantial effects on steroid hormone concentrations. In this study, we specifically explored which enzymatic activities were affected before a first treatment versus after a time of metformin treatment. Material and Methods: Twelve male subjects (54.2 ± 9.1 years, 177.3 ± 4.1 cm, 80 ± 10.4 kg) and seven female subjects (57.2 ± 18.9 years, 162.7 ± 4.1 cm, 76.1 ± 10.4 kg) were recruited based on an indication of metformin. Prior to the first intake of metformin and after 24 h, urine collections were performed. Urine steroid analysis was completed using gas chromatography-mass spectrometry. Results: The average reduction in steroid hormone concentrations after the metformin treatment was substantial and relatively equally distributed in all metabolites and the sum of all metabolites with 35.4%. An exception was dehydroepiandrosterone, with a decrease of almost three hundred percent of average concentration. In addition, the sum of all cortisol metabolites and 18-OH cortisol (indicative of oxidative stress) were lower after the metformin treatment. Furthermore, significant inhibition of 3ß-HSD activity was detectable. Discussion: Effects prior to and after the metformin treatment on inhibiting 3ß-HSD activity were detected in line with findings from others. Furthermore, the pattern of a reduction, for example, in the sum of all glucocorticoids following the metformin treatment supported an effect on oxidative stress, which was further supported by the reduction in 18-OH cortisol. Nevertheless, we do not understand all steps in the complex pattern of the enzymes that affect steroid hormone metabolism and, consequently, further studies are necessary to improve our understanding.

Unexpected Plasma Gonadal Steroid and Prolactin Levels Across the Mouse Estrous Cycle.

Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (±SEM) concentration of 11 ± 3 ng/mL and occurred coincident with the peak in progesterone levels (22 ± 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 ± 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 ± 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 ± 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.

Novel approaches on melatonin role: Presence of clock-hormone in fish seminal plasma

The study of melatonin is of great importance for the fundamental knowledge of any living system since it displays many different physiological roles, including being a potent natural antioxidant. To the best of our knowledge, there is no information regarding melatonin in fish seminal plasma. This study aimed to determine this clock-hormone levels in the seminal plasma of three aquaculture fish species: European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), and Senegalese sole (Solea senegalensis) (both wild and F1 breeders), and to explore melatonin potential role in fish reproduction and spermatozoa antioxidant status. Blood and seminal plasma were collected from fish during their reproductive season, at two different times of the day [mid-light (ML) and mid-dark (MD)], and melatonin concentration was determined by radioimmunoassay (RIA). Testosterone (T), 11-ketotestosterone (11KT), and total antioxidant status (TAS) were also determined, to investigate the putative role of seminal melatonin in fish reproduction, both at endocrine and antioxidant levels. For each species, Pearson's correlation analysis was performed between all possible factors. Blood plasma melatonin showed higher average values at night in the three species: gilthead seabream (808 ± 139 pg/mL), European seabass (364 ± 85 pg/mL), and Senegalese sole (248 ± 40 and 88 ± 11 pg/mL in F1 and wild males, respectively). However, melatonin levels in seminal plasma were species-specific: in European seabass, melatonin levels were not detectable at any time-point, whereas in gilthead seabream it was only found at MD (average of 21 pg/mL), and in Senegalese sole, different melatonin patterns were observed between F1 and wild males, but both had higher melatonin at MD (6.84 and 14.26 pg/mL, respectively). In gilthead seabream, at MD seminal melatonin levels correlated with the antioxidant status of seminal plasma. A relationship between blood melatonin and seminal TAS levels was observed in European seabass at ML: in this species, seminal melatonin could not be detected and the lowest seminal TAS levels were found. Regarding steroid analysis, opposite patterns in the seminal plasma of F1 and wild Senegalese sole were observed: at MD, wild Senegalese sole had substantially greater 11KT levels (2.53 ng/mL), whereas F1 males had higher T levels (1.92 ng/mL). In gilthead seabream, a positive correlation between T and ML blood melatonin and seminal TAS was observed. This study unraveled the species-specificity and daily changes of melatonin in fish seminal plasma.

The effect of etanercept therapy on adrenal steroid metabolism in juvenile idiopathic arthritis: a steroid metabolomics approach

ObjectiveTo evaluate the impact of anti-tumor necrosis factor-alpha (TNFα: etanercept [Etanercept ®]) therapy on adrenal activity in juvenile idiopathic arthritis (JIA) .MethodEleven JIA patients aged 12 ± 6.2 years with a disease duration of 6.3 ± 5.2 years were enrolled. They were treated once weekly with etanercept (0.8 mg/kg) for 3 ± 2.8 years. Urine samples for gas chromatography-mass spectrometry steroid hormone analysis were collected before, and 1 and 3 days after etanercept injection and compared to age- and sex-matched healthy controls.ResultsThe levels of 21 of the 31 metabolites were low before etanercept treatment. Those 21 metabolites included 4 C19 steroids (androgens), 5 C C21 steroid hormone intermediates, 10 cortisol metabolites, and 2 corticosterone metabolites. One day after treatment, only 5 of the 21 metabolite levels remained low. They included 2 C19 metabolites, 2 C21 steroid metabolites and 1 cortisol metabolite β –Cortol (β-Cl). Three days after treatment, the only metabolites levels that continued to be low were 2 C19 metabolite, 2 C21 steroid hormone intermediates and 1 cortisol metabolite α-Cortol (a-Cl), while the remaining 15 metabolites had already normalized after 1 day. Dehydroepiandrosterone-sulfate and 17-hydroxypregnenolone metabolite levels were the last ones to recover. Urinary metabolite ratios reflecting cytochrome P450 CYP21A2 (21-hydroxylase) and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzymatic activitieswere lower in JIA patients than in controls, although significant was not reached.ConclusionAlmost all of the pre-etanercept treatment cortisol urinary metabolite levels were significantly lower than normal, and almost all rose to normal values by 1 day after treatment. The therapeutic effect of anti-TNFα treatment in JIA may be related to its effect on the restoration of adrenal function and cortisol levels.

Quantitation of Ecdysterone and Targeted Analysis of WADA-Prohibited Anabolic Androgen Steroids, Hormones, and Metabolic Modulators in Ecdysterone-Containing Dietary Supplements

The aim of our research was to perform a comprehensive study of ecdysterone (ECD)-containing dietary supplements (DSs). Two analytical methods were optimised according to the expected concentration of the target compounds: quantitation of ECD by liquid chromatography (LC) coupled to diode array detector (DAD), and limit test for 47 World Anti-Doping Agency prohibited by LC coupled with tandem mass spectrometer (MS/MS). For quantitation of ECD, the method was fully validated with outstanding performance characteristics (LOD: 35 µg·g−1, LOQ: 115 µg·g−1, CV% &lt; 5%), resulting in significantly lower LOD and shorter runtime than published previously. For limit tests, a chromatographic method was developed to obtain excellent separation, while MS/MS parameters were optimised to allow the lowest possible reporting limit (RL: 0.6–10 ng·g−1 or mL−1). Twenty-one ecdysterone-containing DSs from ten brands were analysed. In all cases, the measured ECD content was much lower than labelled, and 20% of the samples contained a prohibited substance. The concentration of ecdysterone and contaminations varied randomly from batch to batch. The developed methods help to prevent the use of contaminated or useless DSs.

Association between days alive without life support/out of hospital and health-related quality of life.

Trials in critically ill patients increasingly focus on days alive without life support (DAWOLS) or days alive out of hospital (DAOOH) and health-related quality of life (HRQoL). DAWOLS and DAOOH convey more information than mortality and are simpler and faster to collect than HRQoL. However, whether these outcomes are associated with HRQoL is uncertain. We thus aimed to assess the associations between DAWOLS and DAOOH and long-term HRQoL. Secondary analysis of the COVID STEROID 2 trial including adults with COVID-19 and severe hypoxaemia and the Handling Oxygenation Targets in the Intensive Care Unit (HOT-ICU) trial including adult intensive care unit patients with acute hypoxaemic respiratory failure. Associations between DAWOLS and DAOOH at day 28 and 90 and long-term HRQoL (after 6 or 12 months) using the EuroQol 5-dimension 5-level survey (EQ VAS and EQ-5D-5L index values) were assessed using flexible models and evaluated using measures of fit and prediction adequacy in both datasets (comprising internal performance and external validation), non-parametric correlation coefficients and graphical presentations. We found no strong associations between DAWOLS or DAOOH and HRQoL in survivors at HRQoL-follow-up (615 and 1476 patients, respectively). There was substantial variability in outcomes, and predictions from the best fitted models were poor both internally and externally in the other trial dataset, which also showed inadequate calibration. Moderate associations were found when including non-survivors, although predictions remained uncertain and calibration inadequate. DAWOLS and DAOOH were poorly associated with HRQoL in adult survivors of severe or critical illness included in the COVID STEROID 2 and HOT-ICU trials.

Recommendations for the measurement of sexual steroids in clinical practice. A position statement of SEQCML/SEEN/SEEP.

The proper clinical approach to a wide range of disorders relies on the availability of accurate, reproducible laboratory results for sexual steroids measured using methods with a high specificity and sensitivity. The chemiluminescent immunoassays currently available have analytical limitations with significant clinical implications. This position statement reviews the current limitations oflaboratory techniques for the measurement of estradiol and testosterone and their impact on diverse clinical scenarios. A set of recommendations are provided to incorporate steroid hormone analysis by mass spectrometry in national health systems. International societies have recommended this methodology for a decade.

Steroid profiling for the diagnosis of congenital adrenal hyperplasia by microbore ultra-performance liquid chromatography–tandem mass spectrometry

BackgroundA rapid and accurate measurement approach for 17α-hydroxyprogesterone (17-OHP) and related steroids in amount/volume-limited clinic samples is of importance for precise newborn diagnosis of congenital adrenal hyperplasia (CAH) and its subtypes in clinic. MethodsSixteen steroids (17-OHP, androstenedione, cortisol, tetrahydro-11-deoxycortisol, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, dehydroepiandrosterone, testosterone, aldosterone, 17α-hydroxypregnenolone, dihydrotestosterone and 18-hydroxycorticosterone) were included in the panel of high-throughput microbore ultra-performance liquid chromatography-tandem mass spectrometry. Samples were collected from 126 normal subjects and 65 patients including different subtypes of CAH. ResultsThe method was validated with satisfactory analytical performance in linearity, repeatability, recovery and limit of detection. Reference intervals for 16 steroids were established by quantifying the level of steroids detected in normal infants. The applicability of the method was tested by differentiating steroid metabolic characteristics between normal infants and infants with CAH, as well as between infants with different CAH subtypes. The relevance of 17-OHP, 21-deoxycortisol, and 17-OHP/11-deoxycortisol for 21-hydroxylase deficiency screening was demonstrated. The level of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and androstenedione can be used for the diagnosis of different rare subtypes of CAH. ConclusionThis study provides a strategy for highly efficient steroid analysis of amount/volume-limited clinic samples and holds great potential for clinical diagnosis of CAH.

Dietary aromatase inhibitor treatment converts XY gsdf homozygous mutants to sub-fertile male in Nile tilapia (Oreochromis niloticus)

Gonadal soma-derived factor (gsdf) belongs to the TGF-β superfamily and is critical for sex determination and differentiation in fish. XY gsdf−/− Nile tilapia (Oreochromis niloticus) show male-to-female sex reversal 1 month after hatching (mah) and are infertile at the adult age; however, the functions of gsdf in spermatogenesis in fish are unclear. Herein, XY gsdf−/− Nile tilapia were converted to males with testis by feeding 200 μg/g letrozole (LE), an aromatase inhibitor, from 1 to 4 mah. The testis somatic index of XY gsdf−/− LE fish was significantly lower than that of XY gsdf+/+ LE fish at 6 mah. The artificial fertilization ratio of XY gsdf−/− LE fish sperm (53.22 ± 4.35%) was significantly lower than that of XY gsdf+/+ LE fish (86.33 ± 1.53%), indicating that XY gsdf−/− LE male fish were sub-fertile. Testes of LE XY gsdf−/− and gsdf+/+ fish at 6 mah were used for a transcriptome analysis. Compared with the testis of XY gsdf+/+ LE fish, 1426 and 1654 genes were up-regulated and down-regulated in the testis of XY gsdf−/− LE individuals, respectively. A KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that the differentially expressed genes (DEGs) were engaged in 21 pathways, including cancer, cell growth and death, and endocrine system pathways. Genes related to steroidogenesis and meiosis, such as cyp19a1a, foxl2, and cyp17a1, were significantly up-regulated, whereas sycp3, dmc1, and aldh1a1 were significantly down-regulated in the testis. In addition, a serum steroid hormone analysis showed that 17β-estradiol and 11-ketotestosterone levels of XY gsdf−/− LE fish were significantly higher and lower than those of XY gsdf+/+ LE fish. These findings demonstrated that endocrine system disruption in XY gsdf−/− LE might explain the decrease in fertility. This research establishes the groundwork for future research on the function of gsdf in spermatogenesis in fish.

Analysis of salivary steroid hormones in boys with autism spectrum disorder

BackgroundAutism spectrum disorders (ASD) is a neurodevelopmental disorder with high incidence rate and difficult diagnosis. The purpose of this study was to explore whether salivary cortisol, dehydroepiandrosterone (DHEA) and pregnenolone can be used as biomarkers of ASD children.MethodsThe saliva samples of 55 boys with ASD were collected as the experimental group, and the saliva samples of 24 neurotypical boys were collected as the control group. The Child Behavior Checklist (CBCL), Autism Behavior Checklist (ABC), Social Responsiveness Scale (SRS), Repetitive Behavior Scale (RBS) were used to assess the severity of symptoms in boys with ASD. Cortisol, DHEA and pregnenolone concentrations in saliva were measured using an ABSSCIEX QTRAP® 6500 + LC/MS/MS system. SPSS 23.0 was used for statistical analysis. Comparisons between the two groups which conform to normal distribution were performed by T-test, and those which don’t conform to normal distribution were performed by Mann–Whitney U test. Correlation analysis between two variables was performed using Spearman's correlation analysis. Receiver operating characteristic curve (ROC) analysis was performed to evaluate the discriminatory sensitivity of each hormone between ASD and normal control groups. Logistic regression models were used to analyze whether DHEA and salivary pregnenolone can be used as a biomarker of ASD.ResultsThere were no significant differences in age, and weight between the ASD group and the normal control group. The ABC, SRS, RBS and CBCL scale scores in the ASD group were significantly higher than those in the normal control group. The salivary DHEA and pregnenolone concentrations in the ASD group were significantly higher than those in the normal control group, but there was no significant difference in cortisol. Spearman's correlation analysis showed that only pregnenolone associated with ABC. Logistic regression model analysis suggested that pregnenolone in saliva was an independent predictor of ASD. ROC analysis found that pregnenolone had good discrimination sensitivity between ASD and normal controls.ConclusionGave salivary preoperative a space for utilization as biomarker as number of cases are limited to this high expectation.

Using LC-MS/MS to Determine Salivary Steroid Reference Intervals in a European Older Adult Population.

A number of steroids, including glucocorticoids and sex hormones, have been associated with neurodegenerative and cardiovascular conditions common in aging populations. The application of liquid chromatography tandem mass spectrometry (LC-MS/MS) steroid analysis offers an opportunity to conduct simultaneous multiplex steroid analysis within a given sample. In this paper, we describe the application of an LC-MS/MS steroid analysis method for the assessment of reference ranges of steroids in human saliva samples (200 µL) collected from older adults (age 50 years and above) enrolled in a European cohort investigating the risk for Alzheimer's dementia. Saliva samples were prepared using supported liquid extraction (SLE) along with a calibration curve and analysed using a Waters I-Class UPLC (Ultra Performance Liquid Chromatography) and a Sciex QTrap 6500+ mass spectrometer. Mass spectrometry parameters of steroids were optimised for each steroid and a method for the chromatographic separation of 19 steroids was developed. Lower limits of quantitation (LLOQs), linearity and other method criteria were assessed. In total, data from 125 participants (500 samples) were analysed and assessed for reference ranges (64 male, 61 female). A total of 19 steroids were detected in saliva within the range of the method. There were clear diurnal patterns in most of the steroid hormones detected. Sex differences were observed for androstenedione (A4), testosterone (T), cortisone (E) and aldosterone (Aldo). In the first sample of the day, dehydroepiandrosterone (DHEA) was significantly higher in healthy volunteers compared to those with Alzheimer's disease biomarkers. This LC-MS/MS method is suitable for the analysis of 19 steroids in saliva in adults.

A New Heart-Cutting Method for a Multiplex Quantitative Analysis of Steroid Hormones in Plasma Using 2D-LC/MS/MS Technique.

The aim of the current research was to develop a simple and rapid mass spectrometry-based assay for the determination of 15 steroid hormones in human plasma in a single run, which would be suitable for a routine practice setting. For this purpose, we designed a procedure based on the 2D-liquid chromatography-tandem mass spectrometry with a minimalistic sample pre-treatment. In our arrangement, the preparation of one sample takes only 10 min and can accommodate 40 samples per hour when tested in series. The following analytical run is 18 min long for all steroid hormones. In addition, we developed an independent analytical run for estradiol, significantly increasing the assay accuracy while taking an additional 10 min to perform an analytical run of a sample. The optimized method was applied to a set of human plasma samples, including chylous. Our results indicate the linearity of the method for all steroid hormones with squared regression coefficients R2 ≥ 0.995, within-run and between-run precision (RSD < 6.4%), and an accuracy of 92.9% to 106.2%. The absolute recovery for each analyzed steroid hormone ranged between 101.6% and 116.5%. The method detection limit for 15 steroid hormones ranged between 0.008 nmol/L (2.88 pg/mL) for aldosterone and 0.873 nmol/L (0.252 ng/mL) for DHEA. For all the analytes, the lowest calibration point relative standard deviation was less than 10.8%, indicating a good precision of the assay within the lowest concentration of interest. In conclusion, in this method article, we describe a simple, sensitive, and cost-effective 2D-LC/MS/MS method suitable for the routine analysis of a complex of steroid hormones allowing high analytical specificity and sensitivity despite minimal sample processing and short throughput times.

Phytochemical Evaluation of Different Plant Sources of Vaasa (Adhatoda Species)

Introduction: Vaasa, a valuable medicinal plant widely used in Ayurvedic medicine, has experienced growing demand, resulting in the availability of both wild and cultivated sources. Experts have identified morphotypes of Adhatoda species in Kerala, indicating variations in active constituents and clinical efficacy. This study specifically focused on assessing physicochemical parameters and phytochemical constituents of different sources of Vaasa. Materials and Methods: The parameters such as foreign matter, total ash, acid insoluble ash, water insoluble ash, moisture content, volatile oil content, fiber content, tannin content, sugar content, phenol content, pH, qualitative analysis of ash, extractive values, alkaloids, saponins, flavonoids, tannins, steroids, phenols, carbohydrates, and proteins were estimated in different sources of Vaasa (Adhatoda species): A1 (Valiya adalodakam), A2 (Cheriya adalodakam), A3 (Adhatoda species - Vasika), A4 (Adhatoda species - Ajagandhi), and A5 (Adhatoda beddomei C B Clarke). Results and Discussion: The results revealed comparable physicochemical parameters between sample A1 and Adhatoda vasica Nees, as well as between sample A5 and Adhatoda beddomei C B Clarke, as documented in authentic texts and research articles. In qualitative analysis, no differences were found between samples A2, A3, A4, and A5 compared to sample A1. The study revealed quantitative differences in physicochemical and phytochemical evaluation among the powdered leaf samples. Conclusion: Qualitative analysis did not reveal significant differences among samples A2, A3, A4, and A5 compared to sample A1. However, quantitative variations were observed in both physicochemical and preliminary phytochemical evaluation of the powdered leaf sample. The findings in this study contribute to a better understanding and identification of various Vaasa sources in Ayurvedic medicine, essential for quality control and further research.

PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF Zingiber officinale AND Syzygium aromaticum ON BACTERIA PATHOGENS ISOLATED FROM KUNUN-AYA

The increased use of antibiotics in agricultural practices is accompanied with the development of resistant foodborne pathogens. Spices and herbs have been reported to be potent source of natural antioxidants. This study aimed to assess the antibacterial activity of Zingiber officinale (Ginger) and Syzygium aromaticum (Clove) against bacterial food pathogens. Fresh ginger andclove were collected, identified, air dried and extracted the bioactive compound using distilled water. The extract was used for phytochemical analysis of flavonoids, tannins, saponins, steroids, glycosides, alkaloids and total phenols and determination of inhibition zone. Bacteria and fungi were isolated and identified from locally prepared Pap (Kunun aya) and later used for theantimicrobial analyses. Phytochemical screening shows clove contain flavonoids, tannins, saponins, steroids, glycosides, alkaloids and total phenols whereas glycosides and tannins were absent in ginger. Ginger has more antibacterial activity with the highest zone of inhibition observed in E coli at 16.73mm at 2000µg and the lowest in Salmonella at 13.83mm at 2000µg, clove also shows antibacterial activity with the highest zone of inhibition (14.8mm) in S. aureus at 2000µg and the lowest 11.5mm in E. coli at 2000µg. Thus, from the result obtained, it can be concluded that Zingiber officinale and Syzygium aromaticum extracts possesses effective antimicrobial agents against food pathogens.

Semi-automated serum steroid profiling with tandem mass spectrometry

ObjectivesHighly selective and sensitive multi-analyte methods for the analysis of steroids are attractive for the diagnosis of endocrine diseases. Commercially available kits are increasingly used for this purpose. These methods involve laborious solid phase extraction, and the respective panels of target analytes are incomplete. We wanted to investigate whether an improvement of kit solutions is possible by introducing automated on-line solid phase extraction (SPE) and combining originally separate analyte panels. MethodsSample preparation was performed using automated on-line SPE on a high-pressure stable extraction column. Chromatographic separation, including isobaric compounds, was achieved using a 0.25 mM ammonium fluoride-methanol gradient on a small particle size biphenyl column. Standard compounds and internal standard mixtures of two panels of a commercially available kit were combined to achieve an optimized and straightforward detection of 15 endogenous steroids. Validation was performed according to the European Medicines Agency (EMA) guidelines with slight modifications. ResultsValidation was successfully performed for all steroids over a clinically relevant calibration range. Deviations of intra- and inter-assay accuracy and precision results passed the criteria and no relevant matrix effects were detected due to highly effective sample preparation. External quality assessment samples showed the applicability as a routine diagnostic method, which was affirmed by the analyses of anonymized clinical samples. ConclusionsIt was found possible to complement a commercially available kit for quantitative serum steroid profiling based on isotope dilution LC-MS/MS by implementing automated on-line SPE, thereby improving the practicality and robustness of the measurement procedure.

Ultra-performance convergence chromatography tandem mass spectrometry analysis of adrenal and gonadal steroid hormones in southern white rhinoceros (Ceratotherium simum simum) faeces and serum

Steroid hormone analysis is routinely undertaken in the assessment of stress response and reproductive function in the management of both captive and free-ranging wildlife species. Faecal samples have become the preferred sample type for analysis as collection is non-invasive and easily assessable. These investigations are generally aimed at aiding successful translocations, enhanced survival outcomes in captivity and improvement of reproductive rate. Immunoassays are the most common approach in the analysis of hormones, particularly in the case of the southern white rhinoceros (Ceratotherium simum simum). Non-specificity, attributed to structural similarity of steroid metabolites impedes accurate evaluations which can be eliminated by chromatographic techniques which are more specific, selective and provide comprehensive analyses.This study developed and validated three methods using ultra-performance convergence chromatography tandem mass spectrometry for the assessment of classical androgens, progestogens and adrenal steroids, as well as the C11-oxy androgens and C11-oxy progestogens in serum and faeces from white rhinoceros. The limit of detection and quantification were determined for each steroid, parameters such as accuracy (<19.8 % RSD) and precision (<20.2 % RSD) were established with recovery, matrix effect, and process efficiency within acceptable limits. Subsequent analysis of serum and faecal samples from five white rhinoceros identified novel steroids for the first time in this species. In addition to the classical adrenal steroids, the following C11-oxy steroids were detected in faecal samples: 11α-hydroxydihydroprogesterone (168 ng/g), 11α-hydroxyprogesterone (125.9 ng/g), 11β-hydroxyprogesterone (210.2 ng/g) and 11-ketoandrostenedione (3.3–19.6 ng/g) with 11-deoxycortisol being the major glucocorticoid (24.2–67.3 ng/g) together with 21-deoxycortisone (40.7 ng/g) and deoxycorticosterone (7.6–14.6 ng/g). In serum samples 11β-hydroxyandrostenedione (0.35–2.34 ng/mL) and 11β-hydroxytestosterone (0.18–1.62 ng/mL) were the predominant androgens with cortisol (5.8–20.5 ng/mL), the predominant glucocorticoid, while corticosterone, 18-hydroxycorticosterone and aldosterone were also detected.These methods can be applied independently to assess either androgens, progestogens, or adrenal steroid panels or in combination to assess the cohort of gonadal and adrenal steroids in faeces and/or serum, in southern white rhinoceros as well as other wildlife species. Analysis would enable the accurate assessment of reproductive health and stress responses while also distinguishing between stress and distress thus contributing to the conservation of wildlife species.

Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17β-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.