Semi-quantitative determination of designer steroids by high-performance liquid chromatography with ultraviolet detection in the absence of reference material.

New designer steroids are continually being encountered in dietary supplements that claim to increase muscle mass, but quantitative analysis of such ingredients is challenging due to the availability, quality, or cost of commercial reference materials. Although standard reference material typically becomes available for these emerging compounds, laboratories often face the challenge of finding properly certified materials from accredited suppliers, due to traceability requirements. Several of these designer steroids have been isolated and identified using multiple structural elucidation tools. Structural characteristics of these compounds of interest were evaluated and molar absorptivity data was collected and compared to several readily available steroid standards using ultraviolet/visible spectroscopy. This approach was used to find suitable compounds for use as surrogate reference materials in the semi-quantitative determination of two designer steroids, 1-dehydroepiandrosterone (1-androsterone) and 6ß-chloro-4-androsten-17ß-ol-3-one (6ß-chlorotestosterone). Laboratory-fortified matrix samples and dietary supplement samples were analyzed using this method for the estimation of 1-androsterone and 6ß-chlorotestosterone by HPLC-UV. Assay values obtained for the estimation of 1-androsterone in a dietary supplement sample using a prasterone or dehydroepiandrosterone (DHEA) standard curve were 100% of those obtained using a 1-androsterone reference standard, once it became commercially available. Estimations for 6ß-chlorotestosterone in laboratory-fortified matrix samples using a testosterone standard curve were 92%-93% of those obtained using isolated 6ß-chlorotestosterone as "reference material."

Optimization of DHEA Extraction from Sweet Potato Pomace by Ultrasonic-Microwave Synergistic Employing Response Surface Methodology.

Background: A lot of sweet potato residues (SPR) were discarded and wasted. Objective: To make full use of the SPR. Methods: Ultrasonic microwave synergistic (UMS) extraction method was used to extract dehydroepiandrosterone (DHEA) in SPR. The extraction conditions were optimized by response surface methodology based on single factors. Results: The optimum extraction conditions were 1:25 (solid-liquid ratio), 300 W (microwave power), 30 min (extraction time), and 30°C (extraction temperature). The extraction yield of DHEA from SPR reached 117.25 µg/100 g. Conclusions: The advantage of UMS extraction technology is to make full use of the synergistic effect of ultrasound and microwave to improve extraction efficiency. Highlights: The technology provides an effective way to improve the DHEA extraction yield from the SPR in industrial production.

The origin of 7alpha-hydroxy-dehydroepiandrosterone and its physiological role: a history of discoveries.

Nearly 60 years has elapsed since the first isolation and identification of 7alpha-hydroxy-dehydroepiandrosterone, and in that time much information has been gained on its occurrence, metabolism, ontogeny, immunomodulatory activity, cell proliferation, cortisol control in local tissues and neuroactivity. Additional knowledge about this steroid may elucidate its role in obesity, neurodegenerative disturbances such as Alzheimer's disease, or psychiatric disorders such as schizophrenia or depression. This review aims to provide a comprehensive summary of the available literature on 7alpha-hydroxy-dehydroepiandrosterone.

A direct assay for the routine measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone by liquid chromatography tandem mass spectrometry.

BACKGROUND: The measurement of androgens in many laboratories is often limited to testosterone. To more accurately determine the androgen status in both sexes, the measurement of other androgens such as dihydrotestosterone, the more potent metabolite of testosterone, and androstenendione and dehydroepiandrosterone, the most abundant circulating androgens in women would be informative. We report a combined liquid chromatography tandem mass spectrometry method for the measurement of these androgens. METHODS: Internal standards in methanol (10 µL) were added to 100 µL serum followed by the addition of zinc sulphate (100 µL). After mixing, 100 µL of acetonitrile was added and was further mixed. The samples were centrifuged and the steroids extracted using an automated online solid phase extraction on a C18 cartridge by a Waters Acquity with online sample manager coupled to a TQS mass spectrometer. RESULTS: Separation of the androgens was achieved by liquid chromatography. The run time was 6.5 min per sample. The lower limit of quantitation was 0.1 nmol/L for testosterone, androstenedione and dihydrotestosterone and 1 nmol/L for dehydroepiandrosterone. The coefficient of variation of the assay in serum for testosterone was <6%, androstenedione <8% and dihydrotestosterone and dehydroepiandrosterone <10%. DISCUSSION: We have developed a rapid assay for the liquid chromatography tandem mass spectrometry measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone in a routine clinical laboratory. The assay requires a small volume of serum, and all analytes are measured simultaneously. The assay is rapid and simple to execute offering the potential for routine clinical application.

Domestication Effects on Stress Induced Steroid Secretion and Adrenal Gene Expression in Chickens.

Understanding the genetic basis of phenotypic diversity is a challenge in contemporary biology. Domestication provides a model for unravelling aspects of the genetic basis of stress sensitivity. The ancestral Red Junglefowl (RJF) exhibits greater fear-related behaviour and a more pronounced HPA-axis reactivity than its domesticated counterpart, the White Leghorn (WL). By comparing hormones (plasmatic) and adrenal global gene transcription profiles between WL and RJF in response to an acute stress event, we investigated the molecular basis for the altered physiological stress responsiveness in domesticated chickens. Basal levels of pregnenolone and dehydroepiandrosterone as well as corticosterone response were lower in WL. Microarray analysis of gene expression in adrenal glands showed a significant breed effect in a large number of transcripts with over-representation of genes in the channel activity pathway. The expression of the best-known steroidogenesis genes were similar across the breeds used. Transcription levels of acute stress response genes such as StAR, CH25 and POMC were upregulated in response to acute stress. Dampened HPA reactivity in domesticated chickens was associated with changes in the expression of several genes that presents potentially minor regulatory effects rather than by means of change in expression of critical steroidogenic genes in the adrenal.

[Hydroxylation of steroids by Curvalaria lunata mycelium in the presence of methyl-beta-cyclodextrine].

Transformation of 16 delta5-3beta-hydroxy- and delta4-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-beta-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using specters of proton magnetic resonance and mass-specters, have been isolated. Hydroxylation of delta5-3beta-hydroxy-steroids occurred mostly in the C-7alpha position whereas hydroxylation of delta4-3-ketosteroids was in the C-11beta position. Only androst-4-en-3,17-dione, 9alpha-hydroxyl-androstenedione, and androsts-1,4-diene-3,17-dione were hydroxylated at C-14alpha position. Besides main 11beta-derivatives, the 6beta- and 7beta-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienon. The ratio of MCD to transforming steroid was 1 : 1 (mol/mol). Hydroxycortisone and 7alpha-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.

Gradient HPLC separation of dehydroepiandrosterone (DHEA) from its metabolites and biological congeners: role of tetrahydrofuran in the chromatographic mechanism.

A three-step gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the separation of dehydroepiandrosterone (DHEA), its sulfate ester (DHEA-S), its three C7-oxidized metabolites (7alphaOH-DHEA, 7betaOH-DHEA, 7-keto-DHEA), and its biosynthetic congeners (androstenedione, testosterone, estradiol, pregnenolone). This new method allows the quantitative characterization of DHEA metabolism and biosynthetic transformation under given physiological, pathological, or therapeutically influenced circumstances. Tetrahydrofuran probably acts as a proton acceptor coadsorbent, while isopropanol behaves as a proton donor during the separation of testosterone, estradiol, and the stereoisomers of 7-OH-DHEA.

Isolation and quantification by high-performance liquid chromatography-ion-trap mass spectrometry of androgen sulfoconjugates in human urine.

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography-ion-trap mass spectrometry (LC-MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5 microm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89-107%), repeatability (3.4-22%) and intermediate precision (5.8-22%) over the range of 2-200 ng/mL (corresponding to 1.5-147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

Simultaneous determination of dehydroepiandrosterone, its 7-hydroxylated metabolites, and their sulfates in rat brain tissues.

A method is described for simultaneous assessment of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and their 7-hydroxylated metabolites in cortex and subcortex of the rat brain. The procedure for determination of unconjugated steroids and DHEAS involved diethyl ether extraction of the homogenized tissue, solvent partition of the dry extract, and final quantification by specific radioimmunoassays. In addition, determination of 7-hydroxy-dehydroepiandrosterone sulfates required solvolysis, followed by high-performance liquid chromatography for separation of 7-hydroxylated metabolites from their precursor. The losses during this process were monitored by measurement of spiked radioactivity of [(3)H]testosterone or [(3)H]dehydroepiandrosterone sulfate. The content of dehydroepiandrosterone sulfate in both brain tissues was of the order of ten(s) nmol/g tissue irrespective its type (cortex or subcortex), while concentrations of other steroids were about 10 times lower in both tissues. In contrast to the ratio of sulfated/unconjugated DHEA, the levels of unconjugated 7-hydroxylated metabolites and their sulfates were close to each other. The reproducibility of the method with respect to coefficients of variation varied from 12 to 25%. An age-related decrease of sulfated dehydroepiandrosterone in the cortex of animals was also observed.

Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions.

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [(3)H]-labeled 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA substrates from 50 microM [1,2,6,7-(3)H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7 alpha-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2h/2 ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), more 7-oxo-DHEA than 7 alpha-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP(+), a mixture of 7 alpha-OH-DHEA, 7 beta-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.

Dehydroepiandrosterone metabolism in human plasma.

The possibility that dehydroepiandrosterone (DHEA) is metabolized in human plasma was studied by column and thin-layer chromatography. The results obtained indicate that a time-dependent disappearance of DHEA is matched by the appearance of newly-formed species that may represent DHEA conversion by-products. Neither disappearance of DHEA, nor formation of the alleged conversion by-products was observed when reactions were performed under conditions in which plasma enzymes were removed or inactivated. These results suggest that, in plasma, DHEA is partially transformed into different substances, and that the conversion reactions are catalyzed by enzymes present in this tissue. The observed kinetics of appearance and partial disappearance of the radiolabeled species can be interpreted as indicating that some of the by-products formed are further converted into other substances. The data shown appear to indicate that plasma can be added to the list of the already known compartments that are involved in steroid metabolism.

Augmentation of the pharmacological action of corydalis tuber by saussurea root in isolated mouse ileum.

To understand the meaning of blending crude drugs in Chinese medicinal prescriptions, the influence of Saussurea root on the pharmacological action of Corydalis tuber was examined. Saussurea root increased the depression of acetylcholine-induced contraction caused by the hot water extract solution of Corydalis tuber in mouse ileum at low dosage, which showed no direct influence on acetylcholine. Dehydrocostuslactone in Saussurea root was characterized as the component having increasing activity and the relationship between the concentration of acetylcholine and the variation in the contraction depressed by Corydalis tuber alone or a mixture of the Corydalis tuber and dehydrocostuslactone was investigated for clarification of the mode of action.

Physiological concentrations of DHEA in human hair.

In 1974, steroids were added to the list of doping agents banned by the International Olympic Committee because of their effects on the performance of the athletes. Dehydroepiandrosterone (DHEA) is a steroid hormone naturally produced by the adrenal glands and by the ovaries. DHEA can be converted into other hormones, including estrogen and testosterone. In the United States, DHEA is classified as a nutritional supplement. This is not the case in France, where the drug is listed as a doping agent. As athletes can abuse DHEA to benefit from its conversion to testosterone, there is a need to establish the physiological range of DHEA concentrations in human hair. DHEA was investigated in hair obtained from 27 control subjects, including 15 males and 12 females aged 17-42 years. After decontamination with dichloromethane, 100 mg of hair was incubated in 1 M NaOH in presence of 1 ng of testosterone-d3. After neutralization, the extract was purified using solid-phase extraction with Isolute C18 columns and subsequent liquid-liquid extraction with pentane. After silylation, DHEA was analyzed by gas chromatography-mass spectrometry. Results were linear in the range 1-20 pg/mg. Relative extraction recovery was 91.6% with a limit of detection of 0.5 pg/mg. Concentrations were in the range 1.2-6.7 pg/mg (mean value of 4.3 pg/mg) and 0.5 to 10.6 pg/mg (mean value of 5.3 pg/mg) for the males and females, respectively. Extensive chromatographic procedures (two purification steps by solid-phase and liquid-liquid extraction, combined with injection of 4 microL through the column in pulsed mode) were analytical prerequisites for successful identification of DHEA in hair because of the low target concentrations. This new technology may find useful applications in anabolic abuse control.

Evaluation and separation of steroid-bovine serum albumin conjugates by high-performance liquid chromatography.

The conventional methods for characterization of steroid immunogens are based on the determination of the total amount of hapten bound to the protein carrier either by the UV spectroscopy or titration of unsubstituted amino groups. These methods do not allow more detailed insight into the immunogen composition. HPLC of the immunogen combined with UV detection is a relatively rapid and convenient method enabling determination of the hapten content in each fraction and, eventually, separation of individual fractions differing in the hapten content or purification of crude product.

Analysis of steroids Part 50. Derivatization of ketosteroids for their separation and determination by capillary electrophoresis.

4-Ene-3-ketosteroids and 17-ketosteroids were quantitatively transformed into the corresponding hydrazones using Girard P and T reagents, respectively. The positively charged derivatives were separated by capillary electrophoresis. The spectrophotometric characteristics of the derivatives permitted their sensitive detection in the 230-280 nm range. The steroids investigated included nortestosterone and its phenylpropionate, norethisterone and its oenanthate, d,l-norgestrel, dehydroepiandrosterone, androstenedione and ethisterone.

Metabolism of dehydroepiandrosterone by cultured human adipose stromal cells: identification of 7 alpha-hydroxydehydroepiandrosterone as a major metabolite using high performance liquid chromatography and mass spectrometry.

Studies of the metabolism of dehydroepiandrosterone (DHA) by cultured human adipose stromal cells revealed that the most abundant metabolite detected by HPLC was a polar compound accounting for up to 45% of total radioactivity. This metabolite was isolated by chromatography on Lipidex 5000 from the culture medium of breast adipose stromal cells cultured with unlabelled DHA (5 microM) and identified by combined capillary gas chromatography and mass spectrometry as 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA). In breast adipose stromal cells, the conversion of DHA to 7 alpha-OHDHA was linear from a substrate concentration of 10 nM to 1 microM. At 1 microM substrate concentration, the formation of 7 alpha-OHDHA in four patients ranged from 6.1 to 22.5 ng/10(5) cells/24 h. Incubations carried out in primary culture and up to the fifth subculture revealed continued formation of 7 alpha-OHDHA. Adipose stromal cells from abdomen, flank and perinephric fat also produced 7 alpha-OHDHA from DHA. These studies have shown that 7 alpha-OHDHA is a major metabolite of DHA in human adipose stromal cells. The variability from patient to patient and the magnitude of this conversion suggests that this pathway may play an important role in the peripheral metabolism of DHA.

A new reversed phase, paired ion thin-layer chromatographic method for steroid sulfate separations.

The sulfoconjugated steroids estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were separated in the reversed phase mode on polyamide-coated TLC plates. Baseline resolution was obtained between tritiated ES and DS standards when run with a mobile phase of 20% acetonitrile in 5mM aqueous triethylamine, triethanolamine, tris-hydroxymethylaminomethane, tributylamine or ammonia. ES and DS showed no mobility in the absence of an ion-pair reagent. The radioactive peaks were detected and integrated non-destructively by scanning. Quantitation was confirmed by elution of cut-out peak areas and liquid scintillation counting. Similar results were obtained with washed ethanol extracts of serum labeled with tritiated ES and DS. The extracts were defatted on the plate with hexane: ethyl acetate (1:1) prior to the reversed phase development.

Isolation and identification of steroids from gonadal vein blood of the fetal horse.

Direct connection of the artery of a fetal ovary to the carotid artery of the mare allowed collection of a large volume of blood over a 30-min period. Extraction of steroids and their fractionation was followed by separation of the steroids by alumina adsorption chromatography, and Sephadex LH-20 and Celite partition chromatography. Further resolution of the material by HPLC led to the identification of dehydroepiandrosterone (DHA) by nuclear magnetic resonance and mass spectrometry. Other compounds were isolated, which remain to be identified fully, but in the 8th month of pregnancy the principal steroid secreted by the fetal ovary was apparently DHA.

Separation of androsterone from epiandrosterone, and dehydroepiandrosterone from its 3-hydroxy epimer by thin-layer chromatography.

The application of thin-layer chromatography to the separation of 13 steroids, including androstanes, 4-androstenes and 5-androstenes, using silica gel and 1,2-propanediol-impregnated cellulose is described. After group-wise separation of various C19 steroids on silica gel, the 3-hydroxy epimers of 5alpha-androstanes and 5-androstenes can be separated by thin-layer chromatography on impregnated cellulose plates. The chromatographic procedure is rapid and makes the prior formation of steroid derivatives unnecessary.

Dehydroepiandrosterone sulfate in plasma: hydrolysis, extraction and radioimmunoassay.

At pH 4.5, the hydrolysis of 3beta-hydroxy-5-androsten-17-one 3-sulfate (DHA-SO4) to DHA was complete within 75 min at 120 degrees or 4h at 100 degrees. In the same conditions, the 3alpha-SO4 of androsterone was stable, and only 8.5% of the 3beta-SO4 of epiandrosterone hydrolysed to epiandrosterone. Of the sulfates of 5-androstene-3beta, 17beta-diol, 100% of the 3beta-mono-SO4, 2% OF THE 17BETA-MONO-SO4 and none of the 3beta, 17beta-di-SO4 was converted to 5-androstenediol. Denatured plasma proteins adsorbed DHA. The recovery of DHA from plasma diluted 1:100, 58.7 +/- 6.2% (mean +/- S.D.). In similar conditions the recovery of cholesterol from plasma diluted 1:20, was 0.12 - 1.76% (mean, 0.44%). A radioimmunoassay for DHA in extracts of hydrolysed plasma is described. Results for normal subjects in the age range 17-45y were 192 +/- 73mug/dl (22 men) and 158 +/- 57mug/dl (40 women).