Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE®) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS-MS)

Illegal steroid administration to enhance growth performance in veal calves has long been, and still is, a serious issue facing regulatory agencies. Over the last years, stating undisputable markers of illegal treatment has become complex because of the endogenous origin of several anabolic steroids. Knowledge on the origin of an analyte is therefore of paramount importance. The present study shows the presence of steroid analytes in wooden crates used for housing veal calves. For this purpose, an analytical procedure using accelerated solvent extraction (ASE®), solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-MS-MS) is developed for the characterisation of androstadienedione (ADD), boldenone (bBol), androstenedione (AED), β-testosterone (bT), α-testosterone (aT), progesterone (P) and 17α-hydroxy-progesterone (OH-P) in wood samples. In samples of wooden crates used for housing veal calves, ADD, AED, aT and P could be identified. Using the standard addition approach concentrations of these analytes were determined ranging from 20?±?4 ppb to 32?±?4 ppb for ADD, from 19?±?5 ppb to 44?±?17 ppb for AED, from 11?±?6 ppb to 30?±?2 ppb for aT and from 14?±?1 ppb to 42?±?27 ppb for P, depending on the sample type. As exposure of veal calves to steroid hormones in their housing facilities might complicate decision-making on illegal hormone administration, inequitable slaughter of animals remains possible. Therefore, complete prohibition of wooden calf accommodation should be considered.     

Endogenous Urinary Steroids in Postmenopausal Women with Epithelial Ovarian Cancer

The profile of endogenous steroids in the presence of ovarian cancer was studied. Urine samples from 15 postmenopausal women with epithelial ovarian cancer and ten age-matched healthy women were collected for 24 h. The concentration of 23 androgen, progesterone and corticoid metabolites in the urine samples of the two groups were quantitatively determined by gas chromatography–mass spectrometry with selected ion-monitoring. In the course of the urinary steroid determination we observed changes in the steroid profiles in the diseases examined compared to the same age and same sex control groups. Profiling urinary steroids has to give a comprehensive information about the synthesis of steroids including the glandular and peripheral steroid metabolisms. The concentrations of An, Et, 11-OH-An, 11-OH-Et, Δ5-PT, THE, THF, α-CL, β-CL and α-C were not significantly different in the two groups. The concentrations of DHEA, 16-OHD, PD, PT, Δ5-AT, THA, THB, aTHB and aTHF were significantly higher in the postmenopausal women with epithelial ovarian cancer than in the controls. The levels of Δ5-PD, THS, 11-OPT and F could not be determined because the concentrations of these compounds were below the limit of detection or the lower limit of quantification. The changes in the levels of single metabolites point out the important role of steroid groups, thus providing help in the recognition and treatment of diseased states.

     

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies.

Sex and age related Ca/P ratio in cortical bone of iliac crest of healthy humans

The mechanical strength of bone depends first of all on the condition of the cortical bone. In the present study the Ca/P ratio was estimated in intact cortical bone samples from iliac crest of healthy humans, 36 women and 44 men, aged from 15 to 55 years using INAA. No statistically significant differences (p>0.05) were observed of age- or sex-related Ca/P ratio. The mean value (M±SD) of the Ca/P ratio for the whole group studied, 2.21±0.29, was within the range of published data and close to the median value. This revised version was published online in August 2006 with corrections to the Cover Date.     

HPLC‐fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC‐mode column

High‐performance liquid chromatography–fluorescence detection using a hydrophilic interaction chromatography‐mode column (ZIC®‐HILIC) was used to determine four kinds of thiol compounds in human serum. Sera were obtained from 34 subjects for this study (17 male subjects aged 22–38 years and 17 female subjects aged 18–38 years). Serum cysteine, cysteinylglycine, glutathione, and γ‐glutamylcysteine, derivatized with ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulfonate, were separated on the ZIC®‐HILIC column and quantified. The serum concentrations of cysteine, cysteinylglycine, glutathione and γ‐glutamylcysteine were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 μm , respectively. In addition, the concentrations of serum thiol compounds from male subjects were significantly higher than those of the female subjects (p < 0.05). Copyright © 2014 John Wiley & Sons, Ltd.     

Under‐reporting of food intake is frequent among Brazilian free‐living older persons: a doubly labelled water study

The assessment of food intake is essential for the development of dietetic interventions. Accuracy is low when intake is assessed by questionnaires, the under‐reporting of food intake being frequent. Most such studies, however, were performed in developed countries and there is little data about the older population of developing nations. This study aimed to verify the total energy expenditure (TEE) of independent older Brazilians living in an urban area, through the doubly labelled water (DLW) method and to compare it with the reported energy intake obtained through the application of a food frequency questionnaire (FFQ). Initially, 100 volunteers aged from 60 to 75 years had their body composition determined by dual‐energy X‐ray absorptiometry (DEXA). Five volunteers of each quartile of body fat percentage had their energy expenditure determined by DLW. The mean age of the subjects included in this phase of the study was 66.4 ± 3.5 years, and ten of the subjects were men. The mean TEE was 2565 ± 614 and 2154 ± 339 kcal.day^?1 for men and women, respectively. The Physical Activity Level (PAL) was 1.58 ± 0.31 and 1.52 ± 0.22, respectively. Under‐reporting of food intake was highly prevalent, with a mean percentage of reported intake in relation to measured TEE of ?17.7%. Thus, under‐reporting of food intake is highly prevalent among Brazilian independent older persons. The DLW method is an important tool in nutritional studies and its use is to be recommended in developing countries. Copyright © 2010 John Wiley & Sons, Ltd.     

Detection of catecholamine metabolites in urine based on ultra-high-performance liquid chromatography–tandem mass spectrometry

The excretion of neurotransmitter metabolites in normal individuals is of great significance for health monitoring. A rapid quantitative method was developed with ultra-performance liquid chromatography–tandem mass spectrometry. The method was further applied to determine catecholamine metabolites vanilymandelic acid (VMA), methoxy hydroxyphenyl glycol (MHPG), dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) in the urine. The urine was collected from six healthy volunteers (20–22 years old) for 10 consecutive days. It was precolumn derivatized with dansyl chloride. Subsequently, the sample was analyzed using triple quadrupole mass spectrometry with an electrospray ion in positive and multireaction monitoring modes. The method was sensitive and repeatable with the recoveries 92.7–104.30%, limits of detection (LODs) 0.01–0.05 μg/mL, and coefficients no less than 0.9938. The excretion content of four target compounds in random urine samples was 0.20 ± 0.086 μg/mL (MHPG), 1.27 ± 1.24 μg/mL (VMA), 3.29 ± 1.36 μg/mL (HVA), and 1.13 ± 1.07 μg/mL (DOPAC). In the urine, the content of VMA, the metabolite of norepinephrine and adrenaline, was more than MHPG, and the content of HVA, the metabolite of dopamine, was more than DOPAC. This paper detected the levels of catecholamine metabolites and summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.     

Urinary steroids in premenopausal women with breast cancer at the time of surgery

Summary Urinary steroid metabolites were measured by capillary gas chromatogaphy in 10 premenopausal women with operable breast cancer and 16 control women a day before operation. There were significant decreases in the levels of androsterone (A), main cortisol (F) metabolites/tetrahydrocortisone (THE), tetrahydrocortisol (THF), allo-tetrahydrocortisol (aTHF)/and β-cortolone (βCL). The lower adrenal function accompanies breast cancer in premenopausal women. As the number of patients included in the study is small our conclusions must be considered as preliminary. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

慢性心功能衰竭患者尿液的代谢组学研究

采用基于液相色谱-质谱联用的方法对慢性心力衰竭(Chronic heart failure, CHF)患者和正常对照(Control)人群的尿液进行分析, 筛选慢性心力衰竭患者尿液中的差异代谢物, 研究其发病机制, 并为临床治疗提供科学依据.选择15个慢性心力衰竭患者(年龄(62.27±3.14)岁)及15个正常人(年龄(65.41±4.63)岁), 采用高分辨度快速液相色谱-四极杆-飞行时间串联质谱(RRLC-QTOF/MS)技术对尿液代谢物进行分析, 采用主成分分析(PCA)对两组代谢物进行分类, 并筛选潜在生物标记物;运用偏最小二乘判别分析法(PLS-DA)建模, 考察生物标记物对疾病筛选的预测能力.研究结果表明, CHF组和Control组尿液代谢物谱能得到很好的区分, 发现并鉴定了2种潜在生物标记物尿苷及丙氨酰色氨酸, 提示嘧啶代谢和色氨酸代谢可能在心力衰竭发生发展中有重要作用.     

Urinary metabolic profiling of volatile organic compounds in acute exposed volunteers after an oil spill in Republic of Korea

The Herbei Spirit oil spill occurred in western Korea. A large number of people who participated in the cleanup tasks of the contaminated area were exposed to crude oil component. We developed a method to monitor volatile organic compound (VOC) metabolites in urine, and evaluate the acute exposure caused by the oil spill in exposed volunteer workers (n = 100, 20.7 ± 2.1 years, mean ± SD). Acidified urine samples were extracted by SPE, derivatized with trimethylsilyl, and analyzed using gas chromatography–mass spectrometry. Calibration curves were found to be linear from 3 to 1000 ng/mL (r² > 0.993). Accuracy was over 82.4%, and precision was less than 24.8%. Using this method, the VOC metabolites, except hippuric acid, were present at higher levels in the urine samples of volunteers after cleanup work. The levels of mandelic acid (MA) and trans,trans‐muconic acid (t,t‐MU) were increased significantly (p < 0.001). The exposure effect was greater in women than in men. The effect of smoking was analyzed in all exposed and non‐exposed groups, with non‐smokers showing increased MA and t,t‐MU levels related to exposure. The present method was reliable to determine VOC metabolites in urine and could be useful for biomonitoring of acute exposure effects of VOCs. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolism of methylstenbolone studied with human liver microsomes and the uPA+/+‐SCID chimeric mouse model

Anti‐doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono‐hydroxylated derivatives (U1–U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di‐hydroxylated metabolites (U11–U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC‐MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of imaging mass spectrometry for the analysis of Oryza sativa rice

Rice is one of the most important food crops in the world and new varieties have been bred for specific purposes, such as the development of drought‐resistance, or the enrichment of functional food factors. The localization and composition of metabolites in such new varieties must be investigated because all artificial interventions are expected to change the metabolites of rice. Imaging mass spectrometry using matrix‐assisted laser desorption/ionization (MALDI‐IMS) is a suitable tool for investigating the localization and composition of metabolites; however, suitable methodologies for the MALDI‐IMS analysis of rice have not yet been established. In this study, we optimized the methods for analyzing rice grains by MALDI‐IMS using adhesive film and found the characteristic distribution of metabolites in rice. Lysophosphatidylcholine (LPC) was localized in the endosperm. Phosphatidylcholine (PC), γ‐oryzanol and phytic acid were localized in the bran (germ and seed coat), and α‐tocopherol was distributed in the germ (especially in the scutellum). In addition, MALDI‐IMS revealed the LPC and PC composition of the rice samples. The LPC composition, LPC (1‐acyl 16:0), LPC (1‐acyl 18:2), LPC (1‐acyl 18:1) and LPC (1‐acyl 18:0), was 59.4 ± 4.5%, 19.6 ± 2.5%, 14.2 ± 4.5% and 6.8 ± 1.4%. The PC composition, PC (diacyl 16:0/18:2), PC (diacyl 16:0/18:1), PC (diacyl 18:1/18:3), PC (diacyl 18:1/18:2) and PC (diacyl 18:1/18:2), was 19.6 ± 1.0%, 21.0 ± 1.0%, 15.0 ± 1.4%, 26.7 ± 0.7% and 17.8 ± 1.9%. This approach can be applied to the assessment of metabolites not only in rice, but also in other foods for which the preparation of sections is a challenging task. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative metabolic profiling of Costus speciosus leaves and rhizomes using NMR,GC-MS and UPLC/ESI-MS/MS

Costus speciosus had been used in oriental systems of medicines, to treat diverse ailments. The present study was focused on NMR, GC-MS and UPLC/ESI-MS/MS-based metabolic profiling of C. speciosus. This metabolic study resulted in the identification of 91 and quantification of 69 metabolites. Caffeic acid derivatives previously unreported in C. speciosus were also identified. High quantity of steroidal saponins namely methyl protogracillin (297.97 ± 0.07 mg/g dried wt.) and dioscin (158.72 ± 0.27 mg/g dried wt.) were observed in butanol fraction of rhizomes. Health care metabolites including caffeic acid (37.88 ± 0.04 mg/g dried wt.) and trehalose (75.12 ± 0.08 mg/g dried wt.) were also detected in ethyl acetate and aqueous fractions of rhizomes, respectively. Metabolites of nutraceutical and biological significance including eremanthine (5.14 ± 0.68%, peak area), tocopherols (~22%), sterols (~25%) were also identified from hexane fractions of rhizomes and leaves using GC-MS. The analytical techniques used had successfully differentiated metabolites composition among leaves and rhizomes.     

Application of INAA in the assessment of selected elements in cancellous bone of human iliac crest

The effect of age and sex was investigated on the concentration of chemical elements in intact cancellous bone of iliac crest of 74 relatively healthy, 15–55 years old women (n = 29) and men (n = 45). Concentrations of Ca, Cl, K, Mg, Mn, Na, P, and Sr in bone samples were determined by instrumental neutron activation analysis using short-lived radionuclides. Mean values (M±S.E.M.) of the mass fraction of the investigated elements (on dry weight basis) for female and male all together were: 127±4 g/kg, 1620±80 mg/kg, 1310±70 mg/kg, 1550±50 mg/kg, <0.32±0.02 mg/kg, 4240±110 mg/kg, 61.8±1.8 g/kg, and 235±18 mg/kg, respectively. The statistically significant (≤0.05) decrease of Ca, Mg, and P concentrations in the iliac cancellous bone with age was found only for women. Sex-related comparison has shown that the mean values of Mg mass fractions in male bone samples were less than in female ones.     

High-Energy Collision-Induced Dissociation by MALDI TOF/TOF Causes Charge-Remote Fragmentation of Steroid Sulfates

A method for structural elucidation of biomolecules dating to the 1980s utilized high-energy collisions (~10 keV, laboratory frame) that induced charge-remote fragmentations (CRF), a class of fragmentations particularly informative for lipids, steroids, surfactants, and peptides. Unfortunately, the capability for high-energy activation has largely disappeared with the demise of magnetic sector instruments. With the latest designs of tandem time-of-flight mass spectrometers (TOF/TOF), however, this capability is now being restored to coincide with the renewed interest in metabolites and lipids, including steroid-sulfates and other steroid metabolites. For these metabolites, structure determinations are required at concentration levels below that appropriate for NMR. To meet this need, we explored CRF with TOF/TOF mass spectrometry for two groups of steroid sulfates, 3-sulfates and 21-sulfates. We demonstrated that the current generation of MALDI TOF/TOF instruments can generate charge-remote fragmentations for these materials. The resulting collision-induced dissociation (CID) spectra are useful for positional isomer differentiation and very often allow the complete structure determination of the steroid. We also propose a new nomenclature that directly indicates the cleavage sites on the steroid ring with carbon numbers.

Radioimmunoassay of Plasma 16a-Hydroxyprogesterone

Abstract

This report describes a radioimmunoassay method for the measurement of unconjugated 16a-hydroxyprogesterone (16a-P) in plasma. Utilizing an antiserum against 16a-P combined with a one step celite microcolumn chromatographic system, we have measured this steroid accurately in small a liquors of plasma from both male and female subjects. The mean value ± S.D. for nine adult male subjects was 0.69 ± 0.16 ng/ml. Plasma 16a-P was significantly higher (p <0.01) during the luteal (1.20 ± 0.24 ng/ml) than during the follicular phase (0.57 ± 0.18 ng/ml).     

Gas chromatography coupled to mass spectrometry‐based metabolomic to screen for anabolic practices in cattle: identification of 5α‐androst‐2‐en‐17‐one as new biomarker of 4‐androstenedione misuse

The use of anabolic steroids as growth promoters for meat‐producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC‐MS aiming to reveal potential biomarkers signing a fraudulent administration of 4‐androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono‐conjugates urinary metabolites were acquired by GC‐MS in the full‐scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4‐AED. After a first evaluation of the signal robustness using additional and independent non‐compliant samples, 17 steroid‐like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC‐MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α‐androstane‐3β,17α‐diol and 5α‐androst‐2‐en‐17‐one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of oxocarboxylic and hydroxycarboxylic acids in normal individuals and ketoacidotic patients

Oxo- and hydroxycarboxylic acids as metabolites of valine, leucine, and isoleucine and of ketogenesis are simultaneously quantitated in the form of their methyl esters and methyl esters/O-methyloximes by gas chromatography using internal and external standards. Normal values for the urinary excretions of the amino acid metabolites are between 3 ± 2 μmol/24 h (mean value ± standard deviation) for 2-oxoisocaproic acid and 122 ± 58 μmol/24 h for 3-hydroxyisobutyric acid. In diabetic ketoacidosis the values are increased by a factor of 2-10.     

In vitro and in vivo metabolism studies of dimethazine

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti‐doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA^+/+‐SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called ‘Xtreme DMZ’. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC‐High Resolution Mass Spectrometry (HRMS) and GC‐MS(/MS) analysis methasterone and six other dimethazine metabolites (M1–M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro‐conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.     

In Vivo Metabolism Study of Timosaponin BIII in Rat Using HPLC-QTOF-MS/MS

Timosaponin BIII, as one of the steroid saponins isolated from Anemarrhena asphodeloides Bge., was proved to have many pharmacological activities in recent years and became a natural active compound with good development prospect. In the present study, a simple and rapid method using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry was developed for the determination of the structures of timosaponin BIII and its metabolites in rats after administrating intragastrically at 300 mg kg⁻¹. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent compound, nine metabolites were detected and identified in urine, and eight in plasma as well as four in brain. It is also indicated that the deglycosylation and oxidation reactions were the main metabolic pathways in the biotransformation of timosaponin BIII in vivo and the structures of the nine metabolites were identified and proposed to be timosaponin BII(M1), the hydroxylated metabolite of TBII(M2), the hydroxylated metabolites of TBIII(M3 and M4), deglycosylation and monooxygenation product of TBIII(M5), the deglycosylation product of TBII(M6), timosaponin AIII(M8), the isomers of timosaponin AIII(M7 and M9).